SYSTEMATICS, BIOLOGY, AND BEHAVIOR OF FRUIT-PIERCING AND BLOOD-FEEDING MOTHS IN THE SUBFAMILY CALPINAE (LEPIDOPTERA: NOCTUIDAE)
By
JENNIFER MICHELLE ZASPEL
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2008
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© 2008 Jennifer M. Zaspel
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To Dr. Hans Bänziger for assistance with this project and for his discovery of blood-feeding moths in the genus Calyptra.
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ACKNOWLEDGMENTS
First and foremost, I thank my advisor and chair of my graduate committee, Dr. Marc A.
Branham and the members of my graduate committee, Dr. Marjorie A. Hoy, Dr. Jacqueline
Miller, and Dr. David Reed for their professional advice, scientific guidance, and financial
support. I also thank Dr. Hans Bänziger and Michael Fibiger for many helpful discussions about
Calyptra. I would like to thank Drs. A. Jeyaprakash and J. Meyer for their technical advice and
laboratory training in molecular biology.
Vladimiar S. Kononenko was instrumental in organizing the expeditions to far eastern
Russia and for the acquisition of the specimens used in several studies in my dissertation. I
would also like to thank my field guide on both expeditions in Russia, Boris Popkov, the staff of
the Hunting Area, and the research scientists at Gornotayeznaya Biological Station. I also
greatly appreciate the assistance of Ms. Valentina Kolesnikova from the Russian Academy of
Sciences Far Eastern Branch for her assistance in obtaining permits for collecting. I also thank
Susan Weller and Harald Krenn for suggestions on the comparative mouthpart survey of calpine
noctuids (Chapter 2); Hans Bänziger, Roland Hilgartner, and Harry Fay kindly provided adult
feeding images figured in the chapter. Drs. R. Rougerie, M. Hajibabaei, D. Janzen, W.
Hallwachs, and P. Hebert assistanced in obtaining barcode sequences for some taxa used in the
molecular phylogenetic study. I also acknowledge all individuals and institutions listed in
Chapter 3 for their assistance in obtaining specimens for the morphological and molecular
phylogenetic study. I gratefully acknowledge the Florida Department of Agriculture, Division of
Plant Industries, Gainesville, FL for the use of their SEM and Dr. Paul Skelly for his assistance
with the imaging equipment as well as Branden Apitz for assistance with SEM and figure
formatting. The following people are acknowledged for their assistance in data collection for
chapter 7: M.E. Sharf and his graduate students for the use of the QT-RT PCR machine in his
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laboratory, Paul Shirk for supplying specimens, and D.G. Boucias for the use of his
spectrophotometer. Finally, I would like to thank past and present members of Branham, Hoy,
McGuire Center, Reed, and Weller laboratories over the years for their support and numerous
scientific discussions.
This work would not have been possible without the support of my family and their
encouragement throughout my graduate studies. This work was supported in part by the
American Philosophical Society, Davies, Fischer, and Eckes Endowment in Biological Control
to Marjorie A. Hoy at the University of Florida, Explorer’s Club, Florida Entomological Society,
National Geographic Society’s Fund for Research and Exploration, National Park Service
Inventories and Monitoring Program, National Science Foundation (DDIG, DEB-0807975),
Systematics Research Fund, and the Vam York Scholarship Fund.
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TABLE OF CONTENTS page
ACKNOWLEDGMENTS ...............................................................................................................4
LIST OF TABLES...........................................................................................................................9
LIST OF FIGURES .......................................................................................................................11
ABSTRACT...................................................................................................................................13
CHAPTER
1 INTRODUCTION ..................................................................................................................15
Literature Review ...................................................................................................................15
Systematics of Calpini .....................................................................................................18Overview of Fruit-Piercing and Blood-Feeding Moths in Tribe Calpini ........................15
Proboscis morphlogy in piercing moths ..........................................................................20
Blood feeding and endosymbionts in Calpini .................................................................21
Research Objectives................................................................................................................24
2 A COMPARATIVE SURVEY OF PROBOSCIS MORPHOLOGY AND ASSOCIATED STRUCTURES IN FRUIT-PIERCING AND BLOOD-FEEDING MOTHS IN THE SUBFAMILY CALPINAE (LEPIDOPTERA: NOCTUIDAE) ...............31
Introduction.............................................................................................................................31
Terminology ....................................................................................................................34Materials and Methods ...........................................................................................................34
Characterization of Functional Feeding Catergories.......................................................35
Specimen Preparation......................................................................................................39
Results.....................................................................................................................................41
Description of the piercing structures of the proboscis visible by light microscopy ......41
Description of the structures visible by scanning electron microscopy ..........................43
Discussion...............................................................................................................................51
3 RECONSTRUCTING THE EVOLUTIONARY RELATIONSHPS OF THE VAMPIRE MOTHS AND THEIR FRUIT-PIERCING RELATIVES USING MORPHOLOGICAL AND MOLECULAR DATA (LEPIDOPTERA: NOCTUIDAE: CALPINAE: CALPINI) .........................................................................................................74
Introduction.............................................................................................................................74
Taxon Sampling...............................................................................................................76Materials and Methods ...........................................................................................................76
Morphological Data.........................................................................................................78
Molecular Data ................................................................................................................80
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Phylogenetic Analyses.....................................................................................................81
Results and Discussion ...........................................................................................................83Evolution of Feeding Behaviors and Complementary Analyses.....................................82
Summary of Morphological Character Variation............................................................83
Phylogenetic Analysis of Morphological Data................................................................88
Molecular Data and Combined Analyses ........................................................................89
Evolution of Feeding Behaviors and Complementary Analysis .....................................91
Conclusions.............................................................................................................................92
4 WORLD CHECKLIST OF TRIBE CALPINI (LEPIDOPTERA: NOCTUIDAE: CALPINAE) .........................................................................................................................125
Introduction...........................................................................................................................125
Calpini ...........................................................................................................................127Checklist ...............................................................................................................................127
Genus and tribal placement undetermined ....................................................................140
5 ANOTHER BLOOD FEEDER? EXPERIMENTAL FEEDING OF A FRUIT-PIERCING MOTH SPECIES ON HUMAN BLOOD IN THE PRIMORYE TERRITORY OF FAR EASTERN RUSSIA (LEPIDOPTERA: NOCTUIDAE: CALPINAE) .........................................................................................................................141
Introduction...........................................................................................................................141
Description of observation sites ....................................................................................142Materials and Methods .........................................................................................................142
Experimental methods ...................................................................................................144
Results...................................................................................................................................145
Discussion.............................................................................................................................148
5 MICROBIAL DIVERSITY ASSOCIATED WITH THE FRUIT-PIERCING AND BLOOD-FEEDING MOTH Calyptra thalictri (LEPIDOPTERA: NOCTUIDAE: CALPINAE) .........................................................................................................................164
Introduction...........................................................................................................................164
Specimens......................................................................................................................165Materials and Methods .........................................................................................................165
Surface Sterilization ......................................................................................................166
DNA Extraction.............................................................................................................167
High Fidelity Polymerase Chain Reaction ....................................................................168
Cloning and Restriction Fragment Length Polymorphism Analysis.............................168
Results and Discussion .........................................................................................................169
High-fidelity PCR Amplification of Microbial Associates in C. thalictri ....................169
Microbial Associates of C. thalictri...............................................................................170
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7 COMPARISON OF SHORT-TERM PRESERVATION AND ASSAY METHODS FOR THE MOLECULAR DETECTION OF WOLBACHIA IN THE MEDITERRANEAN FLOUR MOTH EPHESTIA KUEHNIELLA.....................................177
Scientific Note ......................................................................................................................177
Results...................................................................................................................................181Materials and Methods .........................................................................................................178
Discussion.............................................................................................................................182
8 PERSPECTIVES ..................................................................................................................186
APPENDIX
A DATA MATRIX USED TO PRODUCED TREES BASED ON MORPHOLOGICAL DATA ...................................................................................................................................191
B DATA MATRIX USED TO PRODUCE TREES BASED ON MORPHOLOGICAL AND MOLECULAR DATA................................................................................................194
LIST OF REFERENCES.............................................................................................................214
BIOGRAPHICAL SKETCH .......................................................................................................239
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LIST OF TABLES
Table page 2-1 Sensilla and other structures associated with calpine proboscides and their proposed
function ..............................................................................................................................54
2-2 Specimens examined A.....................................................................................................55
3-1 Specimens examined B. .....................................................................................................95
3-2 PCR conditions and sequences of primers used. .............................................................100
3-3 Known feeding behavior reports for Calpini and related genera included in complementary analyses ..................................................................................................101
3-4 Character support for major clades. .................................................................................104
5-1 Summary of feeding behaviors for moth specimens collected in Primorye Terriotry of Far Eastern Russia from July 14t-15th 2006 and July 17-20th 2006. ...........................151
6-1 Original and phylotype-specific forward and reverse primers designed to detect microbial sequences in DNA isolated from C. thalictri. .................................................175
6-2 Pairwise sequence divergences (uncorrected p) between eubacterial phylotypes from C. thalictri and closely related 16S rRNA sequences using 1410-1504 bp of sequences. ........................................................................................................................176
7-1 Summary of RTQ PCR and spectrophotometry data for E. kuehniella specimens stored for a 2-yr. period compared to fresh specimens. ...................................................184
2-1 Sensilla and other structures associated with calpine proboscides and their proposed function ..............................................................................................................................54
2-2 Specimens examined A.....................................................................................................55
3-1 Specimens examined B. .....................................................................................................95
3-2 PCR conditions and sequences of primers used. .............................................................100
3-3 Known feeding behavior reports for Calpini and related genera included in complementary analyses ..................................................................................................101
3-4 Character support for major clades. .................................................................................104
5-1 Summary of feeding behaviors for moth specimens collected in Primorye Terriotry of Far Eastern Russia from July 14t-15th 2006 and July 17-20th 2006. ...........................151
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6-1 Original and phylotype-specific forward and reverse primers designed to detect microbial sequences in DNA isolated from C. thalictri. .................................................175
6-2 Pairwise sequence divergences (uncorrected p) between eubacterial phylotypes from C. thalictri and closely related 16S rRNA sequences using 1410-1504 bp of sequences. ........................................................................................................................176
7-1 Summary of RTQ PCR and spectrophotometry data for E. kuehniella specimens stored for a 2-yr. period compared to fresh specimens. ...................................................184
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LIST OF FIGURES
Figure page 1-1 Calyptra thalictri male feeding on human thumb (JMZ) in Russia 2008..........................30
2-1 Feeding behaviors of adult moths in the subfamily Calpinae............................................58
2-2 Description of proboscis regions; Oraesia rectistria.........................................................59
2-3 Examples of proboscis structures visible by light microscopy in selected feeding categories. ..........................................................................................................................60
2-4 Proboscis of non-calpine classical tear feeder ...................................................................61
2-5 Proboscides of taxa in the non-piercing fruit-sucking group.............................................62
2-6 Examples of proboscis structures visible by scanning light microscopy in primary piercers of thick-skinned fruit but secondary piercers of hard-skinned fruit.....................63
2-7 Examples of proboscis structures visible by scanning light microscopy in primary piercers of thick-skinned fruit but secondary piercers of hard-skinned fruit II. ................66
2-8 Examples of proboscis structures visible by scanning light microscopy in primary piercers of thick-skinned fruit but secondary piercers of hard-skinned fruit III................68
2-9 Proboscis of taxa in the primary piercing of hard-skinned fruits group. ...........................69
2-10 Proboscides of taxa in the mammalian skin-piercing and blood-feeding group................70
2-11 Proboscides of taxa in the mammalian skin-piercing and blood-feeding group II ............71
2-12 Proboscides of taxa in the tear-drinking group. .................................................................72
2-13 Uncertain taxa. ...................................................................................................................73
3-1 Blood-feeding moth, Calyptra thalictri. ..........................................................................106
3-2 Proposed hypotheses for the evolution of tear feeding and blood feeding within Lepidoptera. .....................................................................................................................107
3-3 Shape of labial palp segment II, Character 17 .................................................................108
3-4 Shape of labial palp segment III, Character 18................................................................109
3-5 Shape of saccular process (entire), Character 22 .............................................................110
3-6 Shape of saccular process = SaP (branched), Sa = saccus, Character 23 ........................111
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3-8 Shape of saccus, Character 27 .........................................................................................113
3-9 Shape of dorsal tegumen, Character 37 ...........................................................................114
3-10 Shape of uncus base 38 ....................................................................................................115
3-11 Shape of the posterior edge of the antevaginal plate (segment VII)................................116
3-12 Shape of the posterior edge of segment VIII, Character 46.............................................117
3-13 Shape of cervical sclerites of the corpus bursa, Character 50..........................................118
3-14 Shape of the corpus bursa, Character 52..........................................................................119
3-15 Shape of appendix bursa (AB), Character 60 ..................................................................120
3-17 Evolution of adult feeding behaviors in Calpini. .............................................................122
3-18 Preliminary strict consensus tree of six most parsimonius rearrangements for 34 taxa based on combined data set..............................................................................................123
3-19 Preliminary Bayesian analysis resulting from a simultaneous analysis of all data partitions for 34 taxa. .......................................................................................................124
5-1 Adult habitus image. Calyptra thalictri, Male................................................................153
5-2 Adult habitus image. Calyptra lata, Male. .....................................................................154
5-3 Adult habitus image. Calyptra hokkaida, Male. .............................................................155
5-4 Proboscis of Calyptra thalictri: TH = Tearing Hooks. ....................................................156
5-5 Map of Primorye Region of Far Eastern Russia. .............................................................157
5-6 Primary collecting site 1. ................................................................................................158
5-7 Primary collecting site 2. .................................................................................................159
5-8 Map of primary collecting site 1. ....................................................................................160
5-9 Map of primary collecting site 2. ....................................................................................161
5-10 Image of Calyptra thalictri feeding on human thumb (JMZ)..........................................162
5-10 Calyptra thalictri feeding on raspberry during night observations. ................................163
7-1 Examination of DNA preservation and amplification by HF PCR of the wspA gene fragment (605 bp) in E. kuehniella. .................................................................................185
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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
SYSTEMATICS, BIOLOGY, AND BEHAVIOR OF FRUIT-PIERCING AND
BLOOD-FEEDING MOTHS IN THE SUBFAMILY CALPINAE (LEPIDOPTERA: NOCTUIDAE)
By
Jennifer Michelle Zaspel
December 2008
Chair: Marc A. Branham Major: Entomology and Nematology
A phylogenetic review of the fruit-piercing and blood-feeding tribe Calpini [Lepidoptera:
Noctuidae: Calpinae] was conducted to determine the evolutionary relationships among the
genera. The evolution of feeding behaviors was investigated by using the resulting phylogeny.
A set of morphological characters was compiled and included characters from the head,
appendages, male and female genitalia. Structures associated with the proboscis were examined
in these moths using both light microscopy and SEM methods. A morphological data matrix
with 66 characters was compiled for 65 taxa. A reduced molecular data set includes nearly
complete sequences of the cytochrome oxidase I mitochondrial gene and a fragment of the
nuclear large subunit 28S rRNA for 34 taxa. Phylogenetic trees of separate and combined data
sets were constructed using parsimony and Bayesian analyses. Binary feeding behavior
characters were coded for all taxa in the morphological matrix and mapped onto the resulting
topology using parsimony optimizations. Results from the analysis based on morphological data
suggest Calpini is monophyletic and is supported by five synapomorphies. Three of these are
unreversed, shared characters of the proboscis. Tearing hooks are restricted to the Calpini and
little additional variation within the tribe exists suggesting proboscis morphology may not be
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strongly correlated with feeding behavior. The results from this study support the hypothesis
that hematophagy in the genus Calyptra evolved from the fruit-piercing habit as opposed to tear
feeding. A preliminary checklist of Calpini is also provided, incorporating corrections and
changes to publication dates and nomenclature as presented in recent checklists.
A microbial survey of Calyptra thalictri Borkhausen using polymerase chain reaction
[PCR] primers for 16S rRNA sequences for Eubacteria. High-fidelity PCR and subsequent DNA
analyses indicated that at least five microorganisms belonging to the α, β, and γ Proteobacteria
were present. Two eubacterial sequences, related to a Klebsiella sp. and a Sinorhizobium sp.,
were detected in the abdomens individuals sampled, and three additional sequences were found
in some samples, suggesting all five could be associated with abdominal structures. No Archaea,
Fungi including yeast-like organisms, Microsporidia, or Wolbachia were detected. These results
document the first microbial associates in a fruit-piercing and blood-feeding moth.
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CHAPTER 1 INTRODUCTION
Literature Review
This chapter reviews the basic biology, adult feeding behavior, and proboscis
morphology of fruit-piercing and blood-feeding moths with an emphasis on moths in the tribe
Calpini. The present state of the taxonomy and classification of Calpini is summarized and
hypotheses of the evolution of feeding behaviors within the tribe are discussed. Finally, the
significance of the diversity and importance of insects and their microbial associations is
discussed, including what is known about Lepidoptera and their microbial associates. The final
portion of this chapter introduces my research objectives, hypotheses, and how they were tested.
Overview of Fruit-Piercing and Blood-Feeding Moths in Tribe Calpini
The introduction of pest species to the United States from 1906 to 1991 has resulted in
the loss of approximately 97 billion dollars (Pimentel et al. 2005). Additional costs are
associated with controlling the pests after they have become established. Due to recent efforts to
manage agricultural pests in a way that is environmentally responsible, greater emphasis has
been placed on biological control and sustainable agricultural methods (Miller and Rossman
1995). Unfortunately some programs have been unsuccessful due to a paucity of information
about the life history, taxonomy, evolutionary relationships, and basic biology of the target pests.
When fundamental information about the systematics and behavior of a group of pest organisms
is lacking, biological control efforts and agricultural system development may fail, thereby
wasting time and precious research dollars. Successful control programs require basic research
on the pest and natural enemies so that problems with species identification, undescribed species,
polymorphic species, and cryptic species can be overcome (Debach and Rosen 1991). When
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coupled with phylogenetic information, biosystematic studies of pest organisms can provide
predictive power for identifying invasive pests and potentially enhance control efficiency.
Many moth species in the insect order Lepidoptera are serious pests of crops. The largest
moth family, Noctuidae, comprises 45,000 species (Poole 1989, Holloway et al. 2001), and
includes many introduced pest species in the U.S. such as cutworms (Agrotis, Prodenia, and
Euxoa), corn earworms and budworms (Helicoverpa), and armyworms (Pseudaletia). Although
much attention has been paid to the taxonomy and systematics of these moth pests and their
control, negligible research has been directed at the systematics of a subgroup of these moths that
are pests of agriculture, and potentially, medical importance during the adult stage: the fruit
piercers and blood feeders.
Within the order Lepidoptera, the ability to pierce mammalian skin and take a blood meal
is restricted to the moth genus Calyptra Ochsenheimer (Lepidoptera: Noctuidae: Calpinae)
(Bänziger 1968-2007, Zaspel et al. 2007, Zaspel 2008). Blood-feeding moths are subcutaneous
pool-feeders, severing the capillaries below the surface of the skin in order to form a pool of
blood from which they can feed (Bänziger 1971, 1980). The moths insert the tip of the proboscis
into the host, cutting the tissues by moving the two straw-like tubes, or galae, of their proboscis
back and forth in opposing directions; this movement has been termed “antiparallel motion of the
proboscis” (Bänziger 1971, 1980). This saw-like movement is often performed at short intervals
followed by intermittent uptake of the blood meal. Blood-feeding moths can feed on all parts of
the animals for ten minutes or longer (Zaspel et al. 2007). Penetration of the host skin by the
moth’s barbed proboscis can be quite painful, and the resulting wound(s) are large in diameter
when compared to the wound of a mosquito bite or bee sting (Zaspel, Chapter 5). The first
species recorded piercing the skin of a mammal and feeding on its blood was Calyptra eustrigata
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Hampson; it was discovered feeding on water buffalo, deer, tapir, and antelope in Malaysia by
Bänziger (1968). Since the description of the first blood-feeding Calyptra species, males of ten
additional Calyptra species have been observed piercing mammalian skin and feeding on blood
under both laboratory and natural conditions (Bänziger 1989, Bänziger 2007, Zaspel et al. 2007).
While fruit feeding is obligatory in both males and females of Calyptra (Bänziger 2007), blood
feeding is restricted to males and is facultative; they are able to survive without ingestion of a
blood meal (Bänziger 2007). Males of Calyptra have been recorded feeding on a broad range of
ungulate hosts and occasionally elephants and humans are attacked (Bänziger 2007, Zaspel et al.
2007).
It has been hypothesized that the ability to pierce mammalian skin and suck blood in
Calyptra spp. evolved from the fruit-piercing habit, a likely scenario given fruit-piercing and
blood-feeding moths share the proboscis and behavioral modifications observed in both feeding
types (Bänziger 2007). It is possible that the moths may seek out mammalian hosts to obtain
additional nutrients such as amino acids or sugars, thereby increasing fitness (Bänziger 2007).
Blood-feeding Calyptra males have not tested positive for proteases; however, male moths do
appear to be in search of salts (Bänziger 2007). It is possible that males are sequestering salts
and transferring them to the females during mating for egg production (Smedley and Eisner
1995) or to replenish salt supplies depleted during oviposition (Adler and Pearson 1982).
Alternative hypotheses regarding the evolution of feeding in Calyptra have been
proposed (Downes 1973, Hilgartner et al. 2007) and suggest the skin-piercing and blood-feeding
behavior is derived from other animal-associated feeding behaviors such as dung, urine, or tear
feeding. Although one Calyptra species, C. minuticornis (Guenée), has been recorded feeding
on tears (Bänziger 2007), this evolutionary trajectory is an unlikely one given the proboscis
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structures of fruit piercers and blood feeders are not homologous with those of tear-feeding
moths, and such lachrophagous moths do not pierce fruit (Bänziger 2007). This hypothesis is
also problematic given the shared behavioral modifications found in both fruit-piercing and
blood-feeding moths. The tearing structures involved in the piercing of fruits or mammalian skin
is restricted to a small group of taxonomically associated noctuid genera (Zaspel, Chapter 3),
while animal-associated feeding behaviors, including the imbibing of blood droplets found on
the bodies of mammals, are widespread within Lepidoptera (Bänziger 1982, Scoble 1992).
These hypotheses have never been tested within an empirical phylogenetic framework, and a
hypothesized directional progression of feeding types cannot be tested formally until the
relationships of Calyptra and related genera are known.
Calyptra consists of 17 described species and two subspecies (Bänziger 1983). These are
medium-sized moths, with wingspans ranging from 35-72 mm in size. These moths typically fly
during the rainy season in the tropics and in the midsummer months in temperate regions. Eggs
are typically laid on the underside of the leaves of host plants in the Menispermaceae and the
Ranunculaceae (Bänziger 1982, Fay 2005, Zaspel pers. observation). All Calyptra species have
modified proboscides equipped with strongly sclerotized tearing hooks used for piercing the skin
of hard fruits such as peaches and citrus, and of mammals, the number of apical tearing hooks of
the proboscis varies between species and among specimens (Zaspel, Chapter 2). This variation
does not appear to be in any way associated with one piercing behavior or the other (e.g., sucking
fruit juices versus sucking blood) (Zaspel, Chapter 2).
Systematics of Calpini
The extent to which invasive species impact the environment is difficult to assess when
knowledge regarding their biodiversity in the U.S. is lacking (Pimentel et al. 2005). A major aim
of systematics is to describe and organize this diversity into a classification scheme that is
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hierarchical, providing stability of species names that reflect history, and providing predictive
power of relevance to biological control and pest management programs. The most recent
classifications place Calyptra and other fruit-piercing genera in the subfamily Calpinae (Kitching
and Rawlins 1998, Fibiger and Lafontaine 2005, Lafontaine and Fibiger 2006, Mitchell et al.
2006). Presently, Calpinae consists of four tribes: Anomini Grote 1882, Calpini Boisduval 1840,
Phyllodini Hampson 1913, and Scoliopterygini Herrich-Schäffer [1852] (Fibiger and Lafontaine
2005, Lafontaine and Fibiger 2006, Holloway 2005). The tribe was catalogued by Fibiger and
Lafontaine (2005) wherein they assigned genera to the tribe, suggesting Calpini is comprised of
eleven genera. Fibiger and Lafontaine (2005) focused on the Palearctic region and therefore was
not inclusive of all genera comprising the tribe. In addition, the fruit-piercing genus Oraesia,
which has proboscis armature identical to that of Calyptra and Gonodonta (Zaspel, Chapter 2),
was excluded while seven genera lacking the diagnostic characteristics of the proboscis were
included. All genera in Calpini contain fruit-piercing species (Fibiger and Lafontaine 2005,
Holloway 2005), and a high concentration of economically important fruit-piercing species is
found within the tribe.
The tribe Calpini is cosmopolitan in its distribution; however, many calpine genera have
geographic distributions that are more restricted. The genus Calyptra is considered to be Old
World in its distribution with a high concentration of diversity in South and Southeast Asia, yet
one species, C. canadensis, occurs in the northeastern United States and Canada (Poole 1989,
Bänziger 1989a, Fibiger and Lafontaine 2005). In the Old World tropics, species of Eudocima
are common pests of hard-skinned fruits (e.g., longan), with the widespread E. fullonia (Clerck)
the target of a biological control project in the region (Fay 2002, Sands and Liebregts 2005).
Gonodonta species can be found in subtropical and tropical regions, with seven species occurring
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in Florida, Texas, and Arizona; Gonodonta species pierce citrus fruits, including tangerines,
grapefruits and oranges, at times causing extensive losses in subtropical and tropical regions
(Todd 1959). Serious outbreaks of Gonodonta species occurred in Mexico and Cuba in the
1940’s and 1950’s, respectively, and one report from the late 1950’s stated that 20 percent of the
fruit in two orange groves in St. Lucie County, Florida was lost due to outbreaks of G. nutrix
(Todd 1959). Oraesia and Plusiodonta species are common in the Old and New World tropics
(Poole 1989, Holloway et al. 2001). Plusiodonta species have been observed feeding on fruits
such as plum in South and Southeast Asia (Zaspel pers. obs. 2005). Adult Oraesia species have
reportedly damaged thick-skinned fruit in Korea (Yoon and Lee 1974). Calpine larvae primarily
feed on plants in the family Menispermacae. Although calpine larvae are not agricultural pests,
all adult moths are piercers of fruits (Bänziger 1982, Holloway et al. 2001); punctures made in
fruits by the moths cause agricultural loss through fermentation and rotting of the fruit or
secondary invasions by microorganisms that result in early fruit fall (Todd 1959, Sands 1993).
Proboscis morphlogy in piercing moths
The structure and function of the lepidopteran proboscis has been examined for a broad
range of taxa across multiple families (Bänziger 1971, 1973, Büttiker et al. 1996, Krenn 1990,
Krenn 1997, Speidel et al. 1996). Previous work utilized light microscopy to evaluate proboscis
structures (Hattori 1969, Bänziger 1970, 1973, 1980) followed by scanning electron microscopy
(SEM) (Cochereau 1977, Büttiker et al. 1996, Speidel et al. 1996). Taxon sampling for these
studies ranged from higher-level exemplars from distantly related lepidopteran families (e.g.,
Noctuidae, Geometridae, and Pyralidae) based on adult feeding behavior (Bänziger 1973,
Büttiker et al. 1996) to more focused proboscis studies on related species (Bänziger 1970, 1986),
subfamilies (Speidel et al. 1996), and families (Krenn 1998, Krenn and Kristensen 2000, Krenn
and Mühlberger 2002, Krenn and Penz 1998, Krenn et al. 2001). From this work, several
20
notable differences in proboscis morphology were observed, including a wide variety of sensilla
types and other specialized feeding structures (Bänziger 1970, 1973, Büttiker et al. 1996, Speidel
et al. 1996). Uniquely specialized proboscis structures (cutting ridges, erectile barbs and
eversible tearing hooks moved by blood-pressure) and concomitant stylet dynamics (anti-parallel
movements, oscillatory torsion, spindle movements) occur in the piercing moths in the family
Noctuidae (Künckel 1875, Breitenbach 1877, Bänziger 1970, 1973, 1980). Within the Calpinae,
the tearing hooks are unique to a tribe of apparently closely related fruit-piercing moths
consisting of at least nine genera: Africalpe Krüger, Calyptra Ochsenheimer, Eudocima Billberg,
Ferenta Walker, Gonodonta Hübner, Graphigona Walker, Oraesia Guenée, Plusiodonta
Guenée, and Tetrisia Walker (Zaspel, Chapter 3). The tearing structures observed in these
genera arise from a socket and thus are not homologous with other forms of modified sensilla of
the proboscis found among fruit-sucking and tear-drinking moths within the Lepidoptera (Zaspel,
Chapter 2).
Blood feeding and endosymbionts in Calpini
Hematophagy is believed to have arisen independently in arthropods during the Jurassic
and Cretaceous periods at least six, and potentially as many as 21 times (Balashov 1984, Ribeiro
1995). Adams (1999) estimates that 14,000 insect species from five orders (Pthiraptera, Diptera,
Hemiptera, Lepidoptera, and Siphonaptera,) are hematophagous. All blood-feeding arthropods
are capable of transmitting pathogens (Durden and Mullen 2002). Arthropod-transmitted
diseases are of great medical and veterinary significance worldwide, and new pathogens are
rapidly emerging (Gratz 1999). At least 27 major human and animal diseases are vectored by
insects (Faust et al. 1962, Gratz 1999). It is known that many fruit-piercing Calyptra species and
species in related genera are of great economic importance in many countries, but their potential
as vectors of human or animal disease remains unknown. Many associations between insects
21
and microorganisms that are considered ‘significant’ are often involved in animal and plant
disease transmission (Daly et al. 1998). Symbiotic relationships are defined as an organism
living on or in another organism, from which nutrients, protection, and assistance in the
metabolism of various biological compounds are either obtained or exchanged (Bourtzis and
Miller 2006). Mutualistic symbiotic relationships involve the dependence of each organism in
the system on one another for survival. In some cases, this mutualism will result in the
production of unique structures and novel metabolisms in an arthropod host. In many insect
species, endosymbiotic bacteria and even some eukaryotic microorganisms (e.g., fungi,
microsporidia, yeasts) occur in the gut, salivary glands, or reproductive tract. Although
relatively little is known about the specific biological role of many insect endosymbionts,
evidence suggests they are involved in reproduction, digestion, nutrition, pheromone production,
and protection of the insect from pathogens (Broderick et al. 2004, Dillon and Dillon 2004).
Endosymbionts are found internally associated with the gut, reproductive tract,
mouthparts, or salivary glands of insects. Insect endosymbionts can be classified as obligatory or
facultative; infection status with facultative endosymbionts can vary between specimens of a
single species depending on the host diet or the environment in which the host lives (Broderick et
al. 2004). Facultative relationships between the endosymbiont and its insect host are often
referred to as ‘microbial associations’ or ‘secondary endosymbiosis’, because the biological role
and dependence of the host on the endosymbiont is unclear or unknown. Some microorganisms
are transient inhabitants of the surface of the gut of insects and are present only sporadically or
temporarily, while the host depends on obligatory or primary endosymbionts for its survival. In
the aphid-Buchnera system, for example, the aphid host has specialized internal structures called
mycetocytes that contain the bacteria, and the aphid host is provided with essential amino acids
22
(Moran et al. 1993). Some insects and their primary endosymbionts are phylogenetically linked
and, in some cases, have been associated for over 200 million years (Bauman et al. 2005,
Downie and Gullan 2005, Gruwell et al. 2007, Takiya et al. 2006).
Relatively little is known about the microbial associates of lepidopteran insects. The leek
moth, Acrolepiopsis assectella Zeller, harbors endosymbiotic bacteria that produce attractants in
the frass that attract specific parasitoids (Thibout et al. 1995). Broderick et al. (2006)
demonstrated the biological insecticide Bacillus thuringiensis (Bt) is ineffective against gypsy
moth larvae, Lymantria dispar (L.), in the absence of their normal midgut microbiota, and the
insecticidal activity of the Bt was restored when the naturally occurring bacteria were
reintroduced to aposymbiotic hosts. Other studies with Lepidoptera have primarily focused on
the occurrence of Wolbachia and the role it plays in reproduction and distortion of sex ratios.
Within butterflies and moths, Wolbachia has been correlated with reproductive anomalies such
as male-killing, cytoplasmic incompatibility, and sex-role reversal in males (Jiggins et al. 1998,
2000, 2001). Wolbachia is prevalent in both butterfly and moth populations in some regions;
Tagami and Miura (2004) surveyed 49 species (500 individuals) in nine lepidopteran families in
Japan for Wolbachia, and found seven of nine families (78%) and 22 of 49 (44.9%) species were
positive.
Few studies have focused on the complete microbial community of lepidopteran hosts.
Broderick et al. (2004) surveyed the midgut of third-instar gypsy moth larvae from laboratory
and field populations in order to better understand the relationship between a lepidopteran host
and its midgut associates. The effects of diet on the bacteria in the midgut were examined using
culture and culture-independent methods using larvae reared on four different tree hosts, and on
an artificial diet. A total of 23 eubacterial strains, or ‘phylotypes’ were detected among the
23
larvae sampled, and it was determined that the eubacterial diversity present in individual larvae
was dependent on the diet. These bacteria could be derived from epiphytic bacteria found on
foliage or from other environmental sources and may be transient inhabitants of the gut.
Enterobacter sp. and E. faecalis, which are typically found in insect guts, were present in all
larvae, suggesting an important role in gypsy moth biology (Broderick et al. 2004).
A complete understanding of the biology and feeding behaviors of fruit-piercing and
blood-feeding moths requires basic knowledge of both their internal and external environments.
This includes, but is not limited to detection and identification of the microbes associated with
these moths. Recent findings suggest that naturally occurring gut microbes play an important
role in the ability of insect vectors to transmit disease to their host (St. André et al. 2002).
Studies have shown insect vector capabilities to be highly influenced by the composition of the
microorganisms found naturally in the midguts of some vectors (Dillon and Dillon 2004). While
it is unclear whether these moths ingest microbial parasites during the blood meal, the survival of
disease organisms in Calyptra species could depend on the presence of other microorganisms in
the midgut.
Research Objectives
Chapter 2. The primary goal of this chapter was to survey the structures associated with
the proboscis in fruit-piercing, blood-feeding, and tear-feeding moths of the subfamily Calpinae
using both light microscopy and scanning electron micrograph (SEM) methods. I hypothesized
that specific proboscis structures in piercing moths could be associated with specific adult
feeding behaviors. The secondary goal of this study was to homologize the structures of the
proboscis across the subfamily using exemplar taxa selected from available checklists and
faunistic guides. The resulting products of this study will include complete descriptions of
structures for all included calpine taxa and glossary of terms for proboscis morphologies;
24
presently, no such glossaries are available. This study will also be the first to compare structures
visable using light microscope imaging and SEM imaging. Finally, this study will be used to
determine whether proboscis morphology can be used for reconstructing a natural classification
and predicting differences in adult feeding behavior in these moths by directly comparing
proboscis structures of taxa in each assigned feeding category.
Chapter 3. Previous research on the proposed relationships between calpine genera was
not based on formal analyses of empirical data and no phylogenetic hypotheses for the tribe
Calpini are available. Thus, the primary objective of this chapter was to reconstruct a phylogeny
of Calpini using morphological and molecular data. The resulting phylogeny will be used to test
the hypothesis of a directional progression of feeding types from nectar feeding to fruit piercing
to skin piercing and blood feeding in these calpine moths. The specific questions addressed by
this research were as follows: 1) What are the evolutionary relationships among the genera in
Calpini? 2) Are the genera within Calpini monophyletic, and which morphological characters
can be used to diagnose clades within the tribe? 3) How many origins of blood feeding occurred
in Calyptra? 4) Is there a directional progression of feeding types in these moths? 5) Is feeding
behavior correlated with mouthpart morphology?
The evolutionary relationships of fruit-piercing and blood-feeding moths in the tribe
Calpini will be investigated using the following character systems: head appendages including
the surface microstructure(s) of the proboscis, abdominal sternites and tergites, and male/female
genitalia. In addition, data from one mitochondrial gene (COI) and a fragment of the nuclear
large subunit (28S, D2 region) rRNA will be used. This study will document the first test of the
hypothesis of a directional progression of feeding types in Lepidoptera. This study also formally
tested whether proboscis morphology is correlated with feeding behavior in fruit-piercing and
25
blood-feeding moths (e.g., presence of tearing hooks with primary fruit-piercing of hard skinned
fruits, shape of sensilla styloconica with blood feeding, and position of erectile barbs with
secondary piercing of hard skinned fruits).
Chapter 4. The tribe Calpini was recently catalogued by Fibiger and Lafontaine (2005)
wherein they assigned additional genera to the tribe, suggesting Calpini consisted of
approximately 200 species in 11 genera. This publication focused on the Palearctic region and
was therefore not inclusive of all genera comprising the tribe. In addition, the fruit-piercing
genus Oraesia Guenée, which has proboscis armature identical to that of Calyptra and
Gonodonta (Zaspel, Chapter 2), was excluded while seven genera lacking these diagnostic
characteristics of the proboscides were included. The primary objective of this chapter was to
combine the works of Holloway (2005) and Fibiger and Lafontaine (2005) into an updated
checklist to complement recent taxonomic studies (Bänziger 1983, Zilli and Hogenes 2002), a
survey of calpine proboscis morphology (Zaspel et al. 2008), and phylogenetic research (Zaspel,
Chapter 3). This checklist also serves to correct minor taxonomic errors in the checklist of Poole
(1989). All original descriptions for each genus, species, subspecies, and their synonyms were
studied. The checklist includes type localities if available, a complete references list, and
corrections and changes to the nomenclature presented in the checklists of Poole (1989), Fibiger
and Lafontaine (2005), and Holloway (2005).
Chapter 5. Comparative studies elucidate evolutionary trends by comparing certain
characteristics, i.e. descriptions of the environments inhabited by the organisms, phenotypic
characters, and behaviors across taxa represented in a phylogeny (Harvey and Pagel 1991).
Comparative analyses of feeding behaviors require that both the behaviors and hosts of the fruit-
piercing and blood-feeding moths for each taxon are known (Harvey and Pagel 1991).
26
Acquisition and characterization of feeding behavior data for the moth species under
investigation was the primary goal of this study. Of the 17 known Calyptra species (Bänziger
1983), C. eustrigata (Hampson), C. minuticornis minuticornis (Guenée), C. orthograpta (Butler),
C. bicolor (Moore), C. fasciata (Moore), C. ophideroides (Guenée), C. parva Bänziger and C.
pseudobicolor Bänziger have been reported to pierce mammalian skin, with the latter five also
able to pierce humans, under natural conditions. Calyptra fletcheri (Berio) has pierced humans
in experiments (Bänziger 1968, Bänziger 1989). These species are considered facultative or
opportunistic blood-feeders primarily in subtropical areas in southern Asia and tropical Southeast
Asian countries (Bänziger 1989b). At least four additional closely related genera (Eudocima,
Gonodonta, Oraesia, and Plusiodonta) have apparently homologous proboscides modifications
used for fruit-piercing, but the occurrence of blood-feeding in those species has not been
observed (Bänziger 1979, Zaspel, unpublished data). The purpose of this chapter was to
document the feeding behavior of two species (C. thalictri and C. lata) occurring in a temperate
region under experimental and semi-natural conditions to determine whether they were also
facultative blood feeders or strictly fruit piercers.
Chapter 6. Until recently, the moth Calyptra thalictri was considered to be an obligate
fruit piercer that never took blood meals from mammals. On an expedition to the Primorye
Territory of Far Eastern Russia, specimens from a remote population of C. thalictri were
documented piercing human skin and feeding on the blood for the first time, suggesting these
moths will feed on mammalian hosts when fruits are unavailable (Zaspel et al. 2007). This
chapter surveys males of C. thalictri collected in Russia in 2006 for potential microbial
associates and discusses their potential biological role. This moth species provides a recent
example of the acquisition of hematophagic behavior within the Lepidoptera. Heads and
27
abdomens of adult males of fruit-piercing and blood-feeding C. thalictri were used to determine
whether or not microorganisms are associated with C. thalictri. I hypothesized that blood-
feeding moths could be associated with microorganisms that were known animal pathogens, or
could potentially play a biological role in its insect host. The results from this chapter document
the first case of microbial associates in a fruit-piercing and blood-feeding moth.
Chapter 7. This chapter focuses on storage methods for future amplification of
endosymbiont DNA. Understanding adequate tissue preservation methods was important for
fieldwork during my dissertation and for obtaining specimens used in Chapters 3 and 6.
Methods proposed for the preservation of insect tissue for DNA analysis have included various
concentrations of ethanol, Carnoy’s solution, liquid nitrogen, and acetone (Post 1993; Dessauer
1996; Fukatsu 1999; Mtambo 2006). However, little attention has been paid to appropriate
storage methods for future detection of endosymbiont DNA within an insect host (Fukatsu 1999).
Some studies report successful amplification of bacterial DNA in a host after thousands of years
(Salo et al. 1994; Fricker et al. 1997; Willerslev et al. 2004), but others have reported
inconsistent amplification of bacterial DNA due to low titers of the bacteria in the host,
difficulties with the DNA extraction process, PCR-inhibiting substances present in the insect gut,
or storage method (Fukatsu 1999; Barnes et al. 2000; Bextine et al. 2004; Hoy and Jeyaprakash,
unpublished data). Fukatsu (1999) suggested acetone storage was superior to ethanol as a
preservation method for both the amplification of insect host DNA and the DNA of their
endosymbionts. The goal of this chapter was to compare molecular methods for the detection of
Wolbachia in the Mediterranean flour moth Ephestia kuehniella (Keller) (Lepidoptera:
Pyralidae), and potentially other endosymbiotic bacteria in their insect host, in preserved
specimens over time. I hypothesized that the ability to amplify endosymbiont DNA in a
28
lepidopteran host would decline over time depending on both the method of storage and method
of DNA amplification. For example, after two months storage in ethanol at -80ºC, Wolbachia
DNA could not be amplified consistently using high fidelity PCR in the honeybees Apis
mellifera scutellata and A.m. capensis (Hoy and Jayaprakash, unpublished observation).
Standard, high-fidelity (HF), and real-time quantitative (RTQ) PCR methods were used to detect
and quantify Wolbachia DNA from E. kuehniella specimens stored under 4 treatment conditions
(2 in 95% EtOH and 2 in acetone) over a 2-year storage period. Spectrophotometry readings
were taken at each assay (n = 9 over a 2-year period) to ensure consistency of concentration and
quality of template DNA for each treatment. Stored samples were compared to fresh specimens
at the end of the experiment.
Chapter 8. Finally, in this chapter, entitled “Perspectives”, I evaluate my experience as a
Ph.D. student in the Department of Entomology and Nematology at the University of Florida.
This chapter is meant to be reflective and considers what I learned and what I might do
differently in the future.
29
Figure 1-1. Calyptra thalictri male feeding on human thumb (JMZ) in Russia 2008.
30
CHAPTER 2 A COMPARATIVE SURVEY OF PROBOSCIS MORPHOLOGY AND ASSOCIATED
STRUCTURES IN FRUIT-PIERCING AND BLOOD-FEEDING MOTHS IN THE SUBFAMILY CALPINAE (LEPIDOPTERA: NOCTUIDAE)
Introduction
The structure and function of the lepidopteran proboscis has been examined for broad
range of taxa across multiple families (Bänziger 1971, 1973, Büttiker et al. 1996, Krenn 1990,
Krenn 1997, Speidel et al. 1996). Previous work utilized light microscopy to evaluate proboscis
structures (Hattori 1962, Bänziger 1970, 1973, 1980) followed by scanning electron microscopy
(SEM) (Cochereau 1977, Büttiker et al. 1996, Speidel et al. 1996). Taxon sampling for these
studies ranged from higher-level exemplars from distantly related lepidopteran families (e.g.,
Noctuidae, Geometridae, and Pyralidae) based on adult feeding behavior (Bänziger 1973,
Büttiker et al. 1996) to more focused proboscis studies on related species (Bänziger 1970, 1986),
subfamilies (Speidel et al. 1996), and families (Krenn 1998, Krenn and Kristensen 2000, Krenn
and Mühlberger 2002, Krenn and Penz 1998, Krenn et al. 2001). From this work, several
notable differences in proboscis morphology were observed, including a wide variety of sensilla
types and other specialized feeding structures (Bänziger 1970, 1973, Büttiker et al. 1996, Speidel
et al. 1996). Uniquely specialized proboscis structures (cutting ridges, erectile barbs and
eversible tearing hooks moved by blood-pressure) and concomitant stylet dynamics (anti-parallel
movements, oscillatory torsion, spindle movements) occur in the piercing moths in the family
Noctuidae (Künckel 1875, Breitenbach 1877, Bänziger 1970, 1973, 1980), with a majority of the
taxa currently placed in the Calpinae.
The subfamily Calpinae presently consists of four tribes (Anomini, Calpini, Phyllodini,
and Scoliopterygini; Fibiger and Lafontaine 2005, Holloway 2005) and is defined by the tearing
structures of the proboscis (Goater et al. 2003, Fibiger and Lafontaine 2005, Holloway et al.
31
2001, Holloway 2005, Kitching and Rawlins 1998). Calpinae is cosmopolitan in its distribution;
however, many genera have geographic distributions that are somewhat restricted. Species in the
subfamily exhibit a broad range of adult feeding behaviors including those that can be considered
‘piercers’ of fruits or other hosts. It has been known for some time that fruit piercers can be
primary or secondary depending on whether they are able to penetrate intact skin, or only fruit
damaged previously by primary piercers or other animals, respectively (Jack 1922). Bänziger
(1982) proposed a more precise characterization of which moths can pierce what type of fruits
(see below).
Within the tribe Calpini, males of ten Calyptra species pierce mammalian skin and feed
on blood (Bänziger 1971, 1982, 1989, Zaspel et al. 2007, Zaspel 2008). Some remaining species
appear to be exclusively fruit piercing; however, also hematophagous Calyptra are obligatory
fruit piercers in South and Southeast Asia (Bänziger 2007). Species of Eudocima are common
pests of a wide variety of fruits ranging from hard- skinned longan, to thick-skinned oranges, to
soft or ripening fruits (e.g., peaches, plums, apples; (Fig. 2-1D), with the widespread Eudocima
fullonia (Clerck) being the target of a biological control project in the region (Fay 2002, Sands
and Liebregts 1993). Gonodonta species can be found in subtropical and tropical regions, with
seven species occurring in Florida, Texas, and Arizona; these species pierce citrus fruits,
including tangerines, grapefruits and oranges, at times causing extensive losses in subtropical
and tropical regions (Todd 1959). Other species, such as Calyptra thalictri (Borkhausen) (Fig.
2-1F) and non-calpini Scoliopteryx libatrix L. (Fig. 2-1C), Ophiusa tirhaca (Cramer), and
Dysgonia algira (L.), pierce fruit even in temperate Europe (Bänziger 1969, 2007). Plusiodonta
and Oraesia species are common in both the Old and New World tropics (Poole 1989, Holloway
et al. 2001). Plusiodonta species have been observed feeding on soft-skinned fruits (peaches and
32
plums) in South Asia (Nepal: Kathmandu valley, pers. observation 2005). Oraesia species cause
damage to thick- and soft-skinned fruit in India, Nepal (Fig. 2-1A), Thailand, Korea and Japan
(Ramakrishna Ayyar 1944, Hattori 1962, Yoon and Lee 1974, Bänziger 1982, 1987). Punctures
made in fruits by the moths cause agricultural losses through fermentation and rotting of the fruit
or secondary invasions by various microorganisms that result in early fruit fall (Todd 1959,
Sands and Leibregts 1993).
Phylogenetic analyses indicate that Calpini is monophyletic and is supported by the
presence of tearing hooks of the proboscis (Zaspel, Chapter 3). At least three genera currently
placed in the Calpini and others in related tribes lack tearing hooks, confusing the classification
of the subfamily and the tribes therein. The taxonomy of Calpini and associated genera is further
complicated by the occasional inclusion of apparently tear-feeding taxa (e.g., Hemiceratoides;
Fig. 2-1E) and the assertion that the tear-feeding habit evolved from the fruit feeding habit
(Hilgartner et al. 2007). There is no evidence yet that Hemiceratoides is fruit piercing, nor that
tear drinking is its normal feeding habit; so far, there are more compelling arguments that skin-
piercing blood-sucking in mammals evolved from fruit-piercing while tear drinking developed in
separate lineages (Bänziger 1980, 2007). Bänziger (2007) pointed out discrepancies in Büttiker
et al. (1996) and Hilgartner et al. (2007). Previous workers describing the proboscis morphology
of H. hieroglyphica and associated structures of have applied terms incorrectly (Hilgartner et al.
2007); further, no specific criteria for the application of the terms or glossary that can be used in
describing proboscis morphology exists. The primary focus of this study was to compare and
describe the diversity of proboscis structures across the genera currently placed in the subfamily
Calpinae using both SEM and light microscopy, and to accurately homologize these structures
within the subfamily.
33
Materials and Methods
Terminology
The surface microstructure and visible macro-structures of the proboscis in calpine moths
are described using light microscopy and SEM imaging; comparisons are made between the two
imaging methods for viewing proboscis structures within the Lepidoptera. The terminology used
herein for proboscis morphology follow the work of Bänziger (1970, 1980), Büttiker et al.
(1996) and Speidel et al. (1996). Sensilla terminology follows Altner and Altner (1986),
Faucheux (1985, 1991, 1995), Hallberg (1981), and Hallberg et al. (1994). A list of definitions
and criteria for the various sensilla and other proboscis structures examined is provided in Table
2-1. A proboscis diagram illustrating proximal versus apical regions for calpine moths is
provided (Oraesia rectistria, Fig. 2-2). Because there was little structural variation within a
genus, exemplars of the genera surveyed are figured and discussed.
Proboscis morphologies for representative calpine genera included in the survey are
discussed according to functional feeding group. Functional feeding groups were characterized
based on Bänziger (1982) and Norris (1935). For the sake of convenience, Bänziger (1982)
chose four categories of fruits based on their increasing difficulty to be pierced by moths: very
soft-skinned fruit (e.g. raspberry), soft-skinned fruit (e.g. peach, grape), thick-skinned fruit
(citrus), and hard-skinned fruit (longan, lichi). Moths were grouped according to their ability to
pierce the four categories as primary piercers. For example, a moth like Calyptra minuticornis is
a primary piercer of thick-skinned fruits (oranges) and all softer-skinned fruit, but a secondary
piercer of hard-skinned fruit (longan). The first two categories are omitted here because the
genera analyzed belong only to the latter two, unlike those studied by Bänziger (1982) that
included the ‘catocalines’ that mostly have far weaker and less armored proboscides. The other
feeding types, i.e. non-piercing fruit sucking, nectar sucking, non-piercing blood sucking, skin
34
piercing blood sucking, and the various degrees of lachryphagy, were characterized in Bänziger
(1973, 2007). Some feeding groups include taxa that exhibit polytypic feeding behaviors; thus,
observed continuity or overlap between feeding types will be discussed for those taxa. The
following is a list of the institutional and private collections consulted during this study. The
acronym of the institution or name of private collection is followed by the name of the individual
that prepared the loan. Acronyms follow Heppner and Lamas (1982): AMNH - American
Museum of Natural History, New York (T. Schuh); HB - Personal collection of H. Bänziger,
Thailand (Hans Bänziger); MF - Personal collection of M. Fibiger, Denmark (Michael Fibiger);
FLMNH - Florida Museum of Natural History, Florida (G. Austin, M. Thomas); NMNH -
National Museum of Natural History, Washington D.C. (M. Pogue).
Characterization of Functional Feeding Catergories
Non piercing, fruit sucking. The moths in this feeding group obtain nutrients through
various fruit juice sources. The moths in this category take up fruit juice from cracked or
damaged fruits; no piercing is involved. While fruit sucking is a common phenomenon within
the Lepidoptera, the taxa sampled in this study were limited to moths in the following calpine
genera (sensu Fibiger and Lafontaine 2005): Goniapteryx servia (Stoll), Hypsoropha hormos
Hübner, Phyprosopus callitrichoides Grote.
Primary piercers of thick-skinned fruit but secondary piercers of hard-skinned
fruit. Moths in this category can pierce the intact rind of thick-skinned fruit such as mandarin, as
well as all softer-skinned fruit, but not hard-skinned longan and, by extension the related lichi
(not studied by us). However, softer varieties of longan and lichi possibly may occasionally be
pierced by some of the larger moths that have stronger proboscides (e.g. Calyptra orthograpta,
Oraesia emarginata). Conversely, some genera, such as Plusiodonta, and Anomis (Fig. 2-1B)
(the proboscis of which lacks the tearing hooks), have smaller proboscides and may not always
35
be able to penetrate mandarin. Many noctuid lineages have been described as fruit-piercers
(Bänziger 1982; Yoon and Lee 1974). The following taxa were examined representing the tribe
Calpini (sensu Fibiger and Lafontaine 2005; Holloway 2005): Calyptra albivirgata (Hampson),
C. bicolor (Moore), C. canadensis (Bethune), C. eustrigata (Hampson), C. fasciata (Moore), C.
fletcheri (Berio), C. gruesa (Draudt), C. lata (Butler), C. minuticornis (Guenée), C.
ophideroides (Guenée), C. orthograpta (Butler), C. parva Bänziger, C. pseudobicolor Bänziger,
C. subnubila (Prout), C. thalictri (Borkhausen), Gonodonta nutrix (Cramer), Plusiodonta
coelonota (Kollar), P. compressipalpus Guenée, P. incitans (Walker), Oraesia argyrosigna
Moore, O. emarginata (Fabricius), O. excavata (Butler), O. excitans Walker, O. glaucochelia
(Hampson), O. honesta Walker, O. nobilis Felder and Rogenhofer, O. provocans Walker, O.
rectistria Guenée, O. serpens Schaus, O. striolata Schaus, O. triobliqua (Saalmüller), as well as
two genera representing the calpine tribes Anomini and Scoliopterygini: Anomis mesogona
(Walker), A. privata (Walker), and Scoliopteryx libatrix (L.) (sensu Fibiger and Lafontaine
2005; Holloway 2005). It should be noted that while Scoliopteryx libatrix is likely to be able to
pierce the sound rind of mandarin, so far this has not yet been studied. It is a confirmed primary
piercer of soft-skinned fruit (Bänziger, 1969, and unpubl.). It is uncertain at this time whether
these genera are taxonomically misplaced or if the feeding behavior is the result of ecological
convergence. Presently, the piercing genera placed in the Calpini form a monophyletic
assemblage (Zaspel, Chapter 3), and their associated proboscis modifications are restricted to the
taxa therein (e.g., tearing hooks and sensilla styloconica dorsoventrally flattened into erectile
barbs, both of which are movable by blood pressure; Bänziger 1980).
Primary piercers of hard-skinned fruit. This feeding group pierces intact skin of
longan and lichi, as well as all softer-skinned fruit. The following taxa were examined
36
representing the tribe Calpini (sensu Fibiger and Lafontaine 2005; Holloway 2005): Eudocima
homaena (Hübner) and E. salaminia (Cramer).
Mammalian skin piercers and blood sucking. Of all butterflies and moths, only ten
species from one genus, Calyptra, have been observed using their proboscis to pierce
mammalian skin and imbibe blood. Except for some Calyptra species that are fruit pests in
orchards, the hematophagous species are uncommon to rare in nature and thus blood-feeding
occurrences are also rarely seen (Bänziger 2007). It should be noted that the females are not
hematophagous, that the males are facultatively hematophagous, that they cannot digest proteins
but sequester NaCl (though some unknown component may also be utilized) (Bänziger 2007).
Bänziger (1986, p 122-123 and Table 6) gave detailed data on the proboscis length, width,
number and length of both tearing hooks and erectile barbs of C. eustrigata, C. minuticornis, C.
orthograpta, and C. fasciata. The differences are clear enough to allow a rough identification of
the four species based solely on the armature. Nevertheless, he noted that the armature is
essentially the same and all can pierce mammalian skin. Two of the species, C. thalictri and C.
fasciata, exhibit differential feeding behaviors depending on their geographic region (Bänziger
1989; Zaspel et al. 2007); also, C. fletcheri and C. thalictri, so far, have only been
hematophagous under experimental or semi experimental conditions (Bänziger 1989, Zaspel et
al. 2007, Zaspel 2008a), but for convenience are placed in this category. The remaining moth
species (Calyptra bicolor, C. eustrigata, C. fasciata, C. minuticornis, C. ophideroides, C.
orthograpta, C. parva, and C. pseudobicolor) have been recorded feeding on blood both in either
natural and or laboratory conditions. The proboscides of these species were examined in this
study.
37
Tear drinking. Lachrymal fluid feeding occurs in at least six lepidopteran families:
Geometridae, Notodontidae, Noctuidae, Pyralidae, Sphingidae and Thyatiridae (Fig. 11;
Lobacraspis griseifusa at batend eye; Bänziger, 1973, 1992; Büttiker 1973; Büttiker et al. 1996;
Norris 1935). Most moths in this feeding group place the distal part of the proboscis onto the
eyelid where it joins the eye and imbibe tears from the host. The Noctuidae have the most
advanced lachryphagous species, e.g., Arcyophora spp. and Lobocraspis griseifusa Hampson,
but they belong to Nolinae and are, like all other confirmed tear drinkers, neither fruit piercing
nor skin piercing blood sucking, and do not have piercing armature (Bänziger 1973). However, a
recent study (Hilgartner et al. 2007) reported a moth species from a genus previously associated
with the genus Calyptra by Karsch (1896) apparently drinking fluid from the eyes of a sleeping
bird in Madagascar. This species, Hemiceratoides hieroglyphica (Saalmüller), has some
proboscis structures that are superficially similar to those of the piercing species in the Calpini;
however, it did not place within the Calpini in a preliminary phylogenetic analysis of the tribe
based on DNA and morphological characters (Zaspel, Chapter 3). Despite its uncertain
phylogenetic placement at this time, it is possible that this species is a member of the Calpinae
and might represent an independent tear-feeding origin for the subfamily. Given
Hemiceratoides’ previous association with other calpine taxa and apparently unique proboscis
modifications, we have included this species and a non-calpine classical tear drinker, L.
griseifusa, for comparison in the tear-drinking category in our treatment of the proboscis
morphologies.
Uncertain Taxa. Ferenta spp., Graphigona regina (Guenée), and Tetrisia florigera
Walker are placed in this category because their piercing capabilities have not yet been reported
or tested. The proboscides of species in these three genera are virtually identical to that of
38
species in Eudocima and so we speculate that the species are piercers of hard-skinned fruits, but
until this can be confirmed their feeding behaviors are considered uncertain.
The fourth tribe in this subfamily, Phyllodini (sensu Holloway 2005), is represented in
this survey by a single taxon: Phyllodes consobrina Westwood. Bänziger (1982) found
Phyllodes consobrina and P. eyndhovii piercing Ficus, mandarin, and rambutan but it was not
clear whether as primary or secondary piercers. They were conservatively assessed as primary
piercers of soft-skinned fruit but it is likely that they can pierce sound thick-skinned mandarin.
Species in this tribe are rare, and observations of their feeding behaviors are scarce. It has been
suggested that the tribe Phyllodini is synonymous with the Calpini (Fibiger pers. communication;
Speidel et al. 1996b), but this has not been tested formally through phylogenetic analysis due to
the unavailability of male-female taxon pairs for morphological analysis or fresh material for
molecular analysis at this time.
Specimen Preparation
Microptics ™ imaging. Proboscis preparations of male and female individuals of 42
species from twelve genera representing the four tribes currently placed in the Calpinae (Fibiger
and Lafontaine 2005, Holloway 2005) were included in this study (Table 2-2). Dried
proboscides were removed at the base of the head from pinned specimens using a fine-tip forceps
and were submerged in 10% cold KOH for 18–24 hours, followed by short heating treatments
(30 min.). Structures were cleaned in several rinses of approximately 70% ethanol (Winter
2000). Structures were then placed in a watch glass positioned with K-Y jelly, and covered with
70% ETOH for digital imaging.
All digital images (96 total) were taken on a Microptics ™ (Visionary Digital) laboratory
workstation with a Nikon D1 digital SLR camera, using a K2 microscope lens fitted with a CF-3
objective when photographing whole structures (proximal region proboscis shots); the K2 lens
39
fitted with a 10X objective was used for the distal region proboscis shots. Permanent slide
mounts (Euparol [Bioquip, Garden City, CA]) were made of all proboscides. Slides were placed
on slide driers for 24 – 48 hours and then cured for 6-months while horizontal in a slide cabinet.
SEM imaging. Due to a lack of variation in proboscis features within each genus,
proboscis preparations of both male and female specimens of 17 species representing the same
12 genera were selected as exemplars from the light microscope samples and were used to take
scanning electron micrographs (Table 2-2). Proboscides were removed at the base of the head
from pinned specimens using a fine-tip forceps and were submerged in 10% cold KOH for 18–
24 hours, followed by heating treatments (30 min.). Structures were cleaned in several rinses of
approximately 70% EtOH (Winter 2000). Structures were dehydrated overnight in 95% EtOH,
then further dehydrated using a critical point dryer and positioned on the SEM viewing stubs.
Once mounted, the samples were sputter coated using a Denton Vacuum Desk III LLC sputter
coater (Moorestown, NJ) for 2-4 minutes. A total of 92 images were taken using a JSM-5510LV
scanning electron microscope (JOEL, USA). As pointed out in Bänziger (1971, 2007), drying
tends to generate unequal shrinking of the proboscis. This is slight in sclerotized piercing
proboscis but can be so strong in soft, non-piercing proboscis to become deformed (cf. Figs. 2-
4). While this seriously impairs a correct understanding of how a proboscis and its armature
work in a live insect, here we essentially study only the shape (not position) of the sensilla,
barbs, hooks, etc. which are not deformed by drying.
40
Results
Description of the piercing structures of the proboscis visible by light microscopy
The basic characteristics of the surface microstructure and most other structures of the proboscis
in calpine moths can be visualized using a light microscope. Six proboscis images taken using
the Microptics™ system were selected from the 96 total images and used to describe associated
structures (Figs. 2-3A-3F). The proboscis morphology is described for a group of selected taxa
examined in the Primary piercers of thick-skinned fruit but secondary piercers of hard-
skinned fruit: O. serpans. The surface of the proximal region of the proboscis is simple and
smooth with circular or semi-circular ribs. Chaetiform sensilla and other cuticular processes are
not visible or are absent. Sensilla styloconica are modified into dorsoventrally flattened, ovate
erectile barbs (eb) (Fig. 2-3A). The erectile barbs are abundant in the apical region of the
proboscis, along with distinct rasping spine-like structures. The rasping spines are triangular and
without a visible distal connus. The ventral surface of the apical region is smooth and the tip
bears tear-shaped, socketed tearing hooks (th) (Fig. 2-3B). Furcate sensilla are present along the
lateral margin of the dorsal galeal crosslinkage (Fig. 2-3B). Proximal and apical regions are
without visible sensilla basiconica or sensilla trichoidea. The ligulae of the dorsal galeal
crosslinkage are triangular (Fig. 2-3B). G. indentata. The surface of the proximal region of the
proboscis is simple and smooth with circular or semi-circular ribs (Fig. 2-3C). Chaetiform
sensilla and other cuticular processes are not visible or are absent. Sensilla styloconica are
modified into dorsoventrally flattened, ovate erectile barbs (eb) (Fig. 2-3C). The erectile barbs
are abundant in the apical region of the proboscis, along with distinct rasping spine-like
structures. The ventral surface of the apical region is smooth and the tip is with cone-shaped,
socketed tearing hooks (th) (Fig. 2-3C). Furcate sensilla are present along the lateral margin of
the dorsal galeal crosslinkage. Sensilla basiconica or sensilla trichoidea are not visible in either
41
proximal or apical regions. The ligulae of the dorsal galeal crosslinkage (dgl) are triangular (Fig.
2-3C). Primary piercers of hard-skinned fruit: E. homaena. The surface of the proximal
region of the proboscis is simple and smooth with diagonal circular or semi-circular ribs. Two or
three dorsoventrally flattened, triangular rasping spines (rs) occur just below the junction of the
ribbed and smooth area of the apical region (Fig. 2-3D). Erectile barbs in depressions linked by
endocuticula occur just below the junction of the ribbed and smooth areas of the apical region.
The surface of the apical region is smooth with serrated ridges (sr). The ventral surface of the
apical region is smooth and the tip is with socketted tearing hooks (th) linked by pale, elastic
endocuticula (2-3D). The ligulae (l) of the dorsal galeal crosslinkage (dgl) are long and spike-
like (Fig. 2-3D). Mammalian skin-piercers and blood sucking: C. fasciata. The surface of the
proximal region of the proboscis is simple and smooth with circular or semi-circular ribs (Fig. 2-
3E). Chaetiform sensilla and other cuticular processes are not visible or are absent. Sensilla
styloconica are modified into dorsoventrally flattened, ovate erectile barbs (eb) (Fig. 2-3E).
Individual erectile barbs are positioned in a single row along the lateral side of the proboscis but
in the apical region they are present on all sides, lacking only in the tip region (Fig. 2-3E). They
are distally inclined and set in endocuticular depressions when at rest but, for piercing, they
bulge out by blood pressure, turning the barbs into a proximally inclined position. Furcate
sensilla are present along the lateral margin of the dorsal galeal crosslinkage (dgl) (Fig. 2-3E).
The apical section is lance-like, fully sclerotized and hence stiff. The ventral surface of the
apical region is smooth and dorso-laterally the tip bears curved tear-shaped, tearing hooks (th)
(Fig. 2-3F). They are surrounded by (pale) elastic endocuticula and set in a circular sclerotized
socket. This is more protruded distally to form a collar that prevents overturning of the tearing
hooks. Sensilla basiconica or sensilla trichoidea are not visible in either proximal or apical
42
regions. The ligulae of the dorsal galeal crosslinkage are curved and triangular (Fig. 2-3F).
Tear drinking: L. griseifusa. The surface of the proximal region of the proboscis is simple and
smooth (Fig. 2-4A) with minute triangular spines present (mts). Sensilla styloconica (ss) are
present and with a distal connus (Figs. 2-4A and 2-4B). Sensilla basiconica or sensilla trichoidea
are not visible in either proximal or apical regions. The ligulae of the dorsal galeal crosslinkage
(dgl) are triangular and are incurved (Fig. 2-4A). The apex of the proboscis is pale in
appearance, membraneous and blunt (Fig. 2-4B).
Description of the structures visible by scanning electron microscopy
The proboscis morphology is described in detail for a group of selected taxa examined in
each of the five functional feeding groups by scanning electron microscopy. As pointed out in
Bänziger (1971, 2007), drying tends to generate unequal shrinking of the proboscis. This is slight
in a sclerotized piercing proboscis but can be strong in a soft, non-piercing proboscis to as to
become deformed (cf. Figs. 2-4). While this seriously impairs a correct understanding of how a
proboscis and its armature work in a live insect, here we essentially study only the shape (not
position) of the sensilla, barbs, hooks, etc., which are not deformed by drying.
The proboscides of selected species from genera placed in the Non-piercers fruit-
sucking: G. servia. The surface of the proximal region of the proboscis is fluted with circular
ribs and distinct longitudinal depressions throughout (Fig. 2-5A). Cuticular processes are absent
from the ribs. Sensilla styloconica (ss) are absent from the proximal region (Fig. 2-5A). The
apical region is densely nodulose with asymmetrical nodules throughout (Fig. 2-5B). The
nodules near the ligulae of the dorsal galeal cross linkage are separated by septa (Fig. 2-5B).
Sensilla styloconica (ss) are present in the apical portion of the proboscis, with each sensillum
consisting of a stylus with longitudinal ridges and an apical sensory cone (sc); proximal and
apical regions are without visible sensilla basiconica, sensilla trichodea, or cuticular processes.
43
The ligulae (l) of the dorsal galeal crosslinkage (dgl) are primarily triangluar, with most terminal
ligula slightly twisted, with two lateral prominences (Figs. 2-5A and 2-5B). H. hormos. The
surface of the proximal region of the proboscis is fluted and nodulose with longitudinal
depressions faintly present in most proximal circular ribs (1/4 entire length of proboscis) (Fig. 2-
5C). Sensilla trichoidea or other cuticular processes are absent from the ribs. The apical region
is densely nodulose with the nodules of the apical region asymmetrical and separated by septa
toward the apex (Figs. 2-5C and 2-5D). Sensilla styloconica (ss) are distributed throughout the
proximal and apical portions of the proboscis, with each sensillum consisting of a stylus with
longitudinal ridges and an apical sensory cone (sc); proximal and apical regions are without
visible sensilla trichodea or cuticular processes (Figs. 2-5C and 2-5D). The ligulae (l) of the
dorsal galeal crosslinkage (dgl) are rectangular, slightly twisted, terminating in two lateral
prominences (Fig. 2-5C). P. callitrichoides. The surface of the proximal region of the proboscis
is fluted with circular ribs and distinct longitudinal depressions throughout (Fig. 2-5E).
Cuticular processes are absent from the ribs. Sensilla styloconica are absent from the proximal
region (Fig. 2-5E). The apical region is sparsely nodulose becoming densely nodulose towards
the apex of the proboscis (Fig. 2-5F). Nodules of the apical region are asymmetrical and are
separated by septa (Fig. 2-5F). Sensilla styloconica (ss) are present in the apical portion of the
proboscis, with each sensillum consisting of a stylus with longitudinal ridges and an apical
sensory cone (sc); proximal and apical regions are without visible sensilla trichoidea or cuticular
processes. The ligulae (l) of the dorsal galeal crosslinkage are triangluar, and are restricted to the
middle portion of the galea (Fig. 2-5E).
The proboscides of selected species from genera placed in the Primary piercers of
thick-skinned fruit but secondary piercers of hard-skinned fruit: Anomis mesogona. The
44
surface of the proximal region of the proboscis is simple and smooth with circular or semi-
circular ribs (Fig. 2-6A); depressions and cuticular processes are absent. Sensilla styloconica are
modified into dorsoventrally flattened, ovate erectile barbs (eb), each with two lateral
prominences and a triangular distal connus (Fig. 2-6A). The erectile barbs are singular and
sparsely positioned along the lateral side of the proximal galea (Fig. 2-6A). Erectile barbs are
typically more abundant towards the apical region of the proboscis and are arranged on both
lateral and ventrolateral sides (Fig. 2-6B). The apical region is nodulose with asymmetrical
nodules throughout (Fig. 2-6B). The nodulose area at the tip of the proboscis is without septa
(Fig. 2-6B). Furcate sensilla (fs) are also present in the apical portion of the proboscis, with each
sensillum consisting of short and long triangular branches, some secondarily bifurcated. Sensilla
basiconica are present only in apical region; proximal and apical regions are without visible
sensilla trichoidea, or cuticular processes. The ligulae (l) of the dorsal galeal crosslinkage (dgl)
are triangular (Fig. 2-6A). Calyptra canadensis. The surface of the proximal region of the
proboscis is simple and smooth with circular or semi-circular ribs (Figs. 2-6C and 2-6D);
depressions and cuticular processes are absent. Sensilla styloconica are modified into
dorsoventrally flattened, ovate erectile barbs (eb), with a distinct distal connus (Figs. 2-6C and 2-
6D). Individual erectile barbs are positioned in a single row along the lateral side of the
proboscis and are positioned on ventrolateral sides in the apical region (Fig. 2-6C). Furcate
sensilla (fs) are present along the lateral margin of the dorsal galeal crosslinkage (Fig. 2-6E).
The furcate sensilla are four-pronged and asymmetrical, consisting of two short and two long
prongs, with a large sensory cone set in between (Fig. 2-6E). The surface of the apical region is
smooth and the tip is with tear-shaped, socketed tearing hooks (Fig. 2-6D). Sensilla basiconica
are positioned throughout the tip of the proboscis, typically arranged in groups of two or three
45
(Fig. 2-6D). Proximal and apical regions are without visible sensilla trichoidea. The ligulae (l)
of the dorsal galeal crosslinkage (dgl) are triangular (Fig. 2-6D). Calyptra lata. The surface of
the proximal region of the proboscis is simple and smooth with circular or semi-circular ribs;
depressions and cuticular processes are absent (Fig. 2-6F). Rasping spines (rs) are sparsely
positioned along the lateral margin of the dorsal galeal crosslinkage and on the ventrolateral
sides of the proboscis and are without a distal connus (Fig. 2-6F). The surface of the apical
region is smooth and the tip is with tear-shaped, socketed tearing hooks (Fig. 2-6F). Sensilla
basiconica (sb) are positioned throughout the tip of the proboscis, typically arranged in groups of
two or three (Fig. 2-6F). Proximal and apical regions are without visible sensilla trichoidea. The
ligulae (l) of the dorsal galeal crosslinkage (dgl) are triangular (Fig. 2-6F). Gonodonta nutrix.
The surface of the proximal region of the proboscis is simple and smooth with circular or semi-
circular ribs; depressions are absent. Cuticular processes are absent from the ribs. Sensilla
styloconica are modified into dorsoventrally flattened, ovate erectile barbs (eb), without a
distinct distal connus (Fig. 2-7A). Individual erectile barbs are positioned in a single row along
the lateral side of the proboscis and are abundant in the apical region. Rasping spines (rs) are
present along the lateral margin of the dorsal galeal crosslinkage (Figs. 2-7A and 2-7B). The
surface of the apical region is smooth and the tip is has cone-shaped, socketed tearing hooks (th,
Fig. 2-7B); furcate sensilla are absent. Sensilla basiconica (sb) are positioned throughout the tip
of the proboscis, typically arranged in groups of two or three (Fig. 2-7B). Proximal and apical
regions are without visible sensilla trichoidea. The ligulae (l) of the dorsal galeal crosslinkage
(dgl) are short and conical (Fig. 2-7B). Oraesia rectistria. The surface of the proximal region of
the proboscis is simple and smooth with circular or semi-circular ribs; depressions and cuticular
processes are absent. Sensilla styloconica are modified into dorsoventrally flattened, ovate
46
erectile barbs (eb), with a distal connus (Fig. 2-7C). Erectile barbs are abundant in the apical
region of the proboscis, along with distinct rasping spine-like (rs) structures. The rasping spines
triangular and without a distal connus. The surface of the apical region is smooth and the tip has
tear-shaped, socketed tearing hooks (th, Fig. 2-7D). Furcate sensilla (fs) are present along the
lateral margin of the dorsal galeal crosslinkage (Fig. 2-7C). The furcate sensilla are
asymmetrical, consisting of one short and one long lateral prominence, with a large sensory cone
set in between (Fig. 2-7C). Sensilla basiconica are positioned throughout the tip of the
proboscis, typically arranged in groups of two or three (Fig. 2-7D). Proximal and apical regions
are without visible sensilla trichoidea. The ligulae (l) of the dorsal galeal crosslinkage (dgl) are
flattened and triangular (Fig. 2-7D). Plusiodonta compressipalpus. The surface of the proximal
region of the proboscis is simple and smooth with circular or semi-circular ribs (Fig. 2-7E);
depressions are absent. Cuticular processes or chaetiform sensilla (cs) are present at the base
(Fig. 2-7E). Sensilla styloconica are modified into dorsoventrally flattened, ovate erectile barbs
(eb), occasionally with a distinct distal connus (Fig. 2-7E). Individual erectile barbs are
positioned in a single row along the lateral side of the proboscis, and are more abundant in the
apical region of the proboscis (Figs. 2-7E and 2-7F). Rasping spines (rs) are sparsely positioned
along the lateral margin of the dorsal galeal crosslinkage and are without a distal connus (Fig. 2-
7F). The surface of the apical region is smooth and the tip has tear-shaped, socketed tearing
hooks (Fig. 2-7F). Sensilla basiconica (sb) are positioned throughout the tip of the proboscis,
typically arranged in groups of two or three (Fig. 2-7F). Proximal and apical regions are without
visible sensilla trichoidea. The ligulae (l) of the dorsal galeal crosslinkage (dgl) are forked and
triangular (Fig. 2-7F). Scoliopteryx libatrix. The surface of the proximal region of the proboscis
is simple and smooth with circular or semi-circular ribs (Fig. 2-8A); depressions and cuticular
47
processes are absent. Sensilla styloconica are modified into dorsoventrally flattened, ovate
erectile barbs (eb) with two lateral prominences and a triangular distal connus (Fig. 2-8A). The
erectile barbs of the proximal galea are singular, and sparsely positioned along the lateral side of
the proboscis (Fig. 2-8A). Erectile barbs are abundant in the apical region of the proboscis and
are arranged on both lateral and ventrolateral sides (Fig. 2-8B). The surface of the apical region
is heterogeneous with both nodulose and smooth regions (Fig. 2-8B). The nodulose area at the
tip of the proboscis consists of areas with and without septa (Fig. 2-8B). The tip of the proboscis
is smooth with a thin, band of cuticle with slight ridges extending into the nodulose portion (Fig.
2-8B). Sensilla basiconica are situated beneath the ridges at the tip of the proboscis (Fig. 2-8B).
Furcate sensilla (fs) are present in the apical portion of the proboscis, with each sensillum
consisting of short and long triangular branches, some secondarily bifurcated with setose distal
processes. Proximal and apical regions are without visible sensilla trichoidea. The ligulae of the
dorsal galeal crosslinkage are thin and triangular (Fig. 2-8A).
Primary piercer of hard-skinned fruit: Eudocima homaena. The ventral surface of the
proximal region of the proboscis is simple and smooth with diagonal circular or semi-circular
ribs; depressions are absent. Chaetiform sensilla and other cuticular processes are not visible or
are absent from the ribs. Two or three dorsoventrally flattened, triangular rasping spines (rs)
occur just below the junction of the ribbed and smooth area of the apical region (Figs. 2-9A and
2-9B). Erectile barbs occur just below the junction of the ribbed and smooth areas of the apical
region and are without a distinct distal connus (Fig. 2-9A and 2-9B). The surface of the apical
region is smooth with serrated ridges (sr); the tip is cone-shaped, with socketed tearing hooks (th,
Fig. 2-9A). Rasping spines and furcate sensilla are absent. Sensilla basiconica are positioned
throughout the tip of the proboscis and on the tearing hooks, typically arranged in groups of two
48
or three (Fig. 2-9B). Proximal and apical regions are without visible sensilla trichoidea. The
ligulae (l) of the dorsal galeal crosslinkage (dgl) are long and spike-like (Fig. 2-9B).
The proboscides of selected Calyptra spp. in the Mammalian skin-piercers and blood-
sucking: Calyptra eustrigata. The surface of the proximal region of the proboscis is simple and
smooth with circular or semi-circular ribs (Figs. 2-10A and 2-10B); depressions and cuticular
processes are absent. Sensilla styloconica are modified into dorsoventrally flattened, ovate
erectile barbs (eb), with a distinct distal connus (Figs. 2-10A). Individual erectile barbs are
positioned in a single row along the lateral side of the proboscis and are positioned on
ventrolateral sides in the apical region (Fig. 2-10A). Furcate sensilla (fs) are present along the
lateral margin of the dorsal galeal crosslinkage (Fig. 2-10B). The furcate sensilla are
symmetrical, consisting of two long lateral prominences, with a longer sensory cone set in
between (Fig. 2-10B). The surface of the apical region is smooth and the tip is with tear-shaped,
socketed tearing hooks (th) (Figs. 2-10A-C). Sensilla basiconica (sb) are positioned throughout
the tip of the proboscis, typically arranged in groups of two or three (Fig. 2-10B). Proximal and
apical regions are without visible sensilla trichoidea. The ligulae (l) of the dorsal galeal
crosslinkage (dgl) are triangular (Fig. 2-10B). Calyptra thalictri. The surface of the proximal
region of the proboscis is simple and smooth with circular or semi-circular ribs (Figs. 2-11A);
depressions and cuticular processes are absent. Sensilla styloconica are modified into
dorsoventrally flattened, ovate erectile barbs (eb), with a distinct distal connus (Figs. 2-11A).
The surface of the apical region is smooth and the tip is with sharp tear-shaped, socketed tearing
hooks (th) (Figs. 2-11B and 2-11C). Proximal and apical regions are without visible sensilla
trichoidea. The ligulae (l) of the dorsal galeal crosslinkage (dgl) are triangular (Fig. 2-11B).
49
The proboscis of Tear drinking: Hemiceratoides hieroglyphica. The placement of this
species in this group at present is tentavive because nothing certain is known about its feeding
habits. The most likely assumption is that it may pierce fruit and occasionally suck tears. The
surface of the proximal region of the proboscis is simple and smooth with diagonal semicircular
ribs (Fig. 2-12A). The ribs of the proximal region terminate into lateral plates with shallow
cuticular depressions (Fig. 2-12D). Furcate sensilla (fs) are present in the proximal and apical
regions and occur along the lateral margin of the dorsal galeal crosslinkage (Figs. 2-12A, 2-12B,
and 2-12D). The furcate sensilla are of two types (fs1 and fs2), both seemingly symmetrical: one
consisting of a long, thin, feather-like plate with three small lateral prominences at the base, and
the other with four prongs (two short and two long) (Fig. 2-12D). Smooth sensilla styloconica
(subtype 1, sss1) are present in the apical region and are feather-like (Figs. 2-12A, 2-12B, and 2-
12D). The apical region is with fixed, deltoid-pyramidal hooks (h), without a distal connus
(Figs. 2-12A-C). The surface of the apical region is smooth and the tip is with sclerotized ridges
(sr) (Fig. 2-12C). Sensilla basiconica (sb) are positioned throughout the length of the galea (g),
typically arranged in the center of the plates along the lateral margins of the dorsal galeal
crosslinkage (Fig. 2-12D). Proximal and apical regions are without visible sensilla trichoidea.
The ligulae (l) of the dorsal galeal crosslinkage (dgl) are triangular (Fig. 2-12D).
Uncertain Taxa: Phyllodes consobrinia. The surface of the proximal region (pr) of the
proboscis is simple and smooth with diagonal circular or semi-circular ribs; depressions and
cuticular processes are absent. Sensilla styloconica are modified into thin, highy flattened, tear-
shaped erectile barbs (sensilla styloconica subtype 1, eb; Fig. 2-13A), without a distal connus.
Other sensilla styloconica are modified into a smooth, flattened rectangular shape (subtype 2,
sss2), are abundant in the apical region of the proboscis, and are overlain along the lateral margin
50
of the galea (Fig. 2-13A). The surface of the apical region is smooth and the tip is with socketed
tearing hooks and compressed furcate sensilla (fs) with asymmetrical edges. Sensilla basiconica
are positioned throughout the tip of the proboscis and are located in the center of the smooth
sensilla styloconica (subtype 2, sss2), furcated sensilla, and tearing hooks (Figs. 2-13A and 2-
13B). Proximal and apical regions are without visible sensilla trichoidea. The ligulae (l) of the
dorsal galeal crosslinkage (dgl) are thin and triangular (Fig. 2-13A).
Discussion
This survey indicates it is likely the tearing hooks of the proboscis are restricted to the
Calpini (sensu Zaspel and Branham 2008) and proboscis morphology is not strictly correlated,
but is associated with feeding behavior (fruit piercing, blood feeding, or tear feeding). This
study confirms that species within the Calpini are equipped with piercing mouthparts and use
them to pierce fruits wither as primary or secondary piercers (Bänziger 1982). However, the
reverse is not true for other piercing species distantly related to members of Calpini. Bänziger
(1982) had already noted that mouth-part structure alone is not sufficiently indicative of what
fruit type a moth can pierce, but that it nevertheless provides important clues: a thin, long,
unsclerotized proboscis lacking piercing armature cannot conceivably, and indeed has never been
seen to, penetrate the sound skin of longan. Also, while in Calyptra, there are both
hematophagous and typical fruit-piercing species (e.g. C. lata, C. hokkaida), their proboscides
are essentially the same and, despite minor differences in levels of sclerotization, size and
number of tearing hooks and erectile barbs characteristic for certain species, they are not
predictive of their piercing capability or feeding habits (Bänziger 1986, 2007). This is not
entirely unexpected since hematophagous Calyptra are only facultatively so but at the same time
obligatory fruit-piercers. Both light and scanning electron microscopy can be used to
differentiate most of the characters of the proboscis. For example, the presence of tearing hooks,
51
rasping spines, and shape of dorsal ligulae can be easily visualized using light microscopy. The
surface microstructure, minor structural differences in the shape if the tearing hooks, and the
presence of furcated sensillia are difficult to detect without the availability of scanning electron
micrographs. However, the endocuticula that joins the tearing hooks with the sclerotized socket
and also surrounds the erectile barbs are not visible by SEM but are evident by light microscopy.
Specialized structures for blood feeding were not found in hematophagous Calyptra species.
It is surprising that Büttiker et al. (1996) found significant morphological adaptations in
the proboscis of tear drinking moths. Despite their strongly modified behavior, tear feeders
essentially imbibe fluid from a pool not unlike puddling Lepidoptera, for which no
morphological adaptation is required. Since the purported adaptations to tear feeding are also
found in females of species where only males are lachryphagous, and because of other
reservations (Bänziger 2007), the proboscis morphology in tear drinkers needs reassessment. A
special case is H. hieroglyphica, the biology of which is unknown except for photographs, and
description of observed movements of its proboscis inserted between the eyelids of sleeping
birds in Madagascar (Hilgartner et al. 2007; also see a reinterpretation by Bänziger 2007).
Scanning electron micrographs (SEM) of its proboscis clearly depict s numerous strong
differences in microstructure when compared to those the SEMs of typical the tear-feeding
moths (Büttiker et al. 1996; this report Figs. 2-11, 2-12); the armature is similar to that found in
piercing proboscides. Further, tear-feeding H. heiroglyphica possess modifications of the
proboscis that are similar to those found in fruit-piercing moths; however, it differs from Calpini
proboscides in that the sensilla basiconica modified into hooks are fused to the proboscis, hence
not movable by blood pressure, and in that the sensilla styloconica which are modified into fixed
erectile barbs are not movable by blood pressure. The proboscis of Eudocima spp. is different
52
53
from that of Calyptra (including Oraesia, Plusiodonta, Gonodonta) in that it is larger and is the
ventrally serrated. This ventral serration, plus the stronger, more robust proboscis, is the main
reason why Eudocima is able to pierce hard skinned fruit (longan, litchi) while Calyptra and the
other genera are not.
Some piercing noctuid taxa not included in the survey such as Ercheia spp., Pericyma
spp., Serrodes spp. have proboscis armatures that are in some respects similar to Calyptra, and
other calpines, while others (e.g., Facidina suffumata, Platyja spp., Saroba albopunctata, and
Acherontia spp.) lack ‘normal armature (teeth, hooks, barbs, serrations, ridges)’ altogether but
pierce with a pointed, fully sclerotized terminal section of the proboscis suggesting that fruit-
piercing has evolved multiple times within the Noctuidae. The glossary of proboscis structures
in this study provides criteria for which homologies can be assessed across a broad range of taxa;
the exact function(s) of some structures still needs to be tested (e.g., furcate sensilla), but
proposed function based on the primary literature is mentioned. I conclude that proboscis
morphology needs careful examination in its application for reconstructing a natural
classification of piercing moths and predicting of observed differences in adult feeding behavior.
The above-mentioned armature of tearing hooks and erectile barbs is characteristic for the tribe
Calpini and thus should be restricted to its members rather than the subfamily.
Table 2-1. Sensilla and other structures associated with calpine proboscides and their proposed function Structure (Abbreviation) Definition/Criterion *references for terminology are listed in methods section Proposed function Cuticular Hooks (h): Fixed, deltoid-pyramid to wedge-like structures. Unknown. Dorsal Galeal Ligulae (dgl): Zipper-like structure comprised of glossae and paraglossae. Holds tubes of the proboscis together. Erectile Barbs Subtype 1 (eb): Modified sensilla styloconica, apical connus present, surrounded by endocuticular material. Mechanoreception; possibly contact chemoreception. Ligula (l): Individual zipper-like structures comprised of glossae and paraglossae. Form dorsal and ventral galeal crosslinkages of proboscis. Smooth Sensilla Styloconica Subtype 1 (sss1): Flattened, feather-like with a basiconic sensillum but without distal connus. Contact chemo-mechanoreceptors. Smooth Sensilla Styloconica Subtype 2 (sss2): Flattened, rectangular with a basiconic sensillum but without distal connus. Contact chemo-mechanoreceptors. Furcated Sensilla (fs): Cuticular styloconic sensilla, often assymetrical, branched with hairs or finger-like projections. Unknown, but may perceive mechanical distortions. 54
Rasping Spines (rs): Finger-like, flattened, aporous structures, without hairs or a sensory cone. Structural, possibly contact chemorecpetors. Sensilla Basiconica (sb): Peg-shaped sensilla with minute pores. Olfactory structures, chemoreceptors. Sensilla Styloconica (ss): Cuticular structures consisting of a basiconic peg elevated on a style or cone (sc). Contact chemo-mechanoreceptors. Sensilla Trichodea (st): Cuticular, hairlike projection, aporous. Mechanoreceptors, function in food localization. Serrated Ridge (sr): Ventrally serrated cuticular ridge located on ventral side of proboscis. Structural, used for piercing hard-skinned fruit (e.g., longan, litchi). Tearing Hooks (th): Aporous, cuticular structure with a collar, moveable by blood pressure, with or without a basiconic sensillum, attached to socket via elastic endocuticula. Structural, involved in piercing through fruit or mammalian tissue, possible mechanoreception. Minute Triangular Spines (mts): Cuticular structures, membraneous. Possibly used in brushing eye of host to induce production of tears.
Table 2-2. Specimens examined A. Genus species author Feeding Group Collection Country of Origin SEM Light Microscope Anomis mesogona (Walker) PTS FLMNH Taiwan X X A. privata (Walker) PTS FLMNH Taiwan X X Calyptra albivirgata (Hampson) PTS NMNH China X C. bicolor (Moore) PTS/MSP NMNH Nepal X C. bicolor PTS/MSP NMNH Nepal X C. canadensis (Bethune) PTS AMNH USA X X C. eustrigata (Hampson) PTS/MSP NMNH Thailand X X C. eustrigata PTS/MSP NMNH Malaysia X C. fasciata (Moore) PTS/MSP Fibiger Nepal X C. fletcheri (Berio) PTS/MSP* NMNH Nepal X C. gruesa (Draudt) PTS NMNH China X C. lata (Butler) PTS FLMNH Slovakia X X C. lata PTS NMMH S. Korea X X C. minuticornis (Guenée) PTS/MSP NMNH Thailand X 55
C. minuticornis PTS/MSP Fibiger Nepal X C. ophideroides (Guenée) PTS/MSP NMNH Himalaya X C. ophideroides PTS/MSP NMNH India X C. orthograpta (Butler) PTS/MSP NMNH China X C. orthograpta PTS/MSP NMNH Thailand X C. parva Bänziger PTS/MSP NMNH Thailand X C. pseudobicolor Bänziger PTS/MSP NMNH Nepal X C. pseudobicolor PTS/MSP NMNH Nepal X C. subnubila (Prout) PTS NMNH Indonesia X C. thalictri (Borkhausen) PTS/MSP NMNM Austria X X C. thalictri PTS/MSP* Fibiger Russia (RFE) X X ______________________________________________________________________________________________________
Table 2-2. Continued Genus species author Feeding Group Collection Country SEM Light Microscopy Image Eudocima homaena (Hübner) PHS Fibiger Indonesia X X Eudocima salaminia (Cramer) PHS NMNH Papua New Guinea X Ferenta castula (Dognin) UT NHM Colombia X Goniapteryx servia (Stoll) NP FLMNH USA X X Gonodonta nutrix (Cramer) PTS USNM Brazil X X Graphigona regina (Guenée) UT NHM Costa Rica X Hemiceratoides hieroglyphica (Saalmüller) TD NMNH Malawi X X Hypsoropha hormos Hübner NP FLMNH USA X X Oraesia argyrosigna Moore PTS NMNH Taiwan X O. argyrosigna PTS NMNH Tanzania X O. argyrosigna PTS Fibiger Nepal X O. argyrosigna PTS Fibiger Nepal X O. emarginata (Fabricius) PTS NMNH Malaysia X O. emarginata PTS NMNH Sri Lanka X O. excavata (Butler) PTS NMNH Japan X 56
O. excitans Walker PTS NMNH Mexico X O. glaucochelia (Hampson) PTS NMNH Bolivia X O. honesta Walker PTS NMNH Mexico X O. honesta PTS NMNH Mexico X O. nobilis Felder and Rogenhofer PTS NMNH Brazil X O. provocans Walker PTS NMNH Malawi X O. rectistria Guenée PTS FLMNH India X O. rectistria PTS NMNH Nepal X X O. serpans Schaus PTS NMNH Venezuela X O. serpans PTS NMNH Venezuela X O. striolata Schaus PTS NMNH Peru X O. striolata PTS NMNH Bolivia X _______________________________________________________________________________________________________
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Table 2-2. Continued Genus species author Feeding Group Collection Country SEM Light Microscopy Image O. triobliqua (Saalmüller) PPTS NMNH Malawi X O. triobliqua PTS NMNH Rhodesia X O. wintgensi (Strand) PTS NMNH Unknown X Phyllodes consobrina Westwood PTS FLMNH Assam X X Phyprosopus callitrichoides Grote NP FLMNH USA X X Plusiodonta coelonota (Kollar) PTS NMNH USA X X Plusiodonta compressipalpus Guenée PTS FLMNH USA X X Plusiodonta incitans (Walker) PTS NMNH Mexico X Plusiodonta incitans PTS NMNH Argentina X Scoliopteryx libatrix (L.) PTS Fibiger Denmark X X Scoliopteryx libatrix PTS FLMNH USA X Tetrisia florigera Walker UT NHM Peru X ____________________________________________________________________________________________________________
Figure 2-1. Feeding behaviors of adult moths in the subfamily Calpinae. (A) Oraesia rectistria
Guenée piercing plum in Nepal (photo J.M. Zaspel), (B) Anomis fructusterebrans Bänziger piercing yellow Himalayan raspberry (Rubus ellipticus Sm.) in N. Thailand (photo H. Bänziger), (C) Scoliopteryx libatrix (L.) piercing raspberry in Switzerland (photo H. Bänziger), (D) Eudocima tyrannus Guenée piercing apple in Korea (photo H. Fay), (E) Hemiceratoides hierglyphica feeding on the tears of a bird in Madagascar (photo R.D. Hilgartner), (F) Calyptra thalictri feeding on blood from human thumb in Far Eastern Russia (photo J.M. Zaspel).
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Figure 2-2. Description of proboscis regions; Oraesia rectistria.
59
Figure 2-3. Examples of proboscis structures visible by light microscopy in selected feeding categories. Primary piercers of thick-skinned fruit but secondary piercers of hard-skinned fruit (A), Oraesia serpans; Proximal region, dgl = dorsal galeal ligulae, eb = erectile barbs subtype 1, (B) O. serpans; Apical region, th = tearing hooks, (C) Gonodonta nutrix; Proximal region, dgl = dorsal galeal ligulae, eb = erectile barbs subtype 1. Primary piercers of hard-skinned fruit, (D) Eudocima homanea; Proximal region, dgl = dorsal galeal ligulae, rs = rasping spines; Apical region, sr = serrated ridges, th = tearing hooks. Mammalian skin piercing and blood feeding, (E) Calyptra fasciata; Proximal region, dgl = dorsal galeal ligulae, eb = erectile barbs subtype 1, (F) Calyptra fasciata; Apical region, th = tearing hooks.
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Figure 2-4. Proboscis of non-calpine classical tear feeder (A) Lobocraspis griseifusa; Apical region 1, dgl = dorsal galeal ligulae, ss = sensilla styloconica, (B) L. griseifusa Apical region, ss = sensilla styloconica.
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Figure 2-5. Proboscides of taxa in the non-piercing fruit-sucking group. (A) Goniapteryx servia; Proximal region, dgl = dorsal galeal ligulae, Apical region 1, ss = sensilla styloconica, (B) G. servia; Apical region 2, l = ligula, ss = sensilla styloconica, sc = sensory cone, (C) Hypsoropha hormos; Apical region, ss = sensilla styloconica, (D) H. hormos; Proximal region, dgl = dorsal galeal ligulae, g = galea, l = ligula, ss = sensilla styloconica, sc = sensory cone, (E) P. callitrichoides; Proximal region, l = ligula, (F) P. callitrichoides; Apical region, ss = sensilla styloconica, sc = sensory cone.
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Figure 2-6. Examples of proboscis structures visible by scanning light microscopy in primary piercers of thick-skinned fruit but secondary piercers of hard-skinned fruit. (A) Anomis mesogona; Apical region 1, eb = erectile barbs, fs = furcated senislla, sc = sensory cone. (B) A. mesogona; Apical region 2, eb = erectile barbs, fs = furcated senislla, sc = sensory cone.
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Figure 2-6 (continued.) Examples of proboscis structures visible by scanning light microscopy in
primary piercers of thick-skinned fruit but secondary piercers of hard-skinned fruit. (C) Calyptra canadensis; Apical region 1, eb = erectile barbs, fs = furcated sensilla, l = ligula, th = tearing hooks, (D) C. canadensis; Apical region 2, sb = sensilla basiconica, sensory cone, (E) C. canadensis; Apical region 3, fs = furcated sensilla, (F) C. lata; Proximal region, rs = rasping spines, Apical region, sb = sensilla basiconica, th = tearing hooks.
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Figure 2-6 (continued.) Examples of proboscis structures visible by scanning light microscopy in primary piercers of thick-skinned fruit but secondary piercers of hard-skinned fruit. (F) Calyptra lata; Proximal region, rs = rasping spines, Apical region, sb = sensilla basiconica, th = tearing hooks.
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Figure 2-7. Examples of proboscis structures visible by scanning light microscopy in primary piercers of thick-skinned fruit but secondary piercers of hard-skinned fruit II. (A) Gonodonta nutrix; Proximal region, g = galea, Apical region 1, eb = erectile barbs, rs = rasping spines, th = tearing hooks, (B) G. indentata; Apical region 2, eb = erectile barbs, l = ligula, rs = rasping spines, sb = sensilla basiconica, th = tearing hooks, (C) Oraesia rectistria; Apical region 1, eb = erectile barbs, fs = furcated sensilla, l = ligula, sb = sensilla basiconica, rs = rasping spines, th = tearing hooks, (D) O. rectistria; Apical region 2, l = ligula, sb = sensilla basiconica, th = tearing hooks.
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Figure 2-7. Continued
67
Figure 2-8. Examples of proboscis structures visible by scanning light microscopy in primary piercers of thick-skinned fruit but secondary piercers of hard-skinned fruit III. (A) Scoliopteryx libatrix; Proximal region, eb = erectile barbs, sc = sensory cone, Apical region 1, sb = sensilla basiconica, (B) S. libatrix; Apical region 2, eb = erectile barbs, fs = furcated sensilla, sc = sensory cone.
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Figure 2-9. Proboscis of taxa in the primary piercing of hard-skinned fruits group. (A) Eudocima homaena; Apical region 1, dgl = dorsal galeal ligulae, g = galea, sr = serrated ridges, (B) E. homaena; Apical region 2, l =ligula, rs = rasping spines, sb = sensilla basiconica, sr = serrated ridges, th = tearing hooks.
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Figure 2-10. Proboscides of taxa in the mammalian skin-piercing and blood-feeding group. (A) Calyptra eustrigata; Apical region 1, eb = erectile barbs, sb = sensilla basiconica, sc = sensory cone, th = tearing hooks, (B) C. eustrigata; Apical region 2, fs = furcated sensilla, sb = sensilla basiconica, (C) C. eustrigata; Apical region 3, th = tearing hooks.
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Figure 2-11. Proboscides of taxa in the mammalian skin-piercing and blood-feeding group II. (A) C. thalictri; Apical region 1, eb = erectile barbs, fs = furcated sensilla, sc = sensory cone, (B) C. thalictri; Apical region 2, l = ligula, th = tearing hooks, (C) C. thalictri; Apical region 3, th = tearing hooks.
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Figure 2-12. Proboscides of taxa in the tear-drinking group. (A) Hemiceratoides hieroglyphica; Proximal region, fs = furcated sensilla, g = galea, sb = sensilla basiconica, Apical region 1, sss1 = smooth sensilla styloconica subtype 1, h = cuticular hook, (B) H. hieroglyphica; Apical region 2, h = cuticular hook, sb = sensilla basiconica, sss1 = smooth sensilla styloconica subtype 1, (C) H. hierglyphica; Apical region 3, fs = furcated sensilla, h = cuticular hook, l = ligula, sb = sensilla basiconica, sr = serrated ridge, (D) H. hierglyphica; Apical region, fs = furcated sensilla (1 and 2), l = ligula, sb = sensilla basiconica.
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Figure 2-13. Uncertain taxa. (A) Phyllodes consobrina; Apical region 1, dgl = dorsal galeal ligulae, eb = erectile barbs, l = ligula, sb = sensilla basiconica, sss2 = smooth sensilla styloconica subtype 2, th = tearing hooks, (B) Phyllodes consobrina; Apical region 2, sb = sensilla basiconica, sss2 = smooth sensilla styloconica subtype 2, th = tearing hooks.
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CHAPTER 3 RECONSTRUCTING THE EVOLUTIONARY RELATIONSHPS OF THE VAMPIRE
MOTHS AND THEIR FRUIT-PIERCING RELATIVES USING MORPHOLOGICAL AND MOLECULAR DATA (LEPIDOPTERA: NOCTUIDAE: CALPINAE: CALPINI)
Introduction
Hematophagy is believed to have arisen independently in arthropods during the Jurassic
and Cretaceous periods at least six, and potentially as many as 21, times (Balashov 1984, Ribeiro
1995). Adams (1999) estimates that 14,000 insect species from five orders (Pthiraptera, Diptera,
Hemiptera, Lepidoptera, and Siphonaptera) are hematophagous. Within Lepidoptera, skin
piercing and blood feeding are restricted to the moth genus Calyptra Ochsenheimer (Fig. 3-1).
Calyptra includes what are commonly known as vampire moths, so named because of their
ability to pierce mammalian skin and feed on blood.
Calyptra spp. are medium-sized, with wingspans ranging from 36-72 mm. Species in this
genus occur in Europe, eastern Africa, sub-Himalayan regions of S. Asia, the Manchurian
subregion, and are broadly distributed throughout S.E. Asia. Calyptra species have modified
proboscides equipped with strongly sclerotized barbed hooks used for piercing the skin of hard
fruits such as peaches and citrus, and occasionally of mammals (Bänziger 1982, Zaspel et al.
2007, Zaspel 2008). Of the seventeen species described (Bänziger 1983), ten male Calyptra
species have been observed piercing mammalian skin and feeding on blood (Bänziger 1989,
Zaspel et al. 2007). Males of these ten species are facultative blood feeders; females have not
been documented feeding on blood. It is possible that the male moths may seek out mammalian
hosts to obtain additional nutrients such as amino acids or sugars thereby increasing fitness, but
the blood meal itself does not appear to increase longevity (Bänziger 2007). Blood-feeding
Calyptra males have not tested positive for protesases, indicating amino acids are not
sequestered; however, male moths do appear to be in search of salts (Bänziger 2007). It is
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possible that the males are sequestering salts and transferring them to the females during mating
for egg production (Smedley and Eisner 1995) or to replenish salt supplies depleted during
oviposition (Adler and Pearson 1982).
At least eight additional closely related genera (Africalpe, Eudocima, Ferenta,
Gonodonta, Graphigona, Oraesia, and Plusiodonta, and Tetrisia) have homologous proboscis
modifications used for fruit-piercing, but the occurrence of blood-feeding in those species has
not been observed (Bänziger pers. com., Zaspel 2007).
Bänziger (1982) described categories of fruits based on their increasing difficulty to be
pierced by moths, e.g., very soft-skinned fruit (e.g. raspberry), soft-skinned fruit (e.g. peach,
grape), thick-skinned fruit (e.g. citrus), and hard-skinned fruit (longan, lichi). Moths were
grouped according to their ability to pierce the four categories as primary piercers. Primary
piercers are able to penetrate the skin of the fruit, while secondary fruit piercers are only capable
of piercing fruit damaged previously by primary piercers or other animals (Bänziger 1982). For
example, a moth like Calyptra minuticornis is a primary piercer of thick-skinned fruits (oranges)
and all softer-skinned fruit, but a secondary piercer of hard-skinned fruit (longan). The other
feeding types, i.e. non-piercing fruit sucking, nectar sucking, non-piercing blood sucking, skin
piercing blood sucking, and the various degrees of lachryphagy, were characterized in Bänziger
(1973, 2007). Some feeding groups include taxa that exhibit polytypic feeding behaviors; thus,
continuity or overlap between feeding types has been observed for some species.
It has been hypothesized that an evolutionary progression from secondary to primary fruit
piercing has culminated in skin piercing and blood feeding in these moths (Fig. 3-2: D, Bänziger
1971, 1989). Alternative hypotheses regarding the evolution of feeding in Calyptra have been
proposed (Downes 1973, Hilgartner et al. 2007) and suggest the skin-piercing and blood-feeding
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behavior is derived from other animal-associated feeding behaviors such as dung, urine, or tear
feeding (Figs. 3-2: B and C, respectively). This evolutionary trajectory is an unlikely given the
proboscis structures of fruit piercers and blood feeders are not homologous with those of tear-
feeding moths, and such lachrophagous moths do not pierce fruit. This hypothesis is also
problematic given the shared behavioral modifications found in both fruit piercing and blood
feeding moths. The tearing structures involved in the piercing of fruits or mammalian skin is
restricted to a small group of taxonomically associated noctuid genera (Zaspel, unpublished
data), while animal-associated feeding behaviors, including the imbibing of blood droplets found
on the bodies of mammals, are widespread within Lepidoptera (Bänziger 1982, Scoble 1992).
These feeding hypotheses have never been tested within an empirical phylogenetic framework,
and a hypothesized directional progression of feeding types cannot be tested formally until the
relationships of Calyptra and related genera are known. The primary purpose of this study is to
reconstruct a phylogeny of Calpini to determine evolutionary relationships among the genera in
Calpini and related tribes based on morphological and molecular characters. The evolution of
feeding behaviors is also investigated by using the resulting phylogeny to test the hypothesis of a
directional progression of feeding types from nectar feeding to fruit-piercing to skin-piercing and
blood-feeding in these calpine moths.
Materials and Methods
Taxon Sampling
The most recent classifications place Calpinae in the family Noctuidae (Kitching and
Rawlins 1998, Fibiger and Lafontaine 2005, Lafontaine and Fibiger 2006, Mitchell et al. 2006).
Calpinae consists of four tribes: Anomini, Calpini, Phyllodini, and Scoliopterygini (Fibiger and
Lafontaine 2005, Lafontaine and Fibiger 2006, Holloway 2005). All genera in Calpini contain
fruit-piercing species (Fibiger and Lafontaine 2005, Holloway 2005, Zaspel and Branham 2008);
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a high concentration of economically important fruit-piercing species is found within the tribe.
For the phylogenetic review of blood-feeding moths and related genera, 193 specimens were
dissected, representing species from the following genera: Africalpe, Eudocima, Calyptra,
Ferenta, Goniapteryx, Gonodonta, Graphigona, Hemiceratoides, Hypsoropha, Oraesia,
Phyprosopus, Plusiodonta, and Tetrisia Walker. Representatives from the three other tribes in
the Subfamily Calpinae were also examined: Anomis flava and A. mesogona (Anomini),
Phyllodes consobrina Westwood (Phyllodini), and Scoliopteryx libatrix (Scoliopterygini). The
following is a list of the institutional and private collections consulted during this study. The
acronym of the institution or name of private collection is followed by the name of the individual
that prepared the loan. Acronyms follow Heppner and Lamas (1982): AMNH - American
Museum of Natural History, New York (T. Schuh); MGCL – McGuire Center for Lepidoptera
and Biodiversity, Gainesville (G. Austin); HNM – Hungarian Natural History Museum,
Budapest (L. Ronkay); NHM – Natural History Museum, London (M. Honey); NMNH -
National Museum of Natural History, Washington D.C. (M. Pogue); Queensland, (H. Fay);
UMD – University of Maryland (C. Mitter); MF – Personal collection of Michael Fibiger; HB -
Personal collection of H. Bänziger, Chiang Mai, Thailand (HB has donated the material used in
this study to the NMNH).
Taxa included in morphological phylogenetic analyses. Ingroup taxa were selected
based on checklists of the tribe Calpini (Fibiger and Lafontaine 2005, Lafontaine and Fibiger
2006, Holloway 2005, Zaspel and Branham 2008), generic checklists (Poole 1989, Zilli and
Hogenes 2002), and previous species and generic associations published by other authors
(Hampson, 1926, Bänziger 1983, Hilgartner et al. 2007). Taxa were also selected based on the
availability of material, including the availability of male-female pairs; type species for all
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genera in the analysis were also examined and included. Sixteen of eighteen of Calyptra, twelve
of forty-six species of Eudocima, ten of forty-one Gonodonta, one of one described Graphigona
species, both described species of Hemiceratoides, two of four described Hypsoropha species,
eleven of twenty-four Oraesia species, one of nine Phyprosopus species, and seven of thirty-
seven described Plusiodonta species were included in the study. The following taxa were used
as outgroups: Anomis mesogona, A. flava, and Scoliopteryx libatrix.
Taxa included in molecular phylogenetic analyses. DNA was extracted from as many
species from the morphological dataset as possible. Because many of the taxa studied occur in
remote areas, fresh specimens were not readily available. Museum material was used when
permission was given and if the material was less than 30 years old. Taxa included in the
molecular dataset are listed in Table 3-1.
Taxa omitted from phylogenetic analyses. A single female of Phyllodes consobrina was
examined but was not included in the analysis because it lacked clear similarities to the other
study genera. The placement of this genus is problematic as it shares characteristics with both
Calpini and Ophusini (Holloway 2005). Holloway (2005) assigns this genus to the tribe
Phyllodini; it is possible that the exact placement of Phyllodes in this tribe needs further
assessment. Goniapteryx servia males and females were also examined but were also excluded
from all analyses because they were too divergent with respect to other study genera and
homology statements could not be made with confidence. Complete male-female pairs of
Africalpe, Ferentia and Tetrisia were unavailable at the time of the study.
Morphological Data
Dissection methodology follows Winter (2000) and is fully described in Zaspel and
Weller (2006). Most wings were not cleared and slide-mounted. Euparol mounts were
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transferred from the dehydration series into a final 15-minute treatment in Euparol essence
(Bioquip, Garden City, CA) before slide mounting. Permanent slide mounts (Canada balsam
[Sigma, St Louis, MO] or Euparol [Bioquip, Garden City, CA]) were made of abdominal pelts,
genitalia, legs, wings, labial palps and antennae. Slides were placed on trays and cured in drying
ovens for 24 – 48 hours. Terminology. Terms for abdominal and genitalic morphology follow
Klots (1970), Bänziger (1983), Forbes (1960), Weller et al. (2000), Jacobson and Weller (2002),
Goater et al. (2003), and Kristensen (2003). Terms for proboscis morphology follows Zaspel et
al. (submitted).
A set of morphological characters based on previous studies (Bänziger 1983, Jacobson
and Weller 2002, Goater et al. 2003, Holloway 2005, Zaspel and Weller 2006) was compiled and
included characters from the head, appendages, male and female genitalia. Thirteen new
characters were described from the proboscis and are figured in Chapter 2. A total of sixty-six
characters and two hundred ninety-nine character states were coded. A morphological data
matrix with with twenty-six binary characters and forty unordered multistate characters was
scored. Inapplicable character states were coded as missing (?) (Strong and Lipscomb 1999).
Characters exhibiting intraspecific variation were not coded. The inclusion of the proboscis
characters in the analysis in order to examine the evolution of piercing behaviors is controversial
(Coddington 1988, McLennan et al. 1988); their presence in the data matrix may lead to a lack of
independence between the actual morphological characters and ecological hypotheses thus
biasing the analyses (Luckow and Bruneau 1997, de Queiroz 1996). However, Luckow and
Bruneau (1997) state, “character exclusion can lead to a weaker phylogenetic hypothesis”, and
also, “characters as statements of homology differ from characters as statements of
functionality”. Thus, the characters of the proboscis were coded as statements of homology and
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included in the analysis, and the feeding behaviors as statements of functionality and were
excluded from the analysis and mapped onto the resulting phylogeny. Additionally, the
proboscis structures can be considered independent from the feeding behaviors because
probability of one type of piercing behavior (e.g., fruit-piercing or skin-piericng and blood-
feeding) is not necessarily correlated with particular proboscis structures. The final
morphological data matrix included sixty-five taxa, fifty-seven of which were complete male-
female pairs, five species were represented by females only, and three were represented by males
only.
Molecular Data
Available fresh tissues for as many moth species as possible from the morphological
dataset were stored at -80˚C. DNA extractions from fresh moth legs and dried, pinned material
(10-30 years old than 20 years old) were performed using the DNeasy Tissue Extraction Kit
(Qiagen) and PUREGENE reagents (Gentra Systems, Minneapolis, MN). Samples were
centrifuged for 15 min at 12,000 rpm and the supernatant was removed and transferred into a
clean 1.5-mL centrifuge tube. DNA was precipitated using isopropanol at -80ºC. Samples were
centrifuged for 15 min at 12,000 rpm and the DNA pellets were washed with 70% EtOH, air
dried for 5 min, and re-suspended in 100-μL of sterile water. In order to prevent contamination
of the surface-sterilized samples, all DNA extractions were performed in an area separated from
where the high-fidelity PCR was conducted; voucher labels were assigned and placed in vials or
on pins with the remaining moth bodies (Table 3-1).
A 50-μL high-fidelity PCR kit (Bioline, Randolph, MA) was used to amplify 1-μL of
template DNA with the following PCR reaction conditions and with the primers listed in Table
3-2. Agarose gel electrophoresis (1% TAE gels) was used to separate PCR-amplified DNA,
which was stained with ethidium bromide and visualized with ultraviolet light. Double stranded
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High-Fidelity PCR products were purified using a Qiagen PCR purification kit and sequenced
directly at the ICBR Core facility at the University of Florida. Sequences were downloaded,
trimmed, and aligned using MUSCLE (Edgar 2004).
The molecular data set includes nearly complete sequences for one segment near the 5′
end of the cytochrome c oxidase I (COI) mitochondrial gene and a fragment of the nuclear large
subunit (28S, D2 region) rRNA. Partial COI sequences were included for the following species:
Calyptra albivirgata, Eudocima salaminia, Hemiceratoides sittaca, and Oraesia emarginata.
The relevance of these regions for phylogeny inference has been demonstrated in previous
studies (Brower and Egan 1997, Brower et al. 1997, Caterino et al. 2001, Hajibabaei et al. 2006,
Weller et al. 1992, Mardulyn et al. 1999, Megens et al. 2004, Monteiro and Pierce 2001, Niehuis
et al. 2006, Wahlberg and Zimmerman 2000). The two gene regions span 1427 bp in total: 665
bp, 696 bp, respectively. Due to a lack of suitable specimens, the molecular dataset does not
include all species from the morphological dataset.
Phylogenetic Analyses
Phylogenetic trees of separate and combined morphological and molecular data sets were
constructed using parsimony analyses implemented in TNT Version 1.0 (Goloboff et al. 2003).
Bayesian analyses of molecular datasets were conducted using MRBayes 3.1 (Huelsenbeck and
Ronquist 2001). Models were fit to molecular data using the program MODELTEST (Posada
and Crandall, 1998) and morphological partitions using the procedures described by Lewis
(2001), Nylander et al. (2004), and Ronquist and Huelsenbeck (2005) allowing for comparison
between maximum parsimony (MP) and Bayesian inference (BI) topologies. Resulting
parsimony and Bayesian topologies were compared for overall similarity using procedures
described by Nye et al. (2006). Branch support for morphological data was calculated using
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jackknife resampling and combined datasets were calculated using nonparametric bootstrap
resampling (Felsenstein 1985).
Evolution of Feeding Behaviors and Complementary Analyses
Comparative studies elucidate evolutionary trends by comparing certain characteristics,
i.e. descriptions of the environments inhabited by the organisms, phenotypic characters, and
behaviors across taxa represented in a phylogeny (Harvey and Pagel 1991). Comparative
analyses of feeding behaviors require that those behaviors and hosts of the fruit-piercing and
blood-feeding moths for each taxon are known (Harvey and Pagel 1991). These data for the
moth species under investigation have been summarized (Bänziger 1982, Bosch 1971, Fay 2002,
Fay and Halfpapp 1999, Hargreaves 1934, Hatori 1962, Hilgartner et al. 2007, Huber et al. 1998,
King and Thompson 1958, Maff 1990, Nomura and Hatori 1967, Reddy et al. 2007, Sands and
Shotz 1991, Todd 1959, Whitehead and Rust 1972, Yoon and Lee 1974, Zaspel et al. 2007,
Zaspel Chapter 2; Table 3-3). Observational data for many species in the tribe were extracted
from available literature. In some cases, species were recorded as ‘probable primary piercers’ or
‘established primary piercer’ of various fruit types. Also, observational data for some taxa have
only been recorded under laboratory conditions as opposed to ‘natural conditions’. Lack of
observational data for some species does not mean that the species does not pierce fruit or
mammalian skin. Due to some incomplete feeding behavior data in the literature the following
assumptions were made. When a report described a moth’s feeding behavior as ‘possible’ or if a
feeding behavior was unknown it was coded as absent (0). When a report described a moth’s
feeding behavior under laboratory conditions it was treated as presence (1) of that particular
feeding behavior. Because feeding behaviors are polytypic for many taxa under investigation
(fruit-piercers also suck nectar), they were coded as present (1) for that feeding category.
Feeding behaviors were divided into the following functional feeding categories: A) Non-
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piercing fruit-sucking, B) Primary piercing of thick-skinned fruit, C) Secondary piercing of hard-
skinned fruit, D) Primary piercing of hard-skinned fruit, E) Mammalian skin piercing and blood-
feeding, and F) Tear feeding. When known, binary feeding behavior characters were coded for
all taxa in the morphological matrix in MacClade 4.0 (Maddison and Maddison 2000). Feeding
behaviors for all taxa were mapped onto the resulting topology using parsimony optimizations
(ACCTRAN, DELTRAN), and equivocal cycling in MacClade version 4.08 (Maddison and
Maddison 2000).
Results and Discussion
Summary of Morphological Character Variation
The final data matrix included both non-genital and genital characters. The characters of
the proboscis showed unexpected variation between ingroup and outgroup taxa. Significant
differences in the surface microstructure were observed between Hypsoropha and Phyprosopus
spp. and other calpine genera. Hypsoropha and Phyprosopus have a fluted proximal proboscis
region, while the other taxa included in the analysis have the simple and smooth condition. All
outgroup genera were lacking the tearing hooks, erectile barbs, and rasping spines. These three
characters are unreversed synapomorphies for the tribe Calpini. While there was some variation
in proboscis structures between outgroup and ingroup genera, the characters were typically
similar within genera. The lack of variation in proboscis characters among ingroup genera is
surprising and suggests proboscis morphology is not tightly correlated with feeding type. All
species within Gonodonta, Oraesia, and Calyptra have identical proboscis armature yet blood-
feeding species have only been documented in the genus Calyptra.
In general, characters of the male aedeagus were highly variable between species and thus
few characters were described from this system. Other characters of the male and female
genitalia were variable, but to a lesser degree, and thus were included in the analysis. The shape
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of the male valve and characters of the labial palps were highly informative and diagnostic at the
generic-level (e.g., Plusiodonta and Gonodonta, respectively). Other characters from the male
genitalia were highly informative such as the pattern of sclerotization of sternite VIII (CI = 0.90,
RI = 0.94). Informative characters from the female genitalia include the shape of the antevaginal
plate (Fig. 3-11; CI = 0.57, RI = 0.67) and the shape of segment VIII (Fig. 3-12; CI = 0.74, RI =
0.84). All morphological characters, their states, consistency, and retention indices are described
below.
Head and appendage characters
01 Surface microstructure of proximal region of proboscis. 0, fluted; 1, simple and smooth (CI = 1.00; RI = 1.00).
02 Surface microstructure of proximal region of proboscis. 0, without circular ribs; 1, with circular ribs and longitudinal depressions; 2, with semicircular ribs but without longitudinal depressions (CI = 1.00; RI = 1.00).
03 Circular ribs with cuticular processes. 0, absent; 1, present (CI = 1.00; RI = 1.00).
04 Surface microstructure of apical region of proboscis. 0, densely nodulose; 1, smooth (CI = 1.00; RI = 1.00).
05 Apex of proboscis. 0, smooth; 1, nodulose; 2, serrate; 3, heterogeneous, both smooth and nodulose (CI = 1.00; RI = 1.00).
06 Nodules of near ligulae of the dorsal galeal cross linkage. ?, does not apply; 0, contiguous; 1, separated by septa (CI = 1.00; RI = 1.00).
07 Erectile barbs (eb) occurring along exterior lateral margin of proboscis. 0, absent; 1, present (CI = 0.50; RI = 0.83).
08 Rasping spines (rs) occurring along lateral margin of proboscis. 0, absent; 1, present (CI = 1.00; RI = 1.00).
09 Furcate sensilla. 0, absent; 1, present, symmetrical; 2, present, asymmetrical (CI = 0.50; RI = 0.86).
10 Two or three rasping spines (rs) occurring below junction of ribbed and smooth region of apical region of the proboscis. 0, absent; 1, present (CI = 1.00; RI = 1.00).
11 Shape of dorsal ligulae. 0, flattened and triangular; 1, conical; 2, forked and triangular; 3, spike-like; 4, curved and triangular; 5, rectangular (CI = 0.80; RI = 96).
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12 Tearing hooks (th) occurring in the apical region of the proboscis. 0, absent; 1, present (CI = 1.00; RI = 1.00).
13 Tearing hooks with basiconic sensilla. ?, does not apply; 0, absent; 1, present (CI = 1.00; RI = 1.00).
14 Male antennae. 0, filiform; 1, pectinate; 2, bipectinate (CI = 0.25; RI = 0.68).
15 Female antennae. 0, filiform; 1, pectinate, 2, bipectinate (CI = 0.50; RI = 0.00).
16 Length of labial palp third segment. 0, short (less than half the length of segment 2); 1, medium (half the length of segment 2); 2, long (as long or longer than segment 2) (CI = 0.25; RI = 0.82).
17 Shape of labial palp second segment. 0, ovate; 1, crescent-shape; 2, cylindrical; 3, bent; 4, balloon-shape; 5, boat-shape, wider towards anterior (Fig. 3-3; CI = 0.50; RI = 0.85).
18 Shape of labial palp third segment. 0, rounded; 1, long, finger-like; 2, thumb-like; 3, marble-shaped (Fig. 3-4; CI = 0.44; RI = 0.83).
Thoracic characters
19 Hook of the tornus of forewing. 0, absent; 1, present (CI = 0.15; RI = 0.63).
20 Lobe of forewing. 0, absent; 1, present (CI = 0.20; RI = 0.64).
Characters of the male genitalia
21 Saccular process. 0, absent; 1, entire; 2, branched or split into two processes (CI = 0.40; RI = 0.65).
22 Shape of saccular process (entire). ?, does not apply; 0, finger-like, pointed, fused to valve; 1, conical, apically truncated; 2, hook-like, thin; 3, triangular prominence; 4, T-shape; 5, thumb-like, triangular 6, thumb-like, setose; 7, small flap; 8, finger-like, pointed, free from valve; 9, asymmetrical, thin, finger-like and thumb-like without setae; A, heart-shape; B, cylinder with apical node (Fig. 3-5; CI = 0.85; RI = 0.87).
23 Shape of saccular process (branched). ?, does not apply; 0, one branch U-shape and one branch heart-shape; 1, two small points; 2, two thumb-like projections (Fig. 3-6; CI = 1.00; RI = 0.00).
24 Shape of valve. 0, apically rectangular with triangular prominence, anterior lateral edge heart-shape; 1, rectangular; 2, tear-drop shape; 3, triangular; 4, wavy; 5, forked; 6, rounded ventrally, expanding into triangular shape towards dorsum; 7, W-shape; 8, rounded at sides with protruding point; 9, crescent-shape; A, M-shape; B, trapezoid-shape (Fig. 3-7; CI = 0.65; RI = 0.76).
25 Process of cucullus. 0, absent; 1, present (CI = 0.20; RI = 0.20).
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26 Shape of process of cucullus. ?, does not apply; 0, sharp pointed; 1, asymmetrical, double point; 2, symmetrical, double point; 3, M-shape (CI = 1.00; RI = 0.00).
27 Shape of saccus. 0, concave in center; 1, thin and rounded; 2, U-shape with small flaps; 3, V-shape; 4, U-shape without flaps; 5, V-shape, thin; 6, thick and rounded; 7, W-shape; 8, vase-shape; 9, V-shape with two small ventral prominences; A, V-shape, split; B, compressed triangle (Fig. 3-8; CI = 0.55; RI = 0.55).
28 Manica. 0, membraneous; 1, with fultra; 2, with arellus; with fultra and arellus; 3, setose, with fultra and arellus (CI = 0.50; RI = 0.00).
29 Shape of uncus. 0, hook shape; 1, swollen hook shape; 2, nose-like; 3, long and nose-like (CI = 0.60; RI = 0.78).
30 Hook on uncus apex. 0, absent; 1, present (CI = 0.00; RI = 0.00).
31 Scaphium. 0, membranous; 1, sclerotized (CI = 0.50; RI = 0.50).
32 Shape of scaphium. 0, spoon shape; 1, sclerotized membrane; 2, cross shape; 3, U-shape; 4, wavy parallel lines; 5, crescent shape; 6, sclerotized tube; 7, Y-shape; 8, tongue-shape (CI = 0.67; RI = 0.79).
33 Subscaphium. 0, absent; 1, present, undefined patches of sclerotiation; 2, pieces of sclerotization under anal tube; 3, triangular regions of sclerotization (CI = 0.75; RI = 0.83).
34 Coremata. 0, absent; 1, present (CI = 0.25; RI = 0.45).
35 Pattern of sclerotization of sternite VIII. 0, square; 1, no definite shape; 2, divided into two square plates; 3, U-shape; 4, bowtie-shape; 5, inverted Y-shape; 6, V-shape; 7, rectangular with visible antecosta; 8, shield-shape; 9, solid U-shape (CI = 0.90; RI = 0.94).
36 Pattern of sclerotization of tergite VIII. 0, rectangular; 1, H-shape; 2, V-shape; 3, Y-shape; 4, triangle shape; 5, square; 6, star-shape (CI = 0.55; RI = 0.83).
37 Shape of dorsal tegumen. 0, rounded, interrupted by circular node in center; 1, entire, ring-like; 2, divided with flap in center; 3, thin, M-shape; 4, entire with lateral prominences; 5, rounded, interrupted, without flap in center; 6, wavy (Fig. 3-9; CI = 0.67; RI = 0.91).
38 Shape of uncus base. 0, rounded at sides; 1, thin, square; 2, nose-like; 3, heart-shape; 4, horseshoe shape; 5, triangular; 6, rounded with lateral prominences; 7, W-shape with swollen sides; 8, flattened triangle; 9, flattened square (Fig. 3-10; CI = 0.56; RI = 0.75).
39 Conjunctiva of uncus and dorsal tegumen. 0, membraneous; 1, contiguous; 2, H-shape; 3, bone-shape; 4, broad, surrounding uncus base; 5, square; 6, large rounded plate urrounding uncus base (CI = 0.45; RI = 0.80).
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40 Orientation of phallobase. 0, straight; 1, inflected ventrally at caecum; 2, inflected ventrally at midpoint; 3, inflected dorsally at midpoint; 4, inflected dorsally at caecum (CI = 0.25; RI = 0.48).
41 Shape of caecum. 0, rounded; 1, cylindrical; 2, square; 3, pointed (CI = 0.67; RI = 0.00).
42 Spines of carina. 0, absent; 1, present, extending completely around phallobase; 2, small patch, not extending around phallobase (CI = 0.20; RI = 0.50).
43 Vesica. 0, smooth; 1, rugose, with spiculi; 2, with cone-shaped cornuti (CI = 0.14; RI = 0.50).
44 Abdominal segments 5/6 with specialized scales. 0, absent; 1, present (CI = 0.50; RI = 0.88).
Characters of the female genitalia
45 Shape of the antevaginal plate (segment VII). 0, slightly rounded; 1, heart-shape; 2, Y-shape; 3, M-shape; 4, divided into two triangular plates (Fi. 3-11; CI = 0.57; RI = 0.67).
46 Shape of posterior edge of antevaginal plate. 0, divided into two rectangular plates with ventral edge rounded; 1, divided into two rectangular plates; 2, entire, rectangular; 3, divided into two inverted L-shapes; 4, divided into two triangular plates; 5, divided into two curved plates; 6, trapezoid shape; 7, square with wavy lateral edge; 8, divided into two pentagon shapes; 9, H-shape; A, U-shape; B, bowtie shape; C, divided into two L-shape plates; D, divided into two rectangular plates with anterior corners sharply pointed; E, contiguous, W-shape; F, V-shape (Fig. 3-12; CI = 0.74; RI = 0.84).
47 Shape of ductus bursa. 0, cylindrical; 1, wrinkled, sac-like; 2, V-shape; 3, inverted triangle; 4, S-shape (CI = 0.27; RI = 0.35).
48 Shape of postvaginal plate. ?, does not apply; 0, oval-shape; 1, vase-shape; 2, square; 3, heart-shape; 4, M-shape; 5, T shape; 6, flattened oval shape with rounded prominence in center; 7, V-shape; 8, shield-shape; 9, triangular; A, small circle; B, pentagon shape with stipled base; C, mushroom-shape (CI = 1.00; RI = 1.00).
49 Cervical sclerites of the corpus bursa. 0, absent; 1, present (CI = 0.08; RI = 0.54).
50 Shape of cervical sclerites of the corpus bursa. ?, does not apply; 0, wavy lines; 1, sclerotized patches without definite shape; 2, large rounded area of sclerotization with wavy lines; 3, wavy large sclerotized area; 4, half of posterior sclerotized; 5, oval-shape; 6, sclerotized all around posterior sac of corpus; 7, sclerotized all around anterior sac of corpus; 8, pear-shape; 9, triangular (Fig. 3-13; CI = 0.69; RI = 0.64).
51 Shape of corpus bursa. 0, round, balloon shape; 1, banana shape; 2, peanut shape; 3, thin, teardrop shape; 4, rectangular; 5, heart-shape; 6, clover shape; 7, swollen posterior, sac-like anterior; 8, S-shape; 9, long, thin (CI = 0.75; RI = 0.67).
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52 Membrane of the corpus bursa. 0, smooth; 1, wrinkled; 2, completely sclerotized; 3, divided into wrinkled posterior portion and smooth anterior portion (Fig. 3-14; CI = 0.19; RI = 0.57).
53 Divison of the antrum and ductus. 0, absent; 1, present (CI = 0.14; RI = 0.60).
54 Shape of antrum. ?, does not apply; 0, cylindrical, completely sclerotized; 1, cylindrical, partially sclerotized; 2, square; 3, vase-shape; 4, triangular; 5, V-shape (CI = 0.83; RI = 0.83).
55 Number of signa. 0, absent; 1, one (CI = 0.67; RI = 0.50).
56 Shape of signa. ?, does not apply; 0, triangular, 1, circle (CI = 1.00; RI = 0.00).
57 Appendix bursa. 0, absent; 1, present (CI = 0.17; RI = 0.64).
58 Appendix bursa. ?, does not apply; 0, membranous; 1, sclerotized (CI = 1.00; RI = 1.00).
59 Shape of appendix bursa. ?, does not apply; 0, cylinder; 1, egg-shape with lateral prominences; 2, flap-like; 3, U shape; 4, ball-shape; 5, kidney-shape; 6, cone-shape; 7, swollen crescent-shape; 8, triangular, nose-like; 9, tube-like (CI = 0.73; RI = 0.40).
60 Ductus seminalis terminating at plug of appendix bursa. ?, does not apply; 0, present, small cylinder; 1, triangular (Fig. 3-15; CI = 0.40; RI = 0.40).
61 Plate of intersegmental membrane (IS 9-10). 0, absent; 1, present (CI = 1.00; RI = 1.00).
62 Shape of IS plate. ?, does not apply; 0, triangle; 1, small round node; 2, inverted Y shape; 3, square (CI = 1.00; RI = 0.00).
63 Ornamentation of the ostium bursa. 0, absent; 1, present (CI = 0.13; RI = 0.61).
64 Band of signum. 0, absent; 1, present (CI = 0.50; RI = 0.50).
65 Number of signum bands. ?, does not apply; 0, one; 1, two; 2, three (CI = 1.00; RI = 0.00).
66 Shape of signum band(s). ?, does not apply; 0, ruffled; 1, ring of small lines; 2, wavy lines (CI = 1.00; RI = 0.00).
Phylogenetic Analysis of Morphological Data
The cladistic analysis resulted in three most parsimonious trees (MPTs) with tree length
of 448 steps, a consistency index (CI) of 0.51 and retention index (RI) of 0.74. A strict
consensus tree of the three MPTs collapsed nine internal nodes (Fig. 3-16; L = 524, CI = 0.46, RI
= 0.71). Sixty of sixty-six characters were parsimony informative and 21% of the data matrix
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consisted of missing data. Constant or invariant characters were included to document state
descriptions and distributions for future analyses. The ingroup relationships were well resolved,
except for species relationships within Gonodonta. Despite a poorly resolved Gonodonta clade,
the ingroup genera divided into five assembledges: Graphigona + Eudocima, Plusiodonta,
Gonodonta, Oraesia, and Calyptra (Fig. 3-16). The monophyly of Calpini was supported by five
synapomorphies, three of which were unreversed (CI = 1.00, RI = 1.00) and the jackknife value
for the clade was 41 (Table 3-4). This jackknife value was similar to the average jackknife
values for other major clades in this analysis (41 vs. 45). All ingroup genera (Calyptra,
Eudocima, Graphigona, Gonodonta, Oraesia, and Plusiodonta) were monophyletic and were
supported by no fewer than three synapomorphies. The sister relationship of Graphigona to
Eudocima was supported by two synapomorphies and a jackknife value of 45. Diagnostic
features for the ingroup genera and other major clades are listed in Table 3-4. The following
genera previously associated with Calpini lacked the unreversed synapomorphies present in all
other ingroup genera: Hypsoropha, Phyprosopus, and Hemiceratoides. The results from this
analysis support previous conclusions that these genera are not closely related to the other
ingroup genera. Given the observed differences in habitus and proboscis structures in these
genera, their current placement within the subfamily is also questionable.
Molecular Data and Combined Analyses
A parsimony analysis of combined morphological and molecular data resulted in six most
parsimonious trees with a length of 4572 steps (CI = .28, RI = .27). The strict consensus of six
trees was 4643 steps (Fig. 3-17; CI = .28, RI = .25). The data matrix included 638 parsimony
informative characters and 47% of the data matrix consisted of missing data. The evolutionary
model selected for molecular data partitions based on the MUSCLE (Edgar 2004) alignment was
the GTR+G. This model was used in conjunction with the likelihood-based Mk model for
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morphology (Lewis 2001, Ronquist et al. 2005) in the Bayesian analysis. The first 28,0000 trees
of 10, 500, 000 were discarded as “burn-in” as indicated by graphing the generations in
Microsoft Office Excel 2003. The majority rule topology was recovered from the Bayesian
analysis (Fig. 3-19) in PAUP* 4.0 (Swofford 2000). The three resulting topologies were
compared for overall similarity using the algorithm and software program (implemented as a
Java applet at http://www.mrc-bsu.cam.ac.uk/personal/thomas/phylo-comparison/
comparison_page.html.) described in Nye et al. (2006). Because this approach assumes
topologies under comparison have equal branch numbers, taxa were trimmed from the strict
consensus tree based on morphological data to match the taxon sampling in the morphological +
molecular dataset. The strict consensus toplogy resulting from the parsimony analysis of
molecular and morphological data was 59% similar to the strict consensus topology based on
morphological data. Although both analyses recovered a monophyletic ingroup, differences
between the topologies were observed (compare Figs. 3-16 and 3-18). Plusiodonta and
Eudocima remained monophyletic in the parsimony analysis of combined data; other
relationships between ingroup genera were incongruent with respect to the alternative
phylogenetic hypothesis based on morphological data alone. The topology based on combined
data and parsimony analysis placed Gonodonta and some Oraesia species as sister genera and
Calyptra basal to Eudocima (Fig. 3-18). In the parsimony analysis based on morphological data,
the arrangement is different: Calyptra and Oraesia are sister genera and the Graphigona +
Eudocima clade is sister to the remaining ingroup genera (Fig. 3-16). In the parsimony analyses
of separate and combined data, the composition of the ingroup was stable to removal of outgroup
taxa and the outgroup relationships are largely similar in both resulting topologies. The
parsimony analyses were both 42% similar to the topology resulting from the Bayesian analysis
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of combined data. The Bayesian analysis does not support the monophyly of Calpini and several
species (e.g., Hypsoropha spp. and Phyprosopus callitrichoides) considered to be outgroup taxa
were placed within the ingroup clade (Fig. 3-19). Additionally, support for monophyly of all
ingroup genera is lost in the Bayesian topology and taxa are erratically placed in clades with
genera thought previously to be distantly related (e.g., Eudocima tyrannus). Branch support
values were generally low in both combined analyses, with few nodes supported by values
greater than 80 for all three support measures (Figs. 3-18 and 3-19). Due to large amounts of
missing data and a reduction in taxon sampling [when compared to the morphological matrix],
the combined analyses in this study should be considered preliminary. The topology resulting
from the Bayesain analysis was largely incongruent with respect to the topologies resulting from
parsimony analyses. This suggests that alternative modeling stratageies may need to be explored
with the combined data set prior to analyzing the data in a Bayesian framework in the future.
Evolution of Feeding Behaviors and Complementary Analysis
Examination of adult feeding records revealed that reports were available for 39 taxa
included in the cladistic analysis (60% of all terminal taxa). These feeding reports and fruit hosts
are summarized in Table 3-4. The binary feeding behaviors were optimized onto the strict
consensus phylogeny resulting from the parsimony analysis based on morphological data. This
approach was taken due to the reduction of taxa and large amounts of missing data in the
combined analyses. All taxa included in this analysis of adult feeding behavior have been
reported taking nectar, fruit juice, or feeding at fruit juice baits (Fig. 3-17). Many of the taxa
included in this analysis are also primary piercers of thick-skinned fruits. Outgroup taxa, Anomis
spp. and Scoliopteryx libatrix, are primary piercers of thick-skinned fruits and five out of six
ingroup genera have documented primary piercing reports for at least two species; adult feeding
behaviors for Graphigona have not been published. Primary piercing of thick-skinned fruits has
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been reported for eight Eudocima species included in this analysis (Table 3-4). All other primary
piercers of thick-skinned fruits are considered secondary piercers of hard-skinned fruits until
consistent observational data demonstrating otherwise emerges. According to this analysis, one
independent incident of tear feeding has occurred in the subfamily Calpinae. Tear feeding on
sleeping birds was reported for Hemiceratoides hieroglyphica in Madagascar by Hilgartner et al.
(2007).
The phylogenetic analysis included sixteen of the seventeen described Calyptra species
(Bänziger 1983). Of the sixteen included in this analysis, males of ten Calyptra species have
been reported feeding on blood under experimental or natural conditions. A single report of
Calyptra lata feeding on blood was observed in July (Zaspel, unpublished data 2008). This
record for C. lata has been included in this analysis but it has been noted that it was a single
occurrence (Fig. 3-17). Equivocal cycling in MacClade 4.0 (Maddison and Maddison 2000) was
used to determine that skin piercing and blood feeding is the most derived feeding type within
the Calpini and that hematophagy has evolved four times within Calyptra.
Conclusions
The results from this study support the hypothesis that hematophagy in the genus
Calyptra evolved from the fruit-piercing habit (Bänziger 1971) as opposed to tear feeding
(Downes 1973, Hilgartner et al. 2007). These results also support a directional progression of
feeding types from nectar feeding to fruit piercing, culminating in skin piercing and blood
feeding hypothesized by Bänziger (1971). This work suggests blood feeding has evolved
multiple times within the genus Calyptra. New blood feeding records have been been described
in recent literature (Zaspel et al. 2007) and recorded on recent collecting expeditions (e.g., C.
lata, Zaspel unpublished field observation 2008). Thus, it is possible that blood feeding does
occur in other Calyptra species but has not yet been observed. Although these results are based
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soley on the morphological data matrix, the hypothesis that hematophagy is derived from fruit
piercing in these moths is also supported by the parsimony analysis of combined morphological
and molecular data. The Bayesian analysis produced an alternative phylogenetic hypothesis that
does not support a monophyletic Calpini; both combined analyses should be considered
preliminary at this time.
Results from the analysis based on morphological data suggest Calpini is monophyletic
and is supported by five synapomorphies. Three of these are unreversed, shared characters of the
proboscis. With the exception of the genus Graphigona, all remaining genera within Calpini are
monophyletic and are supported by at least three synapomorphies. Whether or not Graphigona
and Eudocima are synonymous needs further investigation. Tear feeding, or feeding on wounds
and other secretions has also evolved multiple times within Lepidoptera (Fig. 3-2: A); however,
fruit piercing species within the Calpinae have not been observed feeding on tears. If the
observed tear-feeder Hemiceratoides hieroglyphica is in fact a member of Calpinae, it will
represent an independent origin of tear feeding within the the subfamily.
The lack of variation in proboscis characters among ingroup genera is surprising and
suggests proboscis morphology is not completely correlated with feeding type. All species
within Gonodonta, Oraesia, and Calyptra, respectively, have identical proboscis armature, yet
blood-feeding species have only been documented in the genus Calyptra. Thus, there are no
specialized structures present in the ten documented blood-feeders. Additionally, species in
genera from other tribes within the subfamily (e.g., Anomis and Scoliopteryx) used as outgroups
in this study are documented primary piercers, yet they lack the tearing hooks, erectile barbs, and
rasping spines found in all ingroup taxa. Other distantly related noctuid genera presently
considered ‘catocalines’ also pierce fruit yet have far weaker and less armoured proboscides. It
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is likely that fruit piercing in adult moths has evolved multiple times within the family Noctuidae
as a result of ecological convergence.
Table 3-1. Specimens examined B. Taxon Classification Sampling locality Molecular partitions Voucher specimen # (sex, slide number, collection) COI 28S Anomis flava (Walker) Anomini Australia (F-JMZ462, HFay) COI JMZD006 A. mesogona (Walker) Anomini Taiwan (M-JMZ431, FLMNH) COI JMZE002 A. mesogona Anomini Taiwan (F-JMZ432, FLMNH) Calyptra albivirgata (Hampson) Calpini China (M-JMZ359, NMNH) COI - JMZA006 Calyptra albivirgata Calpini Japan (F-JMZ503, NHM) C. bicolor (Moore) Calpini Nepal (M-JMZ348, HB-1755) COI - JMZA004 C. bicolor Calpini Nepal (F-JMZ349, HB) C. canadensis (Bethune) Calpini U.S.A. (M-JMZ374, AMNH) - 28S JMZ020-2 C. canadensis Calpini U.S.A. (F-JMZ374, AMNH) C. eustrigata (Hampson) Calpini Malaysia (M-JMZ331, HB) - C. eustrigata Calpini Thailand (F-JMZ332, HB) C. fasciata (Moore) Calpini Nepal (M-JMZ329, MF) - C. fasciata Calpini Nepal (F-JMZ330, MF) C. fletcheri (Berio) Calpini Nepal (M-JMZ352, HB-1878) C. gruesa (Draudt) Calpini China (M-JMZ357, NMNH) C. gruesa Calpini Japan (F-JMZ505, NHM) C. hokkaida Wileman Calpini Japan (F-JMZ492, UMD) C. hokkaida Calpini China (Bänziger, 1983; Figs. 11, 76-77) C. lata (Butler) Calpini Korea (F-JMZ358, NMNH) COI 28S JMZA003, JMZ018-1 C. lata Calpini Russia (M-JMZ495, FLMNH) C. minuticornis (Guenée) Calpini Nepal (M-JMZ351, MF) C. minuticornis Calpini Thailand (F-JMZ354, HB) C. ophideroides (Guenée) Calpini Himalaya [sic.] (F-JMZ335, NMNH) C. ophideroides Calpini India (Bänziger, 1983; Figs. 15, 78-83)
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Table 3-1. Continued Taxon Classification Sampling locality Molecular partitions Voucher specimen # (sex, slide number, collection) COI 28S C. orthograpta (Butler) Calpini China (M-JMZ347, NMNH) C. orthograpta Calpini Thailand (F-JMZ346, HB) C. parva Bänziger Calpini Thailand (M-JMZ344, HB-2784) C. parva Calpini Thailand (F-JMZ345, HB) C. pseudobicolor Bänziger Calpini Nepal (F-JMZ341, HB-1779) C. pseudobicolor Calpini Nepal (M-JMZ342, HB-1803) C. subnubila (Prout) Calpini Indonesia (F-JMZ350) C. subnubila Calpini Indonesia (M-Bänziger, 1983; Figs. 17, 36-37) C. thalictri (Borkhausen) Calpini Unknown (M-JMZ334, FLMNH) COI 28S JMZA002, JMZ017-1 C. thalictri Calpini Russia (F-JMZ360, NMNH) Eudocima aurantia (Moore) Calpini Australia (M-JMZ417, MF) COI - JMZD012 E. aurantia Calpini Australia (F-JMZ418, MF) E. anguina (Schaus) Calpini Peru (F-JMZ500, NHM) E. boseae (Saal.) Calpini Madagascar (F-JMZ498, NHM) E. boseae Calpini Madagascar (M-JMZ499, NHM) E. cocalus (Cramer) Calpini Queensland (M-JMZ419, HFay) E. cocalus Calpini Queensland (F-JMZ420, HFay) E. dividens (Walker) Calpini Unknown (M-JMZ467, NMNH) E. dividens Calpini Luzon (F-JMZ468, NMNH) E. homaena (Hübner) Calpini Taiwan (F-JMZ429, FLMNH) E. homaena Calpini Taiwan (M-JMZ430, FLMNH) E. jordani (Holland) Calpini Queensland (F-JMZ425, HFay) COI - JMZD009 E. jordani Calpini Queensland (M-JMZ426, HFay) E. materna (L.) Calpini Malawi (F-JMZ400, NMNH) COI - JMZ2091 E. materna Calpini Malawi (M-JMZ401, NMNH)
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Table 3-1. Continued Taxon Classification Sampling locality Molecular partitions Voucher specimen # (sex, slide number, collection) COI 28S E. phalonia (L.) Calpini Queensland (M-JMZ463, HFay) E. phalonia Calpini Queensland (F-JMZ464, HFay) E. procus (Cramer) Calpini Peru (F-JMZ514, HMNH) E. salaminia (Cramer) Calpini Indonesia (M-JMZ395, NMNH) COI - JMZA008 E. salamin Calpini New Guinea (F-JMZ396, NMNH) E. tyrannus (Guenée) Calpini China (M-JMZ465, NMNH) - 28S JMZ023-3 E. tyrannus Calpini Nepal (F-JMZ466, MF) Gonodonta correcta Walker Calpini Brazil (F-JMZ516, NMNH) COI - MHAUA414-60703 G. correcta Calpini Brazil (M-JMZ517, NMNH) G. incurva (Sepp) Calpini U.S.A. (M-JMZ470, FLMNH) COI - MHAUA078-57660 G. incurva Calpini Peru (F-JMZ509, NHM) G. indentata (Hampson) Calpini Venezuela (F-JMZ382, NMNH) COI - MHAUA005 -17237 G. indentata Calpini Brazil (M-JMZ383, NMNH) G. mexicana Schaus Calpini Bolivia (F-JMZ402, NMNH) G. mexicana Calpini Ecuador (M-JMZ403, NMNH) G. nutrix (Cramer) Calpini U.S.A. (M-JMZ445, FLMNH) - 28S JMZ046-3 G. nutrix Calpini U.S.A. (F-JMZ446, FLMNH) G. parens (Guenée) Calpini Panama (F-JMZ455, NMNH) COI - JMZD002 G. parens Calpini Panama (M-JMZ456, NMNH) G. sicheas (Cramer) Calpini Brazil (M-JMZ451, FLMNH) COI - MHAUA015-27415 G. sicheas Calpini D.R. (F-JMZ452, FLMNH) G. sinaldus (Guenée) Calpini U.S.A. (M-JMZ449, FLMNH) COI - BLPBD566 G. sinaldus Calpini U.S.A. (F-JMZ450, FLMNH) G. unica Neumoegen Calpini U.S.A. (F-JMZ447, FLMNH) G. unica Calpini U.S.A. (M-JMZ448, FLMNH)
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Table 3-1. Continued Taxon Classification Sampling locality Molecular partitions Voucher specimen # (sex, slide number, collection) COI 28S G. uxor (Cramer) Calpini Costa Rica (M-JMZ489, UMD) COI 28S MHAU415, JMZ007 G. uxor Calpini Venezuela (F-JMZ405, NMNH) Graphigona regina (Guenée) Calpini Costa Rica (F-JMZ510, NHM) Graphigona regina Calpini Guatemala (M-JMZ511, NHM) Hemiceratoides hieroglyphica (Saal.) I.S. Malawi (M-JMZ61, NMNH) Hemiceratoides hieroglyphica I.S. Liberia (F-JMZ62, NMNH) Hemiceratoides sittaca Karsch I.S. Uganda (M-JMZ518, NMNH) COI - JMZC002 Hypsoropha hormos Hübner I.S. U.S.A. (F-JMZ476, FLMNH) - 28S JMZ006-1 Hypsoropha hormos I.S. U.S.A. (M-JMZ491, FLMNH) Hypsoropha monilis Fabricius I.S. U.S.A. (M-JMZ474, FLMNH) - 28S JMZ002-1 Hypsoropha monilis I.S. U.S.A. (F-JMZ475, FLMNH) Oraesia argyrosigna Moore Calpini Tanzania (M-JMZ391, NMNH) O. argyrosigna Calpini Nepal (F-JMZ394, MF) O. emarginata (Fabricius) Calpini Malaysia (F-JMZ388, NMNH) COI - JMZB11 O. emarginata Calpini Sri Lanka (M-JMZ389, NMNH) O. excavata (Butler) Calpini Japan (M-JMZ365, NMH) O. excavata Calpini Japan (F-JMZ366, NMH) O. excitans (Walker) Calpini Mexico (F-JMZ379, NMNH) COI 28S MHAB508, JMZ008 O. excitans Capini Costa Rica (M-JMZ486, UMD) O. glaucohelia (Hampson) Calpini Bolivia (M-JMZ378, NMNH) O. glaucohelia Calpini Brazil (F-JMZ507, NHM) O. nobilis Felder and Rogenhofer Calpini Brazil (F-JMZ363, NMNH) COI 28S MHAB677, JMZ009 O. nobilis Calpini Costa Rica (M-JMZ487, UMD) O. provocans Walker Calpini Malawi (F-JMZ369, NMNH) O. rectistria Guenée Calpini India (F-JMZ370, NMNH) COI 28S JMZF11, JMZ011-1 O. rectistria Calpini Nepal (M-JMZ485, FLMNH)
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Table 3-1. Continued Taxon Classification Sampling locality Molecular partitions Voucher specimen # (sex, slide number, collection) COI 28S O. serpans Schaus Calpini Peru (M-JMZ508, NHM) COI 28S MHAB514, JMZ010 O. striolata Schaus Calpini Bolivia (F-JMZ376, NHM) O. striolata Calpini Peru (M-JMZ377, NHM) O. triobliqua (Saalmüller) Calpini Zimbabwe (M-JMZ337, NMNH) O. triobliqua Calpini Madagascar (F-JMZ506, NHM) Phyprosopus callitrichoides Grote I.S. U.S.A. (F-JMZ479, FLMNH) - 28S JMZ003-1 Phyprosopus callitrichoides I.S. U.S.A. (M-JMZ484, FLMNH) Plusiodonta casta (Butler) Calpini Japan (M-JMZ511, NHM) - 28S JMZ045-3 P. casta Calpini Japan (F-JMZ512, NHM) P. coelonota (Kollar) Calpini Taiwan (M-JMZ384, NMNH) COI - JMZC008 P. coelonota Calpini Taiwan (F-JMZ385, NMNH) P. compressipalpus Guenée Calpini U.S.A. (F-JMZ458, FLMNH) COI 28S JMZF12, JMZ012-1 P. compressipalpus Calpini U.S.A. (M-JMZ483, FLMNH) P. dimorpha Robinson Calpini Costa Rica (F-JMZ408, NMNH) COI - JMZE004 P. dimorpha Calpini Fiji (M-JMZ409, NMNH) P. incitans (Walker) Calpini Mexico (F-JMZ386, NMNH) P. incitans Calpini Argentina (M-JMZ387, NMNH) P. miranda Schaus Calpini Argentina (F-JMZ406, NMNH) P. miranda Calpini Costa Rica (M-JMZ407, NMNH) P. repellens (Walker) Calpini Costa Rica (M-JMZ415, NMNH) P. repellens Calpini Mexico (F-JMZ416, NMNH) Scoliopteryx libatrix (L.) Scoliopterygini Denmark (M-JMZ381, FLMNH) COI 28S JMZA001, JMZ001-1 Scoliopteryx libatrix Scoliopterygini Denmark (F-JMZ404, FLMNH)
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Table 3-2. PCR conditions and sequences of primers used. ________________________________________________________________________ COI: 94˚C/1 min; 5 cycles of: 94˚C/30 s, 45˚C/40 s, 72.0˚C/30 s; 30 cycles of: 94˚C/30 s;
51˚C/40 s, 72.0˚C/1 min; final extension of: 72.0˚C/10 min. 28S: 94˚C/2 min 30 sec; 94˚C/30 s; 65˚C/30 s; 72.0˚C/30 s; 2 x 40; 72.0˚C/2 min. ________________________________________________________________________ Primers Sequence from 5´ to 3´ ________________________________________________________________________ COI DNA Barcode Segment: LepF1 5' - ATTCAACCAATCATAAAGATATTGG -3' LepR1 5' - TAAACTTCTGGATGTCCAAAAAATCA -3' MLepF1 5' - GCTTTCCCACGAATAAATAATA -3' MLepR1 5' - CCTGTTCCAGCTCCATTTTC -3' 28S D2 Loop: 28S_D2–F1 5' - GAG TAC GTG AAA CCG TTC AG - 3' 28S_D2–R1 5' - CTG ACC AGG CAT AGT TCA C - 3' ________________________________________________________________________
Table 3-3. Known feeding behavior reports for Calpini and related genera included in complementary analyses (U = unknown, NF = nectar/fruit sucking, PTF = primary piercer of thick-skinned fruits, SP = secondary piercer of hard-skinned fruts, PHF = primary piercer of hard-skinned fruits, BF = skin piercer and blood feeder, TF = tear feeder).
Genus species Feeding Behavior(s) Host(s) Reference(s) Anomis flava PTF Grape, raspberry Yoon & Lee 1974, Bänziger 1982, 1987
mesogona PTF Grape, peach, plum Nomura & Hattori 1967, Yoon & Lee 1974, Bänziger 1982, 1987
Calyptra bicolor PTF Mandarin, raspberry Bänziger 2007
bicolor BF Human Bänziger 1989 eustrigata PTF Apple, mandarin Bänziger 2007 eustrigata BF Water buffalo, tapir Bänziger 1968, 1975
fasciata PTF Apple, mandarin Bänziger 2007 fasciata BF Human , elephant Bänziger 1986, 1989
fletcheri PTF Apple, mandarin Bänziger 2007 fletcheri BF Human Bänziger 1989 101 gruesa PTF Peach, grape, apple Hattori 1962 hokkaida PTF Peach, grape Hattori 1962 lata PTF Peach, grape, apple Hattori 1962, Yoon & Lee 1974 lata BF Human Pers. observation 2008
minuticornis PTF Apple, mandarin Bänziger 2007 minuticornis BF Human , elephant Bänziger 1986
ophideroides PTF Apple, mandarin Hargreaves 1934, Bänziger 2007 ophideroides BF Human Bänziger 1989
orthograpta PTF Apple, mandarin Bänziger 2007 orthograpta BF Human , elephant Bänziger 1986, 1989
Table 3-3. Continued Genus species Feeding Behavior(s) Host(s) Reference(s) Calyptra
parva PTF Apple, mandarin Bänziger 2007 parva BF Human Bänziger 1989
pseudobicolor PTF Apple, mandarin Bänziger 2007 pseudobicolor BF Human Bänziger 1989 thalictri PTF, Grape, citrus Hargreaves 1934, Yoon & Lee 1974
thalictri BF Human Zaspel et al. 2007 Eudocima
aurantia PHF Longan, mandarin Bänziger 1982, Fay & Halfpapp 1999 cocalus PHF Longan, mandarin Fay & Halfpapp 1999 homanea PHF Longan, mandarin Fay & Halfpapp 1999 jordani PHF Longan, mandarin Fay & Halfpapp 1999 materna PHF Longan, mandarin Fay & Halfpapp 1999 phalonia PHF Longan, mandarin Fay & Halfpapp 1999 102 salaminia PHF Longan, mandarin Fay & Halfpapp 1999 tyrannus PHF Longan, mandarin Fay & Halfpapp 1999
Gonodonta incurva PTF Citrus Todd 1959 nutrix PTF Citrus Todd 1959
uxor PTF Citrus Bruner et al. 1945
Table 3-3. Continued Genus species Feeding Behavior(s) Host(s) Reference(s) Hemiceratoides hieroglyphica TF Newtonia, Magpie Robin Hilgartner et al. 2007 Hypsoropha hormos NF Fruit bait Pers. observation monilis NF Fruit bait Pers. observation Oraesia argyrosigna PTF Mandarin Bänziger 1982 emarginata PTF Grape Yoon & Lee 1974 excavata PTF Grape Yoon & Lee 1974 provocans PTF Citrus Hargreaves 1934
rectristria PTF Plum Bänziger 1987, pers. observation triobliqua PTF Citrus Hargreaves 1934 Phyprosopus callitrichoides NF Fruit bait Personal observation Plusiodonta 103 casta PTF Peach, grapes Maff 1990 coelonota PTF Peach, grapes, guava Bänziger 1982, Maff 1990 Scoliopteryx libatrix Raspberry, grape Yoon & Lee 1974, pers. observation
Table 3-4. Character support for major clades. * Indicates genera placed in tribe Calpini (sensu Zaspel & Branham 2008). Clade Character: state: CI, RI Jacknife Value A - Anomis + (Scoliopteryx + (Hyproropha + Phyprosopus) + (Hemiceratoides) + [1: 1: 1.00, 1.00] 100 (Graphigona + (Eudocima + (Plusiodonta + (Gonodonta + (Calyptra + Oraesia)))))) [2: 2: 1.00, 1.00]
[25: 0: 0.20, 0.20] B - Scoliopteryx + (Hyproropha + Phyprosopus) + (Hemiceratoides) + [1: 1: 1.00, 1.00] 18 (Graphigona + (Eudocima + (Plusiodonta + (Gonodonta + (Calyptra + Oraesia)))))) [27: 3: 0.55, 0.55] [41: 0: 0.67, 0.00] [53: 0: 0.14, 0.60] C - Hemiceratoides + (Graphigona + (Eudocima + (Plusiodonta + [4: 1: 1.00, 1.00] 48 (Gonodonta + (Calyptra + Oraesia))))) [20: 1: 0.20, 0.64]D - Graphigona + (Eudocima + (Plusiodonta + (Gonodonta + (Calyptra + Oraesia)))) [7: 1: 0.50, 0.83] 41 [8: 1: 1.00, 1.00] [12: 1: 1.00, 1.00] [26: 0: 1.00, 0.00] [43: 1: 0.14, 0.50] 104 E - Plusiodonta + (Gonodonta + (Calyptra + Oraesia)) [5: 0: 1.00, 1.00] 29 [21: 1: 0.40, 0.65] F - Gonodonta + (Calyptra + Oraesia) [16: 0: 0.25, 0.82] 18 G - Hypsoropha sp. + (Hypsoropha sp. + Phyprosopus) [1: 0: 1.00, 1.00] 65 [2: 1: 1.00, 1.00] [6: 1: 1.00, 1.00] H - Graphigona + (Eudocima) [4: 3: 1.00, 1.00] [17: 5: 0.50, 0.85] 45 I - Calyptra + Oraesia [32: 0: 0.67, 0.79] 34 [39: 2: 0.45, 0.80] J - Hemiceratoides [9: 2: 0.50, 0.86] 98
Table 3-4. Continued Clade Character: state: CI, RI Jacknife Value J - Hemiceratoides [27: 7: 0.55, 0.55] [31: 0: 0.50, 0.50] [35: 4: 0.90, 0.94] K - Eudocima [10: 1: 1.00, 1.00] 15 [11: 3: 0.80, 0.96] [13: 1: 1.00, 1.00] [29: 2: 0.60, 0.78] [39: 5: 0.45, 0.80] L - Plusiodonta [3: 1: 1.00, 1.00] 38 [11: 2: 0.80, 0.96] [54: 2: 0.83, 0.83] M - Gonodonta [11: 1: 0.80, 0.96] 66 [18: 3: 0.44, 0.83] [33: 2: 0.75, 0.83] [44: 1: 0.50, 0.88] [47: 2: 0.27, 0.35] N - Oraesia [9: 1: 0.50, 0.86] 64 [24: 8: 0.65, 0.76] [37: 2: 0.67, 0.91] [46: 8: 0.74, 0.84] [57: 1: 0.17, 0.64] [60: 1: 0.40, 0.40] O - Calyptra [22: 5: 0.85, 0.87] 7 [36: 6: 0.55, 0.83] [40: 3: 0.25, 0.48]
105
Figure 3-1. Blood-feeding moth, Calyptra thalictri.
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Figure 3-2. Proposed hypotheses for the evolution of tear feeding and blood feeding within
Lepidoptera.
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Figure 3-3. Shape of labial palp segment II, Character 17: Taxon voucher number (state, condition). A - S. libatrix JMZ381 (0, ovate); B - A. mesogona JMZ431 (1, crescent-shape); C - H. monilis JMZ475 (2, cylindrical); D - P. callitrichoides JMZ478 (3, bent); E - O. emarginata JMZ388 (4, balloon-shape); F - E. homanea JMZ430 (5, boat-shape, wider towards anterior).
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Figure 3-4. Shape of labial palp segment III, Character 18: Taxon (state, condition). A - C. albivirgata JMZ359 (0, rounded); B - S. libatrix JMZ381 (1, long, finger-like); C -C. eustrigata JMZ331 (2, thumb-like); D - G. incurva JMZ470 (3, marble-shaped), E - P. callitrichoides JMZ478 (4, cylinder).
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Figure 3-5. Shape of saccular process (entire), Character 22: Taxon (state, condition). A - P. casta JMZ513 (0, finger-like, pointed, fused to valve); B - S. libatrix JMZ381 (1, cone-shape, apically truncated); C - G. correcta JMZ517 (2, hook-like, thin); D - H. hormos JMZ491 (3, triangular prominence); E - C. canadensis JMZ374 (4, T-shape); F - C. gruesa JMZ357 (5, thumb-like, triangular); G - P. compressipalpus JMZ483 (6, thumb-like, setose); H - P. coelonota JMZ384 (7, small flap); I - C. thalictri JMZ482 (8, finger-like, pointed, free from valve); J - C. parva HB2784 (9, asymmetrical, thin, finger-like and thumb-like without setae); K - C. pseudobicolor HB1803 (A, heart-shape); L - O. argyrosigna JMZ391 (B, cylinder with apical node).
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Figure 3-6. Shape of saccular process = SaP (branched), Sa = saccus, Character 23: Taxon (state,
condition). For C. hokkaida (0, one branch U-shape and one branch heart-shape) see Bänziger 1883, Fig.6; A - O. rectristria JMZ485 (1, two small points); B - P. callitrichoides JMZ478 (2, two thumb-like projections).
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Figure 3-7. Shape of valve, Character 24: Taxon (state, condition). A - P. compressipalpus JMZ483, right valve (0, apically rectangular with triangular prominence, anterior lateral edge
heart-shape); B - O. rectristria JMZ485, right valve (1, rectangular); C - P. casta JMZ513, left valve (2, tear-drop shape); D - E. boseae JMZ499, left valve (3, triangular); E - H. hieroglyphica JMZ361, left valve (4, wavy); F - S. libatrix JMZ381, left valve (5, forked); G - C. orthograpta JMZ346, left valve (6, rounded ventrally, expanding into triangular shape towards dorsum); H - G. sinaldus JMZ449, left valve (7, W-shape); I- O. argyrosigna JMZ391, right valve (8, rounded at sides with protruding point); J - P. callitrichoides JMZ478, left valve (9, crescent-shape); K -
E. tyrannus JMZ465, left valve (A, M-shape); L - A. mesogona JMZ431, left valve (B, trapezoid-shape).
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Figure 3-8. Shape of saccus, Character 27: Taxon (state, condition). A - S. libatrix JMZ381 (0,
concave in center); B - O. serpans JMZ508 (1, thin and rounded); C - G. correcta JMZ517 (2, U-shape with small flaps); D - G. regina JMZ511 (3, V-shape); E - G. sinaldus JMZ449 (4, U-shape without flaps); F - P. compressipalpus JMZ457 (5, V-shape, thin); G - E. boseae JMZ499 (6, thick and rounded); H - H. hieroglyphica JMZ361 (7, w-shape); I - O. triobliqua JMZ338 (8, vase-shape); J - E. cocalus JMZ419 (9, V-shape with two small ventral prominences); K - H. hormos JMZ491 (A, V-shape, split); L - H. monilis JMZ474 (B, compressed triangle).
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Figure 3-9. Shape of dorsal tegumen, Character 37: Taxon (state, condition). A - O. serpans
JMZ508 (0, rounded, interrupted by circular node in center); B - G. correcta JMZ517 (1, entire, ring-like); C - P. casta JMZ513 (2, divided with flap in center); D - A. mesogona JMZ431 (3, thin, M-shape); E - G. regina JMZ511 (4, entire with lateral prominences); F - H. hieroglyphica JMZ361 (5, rounded, interrupted, without flap in center); G - P. callitrichoides JMZ478 (6, wavy).
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Figure 3-10. Shape of uncus base 38: Taxon (state, condition). A - P. casta JMZ513 (0, rounded at sides); B - G. correcta JMZ517 (1, thin, square); C - E. boseae JMZ499 (2, nose-like); D - G. indentata JMZ383 (3, heart-shape); E - S. libatrix JMZ381 (4, horseshoe shape); F - E. cocalus JMZ419 (5, triangular); G - P. compressipalpus JMZ457 (6, rounded with lateral prominences); H - E. phalonia JMZ463 (7, W-shape with swollen sides); I - G. parens JMZ456 (8, flattened triangle); J - A. mesogona JMZ431 (9, flattened square).
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Figure 3-11. Shape of the posterior edge of the antevaginal plate (segment VII), Character 45:
Taxon (state, condition). A - H. hieroglyphica JMZ362 (0, slightly rounded); B - G. invurva JMZ509 (1, heart-shape); C - G. regina JMZ510 (2, Y-shape); D - C. lata JMZ495 (3, M-shape); E - P. miranda JMZ406 (4, divided into two triangular plates).
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Figure 3-12. Shape of the posterior edge of segment VIII, Character 46: Taxon (state, condition). A - C. minuticornis JMZ354, ventral view (0, divided into two rectangular plates with ventral edge rounded); B - H. hieroglyphica JMZ362, ventral view (1, divided into two rectangular plates); C - G. incurva JMZ509, ventral view (2, entire, rectangular); D - P. coelonota JMZ512, ventral view (3, divided into two inverted L-shapes); E - S. libatrix JMZ404, ventral view (4, divided into two triangular plates); F - C. albivirgata JMZ503, ventral view (5, divided into two curved plates); G - E. phalonia JMZ464, ventral view (6, trapezoid shape); H - E. cocalus JMZ420, ventral view (7, square with wavy lateral edge); I - H. monilis JMZ475, ventral view (8, Y-shape); J - E. boseae JMZ498, ventral view (9, H-shape); K - C. canadensis JMZ375, ventral view (A, U-shape); L - A. mesogona JMZ432, ventral view (B, bowtie shape); M - P. compressipalpus JMZ520, ventral view (C, divided into two L-shape plates); N - C. parva JMZ345, ventral view (D, divided into two rectangular plates with anterior corners sharply pointed); O - E. aurantia JMZ418, ventral view (E, contiguous, W-shape); P - E. homanea JMZ429, ventral view (F, V-shape).
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Figure 3-13. Shape of cervical sclerites of the corpus bursa, Character 50: Taxon (state, condition). A - P. miranda JMZ406 (0, wavy lines); B - P. coelonota JMZ512 (1, sclerotized patches without definite shape); C - O. excavata JMZ366 (2, large rounded area of sclerotization with wavy lines); D - G. uxor JMZ405 (3, large sclerotized area); E - E. tyrannus JMZ466 (4, half of posterior sclerotized); F - E. boseae JMZ498 (5, oval-shape); G - C. albivirgata JMZ503 (6, sclerotized all around posterior sac of corpus); H - C. orthograpta JMZ347 (7, sclerotized all around anterior sac of corpus); I -C. pseudobicolor HB1779 (8, pear-shape); J - C. subnubila JMZ350 (9, triangular).
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Figure 3-14. Shape of the corpus bursa, Character 52: Taxon (state, condition). A - O. excavata
JMZ366 (0, round, balloon shape); B - E. salaminia JMZ396 (1, banana shape); C - P. miranda JMZ406 (2, peanut shape); D - P. incitans JMZ386 (3, thin, teardrop shape); E - G. indentata JMZ382 (4, rectangular); F - G. sinaldus JMZ450 (5, heart-shape); G - G. sicheas JMZ452 (6, clover shape); H - C. orthograpta JMZ347 (7, swollen posterior, sac-like anterior); I - O. argyrosigna JMZ394 (8, S-shape); J - P. repellens JMZ416 (9, long, thin).
119
Figure 3-15. Shape of appendix bursa (AB), Character 60: Taxon (state, condition). A - O.
striolata JMZ376 (0, cylinder); B - O. emarginata JMZ388 (1, egg-shape with lateral prominences); C - O. triobliqua JMZ506 (2, flap-like); D - O. rectristria JMZ370 (3, U shape); E - O. argyrosigna JMZ394 (4, ball-shape); F- O. glaucochelia JMZ507 (5, kidney-shape); G - O. provocans JMZ369 (6, cone-shape); H - C. eustrigata JMZ343 (7, swollen, crescent-shape); I - E. materna JMZ400 (8, triangular, nose-like); J - G. correcta JMZ416 (9, tube-like).
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Figure 3-16. Strict consensus tree of three most parsimonius rearrangements for 65 taxa based on sixy-six morphological characters (L = 524, CI = 0.46, RI = 0.71). Numbers above branches are jackknife values for major clades; numbers below branches are synapomorphies for major clades. Black vertical line indicates outgroup taxa of the tribe Calpini, red vertical line indicates ingroup taxa of tribe Calpini; remaining taxa are outgroup taxa representing other genera in the subfamily Calpinae.
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Figure 3-17. Evolution of adult feeding behaviors in Calpini. Characters are mapped onto the
strict consensus phylogeny using presence/absence characters: A = nectar feeding, B = secondary fruit piercing, C = primary fruit piercing (thick-skinned fruits), D = primary fruit piercing (hard-skinned fruits), E = tear feeding F = skin piercing and blood feeding. Skin piercing and blood feeding as a binary character is optimized to show the multiple origins of hematophagy in the genus Calyptra. * Indicates single occurrence of blood feeding.
122
Figure 3-18. Preliminary strict consensus tree of six most parsimonius rearrangements for 34 taxa based on combined data set (66 morphological characters and segments of COI (665 bp) and 28S (696 bp) genes; L = 4643, CI = 0.28, RI = 0.25). Numbers above branches are nonparametric bootstrap values for clades; numbers below branches are jackknife calculations for clades. Solid line indicates ingroup taxa of the tribe Calpini; remaining taxa are outgroup taxa representing other genera in the subfamily Calpinae.
123
Figure 3-19. Preliminary Bayesian analysis resulting from a simultaneous analysis of all data partitions for 34 taxa. Majority rule consensus of topology generated via MrBayes with 10, 500, 000 generations using model GTR + G for molecular data sets and the Mk Model for morphology. Numbers above branches are posterior probabilities.
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CHAPTER 4 WORLD CHECKLIST OF TRIBE CALPINI (LEPIDOPTERA: NOCTUIDAE: CALPINAE)
Introduction
The most recent classification places Calpinae in the family Noctuidae (Goater et al. 2003,
Kitching and Rawlins 1998, Fibiger and Lafontaine 2005, Lafontaine and Fibiger 2006, Mitchell
et al. 2006). Presently, Calpinae consists of four tribes: Anomini Grote 1882, Calpini Boisduval
1840, Phyllodini Hampson 1913, and Scoliopterygini Herrich-Schäffer [1852] (Fibiger and
Lafontaine 2005, Lafontaine and Fibiger 2006, Holloway 2005). Calpini are defined by the
presence of socketted tearing hooks on the proboscis that are used for piercing the skin of fruits
and mammals (Bänziger 1968, 1971, 1979a, 1982, Zaspel et al. 2007, Zaspel in press). Calpini
are cosmopolitan; however, many calpine genera have geographic distributions that are more
restricted (e.g., Gonodonta Hübner, Graphigona Walker, Calyptra Ochsenheimer). The tribe
was recently catalogued by Fibiger and Lafontaine (2005) wherein they assigned genera to the
tribe, suggesting Calpini consisted of approximately 200 species in eleven genera. This
publication focused on the Palearctic region and was therefore not inclusive of all genera
comprising the tribe. In addition, the fruit-piercing genus Oraesia Guenée, which has proboscis
armature identical to that of Calyptra and Gonodonta, was excluded while seven genera lacking
the diagnostic characteristics of the proboscis were included. The primary objective of the
present checklist is to combine the works of Holloway (2005) and Fibiger and Lafontaine (2005)
into an updated checklist to complement recent taxonomic studies (Bänziger 1983, Zilli and
Hogenes 2002), a survey of calpine proboscis morphology (Zaspel et al. 2008), and phylogenetic
research (Zaspel in preparation). This checklist also serves to correct minor taxonomic errors in
the checklist of Poole (1989).
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All original descriptions for each genus, species, subspecies, and their synonyms have been
studied. This checklist includes type localities if available, a complete references list, and
corrections and changes to the nomenclature presented in the checklists of Poole (1989), Fibiger
and Lafontaine (2005), and Holloway (2005). Authorship assignments for taxa described in the
Lepidoptera Atlas of the “Reise der Novara” follow Nässig and Speidel (2007) and for
convenience, are abbreviated “In F.F.R. 1874”.
In summary, the following taxonomic changes are made: Culasta Moore 1881a is removed
from synonymy from Calyptra Ochsenheimer (1816). This genus lacks the proboscis characters
that define Calpini and is not considered a member; the tribal placement of Culasta remains
undetermined. Eudocima talboti (Prout 1922) is placed in synonymy with Eudocima cajeta
(Cramer 1775) and Graphigona antica Walker [1858] 1857b is placed in synonymy with G.
regina (Guenée In Boisduval and Guenée 1852c). Four Eudocima species, E. behouneki, E.
mazzeii, E. prolai, and E. treadawayi described by Zilli and Hogenes (2002) are added to the
checklist. Although Hilgartner et al. (2007) suggest Hemiceratoides Strand 1911 is a member of
the Calpini, this placement remains uncertain as species in the genus are tear feeders lacking
proboscis features found in all other listed genera of Calpini. Phyllodes, in tribe Phyllodini, has
been associated with members of Calpini (e.g., Eudocima) but also with Ophiusini (e.g.,
Miniodes) (Holloway 2005). Given the previous associations of Phyllodes with both calpine and
ophiusine genera, we recommend its continued placement in Phyllodini pending further
morphological and molecular examination of additional species in the genus. Six genera:
Africalpe Krüger, Ferenta Walker, Gonodonta Hübner, Graphigona Walker, Oraesia Guenée,
and Tetrisia Walker, are added to Fibiger and Lafontaine’s (2005) checklist based on characters
of their proboscides. The following genera, previously treated as the tribe Calpini (sensu Fibiger
126
and Lafontaine 2005), are removed based on their lack of proboscides characters present in other
members of this tribe, and categorized as genera whose tribal placement are as yet undetermined:
Cecharismena Möschler, Goniapteryx Perty, Pharga Walker, Phyprosopus Grote,
Psammathodoxa Dyar, and Radara Walker.
Checklist
CALPINI
AFRICALPE Krüger, 1939: 348
Type-species: Africalpe intrusa Krüger, by monotypy.
intrusa Krüger, 1939 (Africalpe) Libya
nubifera (Hampson, 1907) (Calpe) India
vagabunda (Swinhoe, 1884) (Oraesia) Pakistan
anubis (Rebel, 1947) (Pseudocalpe) Egypt
anubia (Poole, 1989); misspelling
CALYPTRA Ochsenheimer, 1816: 78
Type-species: Phalaena thalictri Borkhausen, by subsequent designation of Duponchel 1826:
[3].
Calpe Treitschke, 1825
Hypocalpe Butler, 1883
Percalpe Berio, 1956
albivirgata (Hampson, 1926) (Calpe) China
bicolor (Moore, 1883) (Calpe) India
canadensis (Bethune, 1865) (Calpe) Canada
purpurascens (Walker, 1865) (Plusiodonta) USA
sobria (Walker, 1865) (Oraesia) USA
127
eustrigata (Hampson, 1926) (Calpe) Sri Lanka
fasciata (Moore, 1882) (Calpe) India
labilis (Berio, 1970) (Calpe) India
fletcheri (Berio, 1956) (Calpe) China
gruesa (Draudt, 1950) (Calpe) China
hokkaida (Wileman, 1922) (Calpe) Japan
hokkaido (Poole, 1989); misspelling
hoenei (Berio, 1956) (Calpe) China
imperalis (Grünberg, 1910) (Calpe) India
lata (Butler, 1881) (Calpe) Japan
aureola (Graeser, 1889) (Calpe) Russian Far East
minuticornis (Guenée In Boisduval and Guenée, 1852a) (Calpe) Indonesia
novaepommeraniae (Strand, 1919) (Calpe) India
nyei Bänziger, 1979b (Calyptra) India
ophideroides (Guenée In Boisduval and Guenée, 1852a) (Calpe) East Indies
orthograpta (Butler, 1886) (Calpe) India
striata (Poujade, 1887) (Calpe) China
parva Bänziger, 1979b (Calyptra) India
pseudobicolor Bänziger, 1979b (Calyptra) India
subnubila (Prout, 1928) (Calpe) Indonesia
thalictri (Borkhausen, 1790) (Phalaena) Unknown
centralitalica (Dannehl, 1925) (Calpe) Unknown
pallida (Schwingenschuss, 1938) (Calpe) Turkey
128
sodalis (Butler, 1878a) (Calpe) Unknown
EUDOCIMA Billberg, 1820: 85
Type-species: Phalaena salaminia Cramer, by monotypy.
Elygea Billberg, 1820
Leptophara Billberg, 1820
Trissophaes Hübner, [1823] 1816
Othreis Hübner, [1823] 1816
Maenas Hübner, [1823] 1816
Rhytia Hübner, [1823] 1816
Acacallis Hübner, [1823] 1816
Ophideres Boisduval, 1832
Acacalis Agassiz, [1847] 1846
Ophioderes Agassiz, [1847] 1846
Othryis Agassiz, [1847] 1846
Khadira Moore, 1881b
Adris Moore, 1881b
Purbia Moore, 1881b
Vandana Moore, 1881b
Argadesa Moore, 1881b
Halastus Butler, 1892
Eumaenas Tams, 1924
anguina (Schaus, 1911b) (Trissophaes) Costa Rica
apta (Walker, [1858] 1857b) (Ophideres) Brazil
129
aurantia (Moore, 1877) (Ophideres) Andaman Islands
rutilus (Moore, 1881b) (Adris) Sri Lanka
bathyglypta (Prout, 1928) (Othreis) Indonesia
behouneki Zilli and Hogenes, 2002 (Eudocima) Philippines
boseae (Saalmüller, 1880) (Ophideres) Madagascar
caesar (Felder, 1861) (Ophideres) Indonesia
cajeta (Cramer, 1775) (Phalaena Noctua) India
multiscripta (Walker, [1858] 1857b) (Ophideres) Sri Lanka
talboti (Prout, 1922) (Othreis) Indonesia, NEW SYNONYMY
cocalus (Cramer, 1777) (Phalaena Noctua) East Indies
crepidolata (Lucas, 1894) (Ophideres) Australia
maculata (Weber, 1801) (Noctua) East Indies
plana (Walker, [1858] 1857b) (Ophideres) Indonesia
colubra (Schaus, 1911b) (Trissophaes) Costa Rica
discrepans (Walker, [1858] 1857b) (Ophideres) Singapore
archon (C. and R. Felder, In F.F.R. 1874) (Ophideres) Thailand
dividens (Walker, [1858] 1857b) (Ophideres) Indonesia
divitiosa (Walker, 1869b) (Ophideres) “Congo”
banakus (Plötz, 1880) (Ophideres) “Upper Guinea”
intricatus (Butler, 1892) (Halastus) Nigeria
euryzona (Hampson, 1926) (Khadira) Madagascar
felicia (Stoll, 1790) (Phalaena Noctua) Unknown
formosa (Griveaud and Viette, 1960) (Khadira) Madagascar
130
homaena (Hübner, [1823] 1816) (Othreis) India
ancilla (Cramer, 1777) (Phalaena Noctua) India
bilineosa (Walker, [1858] 1857b) (Ophideres) Sri Lanka
collusoria (Cramer, 1777) (Phalaena Noctua) Unknown
strigata (Donovan, 1804) (Phalaena) India
hypermnestra (Cramer, 1780) (Phalaena Noctua) India
imperator (Boisduval, 1833) (Ophideres) Madagascar
iridescens (Lucas, 1894) (Ophideres) Australia
pyrocrana (Turner, 1908) (Ophideres) Australia
jordani (Holland, 1900) (Ophideres) Indonesia
kinabaluensis (Feige, 1976) (Othreis) Borneo
kuehni (Pagenstecher, 1886) (Othreis) New Guinea
materna (Linnaeus, 1767) (Phalaena Noctua) “Indiis”
chalcogramma (Walker, 1865) (Ophideres) “Zambesi River”
hybrida (Fabricius, 1775) (Noctua) India, Australia.
mazzeii Zilli and Hogenes, 2002 (Eudocima) Philippines
memorans (Walker, [1858] 1857b) (Ophideres) “West Coast of America”
mionopastea (Hampson, 1926) (Othreis) Malaysia
muscigera (Butler, 1882) (Purbia) New Britain
nigricilia (Prout, 1924) (Purbia) Australia
okurai (Okano, 1964) (Adris) East-Palearctic
suthepensis Bänziger and Honey, 1984 (Adris) Burma
paulii (Robinson, 1968) (Othreis) Fiji
131
phalonia (Linneaus, 1763) (Phalaena Noctua) Africa
dioscoreae (Fabricius, 1775) (Noctua) East Indies
fullonia (Clerck, [1764] 1759) (Phalaena) Unknown
obliterans (Walker, [1858] 1857b) (Ophideres) Samoa
pomona (Cramer, 1776) (Phalaena Noctua) India
princeps (Boisduval, 1832) (Ophideres) New Guinea
pratti (Bethune-Baker, 1906) (Lagoptera) New Guinea
prattorum (Prout, 1922) (Othreis) Indonesia
procus (Cramer, 1777) (Phalaena Noctua) Surinam
columbina (Guenée, In Boisduval and Guenée 1852b) (Ophideres) Colombia
scabellum (Guenée, In Boisduval and Guenée 1852b) (Ophideres) Unknown
prolai Zilli and Hogenes, 2002 (Eudocima) Irian Jaya
salaminia (Cramer, 1777) (Phalaena Noctua) China
serpentifera (Walker, [1858] 1857b) (Ophideres) Dominican Republic
sikhimensis (Butler, 1895) (Adris) Oriental
abathyglypta (Prout, 1928) (Othreis) Indonesia
smaragdipicta (Walker, [1858] 1857b) (Ophideres) Borneo
sultana (Snellen, 1886) (Ophideres) Indonesia
splendida (Yoshimoto, 1999) (Othreis) Oriental
srivijayana (Bänziger, 1985) (Othreis) Oriental
talboti (Prout, 1922) (Othreis) Wallacea
toddi (Zayas, 1965) (Othreis) Cuba
treadawayi Zilli and Hogenes, 2002 (Eudocima) Philippines
132
tyrannus (Guenée, In Boisduval and Guenée 1852b) India
amurensis (Staudinger, 1892) (Ophideres) Russian Far East
FERENTA Walker, [1858] 1857a: 961
Type-species: Phalaena stolliana Stoll, by monotypy.
cacica (Guenée, In Boisduval and Guenée 1852c) (Ophideres) Brazil
castula (Dognin, 1912) (Darceta) Venezuela
incaya Hampson, 1926 (Ferenta) Peru
stollii (Hübner, [1823] 1816) (Coronis)
stolliana (Stoll, In Cramer 1782) (Phalaena Noctua) Surinam
GONODONTA Hübner, 1818: 11
Type-species: Gonodonta uncina Hübner, by subsequent designation of Grote 1902: 472.
Revision: Todd 1959:1.
Athysania Hübner, [1823] 1816
Dosa, Walker 1865
aequalis Walker, [1858] 1857a (Gonodonta) Brazil
aeratilinea Todd, 1973 (Gonodonta) Peru
amianta (Hampson , 1924) (Athysania) Guyana
biarmata Guenée, In Boisduval and Guenée 1852a (Gonodonta) Brazil
elegans Druce, In Godman and Salvin 1889 (Gonodonta) Mexico
evadens Walker, [1858] 1857a (Gonodonta) Galapagos Islands
galapagensis Todd, 1959 (Gonodonta) Galapagos Islands
bidens Geyer, 1832 (Gonodonta) Cuba
meridionalis Todd, 1959 (Gonodonta) Brazil
133
miranda Raymundo, 1908 (Gonodonta) Brazil
tenebrosa Todd, 1959 (Gonodonta) Costa Rica
chorinea (Cramer, 1782) (Phalaena Noctua) Surinam
clotilda (Stoll, 1790) (Phalaena Noctua) Surinam
clothilda (Poole, 1989); misspelling
correcta Walker, [1858] 1857a (Gonodonta) Mexico
distincta Todd, 1959 (Gonodonta) Venezuela
ditissima Walker, 1858 (Gonodonta) Brazil
fernandezi Todd, 1959 (Gonodonta) Guyana
fulvangula Geyer, 1832 (Gonodonta) Uruguay
chrysotornus (Hampson, 1926) (Athysania) Guyana
fulvidens Felder and Rogenhofer, In F.F.R. 1874 (Gonodonta) Colombia
flavidens (Hampson, 1926) (Athysania) Brazil
holosericea Guenée, In Boisduval and Guenée 1852a (Gonodonta) Colombia
immacula Guenée, In Boisduval and Guenée 1852a (Gonodonta) Fr. Guiana
panoana (Schaus, 1933) (Athysania) Brazil
incurva (Sepp, 1840) (Phalaena) Surinam
dentata Felder and Rogenhofer, In F.F.R. 1874 (Gonodonta) Brazil
elaborans Dyar, 1914 (Gonodonta) Dominica
temperata Walker, [1858] 1857a (Gonodonta) Venezuela
teretimacula Guenée, In Boisduval and Guenée 1852a (Gonodonta) Fr. Guiana
velata Walker, [1858] 1857a (Gonodonta) Unknown
indentata (Hampson, 1926) (Athysania) Venezuela
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latimacula Guenée, In Boisduval and Guenée 1852a (Gonodonta) Colombia
lecha Schaus, 1911a (Gonodonta) Costa Rica
lincus (Cramer, 1775) (Phalaena Bombyx) Costa Rica
superba Möschler, 1880 (Gonodonta) Surinam
maria Guenée, In Boisduval and Guenée 1852a (Gonodonta) Brazil
avangareza Schaus, 1911a (Gonodonta) Costa Rica
mexicana Schaus, 1901 (Gonodonta) Mexico
nitidimacula Guenée, In Boisduval and Guenée 1852a (Gonodonta) Ile St. Thomas
nutrix (Cramer, 1780) (Phalaena Noctua) Surinam
acmeptera (Sepp, 1848) (Phalaena) Surinam
obsesa (Walker, [1865] 1864) (Dosa) Brazil
camora (Felder and Rogenhofer, In F.F.R. 1874) (Canodia) Brazil
paraequalis Todd, 1959 (Gonodonta) Mexico
parens Guenée, In Boisduval and Guenée 1852a (Gonodonta) Guadeloupe
plumbicincta Dyar, 1912 (Gonodonta) Mexico
primulina Druce, In Godman and Salvin 1887 (Gonodonta) Guatemala
pseudamianta Todd, 1959 (Gonodonta) Venezuela
pulverea Schaus, 1911a (Gonodonta) Costa Rica
pyrgo (Cramer, 1777) (Phalaena Noctua) Surinam
serix Guenée, In Boisduval and Guenée 1852a (Gonodonta) Colombia
separans Walker, [1858] 1857a (Gonodonta) Brazil
sicheas (Cramer, 1777) (Phalaena Noctua) Surinam
hesione (Drury, 1782) (Phalaena Noctua) Brazil
135
uncina Hübner, 1818 (Gonodonta) Brazil
sinaldus Guenée, In Boisduval and Guenée1852a (Gonodonta) Colombia
sitia Schaus, 1911a (Gonodonta) Costa Rica
soror (Cramer,1780) (Phalaena Noctua) Surinam
sphenostigma Todd, 1973 (Gonodonta) Brazil
syrna Guenée In Boisduval and Guenée1852a (Gonodonta) Fr. Guiana
unica Neumoegen, 1891 (Gonodonta) USA
uxor (Cramer, 1780) (Phalaena Noctua) Surinam
marmorata Schaus, 1906 (Gonodonta) Mexico
walkeri Todd, 1959 (Gonodonta) Costa Rica
GRAPHIGONA Walker, [1858] 1857b: 1230
Type species: Ophideres regina Guenée, by subsequent designation of Berio 1966: 59.
regina (Guenée In Boisduval and Guenée 1852b) (Ophideres) Colombia
antica Walker [1858] 1857b (Graphigona) Brazil, NEW SYNONYMY
gubernatrix (Guenée In Boisduval and Guenée 1852b) (Ophideres) “Tropical America”
ORAESIA Guenée, In Boisduval and Guenée, 1852b: 362
Type-species: Noctua emarginata Fabricius, by subsequent designation of Warren In Seitz 1913.
aeneofusa (Hampson, 1926) (Calpe) Panama
albescens (Seitz, 1940) (Calpe) Unknown
argyrolampra (Hampson, 1926) (Calpe) Colombia
argyrosema (Hampson, 1926) (Calpe) Brazil
argyrosigna (Moore, [1884]) (Oraesia) Sri Lanka
basiplaga (Walker, 1865) (Calpe) Dominican Republic
136
camaguina Swinhoe, 1918 (Oraesia) Philippines
cerne (Fawcett, 1916) (Calpe) Ghana
emarginata (Fabricius, 1794) (Noctua) India
alliciens Walker, [1858] 1857a (Oraesia) India
metallescens Guenée, In Boisduval and Guenée 1852a (Oraesia) Unknown
tentans Walker, [1858] 1857a (Oraesia) India
excavata (Butler, 1878a) (Calpe) Japan
excitans Walker, [1858] 1857a (Oraesia) Dominican Republic
glaucocheila (Hampson, 1926) (Calpe) Brazil
honesta Walker, [1858] 1857a (Oraesia) Dominican Republic
igneceps (Hampson, 1926) (Calpe) Br. Guyana
nobilis Felder and Rogenhofer, In F.F.R. 1874 (Oraesia) Brazil
pierronii (Mabille, 1880) (Odontina) Madagascar
provocans Walker, [1858] 1857a (Oraesia) South Africa
cuprea Saalmüller, 1891 (Oraesia) Madagascar
hartmanni Möschler, 1883 (Oraesia) South Africa
rectistria Guenée, In Boisduval and Guenée 1852a (Oraesia) India
serpens Schaus, 1898 (Oraesia) Mexico
striolata Schaus, 1911a (Oraesia) Costa Rica
stupenda Dognin, 1912 (Oraesia) Colombia
subucula Dognin, 1910 (Oraesia) Paraguay
triobliqua (Saalmüller, 1880) (Odontina) Madagascar
wintgensi (Strand, 1909) (Calpe) Rwanda
137
PLUSIODONTA Guenée, In Boisduval and Guenée 1852b: 385
Type-species: Plusiodonta chalsytoides Guenée, by subsequent designation of Desmarest In
Chenu 1857: 123.
Gadera Walker, [1858] 1857a
Deva Walker, [1858] 1857a
Odontina Guenée, 1862
Tafalla Walker, 1869a
Tinnodoa Nye, 1975
aborta Dognin, 1910 (Plusiodonta) Colombia
achalcea Hampson, 1926 (Plusiodonta) South Africa
amado Barnes, 1907 (Plusiodonta) USA
arctipennis Butler, 1886 (Plusiodonta) Australia
auripicta Moore, 1882 (Plusiodonta) India
basirhabdota Hampson, 1926 (Plusiodonta) Kenya
casta (Butler, 1878b) (Platydia) Japan
chalcomera Hampson, 1926 (Plusiodonta) Kenya
clavifera (Walker, 1869a) (Tafalla) Honduras
cobaltina Viette, 1956 (Plusiodonta) Madagascar
coelonota (Kollar and Redtenbacher, 1844) (Plusia) India
agens (Felder and Rogenhofer, In F.F.R. 1874) (Plusia) India
chalsytoides Guenée, In Boisduval and Guenée 1852a (Plusiodonta) Indonesia
conducens (Walker, [1858] 1857a) (Deva) Sri Lanka
commoda Walker, 1865 (Plusiodonta) Sierra Leone
138
compressipalpis Guenée, In Boisduval and Guenée 1852a (Plusiodonta) N. America
insignis Walker, 1865 (Plusiodonta) USA
suffusa Hill, 1924 (Plusiodonta) USA
cupristria Kaye, 1922 (Plusiodonta) Trinidad
dimorpha Robinson, 1975 (Plusiodonta) Fiji
effulgens Edwards, 1884 (Plusiodonta) Mexico
euchalcia Hampson, 1926 (Plusiodonta) Malawi
excavata (Guenée, 1862) (Odontina) Unknown
gueneei (Viette, 1968) (Odontina) Madagascar
incitans (Walker, [1858] 1857a) (Gadera) Unknown
ionochrota Hampson, 1926 (Plusiodonta) Ghana
macra Hampson, 1926 (Plusiodonta) Kenya
malagassy (Viette, 1968) (Odontina) Madagascar
megista Hampson, 1926 (Plusiodonta) Kenya
miranda Schaus, 1911a (Plusiodonta) Costa Rica
multicolora (Bethune-Baker, 1906) (Deva) New Guinea
natalensis Walker, 1865 (Plusiodonta) South Africa
detracta Walker, 1865 (Plusiodonta) South Africa
nummaria Felder and Rogenhofer, In F.F.R. 1874 (Plusiodonta) South Africa
tripartita Walker, 1865 (Plusiodonta) South Africa
nictites Hampson, 1902 (Plusiodonta) South Africa
nitissima Schaus, 1911a (Plusiodonta) Costa Rica
repellens (Walker, [1858] 1857a) (Gadera) Unknown
139
speciosissima (Holland, 1894) (Deva) Cameroon
stimulans (Walker, [1858] 1857a) (Deva) Dominican Republic
theresae Holloway, 1979 (Plusiodonta) New Caledonia
thomae Guenée, In Boisduval and Guenée 1852a (Plusiodonta) Virgin Islands
tripuncta (Bethune-Baker,1906) (Marcipa) New Guinea
wahlbergi (Felder and Rogenhofer, In F.F.R. 1874) (Plusia) South Africa
africana (Holland, 1894) (Deva) Gabon
regina (Guenée, In Boisduval and Guenée 1852b) (Ophideres) Colombia
TETRISIA Walker, 1867: 186
Type species: Tetrisia florigera Walker, by monotypy.
florigera Walker, 1867 (Tetrisia) Colombia
magnifica (Schaus, 1911b) (Graphigona) Costa Rica
roseifer (Felder and Rogenhofer, In F.F.R. 1874) (Graphigona) Brazil
Genus and tribal placement undetermined
1. Cecharismena Möschler, 1890
Checharismena, Fibiger and Lafontaine 2005; misspelling
2. Culasta Moore, 1881a
3. Goniapteryx Perty, In Spix 1833
4. Hemiceratoides, Strand 1911
5. Hypsoropha Hübner, 1818
6. Pharga Walker, 1863
7. Phyprosopus Grote, 1872
8. Psammathodoxa Dyar 1921
9. Radara Walker, 1862
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CHAPTER 5 ANOTHER BLOOD FEEDER? EXPERIMENTAL FEEDING OF A FRUIT-PIERCING
MOTH SPECIES ON HUMAN BLOOD IN THE PRIMORYE TERRITORY OF FAR EASTERN RUSSIA (LEPIDOPTERA: NOCTUIDAE: CALPINAE)
Introduction
The genus Calyptra Ochsenheimer (Lepidoptera: Noctuidae: Calpini) includes what are
commonly known as vampire moths, so named because of their ability to pierce mammalian
flesh and feed on blood. These are medium sized moths, with wingspans ranging from 35-72
mm in size (Bänziger 1983, Table 1) (Figs. 5-1, 5-2, 5-3). Species in this genus occur in S.
Europe, eastern Africa, sub-Himalayan regions of S. Asia, the Manchurian subregion, and are
broadly distributed throughout S.E. Asia. Calyptra species have enjoyed popularity among
members of the entomological community due to their modified proboscides equipped with
strongly sclerotized barbed hooks used for piercing through both thick and hard skinned fruits
such as peaches, plums, and citrus as well as mammals (Bänziger 1982, Zaspel pers. obs., Fig. 5-
4).
Of the 17 known Calyptra species (Bänziger 1983), C. eustrigata (Hampson), C.
minuticornis minuticornis (Guenée), C. orthograpta (Butler), C. bicolor (Moore), C. fasciata
(Moore), C. ophideroides (Guenée), C. parva Bänziger and C. pseudobicolor Bänziger have
been reported to pierce mammalian skin, the latter five also of man, under natural conditions
while C. fletcheri (Berio) has done so in experiments (Bänziger 1968, Bänziger 1989). These
species are considered facultative or opportunistic blood-feeders primarily in subtropical areas in
southern Asia and tropical Southeast Asian countries: their hosts are typically ungulates such as
cattle, tapirs, zebu, and occasionally elephants and humans; female Calyptra adults have not
been documented feeding on blood (Bänziger 1989b). At least four additional closely related
genera (Eudocima, Gonodonta, Oraesia, and Plusiodonta) have apparently homologous
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proboscides modifications used for fruit-piercing, but the occurrence of blood-feeding in those
species has not been observed (Bänziger 1979 and personal communication, Zaspel, unpublished
data). Although it is known that some fruit-piercing moths are of great economic importance,
e.g., Eudocima fullonia and to a lesser degree even some Calyptra spp. (Fay 2002, Fay and
Halfpaff 2006, Sands 1993, Todd 1959, Yoon and Lee 1974), their potential as vectors of human
or animal disease remains a possibility, however, whether a real danger of vectoring disease
exists is unknown (Bänziger 1980, Bänziger 1989). The purpose of this paper is to document the
first case of Calyptra feeding on human blood in Far Eastern Russia and the novel finding of
blood feeding by the species C. thalictri under experimental conditions.
Materials and Methods
Description of observation sites
During an expedition in the Primorye Territory of Far Eastern Russia (Fig. 5-5) in July,
2006, I sought to observe feeding behaviors of the three Calyptra species (C. hokkaida Wileman,
C. lata Butler, and C. thalictri Borkhausen) in this region (Kononenko 1990a; Remm 1980b).
None of these species had been recorded feeding on mammalian blood and have been considered
exclusive fruit piercers. Since the complete geographic occurrence—and precise phylogenetic
origins—of blood feeding Calyptra remain unknown, an objective of this work was to determine
whether Calyptra species that occupy the northern extent of their range were hematophagous.
The southern part of the Russian Far East (Primorye territory) lies in a zone of
Manchurian coniferous and mixed coniferous and broad leaved forests with very rich and diverse
vegetation. The main forest formations in the region are Abies–Picea taiga in the upper and mid
mountain belts, Pinus koraiensis mixed forest in the mid mountain belt, Abies nephrolepsis
mixed forest in the south of the region, deciduous broad leaved forest and forest dominated by
Quercus mongolica (Kurentzov 1965, Richter 1961). The mixed and deciduous forests of the
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south Far East contain many east Asian trees and shrubs that are absent from Siberia, such as
Quercus, Fraxinus, Acer, Tilia, Ulmus, Carpinus, Phellodendron, Maakia, Aralia, Calopanax,
Actinidia, Schisandra (Kurentzov 1965, Richter 1961). The sub-alpine, and mountain tundra
belts are fragmentary, and occur in the Sikhote-Alin mountain range above 1500m in the central
and northern part of the region. The forest–steppe zone occurs mainly in the southwestern
portions of the region (Kurentzov 1965, Richter 1961).
The climate of the Primorye territory is dominated in part by monsoon features. The
annual temperature in Primorye, is + 4° C, temperature in January: 10-15°C, in July it is over
+20° C (Kurentzov 1965, Richter 1961). The annual precipitation is 800–1200 mm, roughly 70–
75% of which falls from July to September. North–westerly winds dominate in winter, while
south–east winds prevail in the summer (Kurentzov 1965, Richter 1961).
We collected Calyptra specimens at two primary sites (designated Sites 1 and 2; Figs. 5-
6, 5-7, 5-8, 5-9) and three subsites (designated subsites 1a and 1b; subsites 2a, 2b, and 2c
respectively). Collecting site 1 (including subsites 1a and 1b) was in the vicinity of the
Kraunouka Village at the Borisovskoe Hunting Area, roughly 20 km west of Ussuriisk, Russia
(Figs. 5-6 and 5-8: N 43˚ 44.577, E 131˚ 38.218; 287 ft.). This is a popular hunting site for wild
boar (Sus scrofa sp.), two species of deer (Cervus nippon and Capreolus capreolus), and bear
(Ursus thibetanus), situated in the easternmost spurs of the East Manchurian montane system
(Fig. 5-6). Although it has not yet been observed in this region, these mammals could potentially
serve as hosts of adult Calyptra species. This area contains low elevation mountains (100-300
m), hills and cliffs in the upper reaches of the Kraunouka River. The vegetation around the
collecting sites consists primarily of broad leaf forests dominated by oak (Quercus mongolica)
(Kurentzov 1965, Richter 1961). The vegetation along the river valley is considerably more
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diverse broad-leafed forest with variously dominant species such as Juglans mandshurica, Ulmus
japonica, and, Vitis amurensis. Mixed broad-leaf coniferous forest with coniferous trees such as:
Abies holophila, A. nephrolepsis, Pinus koraiensis, and P. funebris, cover the upper reach of
Kraunouka River (Kurentzov 1965, Richter 1961). Most relevant, are the presence of larval food
plants of the local species of Calyptra: Menispermum dahuricum, Thalictrum contortum, T.
simplex, and T. amurensis are abundant in meadows (Kurentzov 1965, Richter 1961).
The second collecting site (subsites 2a, 2b, and 2c) was at the Gornotayeznaya Biological
Station Far Eastern Branch of the Russian Academy of Sciences (20 km east of Ussurisk). This
collecting site was in the vicinity of Gornotayeznoe village situated in southwestern spurs of
Sikhote-Alin montane range (Figs. 5-7 and 5-9: N 43˚ 41.917 E 132˚ 09.131). The area is in the
vicinity of low elevation mountains (200-400 m). The vegetation around the collecting site is
similar to first collecting locality and consists of broad-leaf forest in a creek valley. Wild
rosaceous plants are less well represented here than in the former site; however, apple, pear,
cherry and apricot trees are cultivated in gardens. The fruits of Rosaceae are commonly attacked
by many fruit-piercing moths, including some Calyptra spp. (Bänziger 1971, Bänziger 1982).
The southern slopes of the hills are covered by secondary forest dominated by Quercus
mongolica and Corylus mandschurica in the understory; the northern slopes are covered by
mixed broad leaf – coniferous forest with native and planted trees and pure primary forest
dominated Pinus koraiensis situated in the upper reaches of the Krivoi kljuch creek (Kurentzov
1965, Richter 1961), roughly 10 km from the collecting site.
Experimental methods
Moths were collected using standard techniques: suspended white sheets illuminated by a
60 watt mercury vapor lamp (HgVpr, Winter 2000). Live specimens were collected into separate
10 dram plastic vials. Upon return to the field station at primary collecting sites 1 and 2, male
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specimens were retained alive overnight. Females were transferred into 95% EtOH and males
were placed in a small rearing tent with small pieces of wet cotton for about four hours. Each
specimen was carefully removed from the tent and placed in a numbered live vial (Table 5-1).
Feeding trials were conducted to determine which, if any, of the additional specimens collected
would penetrate human skin and feed on blood. The moths were presented with two separate
feeding opportunities conducted over a 24-hour period. The first trial was always the morning
after the moths were collected, and started between 0800h and 1000h second trial began in the
evening between 1900h and 2100h. The trials were conducted by inserting a thumb into the vial
in such as way so that the moth could not escape, and each moth’s behavior was recorded for ten
minutes. If the moth began to feed, or made an attempt to feed beyond the ten minute time
period it was not interrupted. If a moth did take a blood meal, it was eliminated from the
experiment and recorded as a positive blood-feeder. This was done to minimize potential injury
and/or allergic reactions on the part of the subjects. If no feeding activity was observed after ten
minutes, or if previous feeding behavior(s) ceased, the thumb was removed from the vial and the
moth was given another opportunity to feed during trial 2. All species identifications were
confirmed based on external morphology and genitalic dissections.
Results
On July 14th 2006 a male specimen of Calyptra thalictri (Fig. 5-1) was collected in
Kraunouka about 2 km from the field station in the hunting area (subsite 1a) at approximately
2300h. This specimen was placed in a plastic live vial (designated vial #1, Table 5-1) and a
human thumb inserted into the vial to observe potential piercing behavior. After three minutes,
the moth inserted its proboscis into the thumb just below the nail and began to uptake blood for
approximately 2 minutes. Three additional specimens of this species (two males, and one
female) were also collected the same night at subsite 1a. The following morning (7-15-06:
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1047h), Specimen in vial #1 was given another opportunity to feed on human blood (Table 5-1).
During this time, the moth grazed its proboscis across the subject’s thumb, lapping moisture off
of the skin. After approximately five minutes, the moth inserted the tip of its proboscis into a
crease in the skin on the joint of the thumb and pierced through the skin (Fig. 5-10A). The moth
sucked blood briefly (less than 30 seconds) and without removing the tip of the proboscis pierced
further into the wound and then continued sucking blood. This behavior (intermittent piercing
followed by feeding) continued for six minutes before the moth was removed, and the wound
examined (Fig. 5-10B). The same methodology was used to determine whether the additional
male specimens would feed. However, the specimens only licked the skin, presumably
unsuccessful attempts at piercing. All specimens were ultimately placed in 95% EtOH for future
molecular study.
On July 15th at approximately 2030h (dusk) a male C. thalictri was captured outside the
perimeter of the field station in the Borisovskoe Hunting Area. This specimen was resting on the
trunk of a cherry shrub when it was hand collected. The specimen slowly crawled outside the
slightly compressed hand through the opening between the forefinger and thumb, where it rested.
The moth uncoiled its proboscis several times, touching the skin below the thumbnail. The moth
made several attempts to pierce the skin with its proboscis, finally settling on a crease in the skin
area 7-8 mm above the border of the thumbnail (the same location as the previous blood-feeding
specimen). The moth’s proboscis slowly penetrated the skin. As with the first Calyptra thalictri
specimen collected (specimen in vial #1, Table 5-1), this moth (vial #4, Table 5-1) pulled its
proboscis slightly out of the wound, and then pierced further into the wound. When the moth
pulled its proboscis out of from the wound, it was saturated with blood (Figs. 5-10C, 5-10D, 5-
10E). This feeding continued for an additional seven minutes. This incident of blood feeding
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should be considered “semi-natural”, as the moth was not enclosed in a vial, yet did not actively
seek out the host and could have been disoriented given the time of day was earlier than its
typical flight time. The subject observed slight swelling in the area around the wound, and
described a feeling of slight stinging pain for 2-3 hours following the attack. After feeding
ceased, the specimen was placed in a vial and kept alive overnight. At 0130h the live vial
containing this moth had several large drops of blood at the bottom indicating excretion of the
blood meal (Fig. 5-10F). Later that morning all blood droplets were absent, suggesting re-uptake
of the blood meal, and when the moth was removed from the vial and transferred into EtOH,
additional blood droplets were excreted from the tip of the abdomen.
Between the hours of 2230h and 0100h on 15 July, thirteen additional Calyptra
specimens were collected at a third site about 10 km from the hunting area (subsite 1b) using the
same methods. Of these specimens, eight were C. thalictri (Fig. 5-1; three females and five
males), and five were C. lata (Fig. 5-2; one female and four males). All specimens were
collected into separate live vials and were transported to the Gornotayeznaya Biological Station.
At the Gornotayeznaya Biological Station, the females were given the opportunity to feed, but
made no attempts to do so. The lack of attempts to feed or even lick moisture from the subject’s
thumb corroborates previously published work that states female Calyptra species do not feed on
blood (Bänziger 1989 [b]). Females were not included in future feeding trials.
In addition to the two blood feeders mentioned above, one of the male C. thalictri
specimen collected from subsite 1b (vial #5, Table 5-1) pierced the senior author’s thumb and
fed on blood for four minutes and thirty seconds. The remaining C. thalictri specimens only
attempted to pierce but uptook moisture from the skin. The C. lata specimens made no attempt
to feed.
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During the course of these feeding experiments, seven additional Calyptra thalictri
specimens, and six specimens of C. lata were collected in the vicinity of the Gornotayeznaya
Biological Station and subjected to feeding trials. These specimens were presented the same
feeding opportunities those specimens taken from the Borisovskoe Hunting Area. Of the C.
thalictri collected at various sites surrounding the Gornotayeznaya Biological Station, none fed
on blood and only one specimen made a weak attempt to pierce the skin (but did not draw
blood); all specimens, however, did graze the skin with the proboscis, lapping moisture off of the
thumb. No attempts to pierce or lap moisture from skin were observed by any of the C. lata
specimens. Sixteen additional specimens of C. lata and C. thalictri were collected on 21 July
and were either pinned or transferred into tubes with 95% EtOH for future morphological and
molecular study. Of the nine male C. thalictri specimens collected at Primary site 1 (and
subsites therein), 44% were successful at piercing skin and feeding on blood. Calyptra lata
specimens from Primary site 1 did not attempt to pierce. One attempt to pierce was made by a C.
lata specimen from Primary site 2; C. thalictri specimens from Primary site 2 made no attempts
to pierce. A summary of feeding behaviors for the Calyptra specimens subjected to feeding
trials during 14-15 July and 17-20 July is provided in Table 5-1.
Discussion
These are the first recorded experimental observations of blood feeding by a Calyptra
species in the Primorye Territory. Although under experimental and semi-natural conditions,
they represent the first documented occurrence of blood feeding by Calyptra in a temperate
region, and a new blood-feeding species record for the genus. The observed blood feeding
behavior of C. thalictri specimens in this study consisted of spindle movements, head
oscillations, followed by frequent partial withdrawl and re-insertions of the proboscis; thus, the
blood feeding behavior of C. thalictri under the conditions reported in this paper are identical to
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those previously described for the blood feeding species C. eustrigata (Bänziger 1968, Bänziger
1980). It was unclear from the feeding experiments whether saliva was released or if blood was
regurgitated during feeding.
Although indistinguishable by genitalia and proboscis morphology (including
development of armrature), C. thalictri specimens collected in the Primorye Territory vary
phenotypically from those that occur in Palearctic regions: specimens collected in Primorye have
dark green forewings as opposed to the red-orange colored Palearctic specimens (compare Figs.
5-1 and 5-11). It is possible that the two populations represent two different species, but
addressing this question is beyond the scope of this paper. The fact that Calyptra thalictri
specimens from the second collecting site did not pierce skin and feed on blood is also consistent
with previous published feeding behavior records for this species (Bänziger 1970, Bänziger
1971). Similar differential feeding behaviors have also been reported in other Calyptra
species, e.g., C. fasciata (Bänziger 1989 [b]).
Whether or not Calyptra thalictri are feeding on blood under natural conditions in the
Primorye Region is presently under further investigation. It is possible that C. thalictri
specimens’ feeding behavior was different in the enclosed environment as opposed to their
behavior under completely natural conditions; however, these preliminary observations indicate
that C. thalictri can be induced to pierce human skin to suck under experimental conditions,
while C. lata cannot. These findings are also consistent with previous studies reporting the
blood feeding behavior in these moths is restricted to the males. It has been suggested that males
may engage in zoophilous feeding behaviors as a result of sugar, salt, or amino acid deficiency
(Scoble 1992). When the fruit or nectar hosts supplying these nutrients are unavailable,
butterflies and moths will seek alternate substrates on which to feed (puddles, dung, urine, sweat,
149
150
tears, and blood). Bänziger (1972, 1990) demonstrated that the eulachryphagous moth
Lobocraspis griseifusa Hampson has proteinases with which it can digest protein contents in the
tears of mammals while such hemilachryphagous pyralids as Filodes mirificalis Lederer are, like
the very vast majority of adult Lepidoptera, incapable of protein digestion. It is uncertain
whether such a mechanism exists in blood feeding Calyptra spp. It should be noted however,
that the skin piercing behavior followed by sucking blood from the mammalian host is derived
from the fruit piercing habit, as opposed to other zoophilous feeding behaviors, e.g., tear feeding.
No full analysis of the digestive capabilities of Calyptra has yet been published; however, the
observations in this study support previous work that suggests blood-feeding moths may engage
in this behavior facultatively, depending on regional availability of mammalian versus vegetative
hosts.
Table 5-1. Summary of feeding behaviors for moth specimens collected in Primorye Terriotry of Far Eastern Russia from July 14t-15th 2006 and July 17-20th 2006. LM = Licking moisture off skin with proboscis, PA = Piercing attempted, PBF = Piercing and blood-feeding, NA = No attempt to lick moisture, pierce skin, or feed on blood, DT = Dead at time of trial.
Species Site Sex Date Collected Vial Number Feeding Trial Behavior Time C. thalictri Subsite 1a M 7-14-06 1 1 PBF 2 min. 2 PBF 6 min. C. thalictri Subsite 1a M 7-14-06 2 1 LM 10 min. 2 LM 10 min. C. thalictri Subsite 1a M 7-14-06 3 1 LM 10 min. 2 LM 10 min. C. thalictri Primary 1 M 7-15-06 4 1 PBF 11 min. C. thalictri Subsite 1b M 7-15-06 5 1 LM 10 min. 2 PBF 4.5 min. C. thalictri Subsite 1b M 7-15-06 6 1 LM 10 min. 2 LM 10 min. C. thalictri Subsite 1b M 7-15-06 7 1 LM 10 min. 2 LM 10 min. 151 C. thalictri Subsite 1b M 7-15-06 8 1 LM 10 min. 2 LM 10 min. C. thalictri Subsite 1b M 7-15-06 9 1 LM 10 min. 2 LM 10 min. C. lata Subsite 1b M 7-15-06 10 1 NA 10 min. 2 NA 10 min. C. lata Subsite 1b M 7-15-06 11 1 NA 10 min. 2 NA 10 min. C. lata Subsite 1b M 7-15-06 12 1 NA 10 min. 2 NA 10 min. C. lata Subsite 2a M 7-17-06 13 1 LM 10 min. 2 PA 10 min. C. thalictri Subsite 2b M 7-18-06 14 1 LM 10 min. 2 LM 10 min.
Table 5-1. Continued Species Site Sex Date Collected Vial Number Feeding Trial Behavior Time C. thalictri Subsite 2b M 7-18-06 15 1 LM 10 min. 2 LM 10 min. C. thalictri Subsite 2b M 7-18-06 16 1 LM 10 min. 2 LM 10 min. C. thalictri Subsite 2b M 7-18-06 17 1 LM 10 min. 2 LM 10 min. C. thalictri Subsite 2b M 7-18-06 18 1 NA 10 min. 2 NA 10 min. C. thalictri Subsite 2b M 7-20-06 19 1 LM 10 min. 2 DT n/a C. thalictri Subsite 2b M 7-20-06 20 1 LM 10 min. 2 NA 10 min. C. lata Subsite 2b M 7-20-06 21 1 NA 10 min. 2 NA 10 min. C. lata Subsite 2c M 7-21-06 22 1 NA 10 min. 2 NA 10 min. C. lata Subsite 2c M 7-21-06 23 1 NA 10 min. 2 NA 10 min. C. lata Subsite 2c M 7-21-06 24 1 NA 10 min. 2 NA 10 min. C. lata Subsite 2c M 7-21-06 25 1 NA 10 min. 2 NA 10 min. C. lata Subsite 2c M 7-21-06 26 1 NA 10 min. 2 NA 10 min.
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Figure 5-1. Adult habitus image. Calyptra thalictri, Male.
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Figure 5-2. Adult habitus image. Calyptra lata, Male.
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Figure 5-3. Adult habitus image. Calyptra hokkaida, Male.
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TH 4
Figure 5-4. Proboscis of Calyptra thalictri: TH = Tearing Hooks.
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Figure 5-5. Map of Primorye Region of Far Eastern Russia.
157
Figure 5-6. Primary collecting site 1.
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Figure 5-7. Primary collecting site 2.
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Figure 5-8. Map of primary collecting site 1.
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Figure 5-9. Map of primary collecting site 2.
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Figure 5-10. (A) Image of Calyptra thalictri feeding on human thumb (JMZ); (B) Image of
subject’s wound (JMZ) after piercing and feeding by C. thalictri; (C) Image of Calyptra thalictri feeding on human thumb (VK); (D) Image of Calyptra thalictri feeding on human thumb (VK); (E) Image of Calyptra thalictri feeding on human thumb; (F) Live vial showing blood droplets excreted by C. thalictri.
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Figure 5-10. Calyptra thalictri feeding on raspberry during night observations (under natural conditions) in S. Europe, photo by Hans Bänziger.
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CHAPTER 6 MICROBIAL DIVERSITY ASSOCIATED WITH THE FRUIT-PIERCING AND BLOOD-FEEDING MOTH CALYPTRA THALICTRI (LEPIDOPTERA: NOCTUIDAE: CALPINAE)
Introduction
Although blood feeding is common in many insect orders, within the Lepidoptera skin
piercing and blood feeding are restricted to the moth genus Calyptra. Calyptra Ochsenheimer
(Lepidoptera: Noctuidae: Calpini) is comprised of both obligatory fruit-piercing and facultative
blood-feeding species. Of the 17 Calyptra species (Bänziger 1983), half have males that have
been documented to feed on blood under either natural or experimental conditions (Bänziger
1968, Bänziger 1989). Males are opportunistic blood-feeders primarily in subtropical and
tropical areas in Asia; their hosts are typically ungulates such as cattle, tapirs, zebu and,
occasionally, elephants and humans. It has been shown that blood-feeding males sequester up to
95% of the NaCl from the blood (Bänziger 2007). Calyptra females have not been shown to
feed on blood (Bänziger 1989) and it is unclear whether males provide substances from the
ingested blood to them during mating (Bänziger 2007).
The remaining ‘non-blood feeding’ males of Calyptra species are thought to be
obligatory piercers of fruits such as peaches, plums, and citrus (Bänziger 1982). However, males
of C. thalictri, thought to be limited to fruit piercing, were shown to pierce human skin and feed
on a human host for up to 10 min (Zaspel et al. 2007). It is unknown whether these moths ingest
pathogens or parasites during the blood meal; however, if present, the survival of such disease
microorganisms in Calyptra species could be dependent on the presence of other microorganisms
in the midgut (Azambuja et al. 2005). Recent findings suggest that microbial gut endosymbionts
may affect the ability of an insect vector to transmit disease agents to their vertebrate host(s) (St.
André et al. 2002). Thus, the potential of these moths to vector disease may be related to their
microbial gut fauna.
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It has been speculated that blood-feeding Calyptra males are vectors of human or animal
disease, but whether a real danger exists is unknown (Bänziger 1980, Bänziger 1989), and the
occurrence of this C. thalictri population (and other species) in remote areas has hindered study.
An important step in understanding the biology and feeding behavior of the fruit-piercing and
blood-feeding meals of Calyptra will be the identification of their associated microbial
community.
The goals of this study were to conduct a survey for microorganisms associated with
male specimens of C. thalictri obtained in Far Eastern Russia. Males of these moths were
surveyed for Archaea, Eubacteria, fungi including yeast-like organisms, Microsporidia, and
Wolbachia using a high-fidelity PCR assay. Following terminology used in recent bacterial
surveys of Lepidoptera, and because bacterial species are difficult to delineate, organisms
detected in male C. thalictri in this study are refered to as ‘phylotypes’ (Broderick et al. 2004).
Additionally, in an effort to understand which, if any, were consistently associated with C.
thalictri males, the proportion of individuals that contained each phylotype is reported.
Materials and Methods
Specimens
Nine males of C. thalictri were collected at two sites in July 2006 in the Primorye
Territory in Far Eastern Russia. Collecting site one was in the vicinity of the Kraunouka Village
at the Borisovskoe Hunting Area, roughly 20 km west of Ussuriisk, Russia (N 43˚ 44.577, E 131˚
38.218; 84.5 m). The second site was at the Gornotayeznaya Biological Station Far Eastern
Branch of the Russian Academy of Sciences (20 km east of Ussurisk). This collecting site was
in the vicinity of Gornotayeznoe village situated in southwestern spurs of the Sikhote-Alin
montane range (N 43˚ 41.917 E 132˚ 09.131). Moths were collected using suspended white
sheets illuminated by a 60-W mercury vapor lamp (HgVpr, Winter 2000). Live specimens were
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collected into separate 10-dram plastic vials. Specimens were transferred into 95% EtOH and
transported to the United States where they were stored separately at -80ºC prior to DNA
extraction.
Surface Sterilization
A sterilization method to remove the DNA of microorganisms associated with the
external surfaces of C. thalictri was used because it has been demonstrated to both kill surface-
inhabiting microbes and to eliminate their DNA when other solutions fail to do so consistently.
This protocol does not interfere with PCR analysis of gut or reproductive tract endosymbionts
(Meyer 2007, Meyer and Hoy in press). Whole bodies of individual moths each were placed in a
15-mL plastic centrifuge tube filled with 6% sodium hypochlorite solution for 3 min and then
rinsed five times with autoclaved double de-ionized water using sterile filtered pipette tips
(Posada and Vega 2005). The abdomens then were removed from each moth using sterilized
spring scissors and sterilized fine-tip forceps and placed in separate, covered sterile petri plates.
Spring-scissors, cleaned with 6% sodium hypochlorite solution followed by 100% EtOH, and
held in a flame for 5 sec, were used to make a small incision at the tip of the abdomens of C.
thalictri males under a dissecting microscope to examine the genitalia in order to confirm the
identity of each specimen. All abdominal contents, including the digestive and reproductive
tracts, hemolymph, and fat body were removed and placed in sterile 1.5-mL sterile centrifuge
tubes. In order to determine whether the moths had specific microbes associated with the
salivary glands and/or their mouthparts, heads with the proboscis attached and the contents of the
pharynx were removed from each specimen after the bleach treatment using a sterilized fine-tip
forceps under a dissecting microscope. Heads and associated mouthparts were rinsed with sterile
water in the same manner as the abdomens and then placed in sterile 1.5-mL centrifuge tubes.
Head and gut contents were stored at -80ºC prior to DNA extraction. These procedures were
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conducted in a different room from where the DNA extractions and PCR reactions were
conducted.
DNA Extraction
Individual surface-sterilized moth abdomens or heads were homogenized with a
disposable blunt-ended sterile pipette tip for 4 min each. Homogenized tissues were used for
DNA extraction using PUREGENE reagents (Gentra Systems, Minneapolis, MN) according to
the manufacturer’s protocol. Due to the large amounts of tissue and fat body in the moths, two
protein precipitations were performed on all abdominal samples. DNA pellets were dried for 20
min and re-suspended in 100-μL of sterile water. To further purify the samples, they were
subjected to a chloroform extraction: 200-μL of sterile water was added to 200-μL of chloroform
and added to the samples; the samples were then mixed by hand with a disposable sterile blunt
pipette tip until smooth, and then placed on ice for 5 min. Samples were centrifuged for 15 min
at 12,000 rpm and the supernatant was removed and transferred into a clean 1.5-mL centrifuge
tube. DNA was precipitated using isopropanol at -80ºC. Samples were centrifuged for 15 min at
12,000 rpm and the DNA pellets were washed with 70% EtOH, air dried for 5 min, and re-
suspended in 100-μL of sterile water. In order to prevent contamination of the surface-sterilized
samples, all DNA extractions were performed in an area separated from where the high-fidelity
PCR was conducted.
The quantity of DNA in each sample was determined using a spectrophotometer
(BIORAD SmartSpec Plus, Hercules, CA). For the initial survey, 1-μL of DNA from each
abdominal or head extract was pooled into a single 1.5-mL microcentrifuge tube and 1-μL of this
pooled sample was used in PCR analyses with the primers listed in Table 6-1A. These samples
were pooled to reduce the chance of missing potential microbial associates that were not present
in 100% of the population. In order to increase the amount of DNA available for subsequent
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phylotype-specific PCRs on individual specimens, a multiple displacement amplification (MDA)
reaction was performed on 1-μL of DNA of each extract following the recommendations of the
manufacturer (Dean et al. 2001, Dean et al. 2002, Jeyaprakash and Hoy 2004); resulting
Genomphi amplification products (Amersham Biosciences Piscataway, NJ) were stored at -80ºC.
This supply provided us with additional DNA in the event that a large number of microbial
endosymbionts were found in the pooled DNA.
High Fidelity Polymerase Chain Reaction
A 25-μL high-fidelity PCR that contained 50 mM Tris, pH 9.2, 16 mM ammonium
sulfate, 1.75 mM MgCl2, 350 mM dNTPs, 800 pmol primers, 1 unit Pwo DNA polymerase and
five units of Taq DNA polymerase (Roche Molecular Biochemicals, Indianapolis, IN) was used
to amplify 1 μL of template DNA with the PCR reaction conditions listed in Hoy and
Jeyaprakash (2005) with the primers listed in Table 6-1A. Three linked profiles were used (i) 1
cycle of denaturation at 94ºC for 2 min; (ii) 10 cycles of denaturation at 94ºC for 10 s, annealing
at 65ºC for 30 s, and elongation at 68ºC for 1 min; and (iii) 25 cycles of denaturation at 94ºC for
10 s, annealing at 65ºC for 30 s, an elongation at 68ºC for 1 min plus an additional 20 s for each
consecutive cycle. Agarose gel electrophoresis (1% TAE gels) was used to separate PCR-
amplified DNA, which was stained with ethidium bromide and visualized with ultraviolet light.
Cloning and Restriction Fragment Length Polymorphism Analysis
PCR products were purified with the QIAquick PCR Purification Kit (QIAGEN,
Valencia, CA). To enhance cloning efficiency, 10 μL of all PCR products were combined with
1-μL dATP, 1-μL Taq DNA polymerase, 1-μL buffer, and then placed in the thermocycler at
72ºC for 1 h. PCR products were then cloned into pCR2.1 TOPO following the manufacturer’s
protocol (Invitrogen, Carlsbad, CA); colonies were grown on petri plates at 37ºC overnight.
Individual E. coli transformants were selected at random, grown overnight in 5-mL of LB broth
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+ 20 μL of ampicillin, and plasmid DNA was extracted using QIAGEN Plasmid Mini Columns
(QIAGEN, Valencia, CA). The presence and size of inserted DNA was confirmed by gel
electrophoresis of plasmids following digestion with EcoR I for 2 h at 37ºC. Rsa I digests were
used for the restriction fragment length polymorphism (RFLP) analyses (Jeyaprakash et al.
2003). Clones with inserts yielding unique RFLP’s were bidirectionally sequenced at the
University of Florida Interdisciplinary Core Facility, Gainesville, FL. The resulting sequences
were used to design species-specific primers for use in high-fidelity PCR on individual males in
order to determine the frequency of infection with each phylotype. Bacterial phylotypes detected
in all individual C. thalictri specimens might be considered primary endosymbionts while those
found in fewer specimens could be considered secondary endosymbionts or strays from the
environment. Primers for each phylotype detected were designed manually (Table 6-1B).
Results and Discussion
High-fidelity PCR Amplification of Microbial Associates in C. thalictri
16S rRNA was amplified using template DNA isolated from the abdomen, but not the
head (including mouthparts) and yielded the expected ~1.5-kb band. This suggests that no
symbionts occur in the salivary glands and that the mouthparts were not contaminated with
bacteria after surface sterilization. No amplification products were detected using DNA isolated
from the head or abdomen of the C. thalictri specimens using primers for Archaea, Fungi
including yeast-like organisms, or Microsporidia. Wolbachia were not detected using either
eubacterial 16S rRNA or wspA Wolbachia-specific primers. Given the limited number of C.
thalictri samples, false negatives are a possibility, but these results suggest the microbial
associates of this population are limited to Eubacteria.
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Microbial Associates of C. thalictri
The eubacterial 16S rRNA PCR products were cloned using 1-μL of DNA that had been
pooled from nine C. thalictri males. A total of 34 clones were selected at random and analyzed
using the RFLP technique. Analysis of EcoR I digests indicated that all clones contained an
insert approximately 1.4-kb in length. The Rsa I digests of the 34 clones resulted in five unique
banding patterns. Three clones representing each banding pattern were sequenced (15 total). Of
the 15 sequences, five were unique, indicating that there are at least five types of Eubacteria
associated with male specimens of the blood-feeding population of C. thalictri occurring in the
Primorye Territory of Far Eastern Russia. It is always possible that low frequency phylotypes or
specialized Eubacteria will be found in future analyses.
One sequence (1457 bp, GenBank accession EF599757) from C. thalictri produced
significant alignments to Alcaligenes (Achromobacter) xylosoxidans (Yabuuchi and Ohyama)
strain NFRI-A1of the β-proteobacterial family Alcaligenaceae (1485 bp, GenBank accession
AB161691.1, Yan et al. 2004). This sequence (EF599757), designated Alcaligenes phylotype 1
from C. thalictri, exhibited a 10.8% sequence divergence relative to the A. xylosoxidans strain
NFRI-A1 sequence, which was initially identified from the aflatoxin-producing fungus
Aspergillus parasiticus Speare (Yan et al. 2004). The sequence from C. thalictri was 99%
similar to an uncultured eubacterial species (1475 bp, GenBank accession EF509705) from an
“environmental sample” (Flanagan et al. unpublished) in the BLASTn results. Alcaligenes
(Achromobacter) xylosoxidans has been identified as part of the culturable microbial community
found from the fluid of hooded-pitcher plants, with which some insects form close mutualistic
associations (Siragusa et al. 2007). Other Alcaligenes (Achromobacter) species have been
detected in the midguts of malarial mosquitoes (Lindh et al. 2005), and associated with
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entomopathogenic nematodes (Poinar 1966). Alcaligenes xylosoxidans denitrificans (Ruger and
Tan) species have also been used in paratransgenesis experiments, in which these genetically
modified gut bacteria were delivered into the gut of the glassy-winged sharpshooter
Homalodisca coagulata (Say) in order to interrupt transmission of pathogens to crops (Bextine et
al. 2004). Given the occurrence of this genus in the guts of other insects, it is possible that this
phylotype is associated with the gut of C. thalictri males.
A second sequence (1477 bp, GenBank accession EF599759) produced significant
alignments to 16S rRNA sequences from β-protobacterial species in the family Alcaligenaceae.
This sequence (EF599759), designated Alcaligenes phylotype 2 from C. thalictri, was similar to
the homologous portion of the 16S rRNA sequence from Alcaligenes sp. strain IS-67 (9.7%
sequence divergence (Table 6-2), 1503 bp, GenBank accession AY346140.1), isolated from an
“activated sludge system” (Zhang et al. 2004). This phylotype from C. thalictri could be a
transient from the environment, or a gut associate.
A third sequence (1485 bp, GenBank accession EF599758) produced significant
alignments with the γ-proteobacterial family Enterobacteriaceae. This sequence (EF599758),
designated Klebsiella phylotype from C. thalictri, was similar (0.05% sequence divergence,
Table 2) to the homologous portion of the 16S rRNA gene from K. oxytoca (Flügge) isolate GR6
(1504 bp, GenBank accession AY873801.1), which is typically associated with nonleguminous
plants (Jha and Kumar unpublished). However, K. oxytoca isolates also are associated with
insects, and have been detected in the frass of the leek moth Acrolepiopsis assectella Zeller
(Thibout et al. 1995). Both K. pneumoniae (Schroeter) and K. oxytoca have been isolated from
pink bollworm Pectinophora gossypiella (Saunders) larvae and adults (Kuzina et al. 2002).
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Klebsiella species also have been detected in the Noctuidae (Lighthart 1988) and Pyralidae
(Charpentier et al. 1978). It is likely that this phylotype is a gut symbiont of C. thalictri.
A fourth sequence (1410 bp, GenBank accession EF599760), designated Rhizobium
phylotype from C. thalictri, was 99% similar to homologous portions of the 16S rRNA sequence
from a Rhizobium species (Heylen et al. 2006) and from an Agrobacterium tumefaciens (Smith
and Townsend) sequence (1446 bp, Genbank accession DQ468100.1; Wang and Yan
unpublished) in the BLASTn results. Subsequent sequence divergence analyses suggest it is
more similar to the Sinorhizobium phylotype 2 (EF599761) from C. thalictri (4.3% sequence
divergence, Table 6-2), than it was to the Rhizobium sp. sequence (6.9% sequence divergence,
Table 6-2). Species belonging to the Rhizobium-Sinorhizobium group are essential nitrogen-
fixing bacteria found in legumes (Heckman and Drevon 1987).
A fifth sequence (1431 bp, GenBank accession EF599761) designated Sinorhizobium
phylotype from C. thalictri, produced significant alignments to S. morelense of the α-
protobacterial family Rhizobiaceae (1477 bp, GenBank accession AY559079.1). This sequence
displayed 6.8% divergence from the S. morelense 16S rRNA sequence (Table 6-2). Bacterial
species in the Rhizobium-Sinorhizobium group have not been previously associated as permanent
inhabitants of Lepidoptera, but are found in Tetraponera ants (Kneip et al. 2007). However,
recent studies have demonstrated that Rhizobium and Sinorhizobium are closely related to the
genus Reichenowia, a primary endosymbiont found in blood-feeding leeches (Perkins et al. 2005,
Siddall et al. 2004). Siddall et al. (2004) asserted that Reichenowia species might play a role in
nitrogen metabolism in the leech, or provide it with other nutrients lacking in a blood meal. It is
possible that the Rhizobium and Sinorhizobium phylotypes from C. thalictri represent novel
microbial associations similar to those occurring in blood-feeding leeches; however, the
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systematics of these nitrogen-fixing taxa is not resolved and additional sequence data will be
necessary to determine their phylogenetic placement.
In the diagnostic PCR assay of individual C. thalictri samples using phylotype-specific
primers (Table 6-1B), bacterial sequences representing the Klebsiella and Sinorhizobium
phylotypes were detected in 100% of the nine males. Because the Klebsiella phylotype and
Sinorhizobium phylotype were detected in all C. thalictri individuals sampled, it is likely that
these phylotypes qualify as gut associates. Further study, using methods such as fluorescent in
situ hybridization (FISH) are needed to confirm their exact location in C. thalictri.
Eubacterial sequences representing both Alcaligenes phylotypes and the Rhizobium
phylotype were detected in 11%, 33%, and 11% of the nine specimens, respectively. Recent
studies have shown the microbiota of the gut in lepidopteran hosts may vary with diet (Broderick
et al. 2004), thus, these could be transient contaminants from the environment or derived from
the host plants the moths feed on. The phylotypes found in less than 100% of the individual C.
thalictri specimens in this survey are classified as possible, but unconfirmed, gut symbionts
based on their similarity to other known gut symbionts in insects. Due to the limited number of
specimens available for screening, it is possible that the phylotypes detected in fewer than 100%
of the individuals would be more abundant in a larger sample. The screening also could have
failed to detect some phylotypes because they occur in low titer. However, use of a multiple
displacement amplification followed by high-fidelity PCR is at least six orders of magnitude
more sensitive then “standard-allele-specific” PCR, allowing us to detect as few as 100 copies of
bacterial DNA mixed with insect DNA 100% of the time and as few as 10 copies 50% of the
time (Jeyaprakash and Hoy 2004). However, if the initial primers used were inadequate to detect
specific microbial associates, then this survey could have failed to detect phylotypes. PCR
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174
products were not re-amplified due to concerns about contamination. Because negative controls
were used in all PCR assays using phylotype-specific primers and no positive bands were
observed, as expected, it is unlikely these sequences were due to laboratory contamination.
Wolbachia or any eukaryotic organisms in the specimens screened in our survey were not
detected. The failure to find Wolbachia in the C. thalictri specimens is surprising given the
prevalence of this bacterium in other butterfly and moth species (Tagami and Miura 2004).
Some populations of Lepidoptera do consist of both infected and uninfected individuals (Tagami
and Miura 2004), so it is possible that screening a larger sample of C. thalcitri specimens could
produce different results.
It has been speculated that gut endosymbionts in Lepidoptera larvae are involved in
lowering pH, metabolism of toxic plant compounds, and pathogen suppression (Broderick et al.
2004). These microbes may also play an important role in pheromone production, mating
behavior, longevity, and survival of laboratory-reared colonies in adults. This initial survey will
provide the foundation for future microbial analyses in both fruit-piercing and blood-feeding
adult moths.
Table 6-1. Original and phylotype-specific forward and reverse primers designed to detect microbial sequences in DNA isolated from C. thalictri.
A Organism (Reference), Forward primer sequence Expected PCR product Annealing Temperature B Phylotype (Genbank accession number) Reverse primer sequence (bp) ºC A. Primers used in initial survey Archaeabacteria (Arch 21F, Arch 958R) 5’- TTCCGGTTGATCCYGCCGGA - 3’ 1000 51 (DeLong 1992) 5’ - YCCGGCGTTGAMTCCAATT - 3’ Eubacterial 16S rRNA (27F, 1495R) 5’ -GAGAGTTTGATCCTGGCTCAG - 3’ 1400 55 (Weisburg et al. 1991) 5’ - CTACGGCTACCTTGTTACGA - 3’ Fungal SSU rDNA (NS1, FS2) 5’ - GTAGTCATATGCTTGTCTC - 3’ 1500 48 (Nikoh and Fukatsu 2000) 5’ - TAGGNATTCCTCGTTGAAGA - 3’ Helicosporidia (ms-5’, ms-3’) 5’ - GCGGCATGCTTAACACATGCAAGTCG - 3’ 1300 65 (Nedelcu 2001) 5’ - GCTGACTGGCGATTACTATCGATTCC - 3’ Wolbachia wspA (81F, 691R) 5’ - TGGTCCAATAAGTGATGAAGAAAC - 3’ 600 55 (Braig et al. 1998) 5’ - AAAAATTAAACGCTACTCCA - 3’ Yeast-like organisms (LS1, LR5) 5’ - AGTACCCGCTGAACTTAAG - 3’ 2000 50 (Zhang et al. 2003) 5’ - CCTGAGGGAAACTTCG - 3’ 175
B. Phylotype-specific primers Alcaligenes phylotype (1) 5’-GAGAAGAAAAGGTATCCCCTAATACGGGATAC-3’ 589 65 (EF599759) 5’-CTTGCGAGCACTGCCAAATCTCTTCGGC-3’ Alacaligenes phylotype (2) 5’-GGAAAGAAACGTCGTGGGTTAATACCCCGCGA-3’ 591 65 (EF599757) 5’-CTTGCGAGCACTGCCAAATCTCTTCGGGC-3’ Klebsiella phylotype 5’-GAAACTGGCAGGCTGGAGTCTTGTAGAG-3’ 521 65 (EF599758) 5’-CAGTCTCCTTTGAGTTCCCGGCCGGACC-3’ Rhizobium phylotype 5’-GAGACTGGCAGGCTGGAGTCTTGTAGA-3’ 522 65 (EF599760) 5’-CAGTCTCCTTTGAGTTCCCGACCGAATC-3’ Sinorhizobium phylotype 5’-GTGAAGATAATGACGGTAACCGGAGAAG-3’ 565 65 (EF599761) 5’-CGAACTGAAGGAATACATCTCTGTAATCC-3’
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Table 6-2. Pairwise sequence divergences (uncorrected p) between eubacterial phylotypes from C. thalictri and closely related 16S rRNA sequences using 1410-1504 bp of sequences.
1 2 3 4 5 6 7 8 9 10 1 Alcaligenes sp. IS-67 - 2 Alcaligenes phylotype 2 .097 - 3 Alcaligenes xylosoxidans NFRI-A1 .116 .063 - 4 Alcaligenes phylotype 1 .098 .131 .108 - 5 Klebsiella oxytoca isolate GR6 .212 .193 .216 .242 - 6 Klebsiella phylotype .213 .193 .217 .243 .005 - 7 Rhizobium sp. .229 .229 .200 .224 .242 .242 - 8 Rhizobium phylotype .246 .225 .243 .258 .241 .242 .069 - 9 Sinorhizobium morelense .230 .237 .204 .221 .237 .237 .049 .111 - 10 Sinorhizobium phylotype .250 .234 .248 .260 .233 .232 .109 .043 .068 - *Sequences in bold are from C. thalictri.
CHAPTER 7 COMPARISON OF SHORT-TERM PRESERVATION AND ASSAY METHODS FOR THE
MOLECULAR DETECTION OF WOLBACHIA IN THE MEDITERRANEAN FLOUR MOTH EPHESTIA KUEHNIELLA
Scientific Note
Methods proposed for the preservation of insect tissue for DNA analysis have included
various concentrations of ethanol, Carnoy’s solution, liquid nitrogen, and acetone (Post 1993,
Dessauer 1996, Fukatsu 1999, Mtambo 2006). However, little attention has been paid to
appropriate storage methods for future detection of endosymbiont DNA within an insect host
(Fukatsu 1999). Some studies report successful amplification of bacterial DNA in a host after
thousands of years (Salo et al. 1994, Fricker et al. 1997, Willerslev et al. 2004), but others have
reported inconsistent amplification of bacterial DNA due to low titers of the bacteria in the host,
difficulties with the DNA extraction process, PCR-inhibiting substances present in the insect gut,
or storage method (Fukatsu 1999, Barnes et al. 2000, Bextine et al. 2004, Hoy and Jeyaprakash,
unpublished data). Fukatsu (1999) suggested acetone storage was superior to ethanol as a
preservation method for both the amplification of insect host DNA and the DNA of their
endosymbionts.
Historically, standard PCR has been used to detect Wolbachia and other endosymbionts of
arthropods; however, it has been demonstrated that amplification of Wolbachia DNA can be
improved with High-Fidelity (HF) PCR (Jeyaprakash and Hoy 2000). Thus, both the specimen
preservation technique and choice of assay method could be important in determining the
success when attempting to amplify endosymbiotic DNA in an insect host. The goal of this
study was to compare molecular methods for the detection of Wolbachia in the Mediterranean
flour moth Ephestia kuehniella (Keller) (Lepidoptera: Pyralidae), and potentially other
endosymbiotic bacteria in their insect host, in preserved specimens over time.
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Materials and Methods
Standard, High-Fidelity (HF), and Real-Time Quantitative (RTQ) PCR methods were
used to detect and quantify Wolbachia DNA from E. kuehniella specimens stored under 4
treatment conditions (2 in 95% EtOH and 2 in acetone) over a 2-year storage period.
Spectrophotometry readings were taken at each assay (n = 9 over a 2-year period) to ensure
consistency of concentration and quality of template DNA for each treatment. Stored samples
were compared to fresh specimens at the end of the experiment.
A wild-type strain of E. kuehniella was reared on ‘Plodia’ diet (Silhacek and Miller
1972) in a 16L: 8D photoperiod at 26˚C and 70% RH at the USDA Center for Medical,
Agricultural, and Veterinary Entomology, Gainesville, FL. Live E. kuehniella (120 specimens)
were anesthetized for a period of 5 min in a -20˚C freezer. Once the specimens were immobile,
they were placed in 20 mL screw-capped vials (3 per vial). Half of the vials were filled with
95% EtOH, and the remaining vials were filled with 100% acetone (Fisher: Atlanta, GA), with a
specimen to preservative ratio of 1:20 (based on volume). Half of the vials filled with EtOH
were placed in the -80˚C freezer and the remaining vials were placed in a rack, covered with a
plastic bag to prevent evaporation, and stored at room temperature (25-27ºC). This procedure
was repeated for the vials filled with acetone. After weeks 13, 22, 27, 32, 39, 51, 76, 92, and
101, respectively, 6 specimens from each treatment category were removed and placed into
clean, disposable Petri dishes and the abdomens were separated from the thoraces with sterile
razor blades. Each abdomen was placed on a Kimwipe (Fisher: Atlanta, GA) for about 30 sec,
and then transferred into a labeled 1.5-mL centrifuge tubes. Abdomens were macerated for 3
min with a grinding pestle made from a 1 mL pipette tip in a labeled 1.5-mL centrifuge tube.
Genomic DNA was extracted from abdomens using a PureGene kit (Gentra Systems:
Minneapolis, MN) according to the manufacturer’s protocol. The extracted DNA was re-
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suspended in 250 μL of sterile double deionized water. Extractions were stored at -20˚C
overnight. The quality of the genomic DNA for every sample in each of the 4 treatments was
assessed using a spectrophotometer with a dilution factor of 40 (2 μL of extracted DNA per 78
μL water). Total DNA concentration and absorbance (260:280 ratio) readings recorded for each
set of extractions are listed in Table 7-1. At the start of the experiment, 20 live E. kuehniella
specimens (10 males and 10 females) were tested for the presence of Wolbachia using HF PCR,
and all specimens tested positive (100% infection). The HF PCR products were purified using a
QIAquick PCR purification column (QIAGEN inc.: Valencia, CA) and were cloned into the
plasmid pCR2.1-TOPO according to the manufacturer’s protocol (Invitrogen Corporation:
Carlsbad, CA). DNA sequencing was performed at the University of Florida ICBR Core Facility
using a PERKIN-ELMER Applied Biosystems ABI PRISM Automated DNA sequencer.
MacDNASIS software was used to evaluate the sequences (Hitachi Software Engineering
America Ltd., San Bruno, California). Wolbachia sequences were identical to Wolbachia sp.
group A (Accession #AB024570.1) from E. kuehniella by BLAST. For each specimen in all 4
treatments, we amplified 605 bp of the wspA gene using wsp-F, 5’-
TGGTCCAATAAGTGATGAAGAAACTAGCTA-3’ and wsp-R, 5’-
AAAAATTAAACGCTACTCCAGCTTCTGCAC-3’ primers. All PCRs were performed in a
total reaction volume of 50 μL containing 50 mM Tris (pH 9.2), 16 mM ammonium sulfate, 1.75
mM MgCl2, 350 μm dATP, dGTP, dTTP, dCTP, 400 picomoles primers (wsp-F and wsp-R), 1
unit of Pwo and 5 units of Taq DNA polymerases (Jeyaprakash and Hoy 2000). Negative
controls were included in all PCRs to test for contamination. The HF PCR cycling profile was: 1
denaturing cycle at 95 ˚C for 2 min, 10 cycles each of denaturation at 94 ˚C for 10 s, annealing at
65˚C for 30 s, and extension at 68 ˚C for 1 min and 25 cycles each of denaturation at 94 ˚C for
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10 s, annealing at 65˚C for 30 s and extension at 68˚C for 1 min, plus an additional 20 s added
for every consecutive cycle, using a PERKIN-ELMER DNA Cycler 480. A standard PCR
analysis also was conducted at each sample date and results were compared to those of the HF
PCR analyses (Jeyaprakash and Hoy 2000). In order to estimate Wolbachia density in E.
kuehniella, the wspA gene copy number was measured from abdomens of 4 individuals from
each treatment after a 2-year storage period, and compared to the copy number in abdomens
from 4 fresh specimens by RTQ PCR using a MyiQ Single-Color Real-time PCR Detection
System (Bio-Rad: Hercules, CA). Primers and probes for RTQ PCR analysis of the Wolbachia
target DNA (surface protein gene wspA) were designed using the Primer3 Output v. 0.4.0
software (Rozen and Skaletsky 2000). The forward primer WSP-RTF (5’-
CTATCACTCCATATGTTGGTGTTGGTGTTG-3’) corresponded to the region from base 327-
356 of the wspA sequence and the reverse primer WSP-RTR (5’-
CTCCTTTGTCTTTCTCACCAACGCTTTTAT-3’) to the region from 519-548 of the same
sequence. The length of the amplification product was 222 bp. The Taqman real-time PCR
protocol was performed in a final volume of 25 μL and contained: 1 μL of template DNA, 10 μL
of Power SYBR Green PCR Master Mix (Applied Biosystems: Foster City, CA), 7.24 μL double
deionized water, 0.16 μL Accuzyme DNA polymerase (Bioline: Randolph, MA), and 0.8 μL of
each primer, and was carried out in 96-well plates (Applied Biosystems: Foster City, CA) that
had been sealed with film, and centrifuged for approximately 30 sec (100 rpm) prior to RTQ
PCR. The RTQ PCR cycling profile consisted of 10 min at 95ºC followed by 50 cycles of 10 s at
95ºC, 30 s at 60ºC, 30 s at 68ºC, followed by a melt cycle for which conditions were 15 s at
95ºC, 1 min at 65ºC, and 15 s at 95ºC. A standard curve was calculated by using a standard
plasmid sample that contained the wspA gene at concentrations of [102 to 109] copies/μl. The
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number of molecules in the samples was determined from the threshold cycles (CT) in the PCR
based on the standard curve. Negative controls were included in all PCRs to test for
contamination.
Results
Wolbachia (wspA) was detected in all E. kuehniella individuals (n = 6) in each treatment
(n = 4) over the 2-year period (9 total sample times) using both standard and HF PCR (data not
shown). The average wspA copy number (Table 7-1) found in E. kuehniella abdomens (n = 20)
for each of the 4 treatments and from 4 abdomens from fresh specimens using RTQ PCR was
lower than previously reported in the testis of different male strains of E. kuehniella (groEL copy
number in testis: 5.2 – 7.5 x 106 ± 2.4; Ikeda et al. 2003). This is not surprising given the
potential variation in abundance of Wolbachia within an insect host depending on strain, sex, life
cycle, and tissues or organs sampled (Ijichi et al. 2002). The wspA copy number in each of the
20 individuals sampled was not significantly different among preservation treatments based on
CT values from the RTQ PCR (ANOVA, F= 0.53, df= 4, P= 0.72).
The HF PCR amplicon band intensity for the material stored in 95% EtOH at room
temperature for 2 years appeared lower for 3 out of the 6 specimens sampled (compare figures 7-
1A and 7-1B). When compared to HF PCR, a reduction in band intensity for standard PCR after
the 2-year storage period for all treatments was also observed (data not shown).
The average DNA concentration/sample taken at the last sample date (week 101) ranged
from 17 to 23 µg/mL and the 260:280 ratio of the stored samples ranged from 0.8 to 1.2; fresh
specimens had a DNA concentration of 25 and a 260:280 ratio of 1.7 using the same extraction
protocol (Table 7-1). Average total DNA concentrations and 260:280 ratios taken at the last
sample date (week 101) were not significantly different between treatments (ANOVA, F= 1.4,
df= 4, P= 0.28 and ANOVA, F= 1.7, df= 4, P= 0.18, respectively).
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Surprisingly, over the 2-year storage period, a regression analysis showed an increase in
total DNA concentration over the sample dates (data not shown) (slope = 0.340, y-intercept =
8.360, and R2 = 0.05 for specimens stored in acetone at RT; slope = 1.365, y-intercept = 7.203,
and R2= 0.5 for acetone at -80ºC; slope = 1.469, y-intercept = 5.750, and R2 = 0.3 for 95% EtOH
at RT; slope = 1.132, y-intercept = 8.226, and R2= 0.3 for 95% EtOH at -80ºC), perhaps due to
dehydration of the specimens over time allowing for easier maceration of specimens. A decrease
in the quality of the total DNA was observed (data not shown) for all 4 treatments (slope = -
0.005, y-intercept = 1.28, and R2 = 0.005 for specimens stored in acetone at RT; slope = -0.071,
y-intercept = 1.62, and R2= 0.5 for acetone at -80ºC; slope = -0.021, y-intercept = 1.55, and R2=
0.1 for 95% EtOH at RT; slope = -0.070, y-intercept = 1.55, and R2= 0.3 for 95% EtOH at -80ºC)
suggesting the quality of the DNA was compromised over time due to storage in each of the 4
treatments.
Discussion
These results are consistent with previous studies (Fukatsu 1999) suggesting acetone and
95% EtOH are adequate short-term specimen preservation methods for detection of
endosymbiotic bacterial DNA in an insect host. Although not statistically significant, an
apparent reduction in Wolbachia wspA copy number and HF PCR band intensity of the symbiont
for specimens stored in 95% EtOH at room temperature was observed (Fig. 7-1A, Table 7-1).
Averages in the total DNA concentration and purity of the stored specimens at week 101 were
not significantly different among replicates from the same treatment nor were they significantly
different between treatments. Regression analyses revealed an increase in total DNA
concentration over time for each treatment, and a decrease in absorbance over time. Additionally,
a reduction in band intensity was observed for specimens stored in 95% EtOH at room
temperature at the last sample date (week 101), suggesting long-term storage in 95% EtOH at
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183
room temperature may not be an optimal storage method as it may further damage the DNA or
inhibit the PCR. The results reported here are specific to E. kueniella, but they indicate that
when Wolbachia infection density in the host is high and the DNA extraction method is
consistent, both standard and HF PCR are adequate for detection of Wolbachia after a 2-year
storage period in both acetone and 95% EtOH.
Table 7-1. Summary of RTQ PCR and spectrophotometry data for E. kuehniella specimens stored for a 2-yr. period compared to fresh specimens.
Storage method wspA copy number in abdomen Total DNA concentration (µg/mL) 260:280 (mean ± SD) (mean ± SD)1 (mean ± SD)1
Acetone room temperature 4.422 x 105 ± 1.276 x 104 17 ± 4.0 1.2 ± 1.0 Acetone -80°C 4.361 x 105 ± 3.973 x 104 20 ± 1.0 1.2 ± 0.7 95% EtOH room temperature 3.441 x 105 ± 2.295 x 105 18 ± 5.5 1.2 ± 0.4 95% EtOH -80°C 4.245 x 105 ± 6.913 x 104 23 ± 7.3 0.8 ± 0.2 Fresh specimens2 4.202 x 105 ± 2.218 x 104 25 ± 9.0 1.7 ± 0.0 Acetone combined3 4.391 x 105 ± 2.751 x 104 19 ± 3.2 1.2 ± 0.8 95% EtOH combined 3.843 x 105 ± 1.627 x 105 21 ± 6.5 1.0 ± 0.4 Room temperature combined 3.931 x 105 ± 1.592 x 105 23 ± 8.3 1.5 ± 0.2 -80°C combined 4.303 x 105 ± 5.256 x 104 22 ± 5.1 1.0 ± 0.5
*Total DNA concentration and 260:280 values are averages of individuals sampled (n = 6) and assayed over the 2-year period (n = 9 datapoints). Fresh Specimens (n = 5) were analyzed at week 101. Combined values indicate averages of treatments combined (e.g., stored in acetone at room temperature + stored in acetone in -80°C freezer). 184
RT -80ºC 0 _____________ ____________ 0 - IV 3 4 5 6 7 8 9 10 11 12 13 14
A
B
Figure 7-1. Examination of DNA preservation and amplification by HF PCR of the wspA gene
fragment (605 bp) in E. kuehniella. (A) HF-PCR amplification of 12 individual E. kuehniella specimens following a 2-yr storage period in 95% EtOH at room temperature (lanes 3 - 8) and at -80ºC (lanes 9 - 14). Blank lanes = 0, negative control lanes = -. (B) HF PCR amplification of 12 individual E. kuehniella specimens following a 2-yr storage period in acetone at room temperature (lanes 3 - 8) and at -80ºC (lanes 9 -14). IV = HyperLadder (Bioline MA), blank lanes = 0, negative control lanes = -.
185
CHAPTER 8 PERSPECTIVES
This chapter describes important experiences during my time as a Ph.D. student in the
Entomology and Nematology Department at the University of Florida. This section is meant to
be a self-evaluation and discussion of what I learned, challenges I faced, and how I might handle
similar challenges in the future.
I arrived in Florida in the summer of August 2004 having just completed my Master’s
Degree in Dr. Susan Weller’s lab in the Entomology Department at the University of Minnesota.
At the time, my background was in systematics, morphology, taxonomy, and biogeography and
my goals were to augment these skills by learning techniques in molecular biology. My goals
were to reconstruct a phylogeny of fruit-piercing and blood-feeding moths to test the hypothesis
of a directional progression from nectar feeding to blood feeding in the group known as Calpini.
When I came to UF I had a large loan of specimens of calpine moths from the National Museum
of Natural History (Smithsonian) so I could begin the project immediately. I began by dissecting
relevant taxa, coding morphological characters, and had a preliminary phylogeny within the first
year. At this time, Dr. Branham encouraged me to also survey the characters of the proboscis
using the optics system in his laboratory and also SEM equipment at DPI. This was incredibly
challenging, but through trial and error and much patience I finally learned how to use both types
of equipment. I was extremely fortunate to have Dr. Branham’s training in how to use the digital
equipment in his laboratory for my dissertation. The proboscis images taken and descriptions of
the structures observed has now turned into a sizeable chapter in my dissertation and resulted in
an important publication with Dr. Branham and an international collaborator, Dr. Hans Bänziger.
While most of my coursework was completed during my Master’s program, as part of my
Ph.D. at UF I completed the following courses: Advanced Invertebrate Zoology, Medical and
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Veterinary Entomology, Techniques in Insect Systematics, Insect Molecular Biology, Molecular
Systematics, Phylogenetic Methods, Population Genetics Seminar, Insect Symbiosis Seminar,
and Protein Chemistry and Molecular Cloning Summer Laboratory course. These courses
increased my knowledge in entomology, systematics, and molecular biology. The Insect
Molecular Biology course taught by Dr. Hoy was of great interest to me as I gained further
exposure to microbe-insect interactions through topics covered in class discussions. I found this
subject very interesting and immediately decided to discuss the possibility of conducting an
endosymbiont survey on fruit-piercing and blood-feeding moths. Dr. Hoy encouraged me to
pursue this and offered me bench space, training, and supplies in her laboratory. I began
working on this project right away, however, I did not have fresh specimens of blood-feeding
moths at the time and so I used freshly collected material from a previous expedition to Nepal.
Initially, this work was very stressful as I had little to no previous at the bench as was trying to
re-learn molecular biology at fast pace. Additionally, the specimens I was using for the survey
of microorganisms were testing negative and it was unclear whether these results were true or
false positives. We were unsure whether the moths had been stored properly for extraction of
endosymbiont DNA. During this time I developed a good working relationship with Dr. Hoy
where we had numerous discussions about how we could set up an experiment to answer the
question of which method of specimen storage is best for the extraction of microbial DNA. This
experiment would later become a technical chapter of my dissertation and resulted in another
publication. This chapter taught me a lot about the scientific methods and problem solving.
During this study I learned how to conduct both High Fidelity (HF) and real time quantitative
(RTQ) PCR.
187
During the spring of 2006 I was fortunate to receive a grant from the Explorer’s club to
travel to Far Eastern Russia to look study adult feeding behavior in three species from the
vampire moth genus Calyptra. This was a very successful field trip in that I learned which
species in this region actually feed on blood and which do not. I was also able to acquire
specimens for both my phylogenetic study and for the endosymbiont survey. Thus, while I was
working on the technical chapter I began working on an endosymbiont survey of a fruit-piercing
and blood-feeding moth, Calyptra thalictri. Through this study I learned several important skills
in molecular biology including cloning and RFLP analysis. This study became another chapter
of my dissertation and has resulted in another publication. I was fortunate to be supported by Dr.
Hoy for both molecular biology chapters and most importantly, was trained in bench skills that
most systematic students are not exposed during their Ph.D.’s. My experiences in Dr. Hoy’s lab
were extremely positive in that I also had the opportunity to learn from her former student, Jason
Meyer, and also teach other graduate students and postdoctoral researchers methods I learned
while working in her laboratory.
During my Ph.D. I had the opportunity to TA the course Biology of Lepidoptera where I
designed and taught the laboratories and gave a lecture on insect heads and mouthpart
morphology. I also volunteered as a teaching assistant for Dr. Jim Lllyod’s Natural History
honor’s course for two semesters. I plan to co-instruct a course in insect biology and possibly a
summer field course or seminar during the second year of my postdoctoral position at the
University of Minnesota.
During my Ph.D., I always had financial support from both Dr. Branham and Dr. Hoy for
which I am incredibly thankful. Despite the availability of funds through Drs. Branham and Hoy
and the department, I always felt it was important to try and raise my own funds to support my
188
projects. While a Ph.D. student at UF I submitted several small and large grant proposals to try
and support my salary, laboratory work, and fieldwork. I was successful in raising funds from
several granting agencies including the National Park Service, the National Science Foundation,
and the National Geographic Society. I also raised $3,000 from the Systematics Research Fund
to pay for a trip to the British Museum of Natural History. I submitted two proposals to the EPA
to try and cover my student stipend, but unfortunately was unsuccessful. I also had the
opportunity to write and submit a grant proposal to the DEB panel at the National Science
Foundation with Dr. Susan Weller. This experience was incredibly useful as I now feel prepared
for future grant writing in an academic position.
Through my grant writing experiences I learned several valuable skills including
professional communication. This is incredibly important because many funding agencies
require supporting documentation before reviewing and funding proposals. I have been fortunate
to have the support of all my committee members and other members of the scientific
community in this area and I am certain that their agreeing to write good letters of support was
instrumental in my receiving funding over the years. My committee has also taught me the
importance of writing and publishing the results of my research as the data comes in and not to
wait until the end to write everything as the publication process is tedious and time-consuming. I
think this is one of the most important philosophies a committee can pass down to the students
they advise because it prepares students for the real world and teaches us how to be efficient with
our time. This is important given academics is a competitive environment that requires
individuals to be successful in many areas at the same time while continuing to raise grant
money and publish the results in peer-reviewed journals. My committee has supported me from
the very beginning of my Ph.D. and given me solid scholarly advice on all occasions. They have
189
and trained me and prepared me for a future in an academic position. At times all graduate
students can become overwhelmed by the tasks at hand and question whether or not they are
going to make it. My committee members have helped me overcome these fears and gain the
confidence needed to be a successful scientist. I am very pleased that my dissertation chapters
covered the aspects I set out to cover in the beginning of my Ph.D. program and much more. My
time in the Entomology and Nematology Department was a positive one. I am happy with the
research topic that I chose for my dissertation and that I was able to publish results as the
projects were completed. I must extend my greatest appreciation to all members of my
committee for both their personal and professional support; I consider them my academic family
and friends that I hope to carry with me for the remainder of my career.
My long-term career goals include a position at a major university teaching courses in
Entomology and conducting research on economically important moths, especially fruit-piercing
and blood-feeding moths. I intend to apply systematics to other areas of entomology including
Integrated Pest Management (IPM), Risk Assessment, Biological Control, and Conservation
Biology. This work will also apply to other areas of Entomology such as Medical and
Veterinary Entomology, by investigating disease vector potential in skin-piercing and blood-
feeding moth species. My course of study at the University of Florida has allowed me to gain
important insight into the evolution of an environmental, economic, and agricultural model
organism as well as link many fields of entomology.
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191
APPENDIX A DATA MATRIX USED TO PRODUCED TREES BASED ON MORPHOLOGICAL DATA
Taxa Character A. flava 120010001000?0021100????????????????????????0B090?0011110???0?10?? A. mesogona 120010001000?00211000??B0?1001190280391010000B090?0011110???0?1122 S. libatrix 120030101000?202010011?50?000116021014630000040?0?010?220???0?10?? H. hormos 010011000050?202210013?60?A00111009559430000080A0?010?0?0???0?0100 H. monilis 010011000050?20221000??30?B0010?009559430000181?110113110???0?10?? He. hieroglyphica 12012?002000?00111010??41270010?11455040300001140?000?0?0???0?10?? He. sittica 12012?002000?2?111010??41370010?314550400000?????????????????????? Phy. callitrichoides 010011000020?20134002?290?3001110??061100020083?1201100?0???0?0111 C. albivirgata 12010?1100010200102115?60?300110012610330200050?16010?0?0???0?00?? C. bicolor 12010?1100010200102115?20?3001100126103300100?0?11000?0?0???0?00?? C. canadensis 12010?1100010220201114?20?30011200?6153202000A1110010?0?0???0?10?? C. eustrigata 12010?1100010101120115?60?300110012610330210300?0?010?0?11700?00?? C. fasciata 12010?1100010200100115?20?3001100???10330110300?13010?0?0???0?00?? C. fletcheri 12010?1100010200102115?60?300110012610330100?????????????????????? C. gruesa 12010?1100010200122115?20?3001100126103302000A0?10000?0?11200?00?? C. hokkaida 12010?110001000112213?020?3001100???103302100A1?1600110?0???0?00?? C. lata 12010?1100010200122115?20?300110012610300010341?16010?0?0???0?00?? C. minuticornis 12010?1100010001122110?60?300110012610330200300?0?010?0?0???0?00?? C. ophideroides 12010?1100010201122115?20?3001100???10300000300?16010?0?0???0?00?? C. orthograpta 12010?1100010002310115?60?300110012610010000300?17010?0?0???0?10?? C. parva 12010?1100010001122149?20?300110012610330200304?0?00140?0???0?00?? C. pseudobicolor 12010?110001020010211A?20?30011000?610000010300?18010?0?0???0?00?? C. subnubila 12010?1100010200102115?20?3001100??????30210300?19000?0?11600?00?? C. thalictri 12010?1100010200322118?60?300110006610310200000?0?000?0?0???0?00?? E. anguina 12032?1101311?025101????????????????????????001?0?010?0?0???0?10?? E. aurantia 12032?110131100251200??30?4031140120555001000E0?0?110?0?0???0?10?? E. boseae 12032?1101311?0251010??30?602118015452500010091?15030?0?0???0?00?? E. cocalus 12032?110131100251010??30?90111401205511000007060?100?0?0???0?10?? E. dividens 12032?110131100251010??20?30111401245550011000070?110?0?0???0?10?? E. fullonia 12032?110131100251010??A0?40111801705700000006100?100?0?0???0?10?? E. homanea 12032?110131100251010??310302118012?555100000F0?0?000?0?0???0?10?? E. jordani 12032?110131100251010??60?30211401245551001006100?100?0?0???0?10?? E. materna 12032?1101311002510017?30?302114012055500010000005100?0?11800?10?? E. procus 12032?11013110025101????????????????????????00030?000?0?0???0?10?? E. salaminia 12032?110131100251200??21030211401205550001000050?110?0?0???0?00??
192
193
Taxa Character E. tyrannus 12032?110131100251110??30?602114012455520100000814000?0?0???0?10?? G. correcta 12010?1100110000132112?20?200111215111020001022?1300150?01090?00?? G. incurva 12010?1100110020132112?20?200111215105020011122?1000150?0???0?00?? G. indentata 12010?1100110000102112?20?300111015108000011020?0?400?0?0???0?00?? G. mexicana 12010?1100110000132112?60?300110010?10000011022?0?00150?10210?00?? G. nutrix 12010?11001100?0132112?70?200110215100000010020?0?020?0?0???0?00?? G. parens 12010?1100110000132112?20?300110215118000011022?1001150?0???0?00?? G. sicheas 12010?1100110000132012?20?200111215118000001020?0?610?0?00210?00?? G. sinaldus 12010?1100110000131112?70?400110015100000211020?10510?0?0???0?00?? G. unica 12010?1100110000132112?20?400110215108000211022?1001150?0???0?00?? G. uxor 12010?1100110000132112?20?200111215118?????1020?13000?0?0???0?00?? Gr. regina 12?32??????1000251000??111300117003345040010211?0?700?0?0???0?00?? O. argyrosigna 12010?111001000040211B?80?801110012123300000080?0?800?0?11400?00?? O. emarginata 12010?111001001040210??80?801110012123300000080?0?020?0?11110?00?? O. excavata 12010?1110010100402112?80?800110012125300000080?12010?0?11410?00?? O. excitans 12010?1110010002311112?80?300110012123300010080?12010?0?11010?00?? O. glaucohelia 12010?11100100?2312112?80?400110012125340000080?12810?0?11500?00?? O. nobilis 12010?1110010000401112?80?800110012125300000080?0?000?0?11410?00?? O. provocans 12010?1110010?0?4?11????????????????????????080?0?000?0?11610?00?? O. rectristria 12010?111001010040212?180?100110012123300000083?0?02100?11310?00?? O. serpans 12010?11100100?142210??810100110012125310010?????????????????????? O. striolata 12010?111001000040011B?80?400110012120300010080?0?010?0?11010?00?? O. triobliqua 12010?11100101?040210??80?810110012525300000080?0?02100?11210?00?? P. casta 12110?1100210002410110?20?100111005250101110021?1100120?0???0?00?? P. coelonota 12110?1100210002411117?20?300115015?51100000030?0?200?0?0???1110?? P. compressipalpus 12110?1100210222410116?00?5101110??2561300000C1B10010?0?0???1310?? P. dimorpha 12110?1100210002412117?20?500115005050100000030?0?90120?0???0?00?? P. incitans 12110?1100210??2412117?20?300111005201100000080?0?330?0?0???0?00?? P. miranda 12110?110021000241101??20?300111005201120000431210210?0?0???1300?? P. repellens 12110?1100210002412117?20?5001???050???00000030?0?90120?0???0?00??
194
APPENDIX B DATA MATRIX USED TO PRODUCE TREES BASED ON MORPHOLOGICAL AND
MOLECULAR DATA
Aflava 120010001000?0021100????????????????????????0B090?0011110???0?10?? Amesogona 120010001000?00211000??B0?1001190280391010000B090?0011110???0?1122 Slibatrix 120030101000?202010011?50?000116021014630000040?0?010?220???0?10?? Hhormos 010011000050?202210013?60?K00111009559430000080K0?010?0?0???0?0100 Hmonilis 010011000050?20221000??30?B0010?009559430000181?110113110???0?10?? Hsittica 12012?002000?2?111010??41370010?314550400000?????????????????????? Phycallitrichoides 010011000020?20134002?290?3001110??061100020083?1201100?0???0?0111 Calbivirgata 12010?1100010200102115?60?300110012610330200050?16010?0?0???0?00?? Cbicolor 12010?1100010200102115?20?3001100126103300100?0?11000?0?0???0?00?? Ccanadensis 12010?1100010220201114?20?30011200?6153202000A1110010?0?0???0?10?? Gsinaldus 12010?1100110000131112?70?400110015100000211020?10510?0?0???0?00?? Gparens 12010?1100110000132112?20?300110215118000011022?1001150?0???0?00?? Gincurva 12010?1100110020132112?20?200111215105020011122?1000150?0???0?00?? Gcorrecta 12010?1100110000132112?20?200111215111020001022?1300150?01090?00?? Clata 12010?1100010200122115?20?300110012610300010341?16010?0?0???0?00?? Cthalictri 12010?1100010200322118?60?300110006610310200000?0?000?0?0???0?00?? Eaurantia 12032?110131100251200??30?4031140120555001000E0?0?110?0?0???0?10?? Ejordani 12032?110131100251010??60?30211401245551001006100?100?0?0???0?10??
195 Ematerna 12032?1101311002510017?30?302114012055500010000005100?0?11800?10?? Esalaminia 12032?110131100251200??21030211401205550001000050?110?0?0???0?00?? Etyrannus 12032?110131100251110??30?602114012455520100000814000?0?0???0?10?? Gindentata 12010?1100110000102112?20?300111015108000011020?0?400?0?0???0?00?? Gnutrix 12010?11001100?0132112?70?200110215100000010020?0?020?0?0???0?00?? Gsicheas 12010?1100110000132012?20?200111215118000001020?0?610?0?00210?00?? Guxor 12010?1100110000132112?20?200111215118?????1020?13000?0?0???0?00?? Oemarginata 12010?111001001040210??80?801110012123300000080?0?020?0?11110?00?? Oexcitans 12010?1110010002311112?80?300110012123300010080?12010?0?11010?00?? Onobilis 12010?1110010000401112?80?800110012125300000080?0?000?0?11410?00?? Orectristria 12010?111001010040212?180?100110012123300010083?0?02100?11310?00?? Oserpans 12010?11100100?142210??810100110012125310010?????????????????????? Pcasta 12110?1100210002410110?20?100111005250101110021?1100120?0???0?00?? Pcoelonota 12110?1100210002411117?20?300115015?51100000030?0?200?0?0???1110?? Pcompressipalpus 12110?1100210222410116?00?5101110??2561300000L1B10010?0?0???1310?? Pdimorpha 12110?1100210002412117?20?500115005050100000030?0?90120?0???0?00??
[COI] Ematerna AACATTATATTTTATTTTTGGTATTTGAGCCGGTATAGTAGGAACTTCTCTTAGTTTATTAATTCGAGCTGAATTAGGAAATCCAGGATCATTAATTGGAGATGATCAAATTTATAATACTATTGTTACAGCTCATGCTTTCATTATAATTTTTTTTATAGTAATACCTATTATAATTGGAGGATTCGGAAATTGATTAATTCCTCTTATATTAGGTGCTCCTGATATAGCTTTTCCTCGTATAAATAATATAAGTTTTTGACTTCTTCCCCCTTCTTTAACTCTTCTTATTTCAAGAAGAATTGTAGAAAATGGAGCAGGAACAGGATGAACAGTATATCCCCCACTATCATCTAATATTGCACATAGAGGTAGATCTGTAGATTTAGCTATTTTTTCTTTACATTTAGCTGGTATTTCATCAATTTTAGGAGCAATTAATTTTATTACTACAATTATTAATATACGATTAAATAATTTATCATTTGATCAAATACCCCTATTTGTTTGAGCTGTTGGAATTACTGCATTTTTATTACTTCTTTCTTTACCTGTTCTAGCAGGAGCTATTACTATACTTTTAACAGATCGAAATTTAAATACATCATTTTTTGATCCTGCTGGTGGAGGAGATCCCATTCTTTATCAACATTTATTT??????? Aflava AACATTATATTTTATTTTTGGTATTTGAGCAGGAATAGTCGGAACTTCATTAAGTATATTAATTCGAGCAGAATTAGGTAATCCAGGATCTTTAATTGGTGACGATCAAATTTATAATACTATTGTTACTGCCCATGCTTTTATTATAATTTTTTTCATAGTTATACCTATTATAATTGGTGGATTTGGAAACTGATTAGTACCTCTTATATTAGGAGCTCCAGATATAGCTTTTCCTCGAATAAATAATATAAGATTTTGACTTCTTCCCCCTTCTTTAATTCTTTTAACTTCAAGAAGAATTGTAGAAAATGGAGCAGGAACAGGATGAACAGTTTATCCCCCACTTTCATCTAATATTGCTCATAGAGGAAGCTCAGTAGATCTTGCTATTTTTTCCCTTCATTTAGCAGGTATTTCATCAATTTTAGGAGCTATTAATTTTATTACAACAATCATTAATATACGATTAAATAATTTATCATTTGATCAAATACCTTTATTTGTTTGAGCAGTAGGAATCACAGCATTTTTATTATTATTATCACTACCAGTATTAGCAGGAGCTATTACAATATTATTAACAGATCGTAATTTAAATACTTCTTTTTTTGATCCTGCAGGAGGTGGAGATCCTATtCTTTATCAACACTTATTT???????
196
Amesogona ???????????????TTTTGGTATTTGAGCAGGTATAGTTGGAACTTCATTAAGTATATTAATTCGAGCAGAATTAGGAAACCCAGGATCTTTAATTGGAGATGATCAAATTTATAATACTATTGTTACTGCTCATGCTTTTATTATAATTTTTTTTATAGTTATACCTATTATAATTGGAGGATTCGGAAATTGATTAGTACCACTTATATTAGGAGCACCTGATATAGCTTTCCCCCGAATAAATAATATAAGATTCTGACTTCTTCCACCTTCACTTATTTTATTAACTTCAAGAAGAATTGTAGAAAATGGAGCAGGAACAGGATGAACAGTTTATCCCCCACTTTCATCTAATATTGCTCATAGAGGAAGCTCAGTAGATTTAGCAATTTTTTCACTACATTTAGCAGGAATTTCATCCATTTTAGGAGCTATTAATTTTATTACTACAATTATTAATATACGATTAAATAATCTATCATTCGATCAAATACCATTATTTGTTTGAGCTGTTGGTATTACAGCATTTTTATTATTATTATCTCTTCCTGTATTAGCTGGAGCAATTACTATATTATTAACTGACCGTAATTTAAATAC?????????????????????????????????????????????????????????????? Calbivirgata ?????????????????????????????????????????????TTCTCTAAGCTTATTAATTCGAGCTGAATTAGGTAATCCTGGATCTTTAATTGGAGATGATCAAATTTATAATACTATTGTAACAGCTCACGCTTTTATTATAATTTTTTTTATAGTTATACCTATTATAA
TTGGAGGATTTGGAAATTGACTTGTACCTTTAATATTAGGAGCTCCTGATATAGCTTTCCCTCGAATAAATAATATAAGTTTTTGACTTCTTCCCCCTTCTTTAACTCTTCTAATTTCTAGAAGAATTGTA?????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? Cbicolor AACATTATATTTTATTTTTGGAATTTGAGCAGGAATAGTTGGAACCTCATTAAGACTATTAATTCGAGCTGAATTAGGTAATCCTGGATCTTTAATTGGAGATGATCAAATTTATAATACTATTGTAACAGCTCATGCTTTCATTATAATTTTTTTTATAGTTATACCTATTATAATTGGAGGATTTGGAAATTGACTTGTCCCTCTTATATTAGGAGCACCAGATATAGCTTTCCCTCGAATAAATAATATAAGTTTTTGACTTCTTCCCCCCTCTTTAACTCTTCTAATTTCTAGAAGAATTGTAGAAAATGGAGCAGGAACAGGATGAACAGTATACCCCCCCCTTTCATCTAATATTGCACATAGAGGAAGATCAGTAGATTTAGCTATTTTTTCATTACATTTAGCAGGAATTTCATCAATTTTAGGAGCAATTAATTTTATTACAACAATTATTAATATACGATTAAATAATTTATCATTTGACCAAATACCTTTATTTATTTGAGCAGTAGGAATTACAGCATTCTTATTATTATTATCTTTACCTGTTTTAGCTGGAGCTATTACAATACTTTTAACAGATCGAAATTTAAATACATCTTTCTTCGATCCTGCTGGAGGAGGAGAtCcTATTCTTTACCAACATTTATTT??????? Clata AACATTATATTTTATTTTTGGAATTTGATCAGGAATAGTTGGAACTTCATTAAGATTATTAATTCGAGCTGAATTAGGTAATCCAGGATCTTTAATTGGAGATGATCAAATTTATAACACTATTGTAACAGCTCATGCTTTTATTATAATTTTTTTTATAGTTATACCTATTATAATTGGAGGATTTGGAAATTGACTTGTACCCCTCATATTAGGAGCCCCTGATATAGCTTTCCCCCGAATAAATAATATAAGTTTTTGACTACTTCCCCCCTCATTAACCCTTTTAATTTCTAGAAGAATTGTAGAAAATGGAGCAGGAACAGGATGAACAGTGTATCCCCCCCTTTCATCTAATATTGCACATAGAGGAAGTTCTGTAGATTTAGCTATTTTTTCCCTTCATCTAGCAGGAATTTCATCAATTTTAGGAGCAATTAATTTTATTACAACAATTATTAACATACGATTAAATAATTTATCATTTGATCAAATACCTTTATTTATTTGAGCAGTAGGAATTACAGCATTCTTACTTTTATTATCTTTACCTGTTTTAGCTGGAGCTATTACTATACTTTTAACAGATCGAAATTTAAATACATCTTTTTTTGATCCTGCTGGAGGAGGAGATCcTATTCTTTATCAACATCTATTT???????
197
Cthalictri AACATTATATTTTATTTTTGGAATTTGAGCAGGAATAGTTGGAACTTCACTAAGATTATTAATTCGAGCCGAACTAGGAAATCCAGGATCTTTAATTGGAGATGATCAAATTTATAATACTATTGTAACAGCTCATGCTTTCATTATAATTTTTTTTATAGTTATACCTATTATAATTGGAGGATTTGGAAATTGATTAGTACCCCTTATATTAGGAGCTCCTGATATAGCTTTCCCTCGAATAAATAATATAAGTTTTTGACTCCTCCCCCCTTCTTTAACTCTTCTAATTTCCAGAAGAATTGTAGAAAACGGAGCAGGAACTGGATGAACAGTATATCCCCCCCTTTCATCAAATATTGCACATAGAGGAAGTTCTGTAGATTTAGCTATTTTTTCATTACATTTAGCAGGAATTTCATCAATTTTAGGAGCAATTAATTTTATTACAACAATTATTAATATACGACTAAATAATTTATCATTTGATCAAATACCTTTATTTATTTGAGCAGTAGGAATTACAGCATTTCTACTCTTATTATCTTTACCTGTTTTAG
CTGGAGCTATTACTATACTTTTAACAGATCGAAATTTAAATACATCTTTTTTTGATCCTGCTGGTGGAGGAGATCCTATTTTATATCAACATTTATTT??????? Eaurantia ??????????TTTATTTTTGGTATTTGAGCAGGtAtAGTAGGAACTTCACTCAGTTTATTAATTCGAGCTGAATTAGGAAATCCAGGATCATTAATTGGAGATGACCAAATTTATAATACTATTGTTACAGCTCATGCTTTCATTATAATTTTTTTTATAGTAATACCTATTATAATTGGAGGATTTGGAAATTGACTAGTACCTCTTATATTAGGAGCTCCTGATATAGCTTTCCCTCGAATAAATAATATAAGTTTTTGACTTCTCCCCCCTTCTTTAACTTTATTAATTTCAAGAAGAGTTGTAGAAAATGGAGCAGGAACTGGATGAACAGTTTACCCCCCACTATCATCTAATATTGCACATAGAGGAAGTTCAGTAGATTTAGCTATTTTTTCATTACATTTAGCTGGTATTTCATCAATTTTAGGAGCTATTAATTTTATTACAACAATTATTAACATACGATTAAATAACTTATCATTTGATCAAATACCATTATTTATTTGAGCTGTAGGAATTACAGCATTCTTATTATTATTATCTTTACCAGTTTTAGCAGGTGCTATTACCATACTTTTAACAGACCGAAATTTAAATAC?????????????????????????????????????????????????????????????? Ejordani ?????????????ATTTTTGGTATTTGAGCAGGtAtAGTAGGAACTTCACTTAGTTTATTAATTCGAGCTGAATTAGGAAATCCAGGATCACTTATTGGAGATGATCAAATTTATAATACTATTGTTACAGCTCATGCTTTTATTATAATTTTTTTTATAGTAATACCTATTATAATTGGAGGATTTGGAAATTGATTAGTACCTCTTATATTAGGAGCCCCTGATATAGCTTTCCCCCGAATAAATAATATAAGTTTTTGACTTCTTCCCCCTTCTTTAACTCTTTTAATTTCAAGAAGAATTGTAGAAAACGGAGCAGGAACTGGATGAACAGTCTATCCCCCACTTTCATCTAATATTGCTCATAGAGGAAGTTCAGTAGATTTAGCTATTTTTTCACTACATTTAGCTGGTATTTCATCAATTTTAGGAGCCATTAATTTTATTACAACAATTATTAATATACGATTAAATAATTTATCATTCGATCAAATACCACTATTTATTTGAGCTGTTGGAATTACTGCATTCTTATTACTTCTTTCTTTACCTGTCTTAGCAGGTGCTATTACTATACTTTTAACAGATCGAAATTTAAATAC??????????????????????????????????????????????????????????????
198
Esalaminia ???????????????????????TTTGAGCAGGTATAGTAGGAACTTCTCTTAGTTTATTAATTCGAGCTGAATTAGGAAACCCCGGAtCATTAATTGGAGAtGATCAAATTTATAATACTATTGTTACAGCTCATGCTTTTATTATAATTTTTTTTATAGTTATACCTATTATAATTGGAGGATTTGGAAATTGATTAGTACCTCTAATATTAGGTGCCCCTGATATAGCTTTCCCTCGAATAAATAATATAAGTTTTTGACTCCTTCCCCCTTCTTTAACTCTTCTTATTTCGAGAAGAATTGTA?????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? Gindentata ??AACATTATATTTTATTTTTGGAATTTGAGCTGGTATAGTAGGAACCTCCTTAAGTTTATTAATTCGAGCTGAATTAGGTAATCCAGGATCTTTAATTGGAGATGATCAAATTTATAATACTATTGTAACAGCACATGCTTTTATTATAATTTTTTTTATAGTTATACCTATTATAATTGGAGGATTTGGAAATTGATTAGTACCACTTATACTCGGAGCCCCTGATATAGCTTTCCCCCGAATAAATAACATAAGTTTCTGACTTCTTCCCCCATCTTTAACTCTTCTAATTTCAAGAAGTATTGTAGAAAGTGGAGCA
GGAACAGGATGAACAGTTTATCCCCCCCTTTCATCCAATATCGCTCATGGAGGTAGATCAGTTGATTTAGCTATTTTTTCATTACACTTAGCTGGGATCTCATCAATTTTAGGAGCTATTAATTTCATTACAACAATTATTAATATACGATTAAATAACTTATCATTTGATCAAATACCTTTATTTATTTGAGCTGTAGGAATTACCGCATTCTTACTACTTCTTTCTTTACCAGTTTTAGCAGGAGCTATTACTATACTTTTAACTGATCGAAATTTAAATACCTCTTTTTTTGATCCAGCAGGAGGAGGAGATCCTATTCTTTATCAACATTTATTT????? Gsinaldus ??AACATTATATTTTATTTTTGGAATTTGAGCTGGTATAGTAGGAACCTCTTTAAGTTTATTAATTCGAGCTGAATTAGGTAATCCTGGATCTTTAATTGGAGATGATCAAATTTATAATACTATTGTAACAGCACATGCTTTTATTATAATTTTTTTCATAGTTATACCTATTATAATTGGAGGATTTGGAAATTGATTAGTACCCCTTATATTAGGAGCACCTGATATAGCTTTCCCTCGAATAAACAATATAAGTTTTTGACTCCTTCCCCCTTCTTTAACTCTTTTAATTTCAAGAAGAATTGTAGAAAGTGGAGCAGGAACAGGATGAACAGTTTACCCCCCACTTTCATCTAATATTGCTCATGGAGGTAGTTCAGTTGACTTAGCTATTTTTTCCTTACATTTAGCTGGAATTTCATCAATTTTAGGAGCTATTAATTTCATTACAACAATTATTAATATACGATTAAATAATTTATCATTTGATCAAATACCTTTATTTATTTGAGCTGTAGGCATTACTGCATTCTTATTACTTCTCTCTTTACCGGTTTTAGCTGGAGCTATTACAATACTTTTAACTGACCGAAATTTAAATACTTCTTTTTTTGACCCTGCAGGAGGAGGAGATCCTATTTTATATCAACACTTATTC????? Gparens ?????????????ATTTTTGGTATTTGAGCTGGAATAGTAGGAACATCTTTAAGATTATTAATTCGAGCTGAATTAGGAAATCCTGGATCTTTAATTGGAGATGATCAAATTTATAATACTATCGTAACAGCACATGCTTTTATTATAATTTTTTTTATAGTAATACCTATTATAATTGGAGGATTTGGAAATTGATTAGTACCTCTTATATTAGGAGCCCCTGATATAGCTTTTCCTCGAATAAATAATATAAGTTTTTGACTCCTTCCCCCTTCTCTAACTCTTTTAATTTCAAGAAGAATTGTAGAAAGTGGAGCAGGAACAGGATGAACAGTTTACCCCCCACTTTCATCTAATATTGCTCATGGAGGAAGTTCAGTTGATTTAGCTATTTTTTCATTACATTTAGCAGGAATTTCATCAATTTTAGGAGCTATTAATTTTATTACAACAATTATTAATATACGACTAAATAATTTATCATTTGATCAAATACCTTTATTTATTTGAGCTGTAGGTATTACAGCATTTTTATTACTTCTTTCTTTACCAGTTTTAGCAGGAGCTATTACTATACTTTTAACTGATCGAAATTTAAATACTTCATTTTTTGACCCAGCTGGAGGAGG???????????????????????????????????
199
Gincurva ??AACATTATATTTTATTTTTGGTATTTGAGCTGGAATAGTAGGAACATCTTTAAGATTACTAATTCGAGCTGAATTAGGAAATCCTGGATCTTTAATTGGAGATGATCAAATTTATAATACTATCGTAACAGCACATGCTTTTATTATAATTTTTTTTATAGTAATACCTATTATAATTGGAGGATTCGGAAATTGATTAGTACCTCTTATATTAGGAGCCCCCGATATAGCTTTTCCTCGAATAAATAATATAAGTTTCTGACTCCTTCCCCCTTCTCTAACTCTTTTAATTTCAAGAAGAATTGTAGAAAGCGGAGCAGGAACAGGATGAACAGTTTACCCCCCACTTTCATCTAATATTGCTCATGGAGGAAGTTCAGTTGATTTAGCTATTTTTTCACTTCATTTAGCAGGAATTTCATCAATTTTAGGAGCTATTAATTTTATTACAACAATTATTAATATACGATTAAATAATTTATCATTTGATCAAATACCTTTATTTATTTGAGCTGTAGGTATTACAGCATTCTTATTACTTCTTTCTTTACCAGTTTTA
GCGGGAGCTATTACTATACTTTTAACTGATCGAAATTTAAATACCTCATTTTTCGATCCAGCTGGAGGAGGTGATCCTATTCTTTATCAACATTTATTT????? Gcorrecta ??AACATTATATTTTATTTTTGGTATTTGAGCTGGAATAGTAGGAACATCTTTAAGATTATTAATTCGAGCTGAATTAGGAAATCCTGGATCTTTAATTGGAGATGATCAAATTTATAATACTATCGTAACAGCACATGCTTTTATTATAATTTTTTTTATAGTAATACCTATTATAATTGGAGGATTCGGAAATTGATTAGTACCTCTTATATTAGGAGCCCCCGATATAGCTTTTCCTCGAATAAATAATATAAGTTTTTGACTCCTTCCCCCTTCTCTAACTCTTTTAATTTCAAGAAGAATTGTAGAAAGTGGAGCAGGAACAGGATGAACAGTTTACCCCCCACTTTCATCTAATATTGCTCATGGAGGAAGTTCAGTTGATTTAGCTATTTTTTCACTACATTTAGCAGGAATTTCATCAATTTTAGGAGCTATTAATTTTATTACAACAATTATCAATATACGATTAAATAATTTATCATTTGATCAAATACCTTTATTTATTTGAGCTGTAGGTATTACAGCATTTTTATTACTTCTTTCTTTACCAGTTTTAGCAGGAGCTATTACTATACTTTTAACTGATCGAAATTTAAATACTTCATTTTTTGATCCAGCTGGAGGAGGTGACCCTATTCTTTATCAACATTTATTT????? Gsicheas ??AACATTATATTTTATTTTTGGAATTTGAGCTGGAATAGTAGGAACTTCTTTAAGTTTATTAATTCGAGCTGAATTAGGTAATCCTGGATCTTTAATTGGAGATGATCAAATTTATAATACTATTGTTACAGCACATGCTTTTATTATAATTTTTTTTATAGTTATACCTATTATAATTGGAGGTTTTGGTAATTGATTAGTACCTCTTATACTCGGAGCTCCTGATATAGCTTTCCCCCGAATAAATAATATAAGTTTCTGACTTCTTCCCCCCTCTTTAACTCTTCTAATTTCAAGAAGAATTGTAGAAAGTGGAGCAGGAACAGGATGAACAGTTTACCCCCCACTTTCATCTAATATTGCTCATGGAGGAAGTTCAGTTGATTTAGCTATTTTCTCATTACATTTAGCAGGAATTTCATCAATTTTAGGAGCTATTAATTTTATCACCACAATTATTAATATACGATTAAATAATTTATCATTTGATCAAATACCTTTATTTGTTTGAGCTGTAGGTATTACCGCATTTTTATTACTTCTTTCACTACCAGTTTTAGCAGGAGCTATTACTATACTTTTAACTGATCGAAATTTAAATACTTCATTTTTTGACCCCGCAGGAGGAGGAGATCCTATTCTCTACCAACATTTATTT?????
200
Guxor ??AACATTATATTTTATTTTTGGTATTTGAGCTGGAATAGTAGGAACATCTTTAAGATTACTAATTCGAGCTGAATTAGGAAACCCTGGATCTTTAATTGGAGATGATCAAATTTATAATACTATCGTAACAGCACATGCTTTTATTATAATTTTTTTTATAGTAATACCTATTATAATTGGAGGATTCGGAAATTGATTAGTACCTCTTATATTAGGAGCCCCCGATATAGCTTTTCCTCGAATAAATAATATAAGTTTTTGACTCCTTCCCCCTTCTCTAACTCTTTTAATTTCAAGAAGAATTGTAGAAAGTGGAGCAGGAACAGGATGAACAGTTTACCCCCCACTTTCATCTAATATTGCTCATGGAGGAAGTTCAGTTGATTTAGCTATTTTTTCACTACATTTAGCAGGAATTTCATCAATTTTAGGAGCTATTAATTTTATTACAACAATTATCAACATACGATTAAATAATTTATCATTTGATCAAATACCTTTATTTATTTGAGCTGTAGGTATTACAGCATTTTTATTACTTCTTTCTTTACCAGTTTTAGCGGGAGCTATTACTATACTTTTAACTGATCGAAATTTAAATACTTCATTTTTCGACCCAGCTGGAGGAGGTGATCCTATTCTTTATCAACATTTATTT?????
Hsittaca ?????????????ATTTTTGGAATTTGAGCAGGAATAGTAGGAACTTCTTTAAGTTTATTAATTCGAGCTGAATTAGGTAACCCCGGATCATTAATTGGAGATGATCAAATTTATAATACTATTGTTACAGCTCATGCTTTTATTATAATTTTTTTTATAGTTATACCTATTATAATTGGAGGATTTGGTAATTGATTAGTACCTTTAATATTAGGAGCTCCTGATATAGCTTTCCCTCGTATAAATAATATAAGTTTTTGACTCTTACCCCCTTCTTTAACTCTTTTAATTTCTAGAAGAATTGTA?????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? Oemarginata ?????????????ATTTTTGGTATTTGAGCTGGTATAGTAGGAACTTCTTTAAGATTATTAATTCGAGCTGAATTAGGAAATCCTGGTTCTTTAATTGGTGATGACCAAATTTATAACACTATTGTAACAGCCCATGCTTTTATTATAATTTTTTTTATAGTTATACCTATTATAATTGGAGGATTTGGAAACTGATTAGTTCCTCTAATATTAGGAGCACCTGATATAGCTTTTCCTCGTATAAATAATATAAGTTTTTGACTTCTTCCACCTTCTTTAACCCTTTTAATTTCTAGAAGAATTGTA?????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? Onobilis ??AACATTATATTTTATTTTTGGAATTTGAGCAGGTATAGTAGGAACATCTCTAAGATTATTAATTCGAGCTGAATTAGGTAATCCAGGATCTTTAATTGGAGATGATCAAATTTACAATACTATTGTAACAGCTCATGCTTTTATTATAATTTTCTTTATAGTTATACCTATTATAATTGGAGGTTTTGGAAATTGATTAGTACCTTTAATGTTAGGAGCACCTGATATAGCTTTCCCACGAATAAATAATATAAGTTTCTGACTTCTTCCCCCTTCTTTAACTCTTCTAATTTCTAGAAGAATTGTAGAAAATGGAGCAGGAACTGGATGAACAGTTTACCCCCCACTTTCATCTAATATTGCCCATAGAGGTAGATCAGTGGATTTAGCTATTTTTTCACTTCACTTAGCAGGAATCTCATCAATTTTAGGAGCAATTAATTTTATTACTACAATTATTAATATACGACTTAGAAATTTATCTTTTGATCAAATACCTTTATTTGTGTGAGCTGTAGGTATTACAGCCTTTTTACTGCTATTATCTCTTCCTGTTTTAGCTGGAGCTATTACTATACTTTTAACAGACCGAAATCTAAATACATCCTTTTTTGACCCAGCTGGAGGAGGAGATCCAATCTTATATCAACATTTATTT?????
201
Orectristria ???????????TTATTTTTGGTATTTGAGCAGGtATAGTAGGAACCTCTTTAAGATTATTAATTCGAGCTGAATTAGGTAATCCAGGATCCTTAATTGGTGATGATCAAATCTATAATACTATTGTAACAGCTCATGCTTTTATTATAATTTTTTTCATAGTTATACCTATTATAATTGGAGGATTTGGAAATTGATTGGTTCCATTAATATTAGGAGCACCTGATATAGCTTTCCCACGTATAAATAATATAAGTTTTTGACTTCTTCCTCCATCATTAACTCTTTTAATTTCCAGAAGAATTGTAGAAAATGGAGCAGGAACTGGATGAACAGTCTATCCACCACTTTCATCAAATATTGCTCACGGGGGAAGATCTGTTGATTTAGCCATTTTTTCTCTTCATTTAGCTGGAATTTCATCAATTTTAGGAGCAATTAATTTTATTACAACAATTATTAATATACGACTAAATAATTTATCAT
TTGATCAAATACCACTATTTGTATGAGCTGTTGGTATTACTGCTTTCTTACTTTTATTATCTTTACCAGTTTTAGCAGGAGCTATTACAATATTATTAACTGATCGAAATTTAAATACATCATTTTTTGACCCTGC???????????????????????????????????????????? Oserpans ??AACATTATATTTTATTTTTGGTGTATGAGCAGGTATAGTAGGAACATCTTTAAGATTATTAATTCGAGCTGAATTAGGTAATCCAGGATCTCTTATTGGAGATGATCAAATTTATAATACTATTGTAACAGCACATGCTTTTATTATAATTTTTTTCATAGTTATACCTATTATAATTGGAGGATTTGGAAATTGATTAGTTCCTTTAATATTAGGAGCCCCAGATATAGCTTTCCCTCGAATAAATAATATAAGTTTTTGACTTCTTCCCCCATCATTAATTTTACTAATTTCAAGAAGAATTGTAGAAAATGGAGCAGGAACTGGATGAACAGTGTACCCCCCACTATCATCAAATATTGCACACGGAGGAAGATCAGTAGATTTAGCTATTTTCTCCCTTCATTTAGCTGGAATTTCATCAATTTTAGGAGCTATTAATTTTATTACAACAATTATTAATATACGATTAAATAATATATCATTTGATCAAATACCTTTATTTATTTGAGCTGTAGGAATTACAGCTTTCTTATTATTACTTTCTCTTCCTGTTTTAGCAGGAGCTATTACAATACTTTTAACAGATCGAAATTTAAATACATCTTTTTTTGACCCAGCTGGTGGAGGAGATCCTATTTTATATCAACATTTATTT????? Oexcitans ??AACATTATATTTTATCTTTGGTATTTGAGCAGGTATAGTAGGAACATCATTAAGATTATTAATTCGAGCTGAGTTAGGTAATCCTGGATCTCTTATTGGAGATGATCAAATTTATAATACCATTGTAACAGCTCATGCTTTTATTATAATTTTTTTCATAGTTATACCTATTATAATTGGAGGATTTGGAAATTGATTAGTTCCATTAATATTAGGAGCCCCAGATATAGCTTTTCCTCGAATAAATAATATAAGTTTTTGACTTTTACCCCCCTCATTAACTCTTTTAATTTCAAGAAGAATTGTAGAAAACGGAGCAGGAACTGGATGAACAGTGTACCCCCCACTTTCATCTAATATTGCTCATGGAGGAAGATCTGTAGATTTAGCTATTTTTTCCCTTCATCTAGCTGGAATTTCTTCAATTTTAGGAGCTATTAATTTTATTACAACAATTATCAATATACGATTAAATAATTTATCATTTGACCAAATACCTTTATTTGTTTGAGCTGTTGGAATTACAGCTTTCCTATTACTTCTATCCCTTCCTGTTTTAGCAGGAGCTATTACTATACTTTTAACAGACCGAAATTTAAATACATCTTTTTTTGATCCAGCTGGTGGAGGAGACCCTATTTTATATCAACATTTATTT?????
202
Pcoelonota ???????????????????????TCTGAGCAGGtATAGTAGGAACTTCATTAAGATTACTAATTCGAGCAGAATTAGGTAACCCTGGATCTTTAATTGGAGATGATCAAATTTATAATACTATTGTCACAGCTCATGCTTTCATTATAATTTTTTTTATAGTTATACCTATTATAATTGGAGGATTTGGTAATTGATTAGTTCCACTTATATTAGGTGCACCTGATATAGCTTTCCCTCGTATAAATAATATAAGTTTTTGACTCCTTCCCCCCTCTTTAACTCTTTTAATTTCCAGAAGAATTGTAGAAAATGGAGCTGGAACAGGATGAACAGTTTATCCCCCACTATCTTCTAATATTGCTCACGGAGGTAGATCTGTAGATTTAGCTATTTTTTCATTACATTTAGCAGGAATTTCATCAATTTTAGGAGCTATTAATTTTATTACAACAATTATTAATATACGATTAAATAATCTTTCATTTGATATAATACCATTATTTGTATGAGCAGTAGGTATTACTGCATTCTTACTATTATTATCACTCCCAGTCTTAGCTGGTGCCATTACTATACTATTAACTGACCGAAATTTAAATACCTCTTT????????????????????????????????????????????????????????
Pcompressipalpus ??????????TTTATTTTTGGAATTTGAGCTGGTATAATTGGAACCTCATTAAGATTATTAATTCGAGCAGAATTAGGAAATCCTGGCTCTTTAATTGGAGATGATCAAATTTATAATACTATTGTAACAGCCCATGCTTTCATCATAATTTTTTTTATAGTTATACCTATTATAATTGGAGGATTTGGAAATTGATTAGTACCTTTAATACTAGGAGCCCCTGATATAGCTTTCCCCCGAATAAACAACATAAGTTTCTGACTTCTTCCCCCTTCTTTAACTCTTTTAATTTCTAGAAGAATTGTAGAAAACGGAGCAGGAACCGGCTGAACAGTTTACCCCCCTTTATCATCTAATATCGCACATAGTGGAAGATCTGTAGATTTAGCTATTTTTTCCCTACATTTAGCTGGAATCTCTTCCATTTTAGGAGCAATTAATTTTATTACGACAATTATTAATATACGATTAAATAATCTTTCATTTGATATAATACCTTTATTTGTTTGAGCTGTAGGTATTACTGCATTTTTATTATTACTATCTTTACCAGTATTAGCAGGAGCTATTACCATATTATTAACTGATCGTAATTTAAATACTTCTTTTTTCGACCCCGCTGGGGGAGGAG????????????????????????????????? Pdimorpha ?????????????ATTTTTGGAATTTGAGCCGGTAtAGTAGGAACTTCATTAAGATTACTAATTCGAGCAGAATTAGGTAACCCCGGATCTTTAATTGGAGATGATCAAATTTATAATACTATTGTTACAGCTCATGCTTTTATTATAATTTTTTTTATAGTTATACCTATTATAATTGGAGGATTTGGAAATTGATTAGTTCCATTAATATTAGGAGCCCCTGATATAGCTTTCCCTCGAATAAATAATATAAGTTTTTGACTTTTACCCCCCTCTTTAACTCTTTTAATCTCCAGAAGAATCGTAGAAAATGGAGCTGGAACAGGATGAACAGTCTACCCCCCACTATCATCTAATATTGCCCATGGAGGTAGCTCTGTAGATTTAGCTATTTTTTCTTTACATTTAGCTGGAATTTCTTCAATTTTAGGAGCAATTAATTTTATTACAACAATTATTAATATACGACTTAATAATCTTTCATTTGATATAATACCATTATTTGTATGAGCTGTAGGAATTACTGCATTTTTATTATTACTATCTCTTCCAGTTTTAGCCGGAGCTATTACTATATTATTAACTGATCGAAATTTAAATAC??????????????????????????????????????????????????????????????
203
Slibatrix AACTTTATACTTTATTTTTGGTATTTGAGCTGGAATAGTAGGAACTTCTTTAAGTATATTAATTCGAGCTGAATTAGGAAATCCAGGATCATTAATTGGAAATGATCAAATTTATAATACTATTGTAACAGCGCACGCTTTTATTATAATTTTTTtTATAGTTATACCTATTATAATTGGAGGATTTGGTAATTGATTAGTTCCTTTAATATTAGGAGCTCCTGATATAGCTTTCCCTCGAATAAATAATATAAGTTTCTGACTTCTACCCCCTTCTTTAATTTTATTAACCTCAAG????????????????????????????????????????????????????????????????GCCCATAGAGGAAGATCAGTAGATCTTGCTATTTTTTCTCTTCATTTAGCAGGAATTTCTTCAATTTTAGGTGCTATTAATTTCATTACAACAATTATTAATATACGATTAAATAATTTATCATTTGATCAAATACCTTTATTTGTTTGAGCTGTAGGGATCACAGCATTTTTATTATTATTATCTCTTCCAGTATTAGCAGGAGCTATTACAATACTTTTAACTGATCGAAATTTAAATACTTCATTTTTTGACCCTGCAGGAGGAGGTGATCCAATTCTTTATCAACATTTATTT??????? Ccanadensis ????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????
????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? Etyrannus ????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? Gnutrix ????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? Hhormos ?????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????
204
Hmonilis ????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? Phycallitrichoides ??????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????
??????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? Pcasta ????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? [28S] Slibatrix ???TCGAATGAACGA??ACGGAGAGATTCATCGTCATTCCGCGGCGTACGCGATGCGCGACTCGATGTCGT?CGGCCGAGGCC?GGCGTGCACGACGTGCGTCCGTC?ACGTCCGTGGACGGCGTGCACTTCTCTCTTAGTA?AATACATCGCGACCCGTTCGATGTCGGTCTAAGCGCCGTTCGGGAGCCCCAC?GGTGT?CC???????GGCAACGT?ACACCGCGGGACCGCGACGGTGGCCGACCGGCCGTCGGACGGTAGTTCTGATGAAACGCGCACGCGTTTTTAACGCGTCCGGCCCGACGCAAGTCAACGTCGTA??TCC?AACGT????????CTCGCCCAAGTGCGAACGTAGGTGCGGCGCGCCTGC???TGTTGCCGCCGTGCAGTCTCGGACTGTGCGCGTCTCC?GTCTGCGATGATTCA?GTTTCGGGCACTCG??????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????
205
Pcallitrichoides AACTCGAATGAACGA??ACGGAGAGATTCATCGTCACTCCGCGGCGTACG?GACGCGCGTTTCGATGTCGC?CGGCCACGGTC?GGCAGGCACTGCGCGCGTACGTCGACGTCCGCGGACGGCGTGCACTTCTCTCTCAGTATCACAACATCGCGACCCGTTCGATGTCGGTCTAAGCGCCGTCCGGGAGCCCAGA?GGTGA?CG???????GGCGACCGCGCACCTCTGGACCGAGACGGTGGCCGACCGGCCGTCGGACGGTAGTTCTGATGAAACGCGCACGCGTTTACAACGCGTCCGGCCCGACGCAAGTCAACGTCGTA??TCC?CACGTAATTATATACCGCCTATGCGCGGACGCGGGTGCGGCGCGTCTGC???CGTTGCAGCCGTGCAGTCTCGGACTGTGCGCGTCTCTCGTCTGCGATGATTCA?GTTTCGGGCACTCGCAGGACCCGTCTTGAAACACGGACCAAGGAGTCTAGCATGTATGCGAGTCATTGAGATTTGATCAAAACTGAAAGGCGCAACGAAAGTGAAGGCGCGCGCTCGTCGCGCGCTCAGGGAGGATGGAGCCACGATCTAGGTCGTACTCTCGCACTCCCGAGGCGTCTCGTTTCCAATCCGTGAATGTAGGCGCGCTCTGAGCATAAATGCTGGGACCCGAAAGATGGTGAACTATG???????????
Etyrannus ??????AATGAACGA??ACGGAGAGATTCATCGTCACTCCTCGGCGTACG?GGCGTGCGACTCGATGTCGTCCGGCTTCGGCCGGGCGCGCACGACGCGCGCTCGTCGACGCCCGGGGACGGCGTGCACTTCTCTCTTAGTA??AATGCATCGCGACCCGTTCGATGTCGGTCTAAGCGCCGTTCGGGAGCCCCGTGCGTGCGCTCTAATAAAGTCGCGC??????GCGGGACCGCGACGGTGGCCGACCGGCCGTCGGACGGTATCACTGACGAAGCGCGCACGCGTTTACAACGCGTCCGGCCCGACGCAAGCCAACGTCGTATTTCC?GACGT????????ACCGCCAAAGTGCGGACGCCGGTGCGGCGTAGCTGT???CGTTGCTGCCGTGCAGTCTCGGACTGTGCGCGTCTCT?GTCTGCGATGATTCA?GTTTCGGGCACTCGCAGGACCCGTCTTGAAACACGGACCAAGGAGTCTAGCATGTATGCGAGTCATTGAGATT?CATTTAAACTGAAAGGCGCAACGAAAGTGAAGGCGCGCGCTTGCCGCGTGCTCAGGGAGGATGGAGCGTCGATCTCGGTCGATCTCTCGCACTCCCGAGGCGTCTCGTTTCCAATCTGTGAATGCAGGCGCGCTCTGAGCATAAATGCTGGGACCCGAAAGATGGTGAACTATGCAT?GGTCAGA Guxor ??????AATGAACGA??ACGGAGAGATTCATCGTCATTCCGCGGCGTACG?CGCGCGCGCCTCGATGTCGT?CGGCCTCGGTC?GGCGTGCACGACGCGCGCGCGTCGACGTCCGCGGACGGCGTGCACTTCTCTCTTAGTA??TAAACATCGCGACCCGTTCGATGTCGGTCTAAGCGCCGTCCGGGAGCCCCGT?TGCGC?CT?????????TCGCGG?GCGCTTCGGGACCGCGACGGTGGCCGACCGGCCGTCGGACGGTAGTTTCGAAGAAACGCGCACGCGTTCACAACGCGTCCGGCCCGACGCAAGTCAACGTCGTA??TCC?AACGT???????CACCGCCTAAGCGCGGACGTAGGTGCGGCGCGTCTGT???TGTTGCAGCCGTGCAGTCTCGGACTGTGCGCGTCTCT?GTCTGCGATGATTCA?GTTTCGGGCACTCGCAGGACCCGTCTTGAAACACGGACCAAGGAGTCTAGCATGTATGCGAGTCATTGAGATAATA???AAACTGAAAGGCGCAACGAAAGTGAAGGCGCGCGCTCGCCGCGCGCTCAGGGAGGATGGCGCTGCGATCTCGGTCGCACGCTCGCACTCCCGAGGCGTCTCGTTTCCAATCTGTGAATGCAGGCGCGCTCTGAGCATAAATGCTGGGACCCGAAAGATGGTGAACTATGCCT?GGTCAGA
206
Gnutrix ????????????CGA??ACGGAGAGATTCATCGTCATTCCGCGGCGTACG?CGCGCGCGCCTCGATGTCGT?CGGCCTCGGTC?GGCGTGCACGACGCGCGCGCGTCGACGTCCGCGGACGGCGTGCACTTCTCTCTTAGTA??TAAACATCGCGACCCGTTCGATGTCGGTCTAAGCGCCGTCCGGGAGCCCCGT?TGCGC?CT?????????TCGCGG?GCGCTTCGGGACCGCGACGGTGGCCGACCGGCCGTCGGACGGTAGTTTCGAAGAAACGCGCACGCGTTTACAACGCGTCCGGCCCGACGCAAGTCAACGTCGTA??TCC?AACGT???????CACCGCCTAAGCGCGGACGTAGGTGCGGCGCGTCTGT???TGTTGCAGCCGTGCAGTCTCGGACTGTGCGCGTCTCT?GTCTGCGATGATTCA?GTTTCGGGCACTCGCAGGACCCGTCTTGAAACACGGACCAAGGAGTCTAGCATGTATGCGAGTCATTGAGATAATA???AAACTGAAAGGCGCAACGAAAGTGAAGGCGCGCGCTCGCCGCGCGCTCAGGGAGGATGGCGCTGCGATCTCGGTCGCACGCTCGCACTCCCGAGGCGTCTCGTTTCCAATCTGTGAATGCAGGCGCGCTCTGAGCATAAATGCTGGGACCCGAAAGATGGTGAAT??????????????? Ccanadensis ???TCGAATGAACGA??ACGGAGAGATTCATCGTCATTCCGCGGCGTACGTAGCGCGCGACTCGATGTCGT?CGGCCTCGGTC?GGCGCGCACGACGCGCGCTCGTCTACGTCCGCGGACGGCGTGCACTTCTCTCTTAGTA??AATACATCGCGACCCGT
TCGATGTCGGTCTAAGCGCCGTTCGGGAGCCCCAC?GGTGCGCC?????????TCACGGCACACCGTGGGACCGCGACGGTGGCCGACCGGCCGTCGGACGGTAGTTTTGACGAATCGCGCACGCGTTTACAACGCGTCCGGCCCGACGCAAGTCAACGCCGTA??TCCTTGCGT???????CATCGCCTCAGCGCGAACGTAGGTGCGGCGCGTCTGC???TGTTGCCGCCGTGCAGTCTCGGACTGTGCGCGTCTCT?GTCTGCGATGATTCA?GTTTCGGGCACTCGCAGGACCCGTCTTGAAACACGGACCAAGGAGTCTAGCATGTATGCGAGTCATTGAGATTTTATATAAACTGAAAGGCGCAACGAAAGTGAAGGCGCGCGCTCGCCGCGCGCTCAGGGAGGATGGAGCGTCGGTCTAGGTCGATCTCTCGCACTCCCGAGGCGTCTCGTTTCCAATCTGTGAATGCAGGCGCGCTCTGAGCATAAATGCTGGGACCCGAAAGATGGTGAA???????????????? Oexcitans ???TCGAATGAACGA??ACGGAGAGATTCATCGTCATTCCTCGGCGTACG?GACGCGCGGTTAGATGTCGT?CGGCCTCGGTC?GGCTGGCACGACGCGCGCACGTCGACGTCCGGGGACGGCGTGCACTTCTCTCTTAGTA??AATACATCGCGACCCGTTCGATGTCGGTCTAAGCGCCGTTCGGGAGCCCCAT?CGTTC?CT?????????TCACGG?GTTCGGTGGGACCGCGACGGTGGCCGACCGGCCGTCTGACGGTAGTTCTTAAGAAGCGCGCACGCGTTTACAACGCGTCCGGCCCGACGCAAGTCAACGTCGTA??TCC?TACGT????????ACCGCCTAAGCGCGGACGTGGGTGCGGCGCGTCTGCCGTTGTTGCTGCCGTGCAGTCTCGGACTGTGCGCGTCTCT?GTCTGCGATGATTCA?GTTTCGGGCACTCGCAGGACCCGTCTTGAAACACGGACCAAGGAGTCTAGCATGTATGCGAGTCATTGAGATTATA???AAACTGAAAGGCGCAACGAAAGTGAAGGCGCGCGCTCGCCGCGTGCTCAGGGAGGATGGAGCGTCGATCTAGGTCGATCTCTCGCACTCCCGAGGCGTCTCGTTTCCAATCAGTGAATGCAGGCGCGCTCTGAGCATAAATGCTGGGACCCGAAAGATGGTGAACTATGCCTGGACAG?? 207 Orectristria AACTCGAATGAACGA??ACGGAGAGATTCATCGTCATTCCTCGGCGTACG?GACGCGCGGTTAGATGTCGT?CGGCCTCGGTC?GGCTGGCACGACGCGCGCACGTCGACGTCCGGGGACGGCGTGCACTTCTCTCTTAGTA??AATACATCGCGACCCGTTCGATGTCGGTCTAAGCGCCGTTCGGGAGCCCCAT?TGTAC?CT?????????TTACGG?GTTCGGTGGGACCGCGACGGTGGCCGACCGGCCGTCTGACGGTAGTTTTTAAGAAGCGCGCACGCGTTTACAACGCGTCCGGCCCGACGCAAGTCAACGTCGTA??TCC?TACGT????????ACCGCCTAAGCGCGGACGTGGGTGCGGCGCGTCTGTCGTTGTTGCTGCCGTGCAGTCTCGGACTGTGCGCGTCTCT?GTCTGCGATGATTCA?GTTTCGGGCACTCGCAGGACCCGTCTTGAAACACGGACCAAGGAGTCTAGCATGTATGCGAGTCATTGAGATAATA???AAACTGAAAGGCGCAACGAAAGTGAAGGCGCGCGCTTGCCGCGTGCTCAGGGAGGATGGAGCGTCGATCTAGGTCGATCTCTCGCACTCCCGAGGCGTCTCGTTTCCAATCAGTGAATGTAGGCGCGCTCTGAGCATAAATGCTGGGACCCGAAAGATGGTGAACTATGCCCTGGTCAGA Clata AACTCGAATGAACGA??ACGGAGAGATTCATCGTCATTCCGCGGCGTACG?GACGCGCGCTTCGATGTCGT?CGGCCTCGGTC?GGCCGGCACGACGCGCGTACGTCGACGTCCGCGGACGGCGTGCACTTCTCTCTTAGTA??AATACATCGCGACCCGTTCGATGTCGGTCTAAGCGCCGTCCGGGAGCCCCAT?TGTGC?CT?????????TAACGG?GTATAGTGGGACCGCGACGGTGGCCGACCGGCCGTCGGACGGTAGTTCTGACGAAACGCGCACGCGTTTAAAACGCGTCCGGCCCGACGCAAGTCAACGTCGTA??TCC?CACGT????????ACCGCCTCAGCGCGGACGCGGGTGCGGCGCGTCTGC???TGTTGCCGCCGTGCAGTCTCGG
ACTGTGCGCGTCTCT?GTCTGCGATGATTCA?GTTTCGGGCACTCGCAGGACCCGTCTTGAAACACGGACCAAGGAGTCTAGCATGTATGCGAGTCATTGAGACAATA???AAACTGAAAGGCGCAACGAAAGTGAAGGCGCGCGCTTGCCGCGCGCTCAGGGAGGATGGAGCGTCGATCTCGGTCGACCTCTCGCACTCCCGAGGCGTCTCGTTTCCAATCTGTGAATGCAGGCGCGCTCTGAGCATGAATGCTGGGACCCGAAAGATGGTGAACTATGCCC???????? Pcompressipalpus ?????GAATGAACGA??ACGGAGAGATTCATCGTCATTCCGCGGCGTACG?GTCGCGCGTTTCGATGTCGT?CGGCCTCGGTC?GGCTGGCACGACGCGCGTCCGTCGACGTCCGCGGACGGCGTGCACTTCTCTCTTAGTA??AATACATCGCGACCCGTTCGATGTCGGTCTAAGCGCCGTTCGGGAGCCCCAT?TGTAC?CT?????????TAACGG?GTATAGTGGGACCGCGACGGTGGCCGACCGGCCGTCGGACGGTAGTTCTGAAGAAACGCGCACGCGTTCTAAACGCGTCCGGCCCGACGCAAGTCAACGTCGTA??TCC?TACGT????????ACCGCCTAAGCGCGGACGTGGGTGCGGCGCGTCTGC???TGTTGCCGCCGTGCAGTCTCGGACTGTGCGCGTCTCT?GTCTGCGATGATTCAGGTTTCGGGCACTCGCAGGACCCGTCTTGAAACACGGACCAAGGAGTCTAGCATGTATGCGAGTCATTGAGATAATA???AAACTGAAAGGCGCAACGAAAGTGAAGGCGCGCGCTAGCCGCGCGCTCAGGGAGGATGGAGCGTCGGTCTAGGTCGATCTCTCGCACTCCCGAGGCGTCTCGTTTCCAATCTGTGAATGTAGGCGCGCTCTGAGCATAAATGCTGGGACCCGAAAGATGGTGAACTATGC?????????? Pcasta ??????AATGAACGA??ACGGAGAGATTCATCGTCATTCCGCGGCGTACG?GTCGCGCGCTTCGATGTCGT?CGGCCTCGGTC?GGCTGGCACGACGCGCGTCCGTCGACGTCCGCGGACGGCGTGCACTTCTCTCTTAGTA??AATACATCGCGACCCGTTCGATGTCGGTCTAAGCGCCGTTCGGGAGCCCCAT?TGTAC?CT?????????TAACGG?GTATAGTGGGACCGCGACGGTGGCCGACCGGCCGTCGGACGGTAGTTCTGACGAAACGCGCACGCGTTTATAACGCGTCCGGCCCGACGCAAGTCAACGTCGTA??TCC?TACGT????????ACCGCCTAAGCGCGGACGTGGGTGCGGCGCGTCTGC???TGTTGCTGCCGTGCAGTCTCGGACTGTGCGCGTCTCT?GTCTGCGATGATTCAGGTTTCGGGCACTCGCAGGACCCGTCTTGAAACACGGACCAAGGAGTCTAGCATGTATGCGAGTCATTGAGATAATA???AAACTGAAAGGCGCAACGAAAGTGAAGGCGCGCGCTTGCCGCGCGCTCAGGGAGGATGGAGCGTCGATCTAGGTCGATCTCTCGCACTCCCGAGGCGTCTCGTTTCCAATCTGTGAATGTAGGCGCGCTCTGAGCATAAATGCTGGGACCCGAAAGATGGTGAA????????????????
208
Hmonilis ???GGAAATCAGTGACGAAGGTGGGATC??TCGTC?TTCCTCGGCGTACG?GGCGCGCNCCTCGATGTCGT?CGGCCTCGGTC?GGCTGGCACGACGCGCGTTCGTCGACGTCCGCGGACGGCGTGCACTTCTCTCTTAGTA??AATACATCGCGACCCGTTCGATGTCGGTCTAAGCGCCGTTCGGGAGCCCCAT?TGTAC?CT?????????TCGCGG?GTATAGTGGGACCGCGACGGTGGCCGACCGGCCGTCGGACGGTAGTTCTGACGAAACGCGCACGCGTTTATAACGCGTCCGGCCCGACGCATGTCAACGTCGTA??TCC?TGCGT????????ACCGCCTAAGCGCGGACGTAGGTGCGGCGCGTCTGT???TGTTGCAGCCGTGCAGTCTCGGACTGTGCGCGTCTCT?GTCTGCGATGATTCA?GTTTCGGGCACTCGCAGGACCCGTCTTGAAACACGGACCAAGGAGTCTAGCATGTATGCGAGTCATTGAGATAATA???AAACTGAAAGGCGCAACGAAAGTGAAGGCGCGCCCTTGTCGCGCGCTCA
GGGAGGATGGAGCGTCGATCTAGGTCGATCTCTCGCACTCCCGAGGCGTCTCGTTTCCAATCCGTGAATGTAGGCGCGCTCTGAGCATAAATGCTGGGACCCGAAAGATGGTGAACTATGCCTGGGTCAGA Hhormos ??????AATGAACGA??ACGGAGAGATTCATCGTCATTCCTCGGCGTACG?GGCGCGCGCCTCGATGTCGT?CGGCCTCGGTC?GGCTGGCACGACGCGCGTTCGTCGACGTCCGCGGACGGCGTGCACTTCTCTCTTAGTA??AATACATCGCGACCCGTTCGATGTCGGTCTAAGCGCCGTTCGGGAGCCCCAT?TGTAC?CT?????????TCGCGG?GTATAGTGGGACCGCGACGGTGGCCGACCGGCCGTCGGACGGTAGTTCTGACGAAACGCGCACGCGTTTATAACGCGTCCGGCCCGACGCAAGTCAACGTCGTA??TCC?TGCGT????????ACCGCCTAAGCGCGGACGTAGGTGCGGCGCGTCTGT???TGTTGCAGCCGTGCAGTCTCGGACTGTGCGCGTCTCT?GTCTGCGATGATTCA?GTTTCGGGCACTCGCAGGACCCGTCTTGAAACACGGACCAAGGAGTCTAGCATGTATGCGAGTCATTGAGATAATA???AAACTGAAAGGCGCAACGAAAGTGAAGGCGCGCCCTTGTCGCGCGCTCAGGGAGGATGGAGCGTCGATCTAGGTCGATCTCTCGCACTCCCGAGGCGTCTCGTTTCCAATCCGTGAATGTAGGCGCGCTCTGAGCATAAATGCTGGGACCCGAAAGATGGTGAACTATGCCT?GGTCAGA Ematerna ????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????
209
Aflava ???????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? Amesogona ????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????
Calbivirgata ???????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? Cbicolor ???????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? Cthalictri ????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????
210
Eaurantia ???????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? Ejordani ????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????
???????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? Esalaminia ???????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? Gindentata ???????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? Gsinaldus ????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????
211
Gparens ???????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? Gincurva ??????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????
?????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? Gcorrecta ???????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? Gsicheas ????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????
212
Hsittaca ???????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? Oemarginata ????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????
Onobilis ???????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? Oserpans ???????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? Pcoelonota ???????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? Pdimorpha ???????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? ;END;
213
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BIOGRAPHICAL SKETCH
Jennifer Michelle Zaspel was born in North Branch, Minnesota. Upon graduation from
North Branch High School in 1997, she began undergraduate studies at the University of
Minnesota (Duluth). After transferring to the University of Minnesota (Twin Cities) in 2000, she
received her Bachelor of Science degree in science in agriculture (with emphasis in entomology)
in August of 2001. She then obtained a Master of Science degree in entomology from the
University of Minnesota in 2004 under the advisement of Dr. Susan Weller. In August of 2004
she enrolled in a Ph.D. program at the University of Florida under the advisement of Dr. Marc A.
Branham in the Department of Entomology and Nematology. Her advisory committee members
were Drs. Marjorie Hoy, Jacqueline Miller, and David Reed. Dr. Hans Bänziger served as an
unofficial, ad hoc external committee member. Jennifer is currently a member of Sigma Xi
Scientific Research Society, the Entomological Society of America, the Florida Entomolgical
Society, the Lepidopterists’ Society, the Willi Hennig Society, the Society of Systematic
Biologists, the American Association for the Advancement of Science (AAAS), and is on the
Board of Directors for the Center of Systematic Entomology.
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