TAXONOMY, DISTRIBUTION AND PEST
STATUS OF PLUTELLA SPECIES
(LEPIDOPTERA: PLUTELLIDAE) IN
AUSTRALIA AND NEW ZEALAND
Kariyawasam Haputhanthri Kankanamge Tharanga
Niroshini
Submitted in fulfilment of the requirements for the degree of
Master of Applied Sciences (Research)
School of Earth Environmental and Biological Sciences
Science and Engineering Faculty
Queensland University of Technology
2018
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Keywords
Adults, ANOVA, Bayesian analysis, CO1 barcode gene, crops, DNA, genitalia
morphological features, host plant, larvae, light trap, maximum likelihood,
measurements, PCR, phylogenetic analyses, Plutella australiana, Plutella xylostella,
R statistical analyses, Sanger sequencing, taxonomy.
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Abstract
The diamondback moth (DBM), Plutella xylostella L. (Lepidoptera: Plutellidae), is the
most destructive agricultural pest in the word causing damage to brassica crops such
as cabbage, kale, broccoli, and cauliflower. Its global distribution, movement over long
distances and rapid evolution of insecticide resistance make this a key pest of
international importance. P. xylostella was introduced to Australia in 1882 and has
become widely distributed in Australia.
Despite reports of low levels of genetic variation in the Australian population
(Endersby et al., 2006), more recent molecular studies indicated the presence of
variants within the Australian Plutella population. A study of allozymes in P.
xylostella populations from 14 locations worldwide included specimens from 5
different locations in Australia (Adelaide, Brisbane, North Queensland, Melbourne
and Sydney), and found significant differences within the samples from Australia
(Pichon et al., 2006). Similarly, Roux et al. (2007) using the inter simple sequence
repeat (ISSR) marker showed a genetic differentiation between Melbourne and Sydney
P. xylostella populations.
In 2013, a new taxon, Plutella australiana, was described (Landry & Hebert, 2013)
based on 8.6% sequence divergence in the ‘barcode’ region of the mitochondrial
cytochrome c oxidase 1 gene (CO1) and differences in the morphology of the genitalia
in both males and females. The new taxon was identified as broadly distributed in
southern and eastern Australia. However, there were no larval collections, leaving the
host plants and possible pest status unknown. In addition, the description of the new
and potentially endemic taxon created difficulties in the import and release of
biological control applications, and potential difficulties in pest management practices
and market access.
This study identified and addressed gaps in knowledge, with the main aim being to
clarify the differentiation of P. australiana from P. xylostella and to increase
knowledge of its distribution and host plants. CO1 barcode sequence data were used
in the identification of both taxa, including phylogenetic analyses (Maximum
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likelihood and Bayesian inference). Morphological features of female and male
genitalia were measured to determine statistical variance and examined to identify
reliable diagnostic features. Distribution of P. australiana at the regional scale was
evaluated from field collections, including individuals from New Zealand. Collections
of larvae from both crops and wild brassicas were conducted to identify host plants
and contribute to knowledge of the possible pest status of P. australiana. These
examinations contribute to the better understanding of possible risks to Australian
brassica production, and to inform potential pest management strategies.
The results showed that P. xylostella and P. australiana are two distinct taxa based on
CO1 data. Examination of key morphological features showed that only two features,
the curvature of the tubular projection in P. australiana and the presence of raised
folds surrounding the antrum in P. xylostella, and both only in females, are reliable as
diagnostic tools. Although some measured features are statistically significantly
different overall between the two populations, the overlap in the variance indicates that
those features cannot be used as diagnostic tools. Similarly, other characteristic
features proposed by Landry and Hebert (example: sinuation in the ventral margin of
the valva) were found to be not reliable for identification of the two taxa.
Light trap collections of adults show that the two taxa are sympatric in most locations,
including Tasmania. However, larvae of P. australiana were present in only two
collections: on cabbage (Brassica oleracea) in Theresa Park, NSW in 2015 and on
field mustard weeds (Brassica rapa) amongst a kale crop in Werombi NSW in 2015.
This study is the first to describe the occurrence of P. australiana larvae on cabbage
and field mustard. The preference of P. australiana for weedy B. rapa over kale in one
site suggests that emergence of P. australiana as a pest of canola requires
investigation.
The outcomes of this thesis have been to broaden the knowledge of the new taxon, P.
australiana including morphological features that can be used in order to differentiate
P. xylostella and P. australiana. The results contribute to the better understanding of
possible risks of the new taxon to Australian brassica production, and to inform
potential pest management strategies.
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Table of Contents
Keywords .................................................................................................................................. i
Abstract .................................................................................................................................... ii
Table of Contents ......................................................................................................................v
List of Figures ....................................................................................................................... viii
List of Tables ........................................................................................................................ xiii
List of Abbreviations ........................................................................................................... xvii
Statement of Original Authorship ....................................................................................... xviii
Acknowledgements ............................................................................................................... xix
Chapter 1: General Introduction and Literature Review ................................ 1
1.1 Background .....................................................................................................................1
1.1.1 Research problem and aims ..................................................................................2
1.1.2 Thesis outline .......................................................................................................3
1.2 Literature Review ...........................................................................................................4
1.2.1 Life history ...........................................................................................................4
1.2.2 Distribution ...........................................................................................................8
1.2.3 Host plants ..........................................................................................................10
1.2.4 Pest management ................................................................................................14
1.2.5 Taxonomy ...........................................................................................................17
1.2.6 A new Plutella taxon in Australia........................................................................19
Chapter 2: Molecular and morphological examination of Plutella species in
Australia and New Zealand ..................................................................................... 25
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2.1 Introduction .................................................................................................................. 25
2.2 Materials and Methods ................................................................................................. 28
2.2.1 Sampling ............................................................................................................ 28
2.2.2 Molecular analysis ............................................................................................. 31
2.2.3 Morphology ....................................................................................................... 33
2.3 Results .......................................................................................................................... 39
2.3.1 CO1 sequence data............................................................................................. 39
2.3.2 Morphology ....................................................................................................... 43
2.4 Discussion .................................................................................................................... 49
Chapter 3: Host plants and distribution of Plutella species in Australia ...... 53
3.1 Introduction .................................................................................................................. 53
3.2 Materials and Methods ................................................................................................. 56
3.2.1 Sampling ............................................................................................................ 56
3.2.2 Molecular Analysis ............................................................................................ 56
3.2.3 Morphology ....................................................................................................... 57
3.3 Results .......................................................................................................................... 59
3.3.1 CO1 sequence Data ............................................................................................ 59
3.3.2 Host plants of P. xylostella and P. australiana .................................................. 59
3.3.3 Morphology ....................................................................................................... 62
3.4 Discussion .................................................................................................................... 76
Chapter 4: General Discussion ......................................................................... 79
4.1 CO1 ‘barcode’ analysis of Australian and New Zealand Plutella taxa ............................ 80
4.2 Diagnostic morphological features ................................................................................... 82
4.3 Summary and conclusion on the taxonomy of P. xylostella and P. australiana .............. 84
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4.4 Distribution, host range and pest status of the two taxa ....................................................85
4.4.1 Distribution ..........................................................................................................85
4.4.2 Host preference and pest status of P. australiana................................................86
4.4.3 Summary and conclusions on the distribution, host range and pest status of
the two taxa. .......................................................................................................87
4.5 Pest management ..............................................................................................................88
4.6 Limitations and recommendations ....................................................................................88
References ................................................................................................................. 91
Appendices .............................................................................................................. 104
Appendix A ...........................................................................................................................104
Appendix B ...........................................................................................................................105
Appendix C ...........................................................................................................................109
Appendix D ...........................................................................................................................113
Appendix E ...........................................................................................................................114
Appendix F............................................................................................................................115
Appendix G ...........................................................................................................................116
Appendix H ...........................................................................................................................118
Supplementary Materials ...................................................................................... 121
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List of Figures
Figure 1.1 Diamondback moth eggs. ........................................................................... 5
Figure 1.2 Fourth instar larvae of diamondback moth. ............................................... 5
Figure 1.3 Swede crops in a community garden damaged by DBM larvae. ............... 6
Figure 1.4 Diamondback moth pupa on a red cabbage leaf. ....................................... 6
Figure 1.5 A) Lateral view of a DBM adult and B) dorsal view of a DBM
showing the diamond pattern of a DBM adult. .............................................. 7
Figure 1.6 World distribution of P. xylostella, the diamondback moth. Blue =
widespread, Black = present. Although some countries are not marked,
DBM may be present (CABI, 2016). ............................................................. 9
Figure 1.7 Major biological control agents of P. xylostella (Sarfraz et al., 2005).
...................................................................................................................... 15
Figure 1.8 Sites in Australia where specimens of P. xylostella (red) and P.
australiana (blue) have been collected. The circles show the proportion
of the two species at each site. These records only include specimens
identified through DNA barcode analysis (Landry & Hebert, 2013). ......... 20
Figure 1.9 Picture from Dugdale (1973) showing the curved tubular projection
(12) in a female genitalia from a diamondback moth specimen collected
from New Zealand. ...................................................................................... 22
Figure 2.1 Distribution of diamondback moth in Australia. Blue = widespread,
black = present (CABI, 2016). ..................................................................... 25
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Figure 2.2 Neighbor joining tree (nodes collapsed) based on Kimura-2-
parameter distances for the barcode region of the cytochrome c oxidase
1 gene. Specimens are labelled by the Australian state or by country of
origin and bracketed numerals indicate the number of specimens from
each site (taken from Landry and Hebert (2013)). ....................................... 26
Figure 2.3 A) Ranger moth light trap in a cabbage field and B) Insects trapped
in the light trap. Moths of Plutella spp. are circled in yellow. .................... 29
Figure 2.4 Measurements of both female and male adult genitalia characteristics
recorded to determine the statistical variance across taxa (Scale bars =
200 μm). Images were taken by Tharanga Kariyawasam. ........................... 37
Figure 2.5 Distribution of DBM adults caught in light traps at Samford (QLD),
Hobart (TAS), Theresa Park (NSW), Werombi (NSW), Mowbray Park
(NSW), Birkdale (QLD), Gatton (QLD) and Laidley (QLD). Numbers
within bars represent the individuals identified and assigned to relevant
taxa (see Table 2.1. for total number of individuals). .................................. 40
Figure 2.6 Bipartition maximum likelihood (ML) tree with bootstrap values.
The tree was collapsed to remove low supported nodes (≥75%) and the
nodes were further collapsed (shape of the clade) because of the large
number of specimens assigned to each taxon. See Appendix D for the
original phylogenetic tree. ........................................................................... 41
Figure 2.7 Bayesian analysis with posterior probabilities. The tree was collapsed
to remove low supported nodes (≥75%) and the nodes were further
collapsed (shape of the clade) because of the large number of specimens
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assigned to each taxon. See Appendix E for the original phylogenetic
tree. ............................................................................................................... 42
Figure 2.8 Density plots showing the distribution of measurements taken for a)
tubular projection length (TPL) and b) sternite 7 length (S7L)
parameters of females showing an overlap between Australian P.
australiana (AA), Australian P. xylostella (AX) and New Zealand P.
xylostella (NX). ............................................................................................ 46
Figure 2.9 Density plots showing the distribution of measurements taken for (a)
phallus length (PL) and (b) vinculum saccus length (VSL) parameters
of males showing an overlap between Australian P. australiana (AA),
Australian P. xylostella (AX) and New Zealand P. xylostella (NX). .......... 48
Figure 3.1 DBM larvae damaging A) cabbage leaf, B) head formation of red
cabbage. ........................................................................................................ 54
Figure 3.2 Distribution of larvae from field collections in Hobart (TAS), Theresa
Park (NSW), Werombi (NSW), Gatton (QLD), Laidley (QLD),
Currumbin (QLD) and Birkdale (QLD). Numbers within bars represent
the individuals identified and assigned to relevant taxa (for total
numbers see Table 3.1.). .............................................................................. 60
Figure 3.3 Field mustard (B. rapa) (above) and the cabbage field (below) that
P. australiana larvae were collected. In order from left to right are the
field mustard plant, its flower, larvae feeding on the leaf, pupae found
on the stem and the cabbage field where P. australiana larvae were
collected. ...................................................................................................... 61
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Figure 3.4 Density plots showing the distribution of measurements taken for a)
whole length (WL), b) whole width (WW), c) tubular projection length
(TPL) and d) sternite 7 length (S7L) parameters of females showing an
overlap between P. xylostella and P. australiana. However, mean
values are significantly different. ................................................................. 65
Figure 3.5 Density plot showing the distribution of measurements taken for
phallus length (PL) parameter of males showing an overlap between P.
xylostella and P. australiana. However, mean values are significantly
different. ....................................................................................................... 67
Figure 3.6 Density plots showing the distribution of measurements taken for a)
whole length (WL), b) upper part length (UPL) and c) sternite 7 length
(S7L) parameters showing an overlap between Australian P. xylostella
adults (AUS adults), New Zealand P. xylostella adults (NZ adults) and
P. xylostella larvae. However, mean values are significantly different. ...... 69
Figure 3.7 Density plots showing the distribution of measurements taken for
whole width (WW) and tubular projection length (TPL) parameters of
females, showing an overlap between Australian P. australiana adults
from light traps and P. australiana adults reared from larvae. However,
mean values are significantly different. ....................................................... 71
Figure 3.8 Density plot showing the distribution of measurements taken for
phallus length (PL) parameter of males, showing an overlap between P.
xylostella adults from light traps and P. xylostella adults reared from
larvae. ........................................................................................................... 73
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Figure 3.9 Density plots showing the distribution of measurements taken for
whole length (WL), valva length (VL) and phallus length (PL)
parameters of males, showing an overlap between P. australiana adults
from light traps and P. australiana adults reared from larvae. However,
mean values are significantly different. ....................................................... 75
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List of Tables
Table 1.1 Worldwide distribution of Plutella species. (Information taken from
(Robinson & Sattler, 2001). ........................................................................... 9
Table 1.2 Known host plants of P. xylostella (Information taken from Sarfraz et
al. (2006)). .................................................................................................... 12
Table 2.1 Locations of DBM adult collections and number of individuals taken
for genetic and morphological analyses. Specimens not taken for
morphological analyses are marked as ‘/’ and unknown GPS
coordinates are marked as ‘-‘. ...................................................................... 30
Table 2.2 Morphological features of DBM female genitalia examined in this
study to identify diagnostic features. Features are circled in red. Images
were taken by Tharanga Kariyawasam. ....................................................... 35
Table 2.3 Morphological features of DBM male genitalia examined in this study
to identify diagnostic features. Features are circled in red. The ventral
view of P. australiana and lateral views of the valva were taken from
Landry and Hebert (2013). The remaining images were taken by
Tharanga Kariyawasam. .............................................................................. 36
Table 2.4 Images show the appearance of the vinculum saccus in two males
where one had the characteristic morphology of P. australiana but were
identified as P. xylostella from CO1 sequence data and the other had
the characteristic morphology of P. xylostella but were identified as P.
australiana from CO1 sequence data. These specimens were collected
from Hobart and Theresa Park respectively. ................................................ 44
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Table 2.5 Mean values ± SD of parameters for each female population are shown
in micrometers (μm). P-value of the one-way ANOVA (at 0.95
confidence intervals) are presented which showed a significance
difference for the TPL (tubular projection length) parameter and the
S7L (sternite 7 length) parameter. Statistically significant codes: ***P
< 0.001, **P < 0.01. ..................................................................................... 45
Table 2.6 Mean values ± SD of parameters for each male population are shown
in micrometers (μm). P-value of the one-way ANOVA (at 0.95
confidence interval) is presented which showed a significance
difference for the PL (phallus length) parameter and VSL (vinculum
saccus length) parameter. Statistically significant codes: *P < 0.05,
***P < 0.001. ............................................................................................... 47
Table 3.1 Larvae collection details including location, crop type and number of
individuals taken for genetic and morphological analyses. Specimens
not taken for morphological analyses are marked as ‘/’ and unknown
GPS coordinates are marked as ‘-‘. .............................................................. 57
Table 3.2 The image shows the appearance of the vinculum saccus of a male
that had the characteristic morphology of P. australiana but was
identified as P. xylostella from CO1 sequence data. The larva was
collected from B. rapa weeds in Werombi NSW in 2015. .......................... 63
Table 3.3 Mean ± SD values of parameters for each female population are shown
in micrometres (μm). P-value of the one-way ANOVA (at 0.95
confidence intervals) is presented which showed a significance
difference for the whole length (WL), whole width (WW) and tubular
xv
projection length (TPL) parameters. Statistically significant codes: *P
< 0.05, **P < 0.01. ....................................................................................... 64
Table 3.4 Mean ± SD values of parameters for each male population are shown
in micrometres (μm). P-value of the one-way ANOVA (at 0.95
confidence intervals) is presented which showed a significance
difference for the phallus length (PL) parameter. Statistically
significant codes: ***P < 0.001. .................................................................. 66
Table 3.5 Mean ± SD values of parameters for each female population are shown
in micrometres (μm). One-way ANOVA (at 0.95 confidence intervals)
is presented which showed a significance difference for the whole
length (WL) and upper part length (UPL) parameters. Statistically
significant codes: *P < 0.05. ........................................................................ 68
Table 3.6 Mean ± SD values of parameters for each female P. australiana
population are shown in micrometres (μm). One-way ANOVA (at 0.95
confidence intervals) and Kruskal Wallis test are presented which
showed a significance difference for the whole width (WW) and tubular
projection length (TPL) parameters. Statistically significant codes: *P
< 0.05. .......................................................................................................... 70
Table 3.7 Mean ± SD values of parameters for each male P. xylostella
population are shown in micrometres (μm). One-way ANOVA (at 0.95
confidence intervals) is presented which showed a slight difference for
the phallus length (PL) parameter. Statistically significant codes: ‘.’0.1.
...................................................................................................................... 72
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Table 3.8 Mean ± SD values of parameters for each male P. australiana
population are shown in micrometres (μm). One-way ANOVA (at 0.95
confidence intervals) is presented which showed a significance
difference for the whole length (WL), valva length (VL) and phallus
length (PL) parameters. Statistically significant codes: *P < 0.05, **P
< 0.01,***P < 0.001. .................................................................................... 74
xvii
List of Abbreviations
ANOVA analysis of variance
BLAST basic local alignment search tool
bp base pairs
DBM diamondback moth
DDT dichlorodiphenyltrichloroethane
DNA deoxyribonucleic acid
MEGA molecular evolutionary genetics analysis
MUSCLE multiple sequence comparison by log-expectation
PCR polymerase chain reaction
RAxML randomized axelerated maximum likelihood
SD standard deviation
μm micrometre
xviii
Statement of Original Authorship
The work contained in this thesis has not been previously submitted to meet
requirements for an award at this or any other higher education institution. To the best
of my knowledge and belief, the thesis contains no material previously published or
written by another person except where due reference is made.
Signature:
Date: February 2018
QUT Verified Signature
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Acknowledgements
I am thankful to all the people who have encouraged, helped and contributed along the
way to accomplish my goals and completing this thesis.
My biggest thanks is to my husband for the immense encouragement, love and
dedication given during my study - starting from moving to Australia with me; to
staying by my side in all the hardest moments of my candidature including health
conditions I had to resolve along the way; helping in my travelling and field
collections; not complaining when I worked in the lab till late at night and during the
weekends. You are my strength and I also appreciate your patience during the time of
my thesis writing.
My sincere and heartfelt thank you goes to my parents with whom my achievements
would have not been possible without them by my side and their unconditional love,
immense support, encouragement and believing in me. Love to my sister who has
always been my biggest supporter especially throughout the time of this study and
thanks to my brother-in-law for his kind support.
Many thanks go to my principal supervisor associate professor Caroline Hauxwell for
her immense guidance, time, contribution, editing and advice given to me to undergo
this study and towards the completion of my study and thesis writing. Under her
guidance I have learned a lot about arranging, planning and accomplishing tasks and
especially in conducting field work and going to new places in Australia, especially at
times when driving by myself along the way. I discovered many beautiful places in
Australia as an international student.
A big thanks to my associate supervisor Dr Susan Fuller for her immense help,
encouragement and dedication of her time in editing my thesis. A sincere thank you
for my external supervisor, Associate Professor Stephen Cameron for his guidance and
help, initially as my associate supervisor till he moved to the US. Thanks to my former
supervisor Dr Mark Schutze for his help and advice.
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I also thank Professor Acram Taji for her timely and immense guidance given from
the beginning of my candidature whenever I sought her advice. My gratitude to Dr
Tanya Scharaschkin for her help in answering the phylogenetic problems I had as a
new researcher in that area and also her help in the identification of plant materials I
collected during my field collections. My sincere thanks to Dr Mattew Krotch for his
help and guidance given to me whenever I needed to understand or solve issues I had
in my study.
It was a great experience in meeting and connecting to many people in order to find
fields to conduct light trapping and collect larvae and also to gather information needed
and samples to support my study. I was given support from many lovely people who
arranged, and assisted me in field collections in Sydney, Tasmania and Brisbane. For
that thank you to Andy Ryland, Jason Lynch, Lionel Hill, Catherine Byrne, Lara
Senior, Gavin Berry and Achinda. For allowing me to conduct the field collections in
their farms thank you to Matt, Eddie, Franco, David, Wade and Mulgowie farming
company. For providing me with valuable information, document arrangements and
sending samples from New Zealand I extend my appreciation to John Dugdale,
Graham Walker, Philippa Stevens and others who helped in the collections and other
arrangements. For providing information I thank Ted Edwards and Christian Mille. I
wish to appreciate the help given by Desley Tree in showing me how to conduct slide
preparations.
I am thankful to the QUT EEBS laboratory and technical staff for their immense
support in providing necessary equipment and support during my laboratory and field
work. For that thank you to Amy Carmichael, Anne-Marie McKinnon, Karina Pyle,
Mark Crase, and Vincent Chand. My gratitude to QUT members; EEBS research
student support team finance officers and research student centre for the help given to
me during my candidature. My extended appreciation to Karyn, Sophie and Christian
from the QUT Academic Language and Learning Services for their guidance and kind
support in reading and correcting drafts of my thesis. My research group members and
peers in the writing circle sessions helped me develop my writing skills. Thank you to
the QUT International Student Services and disability center for providing me the
support and guidance I needed at the time. I also thank QUT EEBS school and faculty
members for any help given to me during my candidature.
xxi
Thank you for all the members in the invertebrate microbiology group especially
Robert and Andrew for assisting me in the field work, molecular and DBM colony
work. Special thanks to Melodina Fabillo for her great help, guidance and patience in
teaching and showing me phylogenetic analyses methods. Similarly, I thank Purnika,
Joshua and Thita for their great help to tackle the problems in R statistics and
interpretation of the results. Thanks for the members in the fruit fly research group
who have helped me in various ways during my study. Friends whom I have exchanged
life experiences and research experiences were valuable in keeping me going through
my study. For that thanks to Purnika, Thita, Lixin, Rak, Melody, Sarah, Noor, Aisha,
Naimul, Joshua, Savindi, Sasha, Hernan, Christi, Karma and other friends outside
QUT.
Finally, I express my gratitude to the Lion Center for providing the living stipend
support during my study and QUT for the opportunity to pursue my studies and for
providing me the tuition fee waiver.
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Chapter 1: General Introduction and
Literature Review
1.1 Background
The Lepidoptera (butterflies and moths) are one of the largest and most widespread
insect orders, yet most species are awaiting formal description (Zhang, 2011). The
superfamily Yponomeutoidae (clade Ditrysia) contain around 1800 species worldwide
and include notable pest species, including the leek moth (Acrolepiopsis assectella:
Glyphipterigidae), small ermine moths (Yponomeuta spp.) and the diamondback moth
(Plutella xylostella L. Plutellidae) (Heppner, 1998; Zhang, 2011). Around 58 species
are known in the genus Plutella (Encyclopedia-of-Life; Robinson & Sattler, 2001).
The diamondback moth (DBM), P xylostella is the most destructive insect pest of
Brassica crops such as cabbage, kale, broccoli, and cauliflower, with global damage
and control costs estimated to be US$ 4-5 billion annually (Furlong et al., 2013).
Diamondback moths have developed resistance to most insecticides (Atumurirava et
al., 2011; Sun et al., 1986; Zhou et al., 2011) and were reported as the first species to
develop resistance to some toxins of Bacillus thuringiensis (Tabashnik et al., 1987;
Talekar & Shelton, 1993). Natural enemies including parasitoids, arthropod predators,
viruses, microsporidia, pathogenic fungi and bacteria have been explored as biological
controls (Furlong et al., 2013). Classical biological control introduces and establishes
natural enemies where exotic pests have been introduced but without their natural
enemies (Waterhouse & Sands, 2001). These biological controls could potentially
cause damage to native species, and introduction of biological controls into Australia
requires intensive and extensive non-target testing before release under the Biosecurity
act (2015).
Since first reported in Australia (Tryon, 1889), P. xylostella has become common and
widely distributed, and is resistant to synthetic pyrethroid insecticides in all Australian
states (AgricultureVictoria, 1996; Endersby et al., 2011). The release of biological
2
controls against the exotic P. xylostella in Australia has been more challenging with
the description of a new, closely related taxon, Plutella australiana, by Landry and
Hebert (2013), proposed this as a morphologically-cryptic Australian native taxon.
This proposed new taxon was described based on 8.6% sequence divergence in the
barcode region of the mitochondrial cytochrome oxidase 1 (CO1) gene and the
differences in the morphology of genitalia in both females and males.
The distribution of the new taxon was described as southern and eastern Australia but
was not found in Tasmania. Further sampling was required to confirm the presence or
absence of P. australiana in other parts of Australia. The description was based only
on adults from light traps, with no larval collections, and so host plant use and possible
pest status of the new taxon were unknown. The only subsequent host record for P.
australiana was on Lincoln weed from South Australia in 2015 (Perry et al., 2015).
Consequently, the species status, host plants, pest status and wider distribution of the
newly described P. australiana remains largely unknown, and this has created
uncertainty for future biological control applications, pest management practices and
market access.
1.1.1 Research problem and aims
The main aim of this thesis is to clarify the differentiation of P. australiana from P.
xylostella and to increase knowledge of its distribution and host plants. CO1 barcode
sequence data were used in the identification of both taxa, including phylogenetic
analyses (Maximum likelihood and Bayesian inference). Morphological features of
female and male genitalia were examined and measured to identify reliable diagnostic
features. Distribution of P. australiana at the regional scale was evaluated from field
collections, including individuals from New Zealand. Collections of larvae from both
crops and wild brassicas were conducted to identify host plants and contribute to
knowledge of the possible pest status of P. australiana. These examinations contribute
to the better understanding of possible risks to Australian brassica production, and to
inform potential pest management strategies.
3
The following specific research aims were identified for this study;
1) To clarify the identity of the two taxa based on CO1 barcode, with an increased
sample size from the original published study.
2) To identify which morphological features can be used to differentiate the two
taxa by conducting a detailed morphological comparison of the adult genitalia.
3) To add to the current knowledge of distribution by examination of samples
from the East Coast of Australia, Tasmania and New Zealand.
4) To collect larvae to identify the host plants of P. australiana and contribute to
understanding of the possible pest status of the new taxon.
5) To consider the implications for management of Plutella spp. in Australia and
to inform management strategies.
The outcomes of this thesis have been to broaden the knowledge of the new taxon, P.
australiana including its potential threat to Australian brassica production, and to
identify morphological features that can be used in order to differentiate P. xylostella
and P. australiana. The results contribute to the better understanding of possible risks
of the new taxon to Australian brassica production, and to inform potential pest
management strategies.
1.1.2 Thesis outline
This thesis has four chapters;
Chapter 1 (this chapter): Contains the background to the thesis, research questions
and aims, the thesis outline and a literature review.
Chapter 2: This chapter addresses the first three aims of this study. It includes
molecular (CO1 barcode and phylogeny), identification and measurement of key
features of the genitalia of both Plutella taxa in Australia and New Zealand specimens.
This chapter focusses on the description of adult specimens collected from light traps.
Chapter 3: This chapter addresses the fourth and fifth aims of this study. It focuses on
the field collected larvae from brassica crops and weeds from different locations in
4
Australia including Tasmania and New Zealand. It includes molecular (CO1 barcode)
and measurement of morphological features of adults reared from larvae collected in
the field with a comparison with the adult individuals included in chapter 2.
Identification of P. australiana host plants, pest status of the new taxon and its
implication for pest management are included.
Chapter 4: Provides the general discussion, combining the results obtained in chapter
2 and 3 of this study. Limitations and recommendation for future works are included.
1.2 Literature Review
This literature review details the life history, distribution, host plants, pest management
and taxonomy with a special reference to the Australian Plutella taxa. This review
identifies the gaps which need to be addressed leading to the main research problem
of the current study.
1.2.1 Life history
The life cycle of Plutella xylostella, the diamondback moth (DBM) ranges from 14 to
50 days depending on temperature (Waterhouse & Sands, 2001). Adults emerge in
early summer, laying eggs on all parts of the brassica plant but mainly on the upper
surface of the leaves. After hatching, there are four larval instars followed by pupation
in open cocoons. Many overlapping generations can be found in the course of a year
during a single brassica vegetable or canola crop cultivation, with all life stages of the
DBM are present in the crop at the same time, and they complete multiple generations
each year (Furlong et al., 2008; Talekar & Shelton, 1993).
5
Eggs
Figure 1.1 Diamondback moth eggs.
The eggs are pale yellow, approximately 0.5mm in length and laid either singly or as
clusters on all parts of the plant (Figure 1.1). The eggs hatch in 4 to 8 days depending
on temperature (D. Harcourt, 1957).
Larvae
Figure 1.2 Fourth instar larvae of diamondback moth.
First instar larvae are approximately 1-2mm in length whereas the fourth instars
(Figure 1.2) are approximately 12mm in length and pale green in colour. The head
capsule is pale brown and the body, green and segmented. Whenever the host plant is
6
disturbed the larvae retreat backwards and drop down from the host plant using a silky
tread.
Although the first instar larvae are leaf miners and leave pale, tunnel-like patches on
the leaves, later larval stages are surface feeders chewing out small irregular holes.
The larva completes development in 10 to 30 days depending on the temperature and
it is this time that is the problematic stage in terms of causing damage to the Brassica
crops (Figure 1.3).
Figure 1.3 Swede crops in a community garden damaged by DBM larvae.
Pupae
Figure 1.4 Diamondback moth pupa on a red cabbage leaf.
7
Pupae can be seen in white silky cocoons generally fastened to the plant parts on the
host plant (Figure 1.4) (CABI, 2016). Pupae are approximately 10mm in length and
turn green to brown before adult emergence. Emerging will take 4 to 10 days
depending on the temperature. If the cocoon is damaged or removed the pupae survival
is very low.
Adults
Figure 1.5 A) Lateral view of a DBM adult and B) dorsal view of a DBM showing the
diamond pattern of a DBM adult.
Adults are 10-12 mm in size and greyish brown in colour with a distinct beige band
pattern along the inner side of their forewings which when viewed dorsally, appear as
A
B
8
three or four diamond shaped areas when at rest, giving the moth its common name
(Figure 1.5 A, B). The diamond shapes are more distinct in males than those of
females.
Adults become active just before dusk and stay inactive during the day unless
disturbed. Mating begins at dusk on the day of emergence (CABI, 2016). Almost 95%
of females begin laying eggs on the day of emergence (CABI, 2016); a process that
lasts 10 days with the number of eggs laid per female ranging from 159 (D. Harcourt,
1957) to 288 (Ooi & Kelderman, 1979). Sivapragasam and Heong (1984) reported that
temperature has a significant effect on adult survival, oviposition rates and generation
(CABI, 2016). Adults do not fly long distances within a crop, they only fly only around
13 m - 35 m within a crop field (Mo et al., 2003). They migrate long distances carried
by the winds for about 1500 km at 400 km - 500 km per night (Chapman et al., 2002).
1.2.2 Distribution
Global distribution
The origin of P. xylostella (syn. P. maculipennis) is not clear. It may have originated
in the Mediterranean (Hardy, 1938), Africa (Kfir, 1998) or Asia (Liu et al., 2000),
based on the number of endemic brassicas, molecular data and the diversity of
parasitoid species identified from these regions. A recent study favoured an African or
possibly Asian origin over a European origin based on high haplotype diversity,
particularly in the African population, and mismatch analysis supported a more recent
spread into North America, Australia and New Zealand (Juric et al., 2017).
Plutella xylostella now has a cosmopolitan distribution (Figure 1.6, Table 1.1) and can
be found wherever suitable host plants are found (Shelton, 2001). P. xylostella is
known to move over long distances in air currents (CABI, 2016), with distances of
1500 km at 400 km - 500 km per night (Chapman et al., 2002). Moreover, the
movement of insecticide resistant individuals between countries can have serious
implications for its pest control (CABI, 2016).
9
Figure 1.6 World distribution of P. xylostella, the diamondback moth. Blue =
widespread, Black = present. Although some countries are not marked, DBM may be
present (CABI, 2016).
Table 1.1 Worldwide distribution of Plutella species. (Information taken from
(Robinson & Sattler, 2001).
Number Plutella species Countries of presence
1 P. xylostella (P. meculipennis) Cosmopolitan (except extreme
alpine areas and Antarctic region)
2,3 P. antiphona, P. psammochroa New Zealand
4 , 5, 6 P. notabilis, P. omissa , P. armoraciae USA (Washington)
7 P. geniatella Switzerland
8, 9 P. polaris, P. haasi Norway
10 P. mariae Russia
11, 12, 13 P. capparidis, P. noholio, P. kahakaha Hawaii
14 P. porectella Europe, North America and South
America
15 P. balanopis South Africa
16, 17 P. deltodoma, P. diluta Chile
18 P. canaella Italy
19, 20 P. acrodelta, P. nephelaegis Argentina
21 P. rectivittella Colombia
22 P. formicatella Seychelles
10
Australian distribution
Plutella xylostella was first reported in Australia in1882 in Queensland (Tryon, 1889)
and is now common and widely distributed within Australia (French, 1893; Lea, 1895;
Nielsen et al., 1996; Thompson & Moore, 1895). The introduction and distribution of
P. xylostella in Australia was thought to occur from the imported cabbage via
passenger steamers (Tryon, 1889). Plutella xylostella is now a significant economic
pest of brassicas, reported to attack up to 136,000 hectares of brassica vegetable crops
(Shelton, 2001), and in canola (cultivars of Brassica rapa L. and B. napus L.), which
is widely grown in South-east Australia and Western Australia. Australia is the world’s
second largest exporter of canola (RIRDC, 2005-06) and canola is the third largest
winter crop in Australia, thus management of P. xylostella is of considerable
importance. Moreover, resistance to synthetic pyrethroid insecticides has been
detected in populations of DBM in all Australian states (AgricultureVictoria, 1996;
Endersby et al., 2011). This rapid and widespread evolution of resistance supports the
need for an effective integrated resistance management strategy, as well as an
integrated pest management strategy.
1.2.3 Host plants
Plutella xylostella is known as one of the most destructive insect pests of
Brassicaceous crops worldwide (Ahuja et al., 2010; Furlong et al., 2013). The
Brassicaceae family contains 380 genera and over 3000 species of cultivated and wild
plants (Heywood, 1993; Warwick et al., 2003). This family contains economically
important crops such as cole crops (cabbage, cauliflower), oilseeds (canola, mustard)
and root vegetables (radish, turnip) (Muhammad et al., 2005).
Wild cruciferous plants act as a bridging host for P. xylostella between crops, and a
few studies have examined the presence of P. xylostella on wild cruciferous and non-
cruciferous host plants (Barker et al., 2001; Begum et al., 1996; Furlong et al., 2013;
Löhr & Rossbach, 2001; Robinson & Sattler, 2001; Sarfraz et al., 2006; Sarfraz et al.,
2011) (Table 1.2). Wild plants affect the developmental and reproductive parameters
in P. xylostella (Begum et al., 1996; Harcourt, 1986; Muhamad et al., 1994; Sarfraz,
Dosdall, & Keddie, 2010; Shelton & Nault, 2004; Talekar & Shelton, 1993; Yamada,
11
1983), and P. xylostella grown on wild crucifers were found to fly for longer (Begum
et al., 1996). For these reasons, (Sarfraz et al., 2011) suggested that monitoring and
controlling P. xylostella in weed species even before crops are cultivated may help
controlling this pest in the fields.
Brassicas contain glucosinolates and sulphur-containing secondary plant compounds
which stimulate DBM feeding and oviposition (Furlong et al., 2013; Marazzi et al.,
2004). Similarly, plant volatiles, waxes, host plant nitrogen content, leaf morphology
and leaf colour, or a combination of these factors, have been reported to stimulate the
oviposition and feeding of P. xylostella (Sarfraz, Dosdall, & Keddie, 2010; Sarfraz et
al., 2006; Stoner, 1990). Moreover, soil fertility levels are known to affect the
oviposition and herbivory in P. xylostella (Sarfraz et al., 2006) similar to studies
showing that the level of nutrients in the leaves affect parameters such as development
and survival in other insects such as aphids and leaf mining flies (Björkman, 2000;
Bruyn et al., 2002).
Plants such as B. rapa and B. napus were found to be preferred host plants among P.
xylostella from early years until now (Brown et al., 1999; Clarke, 1971; Endersby et
al., 2004; Perry et al., 2015; Talekar & Shelton, 1993). Those host plants including
cabbage are known to contain green leaf volatiles, isothiocyanates, nitriles, dimethyl
trisulfide, and terpenes which may be the reason to be more attractive to P. xylostella
(Girling et al., 2011; Kugimiya et al., 2010).
12
Table 1.2 Known host plants of P. xylostella (Information taken from Sarfraz et al. (2006)).
Species/cultivar Common name(s) Selected reference(s)
(i) Cultivated cruciferous host plants of
P. xylostella
Brassica napus L. Canola, Canadian turnip, rutabaga
Idris and Grafius (1996), and Brown et al.
(1999)
Brassica rapa L. (=B. compestris (L.))
Turnip rape, turnip green, field mustard,
canola
Brown et al. (1999), and Ulmer et al.
(2002) Brassica carinata L. Ethiopian mustard Ayalew et al. (2004) Brassica juncea (L.) Indian mustard, brown mustard Bodnaryk (1997), and Brown et al. (1999) Brassica napa L. Turnip Abro et al. (1994) Brassica nigra (L.) Black mustard Idris and Grafius (1996)
Brassica oleracea L. var. acephala Collard, flowering kale
Idris and Grafius (1996), and Badenes-
Perez et al. (2004) Brassica oleracea L. var. alboglabra Kale Talekar and Shelton (1993)
Brassica oleracea L. var. botrytis Cauliflower
Idris and Grafius (1996), and Reddy et al.
(2004)
Brassica oleracea L. var. capitate Cabbage
Abro et al. (1994), and Idris and Grafius
(1996) Brassica oleracea L. var. gemmifera Brussels sprouts Talekar and Shelton (1993) Brassica oleracea L. var. gongylodes Kohlrabi Reddy et al. (2004)
Brassica oleracea L. var. italic Broccoli
Idris and Grafius (1996), and Reddy et al.
(2004) Brassica rapa L. var. pakchoi Pak choi Talekar and Shelton (1993)
Brassica rapa L. var. pekinensis Chinese cabbage
Talekar et al. (1994), and Liu and Jiang
(2003) Raphanus sativus L. Radish, bier radish Abro et al. (1994) Sinapis alba L. (=Brassica hirta Moench) White mustard, yellow mustard Bodnaryk (1997), and Brown et al. (1999)
13
Table 1.2 continued
(ii) Wild cruciferous host plants of
P. xylostella
Species
Arabidopsis thaliana (L.) Heynh Thalecress, mouse-earcress
Ratzka et al. (2002)s, and Barker et al.
(2004)
Barbarea vulgaris (L.) R. Br. Yellow rocket, rocketcress
Idris and Grafius (1996), Shelton and
Nault (2004), and Badenes-Perez et al.
(2004) Berteroa incana L. DC Hoary alyssum Idris and Grafius (1996) Capsella bursa-pastoris (L.) Shepherd's purse, mother's-heart Idris and Grafius (1996) Cardamine flexuosa With. Flexuous bittercress Muhamad et al. (1994) Descurainia sophia (L.) Flixweed Talekar and Shelton (1993)
Erysimum cheiranthoides L. Wormseed mustard, treacle mustard
Renwick and Radke (1990), and Idris and
Grafius (1996) Lepidium campestre (L.) R. Br. Field pepperweed Idris and Grafius (1996)
Lepidium virginicum L. Virginia pepperweed, peppergrass
Muhamad et al. (1994), and Begum et al.
(1996) Raphanus raphanistrum L. Wild radish, wild rape, wild turnip Idris and Grafius (1996)
Rorippa indica (L.) Hiern Indian marshcress
Muhamad et al. (1994), and Begum et al.
(1996) Rorippa islandica (Oeder) Barbàs Marsh yellowcress Muhamad et al. (1994) Sinapis arvensis L. (=Brassica kaber (DC)
Wheeler) Wild mustard, crunchweed Idris and Grafius (1996) Sisymbrium altissimum L. Tumbling mustard, tall hedge mustard Talekar and Shelton (1993) Thlaspi arvense L. Stinkweed, pennycress, Frenchweed Idris and Grafius (1996)
(iii) Non-cruciferous plants on which
P. xylostella is known to survive/develop
Species Family Glucosinolates
Common
name Reference(s)
Tropaeolum majus L. Tropaeolaceae Yes Nasturtium Renwick and Radke (1990)
Cleome species Capparidaceae Yes Spider plant M. Sarfraz et al. (2005)
Pisum sativum L. Fabaceae No Peas
P. Gupta and Thorsteinson (1960),
Lohr (2001), and Löhr and Gathu
(2002)
Hibiscus esculentis L. Malvaceae No Okra J. Gupta (1971)
14
1.2.4 Pest management
The global standard practice the use of synthetic chemical insecticides to control P.
xylostella, but as a result of heavy and repeated use, the P. xylostella has become
resistant to almost all insecticides (APRD, 2012; Furlong et al., 2013; Muhammad et
al., 2005; Ridland & Endersby, 2011). However, P. xylostella rapidly evolves
resistance to chemical insecticides, and was the first agricultural pest to develop field
resistance to Bacillus thuringiensis (Bt) toxins (Ankersmit, 1953; Johnson, 1953;
Talekar & Shelton, 1993), to which resistance is now widespread. P. xylostella was the
second-ranked in exhibiting resistance to a high number of insecticide in the Arthropod
Pesticide Resistance Database (APRD, 2012). The APRD listed 95 compounds for
which P. xylostella has been reported to show resistance.
The rapid build-up of insecticide resistance has been seen mainly in tropical countries,
where the over use of insecticides aims to control up to 20 P. xylostella generations in
brassica crops per year (CABI, 2016). Although farmers have used insecticides for
over 30 years in P. xylostella control, resistance to existing insecticides and the lack of
new insecticides has led researchers to look for alternative control measures (CABI,
2016). Moreover, the use of non-selective insecticides leads to the destruction of
natural enemies (Furlong et al., 2004). In response, integrated pest management (IPM)
strategies have been developed. These strategies include a combination of chemical,
biological and cultural control methods with an emphasis on maintaining natural
enemies (Sarfraz et al., 2006).
The basic aim of a sustainable IPM program for P. xylostella is the introduction and
conservation of natural enemies, which play a major role in limiting P. xylostella
population growth (CABI, 2016). The establishment of Diadegma semiclausum
(Hymenoptera: Ichneumonidae) and the use of the bacterium B. thuringiensis (Bt) and
its toxins in the highlands in Indonesia, Malaysia, Taiwan and Philippines (Ooi &
Kelderman, 1979; Poelking, 1992; Sastrosiswojo & Sastrodihardjo, 1986; Talekar et
al., 1992) are examples of successful, coordinated control of P. xylostella.
Additionally, a wide range of natural enemies including parasitoids, arthropods
fpredators, viruses, microsporidia, pathogenic fungi and bacteria have been known to
15
attack P. xylostella, though only few of them with significant results on field usage.
(Furlong et al., 2013; Sarfraz et al., 2005) (Figure 1.7). Research over the past two
decades has focused on manipulation and use of these organisms, particularly in
classical biological control programs (see below) (Furlong et al., 2013). The use of
entomopathogens, such as bacteria, virus, fungi and nematodes to control P. xylostella
has been reviewed by Wilding (1986) and more recently by Cherry et al. (2002), and
identified a number of pathogens infecting P. xylostella.
Figure 1.7 Major biological control agents of P. xylostella (Sarfraz et al., 2005).
Classical biological control
Classical biological control is the practice of importing and releasing natural enemies,
to control an introduced pest (Hoffmann & Frodsham, 1993). It is used when an insect
pest is introduced into another geographic area without its natural enemies
(Waterhouse & Sands, 2001). The success of this application requires the correct
identification of the natural enemy and its host strain (Sarfraz et al., 2005). Regions
around the world where the majority of agriculture is based on introduced crops, in
particular Australia, are notable for the high proportion of their exotic arthropod pests
and the success of classical biological control (Waterhouse & Sands, 2001).
Australia’s first involvement with classical biological control was in 1888 and 1891
when A. Koebele visited Australia to find sources of parasitoids and predators of insect
16
pests that had become established in California and Hawaii (Waterhouse & Sands,
2001). Three parasitoids, D. semiclausum, Cotesia plutellae and D. collaris, have been
introduced and then established successfully throughout Australia for control of P.
xylostella (Sarfraz et al., 2005).
The first step in classical biological control is to determine the origin and identity of
the introduced pest and then to collect appropriate natural enemies associated with it
or with closely-related species. The natural enemy is fully described and then subjected
to a rigorous quarantine process, to ensure that no unwanted organisms are introduced.
It is then reared in large numbers and assessed for risk, with particular attention to host
specificity (Hoffmann & Frodsham, 1993). The impact on non-target and native
species in Australia is of particular importance in introduction of biological controls
under the biosecurity act 2015, including in the introduction and registration of
potential biopesticides based on microorganisms (Hauxwell et al., 2010).
Studies following release are conducted to determine if the natural enemy has
successfully established at the site of release, and to assess the long-term benefit of its
presence (Hoffmann & Frodsham, 1993). There are also situations in which biological
control species have been introduced into quarantine but not liberated due to reasons
such as poor breeding, lack of efficacy against the target host and/or lack of specificity
(Waterhouse & Sands, 2001). When classical biological control is used, it is a
requirement to ensure that only the target pest species is affected by the introduction
of the natural enemy (Waterhouse & Sands, 2001).
Baculoviruses are invertebrate-specific virus pathogens that are used as efficient
biopesticides (Asser-Kaiser et al., 2007; Hunter-Fujita et al., 1998). They are widely
used in Australia due to their efficacy, relative stability (they can withstand being
sprayed using standard farm equipment), high host specificity and lack of non-target
infection in invertebrates (Hauxwell et al., 2010).
Plutella xylostella granulovirus (PlxyGV) of P. xylostella was first reported in Japan
(Asayama & Osaki, 1969). Since then several scientists have reported the potential of
granulovirus for use as a biological control agent for P. xylostella in Taiwan, India,
Kenya, China and South Africa (Abdulkadir et al., 2013; Bin Abdul Kadir et al., 1999;
17
Grzywacz et al., 2001; Kadir, 1986; Rabindra et al., 1997). As Australia have not used
this biological control yet, PlxyGV is being investigated as a potential biological control
by QUT (Spence et al., 2016).
1.2.5 Taxonomy
Reliable systematics is necessary to understand taxa of pest species, the traits important
to their management and host range, and to biological control (Cho et al., 2008). The
morphology of genitalia was identified as an important and significant source of
phylogenetically informative morphological characters (Beutel & Kristensen, 2012).
Description of morphology of insect genitalia is now recognised as an essential
standard for species-level taxonomy and inference of phylogenetic relationships
(Schmidt, 2012).
The early classification of P. xylostella was described using morphological
characteristics such as genitalia, wing venation, larval and pupal morphology, pupal
and external morphology of adults (Clarke, 1971; Dugdale, 1973; Moriuti, 1986).
Later, studies described more detailed morphological characters including character
mapping (Baraniak, 2007; Robinson & Sattler, 2001) and one study examined the
measurements of external morphological features of Indian P. xylostella populations
(Chacko & Narayanasamy, 2004). However variation and plasticity in the
morphological characters make it difficult to rely solely on these features and can result
in an unsatisfactory phylogenetic resolution (Beutel & Kristensen, 2012). More
recently, molecular sequence and integration of both molecular and morphology have
been used to resolve the relationship of Plutella populations around the world. Similar
approaches have been used combining both morphology and molecular data to
describe wood white butterfly (Shtinkov et al., 2016), fruit flies (Schutze et al., 2014),
spruce budworm (Lumley & Sperling, 2010) and Niganda moths (Pellinen &
Wahlberg, 2015) to clarify their species status.
18
Molecular marker selection
Molecular systematics is used to infer the phylogenetic relationships among organisms
using molecular data (DNA and RNA) to resolve competing hypotheses of species
relationships and the placement of taxa whose relationships are known to be
problematic (Judd et al., 1999). Caterino et al. (2000) stated that with sufficient
specimens sampling the relationship among subspecies, species and species groups can
be resolved with significant new insights. Similarly, Springer et al. (2001) pointed out
that the value of mitochondrial sequences in phylogenetic analyses is further enhanced
if they are collected in conjunction with nuclear sequences, because mitochondrial
sequences provide an independent estimate of phylogenetic relationships that can be
compared with estimates based on nuclear sequences.
Selecting a suitable marker is important for the accuracy in resolving species status
(Rach et al., 2017). Since the introduction in 2003, the mitochondrial CO1 barcode
gene region (cytochrome c oxidase 1) has become the most widely used molecular
marker among most of the animal phyla (Kress et al., 2015).
Higher mutation rates and more rapid sorting of variation usually results in divergence
of mtDNA sequences among species and a comparatively small variance within
species, and the use of CO1 barcode relies on the principle that genetic variation within
species is smaller than that between species (Lukhtanov et al., 2009). The estimated
rate of divergence at the CO1 locus in insects was reported as 2% per million years
(Brower, 1994; Juan et al., 1995).
COI has been widely used for DNA barcoding of insects (Hebert et al., 2003). DNA
barcoding was proposed as an inexpensive and effective method to identify living
organisms using an approximately 658 bp of mitochondrial DNA (mtDNA) as a
barcode. Several insect studies have used the mitochondrial cytochrome oxidase 1
marker to study taxonomy, population and evolution (Chang et al., 1997; Landry et al.,
1999; Miller et al., 2015; Schmidt et al., 2015). It has been also reported that CO1
barcode based delimitation of species is a good start for taxonomic processes (Hebert
et al., 2004).
19
Mitochondrial DNA (mtDNA) markers are considered to be more sensitive than
nuclear markers for population genetic studies (Loxdale & Lushai, 1998).
Mitochondrial genes in general have several advantages such as the ability to amplify
easily due to the high copy numbers per cell and their haploid character. They also
evolve much faster than the coding regions of nuclear genes because mitochondria lack
a proofreading mechanism (Rach et al., 2017). However, reliance on mitochondrian
sequences and in particular on the COI barcode alone has been questioned.
Mitochondrial DNA alone cannot provide good results in defining new species because of
factors such as reduced effective population size and introgression, maternal inheritance,
recombination, inconsistent mutation rate, heteroplasmy and compounding evolutionary
processes, and COI sequence data should be used in conjunction with nuclear DNA,
morphology, or ecology (Rubinoff et al., 2006; Rubinoff & Holland, 2005).
Integrative Taxonomy
Species definition has now become a highly-debated topic in modern systematics
where new molecular species delimitation methods are being developed (De Queiroz,
2007; Morando et al., 2003; Pons et al., 2006; Puorto et al., 2001; Templeton, 2001;
Wiens & Penkrot, 2002). Integrative taxonomy is the use of many different sources of
data such as molecular, morphological, behavioural and ecological data to delimit
species in a reliable manner (Dayrat, 2005; Padial et al., 2010). Although the selected
species concept will influence the choice, analysis and interpretation of data, to obtain
a good outcome at least three sources of data or ‘disciplines’ must be used for more
rigorous species delimitation hypotheses (Schlick-Steiner et al., 2010). A number of
studies have been conducted under the integrative taxonomic framework to resolve
species status such as the nematode species, Malagasy tree frogs, spruce budworm and
harvestmen, all with promising results (Fonseca et al., 2008; Glaw et al., 2010; Lumley
& Sperling, 2010; Wachter et al., 2015).
1.2.6 A new Plutella taxon in Australia
The Australian Plutellidae contain 26 species in 8 genera, of which several genera,
such as Diathryptica Meyrick and Tritymba Lower, are endemic (Nielsen et al., 1996).
20
The genus Plutella was thought to be represented in Australia by a single introduced
taxon, P. xylostella, until the description of two genetically divergent lineages of this
taxon by Landry and Hebert (2013). Their initial study objective was to identify the
Australian lepidopteran fauna using the CO1 barcode, as a result of which they
described a new ‘Australian’ taxon, Plutella australiana, based on 8.6% sequence
divergence in the barcode region of the mitochondrial CO1 gene and differences in the
morphology of genitalia. The collections and analysis of Plutella taxa in the study were
based on light trapped material and museum specimens from the Australian Capital
Territory (ACT), New South Wales (NSW), South Australia (SA) and Queensland
(QLD). On the basis of these samples, P. australiana was reported to be broadly
distributed in the eastern half of Australia (Figure 1.8) (Landry & Hebert, 2013).
However, their study did not include any larval collections, leaving the information on
its host plant and its pest status unknown, and sampling is required to confirm the
presence or absence of P. australiana in other parts of Australia and the wider region.
Figure 1.8 Sites in Australia where specimens of P. xylostella (red) and P. australiana
(blue) have been collected. The circles show the proportion of the two species at each
site. These records only include specimens identified through DNA barcode analysis
(Landry & Hebert, 2013).
21
COI sequence data showed a clear difference between the two Plutella taxa, but P.
australiana cannot be differentiated from P. xylostella using external morphology.
Genitalia morphological characteristics of both females and males between the two
taxa were described, but based on only a few slide preparations (5 P. australiana males
and 2 P. australiana females) (Landry & Hebert, 2013).
A few records had previously suggested some evidence for the possible presence of a
second taxon or variant of P. xylostella in Australia. An allozyme study of P. xylostella
from 14 locations worldwide included specimens from five different locations in
Australia (Adelaide, Brisbane, North Queensland, Melbourne and Sydney) and found
significant differences among the samples from Australia (Pichon et al., 2006).
Similarly, Roux et al. (2007) using inter sample sequence repeat (ISSR) marker
showed genetic differences between P. xylostella populations from Melbourne and
Sydney. In the region, the description of a curved tubular projection similar to that of
P. australiana females was previously described by Dugdale (1973) from a single
female specimen presumed to be P. xylostella collected from New Zealand in
comparison to that of P. antiphona Meyrick 1901 and P. sera Meyrick 1886 (Figure
1.9). This may suggest the presence of P. australiana in New Zealand, but its presence
in the region needs further examination.
22
Figure 1.9 Picture from Dugdale (1973) showing the curved tubular projection (12) in
a female genitalia from a diamondback moth specimen collected from New Zealand.
As an overall summary, the description of P. australiana in Australia has left many
questions to be answered such as its species status, are the morphological features
reliable when applying it to a wide range of specimens of the two taxa, distribution in
other part of Australia and other countries especially in New Zealand, host plants and
pest status and in particular the implications for the Australian pest management. The
introduction of the new taxon requires additional testing for proposed biological
controls, including biopesticides.
This thesis aims to broaden the knowledge of the description and distribution of new
taxon, P. australiana, including its potential threat to Australian brassica production,
23
and to identify morphological features that can be used to differentiate P. xylostella
and P. australiana. The results contribute to the better understanding of possible risks
of the new taxon to Australian brassica production, and to inform potential pest
management strategies.
24
25
Chapter 2: Molecular and morphological
examination of Plutella species in
Australia and New Zealand
2.1 Introduction
The diamondback moth (DBM), Plutella xylostella L. (Lepidoptera: Plutellidae), was
first reported as an introduced species to Australia in 1882 (Tryon, 1889), and has since
become widespread (French, 1893; Thompson & Moore, 1895) (Figure 2.1).
Figure 2.1 Distribution of diamondback moth in Australia.
Blue = widespread, black = present (CABI, 2016).
In 2013, Landry and Hebert described a new taxon, Plutella australiana, in Australia.
The study examined specimens collected using light traps between 2004-2012 from
the Australian Capital Territory (ACT), New South Wales (NSW), South Australia
(SA) and Queensland (QLD) and pinned museum specimens. The taxon description
was based on an analysis of the mitochondrial cytochrome oxidase 1 (CO1) ‘barcode’
gene sequence (Figure 2.2). The new taxon was described as being broadly distributed
in southern and eastern Australia. Further sampling was required to confirm the
presence or absence of P. australiana in other parts of Australia, specifically
Tasmania, where there was no record of occurrence.
26
Figure 2.2 Neighbor joining tree (nodes collapsed) based on Kimura-2-parameter
distances for the barcode region of the cytochrome c oxidase 1 gene. Specimens are
labelled by the Australian state or by country of origin and bracketed numerals indicate
the number of specimens from each site (taken from Landry and Hebert (2013)).
27
The genital morphology of P. xylostella has been described (Baraniak, 2007; Dugdale,
1973; Landry & Hebert, 2013; Moriuti, 1986; Robinson & Sattler, 2001) but the
description of P. australiana genital morphology is based on only a few specimens
(Landry & Hebert, 2013): five P. australiana males and two P. australiana females.
It has been suggested (Personal communication from John Dugdale, Senior
entomologist, New Zealand) that the morphology of some New Zealand female
specimens based on Landry and Hebert (2013) genitalia descriptions, might indicate
the presence of P. australiana, but this has not been confirmed using CO1 sequence
data. Those specimens were from early 19th century (example: 1907). However, as the
description of P. australiana genitalia were based on few samples there is a need to
further examine and identify the reliable diagnostic genitalia morphological features
using a larger sample number of both taxa.
The described new ‘native’ Australian taxon closely related to P. xylostella, would
potentially be at risk from biological control agents released against the introduced
pest, and thus prevent or delay release or registration of new biological controls that
are essential to an integrated resistance management strategy. Further work is therefore
required to determine the taxonomic status of P. australiana, its geographic
distribution and potential economic importance as a pest. The results using a larger
sample size of both taxa will help to further clarify the identity of the two taxa, to
identify the reliable diagnostic feature that can be used to identify both taxa and to
expand the knowledge of P. australiana distribution in Australia.
This chapter presents the comparison of new adult specimens of P. xylostella and P.
australiana collected from south-eastern Australia (including Tasmania) and from
New Zealand. Mitochondrial CO1 sequence data was used to establish taxon identity
which was then compared with a detailed morphological analysis of the genitalia of
the two Plutella taxa including measurements of the genitalia characteristics to
determine the statistical variance across taxa and populations along with an
examination of morphological characteristics to identify reliable diagnostic features.
28
2.2 Materials and Methods
2.2.1 Sampling
Moths were collected using 40W ranger moth traps (Watkins and Doncaster, UK)
connected to a 12V/15Ah valve regulated lead acid battery (Figure 2.3). New moon
dates were considered when selecting the light trapping dates as moonlight is known
to influence (decrease) monthly moth catch rates (Nowinszky et al., 1979; Williams,
1936). Collections were undertaken in Tasmania and eight different locations in
eastern Australia (Queensland and New South Wales) and conducted between August
to December in 2014 and 2015 and between May to October in 2016. Adult collections
were sorted from light trap samples using an electric pooter (Australian Entomological
Supplies) and transferred into solo cups or take away containers for transportation.
Thirty individuals per site were selected for molecular analysis at each site except
where less than thirty individuals were caught, in which case all the specimens
collected were used.
Specimens were also obtained from ten locations in New Zealand and from a
laboratory reared colony. Samples from New Zealand were received preserved in 40%
ethanol collected between 2005 and 2016 as both adults and larvae. Individuals from
the lab reared colony and from Tuakau (2006) were not included in the analyses as
good quality sequences could not be obtained at the beginning. A summary of the
samples included in the analysis is shown in table 2.1.
29
Figure 2.3 A) Ranger moth light trap in a cabbage field and B) Insects trapped in the
light trap. Moths of Plutella spp. are circled in yellow.
30
Table 2.1 Locations of DBM adult collections and number of individuals taken for
genetic and morphological analyses. Specimens not taken for morphological analyses
are marked as ‘/’ and unknown GPS coordinates are marked as ‘-‘.
Females Males
SAM 201427.34304 °S,
152.90909 °ESamford, QLD 3 0 3
HOB3 201433.98295 °S,
150.63965 °EHobart, Tasmania 16 4 7
SYDT 201433.98295 °S,
150.63965 °ETheresa Park, NSW 8 6 1
SYDW 201434.00338 °S,
150.56716 °EWerombi, NSW 10 10 /
BIR 201527.493653 °S,
153.204733 °EBirkdale, QLD 5 1 4
SYD15T and T 201533.98295 °S,
150.63965 °ETheresa Park, NSW 29 / 10
SYD15W 201534.00338 °S,
150.56716 °EWerombi, NSW 22 9 /
M15 201534.15285°S,
150.55449°EMowbary Park, NSW 13 11 /
G 201627.537202 °S,
152.335257 °EGatton, QLD 5 / /
L 201627.754827 °S,
152.369293 °ELaidley, QLD 4 / /
DBM1 2016 - Nelson, New Zealand 14 5 5
DBM2 2016 -Hobson, Rakaia, New
Zealand3 / /
DBM3 2016 -Hobson, Rakaia, New
Zealand7 / 7
DBM4 2013 - Pukekohe, New Zealand 29 4 5
DBM5 2013 - Levin, New Zealand 10 4 6
DBM6 2014 - Lincoln, New Zealand 3 / /
DBM9 2008 - Southbridge, New Zealand 23 5 5
DBM10 2008 - Chertsey, New Zealand 12 7 1
Sample codeDate
(year)GPS Location
Number of
sequences used in
the phylogenetic
analyses
Number of specimens
used for morphological
identification
31
2.2.2 Molecular analysis
DNA extraction, PCR amplification and sequencing
In total 216 adults were DNA extracted and Sanger sequenced (this sample number
represents only the specimens with more than 75% sequence quality). DNA was
extracted using the ISOLATE II Genomic DNA Kit (Bioline, Australia) from one leg
of each specimen and with an overnight incubation at 56 ˚C. The 658 bp fragment of
mitochondrial CO1 was amplified using primers LepF1 (5’-
ATTCAACCAATCATAAAGATATTGG-3') and LepR1 (5’-
TAAACTTCTGGATGTCCAAAAAATCA-3’) (DeWaard et al., 2007). PCR
amplification was carried out with 10.5 µl 10% Trehalose, 2.625 µl of 5X buffer
(Bioline), 0.125 µl of MyTaq HS Red DNA Taq polymerase (Bioline), 0.25 µl of each
primer, 6 µl of template DNA, 1.25 µl of 25 mM MgCl2 (Bioline), and made up to a
final volume of 25 µl with deionized water (ddH2O). Amplifications were performed
in an Eppendorf Mastercycler® Pro S thermal cycler with an initial denaturing step at
94 ˚C for 2 minutes, followed by 45 cycles at 94 ˚C for 40 seconds, 54 ˚C for 40
seconds, 72 ˚C for 1 minute. PCR products were separated in 1.5% agarose gel using
TBE buffer (40 mM Tris-acetate, 1 mM EDTA) for 50 minutes at 90 volts to confirm
the quality of the PCR product. PCR products were purified using the ISOLATE II
PCR and Gel Kit (Bioline, Australia). Purified PCR product was amplified in a
sequencing reaction containing 2.0 μl of PCR product, 1.0 μl of 3.2 pmol forward
primer, 1 μl of ABI Prism® Big Dye Terminators version 3.1 (Applied Biosystems,
California, USA), 3.5 μl of 5x sequencing dilution buffer adjusted to a total reaction
volume of 20μL with ddH2O. The sequencing cycle protocol involved initial
denaturing at 96 °C for 5 minutes, followed by 30 cycles of 96 °C for 10 seconds, 50
°C for 5 seconds, 60 °C for 4 minutes, before a final hold at 15 °C for 10 minutes. The
purified products were eluted in 20 μl of Elution Buffer C (Bioline, Australia) and then
cleaned using a standard ethanol precipitation protocol prior to sequencing using both
forward and reverse primers, by the QUT Institute for Future Environments-Central
Analytical Research Facility (IFE-CARF) or Macrogen Inc. (South Korea).
Sequencing was performed using an AB 3500 Genetic Analyser in QUT IFE-CARF,
while an automatic sequencer ABI3730XL was used in Macrogen Inc. (South Korea).
Obtained sequence results were used for an initial identification by blasting (BLAST)
against the available sequences in Genbank database using a 100% match.
32
Phylogenetic analyses
Either the forward or reverse sequence with a quality score higher than 75% was used
in the alignment. Low quality sequence ends were trimmed using a modified Mott
algorithm (http://www.unipos.net/download/Geneious7Manual.pdf). Sequences were
aligned using default alignment parameters in MUSCLE within Geneious version
9.0.4 (http://www.geneious.com, Kearse (2012)). Ambiguously aligned blocks were
excluded from the analyses. Further adjustments were made by eye and gaps within
the alignment were treated as missing data.
Two hundred and sixteen (n=216) sequences obtained from adult individuals and 204
sequences obtained from adults reared from field collected larvae mentioned in chapter
3 (section 3.2.2.) were included in the final dataset. Additionally, reference sequences
from Genbank for both P. xylostella and P. australiana (n=12 each, shown with
accession numbers in the phylogenetic tree, see Appendix A) and three outgroups [two
closely related species; Plutella hyperboreella, Plutella porrectella and the closely
related genus Hyperxena scierana were also included in the analysis (see Appendix A
for accession numbers)].
Phylogenetic analyses were conducted using Bayesian and maximum likelihood
methods using the CIPRES Science Gateway (Miller et al., 2010). The substitution
model was set as TIM2+I as found using jModel test 2 (Darriba et al., 2012). The
maximum likelihood (ML) analysis was performed using RAxML 8.2.8 (Stamatakis,
2006). Support values for ML trees were estimated with 1000 bootstrap replicates.
Bayesian analysis was performed using MrBayes 3.2.6 (Ronquist et al., 2012). The
Bayesian analysis was run for 10,000,000 generations with trees sampled every 1000
generations with the default nruns=2 and nchains=4. Trees were visualized using
TreeGraph 2 version 2.13.0 (Stöver & Müller, 2010).
The sequence divergence between the two taxa was quantified using the Kimura 2
parameter in MEGA version 7.0.18 (Kumar et al., 2016).
33
2.2.3 Morphology
Preparation and identification using genitalia morphological features
A subset of both male and female adults (n=120) from three Australian states
(Queensland, New South Wales, Tasmania) and New Zealand were dissected for
comparison of the genitalia. A total of 54 males and 66 females were examined, of
which 25 males and 41 females were Australian specimens, and 29 males and 25
females were from New Zealand (see appendix B and C for measurement data).
Dissection of both female and male genitalia followed the method described in Landry
(2007) with slight modifications. The abdomen was cut at the sixth abdominal segment
in both males and females to separate the genitalia structure. The structure was then
put into an eppendorf tube containing 20% KOH and macerated (softened or separated)
in a hot water bath just below simmering point for 10 minutes. It was then transferred
onto a microscopic slide containing distilled water with a drop of diluted dishwashing
detergent to break the surface tension. While the male genitalia structure was carefully
removed from the macerated abdomen segments, the female structure was carefully
separated at the 7th abdominal segment (sternite 7/S7). Any scales and further
remaining macerated tissues were removed by gently brushing with a fine tipped nylon
artist brush in both male and females. The clean structures were then put into an
eppendorf tube containing 100% ethanol until taken for measurements or slide
preparation.
Genitalia structures were viewed using a Nikon eclipse 50i light compound microscope
and images were captured using a Nikon digital camera attached to a Nikon compound
microscope under 4x magnification and the NIS-Elements BR digital image analysis
software. Measurements were taken using the latter mentioned software.
The clean structures were kept on a glass slide with a water drop mixed with diluted
dishwashing detergent to break the surface tension, and kept at their lateral positions
to be photographed and then measured. Lateral positions of the structures were used
in both male and female preparations; in males because the concaved structure of the
vinculum saccus causes them to turn to the side when in the ventral position, and the
female structures in order to minimize the damage to the fragile structure.
34
The characteristic features described by Landry and Hebert (2013) and observed in
this study are shown in tables 2.2 and 2.3. In females, the tubular projection was
reported to be more curved in P. australiana, and the raised pair of folds forming
surrounding the antrum were reported to form two conical projections in P. xylostella
that are absent in P. australiana (Table 2.2). In males, the appearance of the vinculum
saccus was reported to be more slender (Table 2.3), the ventral margin of the valva to
have a slight sinuation and the ventro-distal margin to be rounded in P. australiana.
In addition, key features were measured. In males, the combined length of the valva
and vinculum saccus as the whole length, the width and length of the valva, the length
of the vinculum saccus and the length of the phallus (Figure 2.4A) were recorded. In
females, the whole width and length, the length of the upper part (S8 including the
ovipositor), the length of the tubular projection, and length of the sternite 7 (S7L) were
recorded (Figure 2.4B).
35
Table 2.2 Morphological features of DBM female genitalia examined in this study to
identify diagnostic features. Features are circled in red. Images were taken by
Tharanga Kariyawasam.
P. australiana P. xylostella
Lateral view
Tubular projection has a curved apical half.
Lateral view
Tubular projection is straight and evenly broad.
Ventral view
Abdominal sternum 7 has a flat surface
surrounding the antrum.
Ventral view
Abdominal sternum 7 has a raised pair of folds
forming surrounding the antrum which forms
two conical projections bracing the tubular
projection of the antrum.
36
Table 2.3 Morphological features of DBM male genitalia examined in this study to
identify diagnostic features. Features are circled in red. The ventral view of P.
australiana and lateral views of the valva were taken from Landry and Hebert (2013).
The remaining images were taken by Tharanga Kariyawasam.
P. australiana P. xylostella
Ventral view
Lateral view
Vinculum saccus is slender.
Lateral view
Slight sinuation in the ventral margin
(indicated by the arrow). Rounded ventro-
distal margin (right).
Ventral view
Lateral view
Vinculum saccus is broader.
Lateral view
Straight in the ventral margin (indicated by
the arrow). Less distinctly angled ventro-distal
margin (right).
37
A)
B)
Figure 2.4 Measurements of both female and male adult genitalia characteristics
recorded to determine the statistical variance across taxa (Scale bars = 200 μm).
Images were taken by Tharanga Kariyawasam.
A) male: WL = whole length, VW = valva width, VL = valva length, PL = phallus
length, VSL = vinculum saccus length.
B) female: WL= whole length, WW = whole width, UPL = upper part length, TPL =
tubular projection length, S7L = sternite 7 length.
38
Statistical analyses
Australian P. xylostella, Australian P. australiana and New Zealand P. xylostella
specimens were compared as three separate populations. All data were tested for
normality and homogeneity of variance using a Shapiro Wilk normality test and
Levene’s test for equality of variances before being analyzed. One-way ANOVA was
conducted to examine the differences of the mean of each parameter between the three
populations. A post hoc Tukey Honest Significant Differences (TukeyHSD) test was
used to assess significant differences between the populations. Statistically significant
parameters were visualized using density plots. All statistical analyses were conducted
using the statistical software R (R Development Core Team, 2013).
Statistical analysis of female genitalia
Measurements of whole length (WL), upper part length (UPL), tubular projection
length (TPL) and sternite 7 length (S7L) data were normally distributed, but whole
width (WW) was not. Normal distribution of whole width data was obtained using a
power of 3 (^3) transformation before analysis. One-way ANOVA was conducted to
examine the differences of the mean of each parameter across the three populations.
When ANOVA showed significant differences for any parameter, TukeyHSD test was
used to assess the significant differences between the three populations. Statistically
significant parameters were visualized using density plots.
Statistical analysis of male genitalia
Measurements of whole length (WL), valva width (VW), valva length (VL) and
vinculum saccus length (VSL) were normally distributed, but the phallus length (PL)
was not. Transformation of the PL data to obtain a normal distribution was performed
using a log10 transformation before analysis. One-way ANOVA was conducted to
examine the differences of the mean of each parameter across the three populations.
When ANOVA showed significant differences for any parameter, TukeyHSD test was
used to assess the significant differences between the three populations. Statistically
significant different parameters were visualized using density plots.
39
2.3 Results
2.3.1 CO1 sequence data
DNA sequencing of CO1 barcode region identified the presence of P. xylostella and
P. australiana. Phylogenetic trees showed two distinct clades with 100% support
values in both maximum likelihood and Bayesian trees (Figures 2.6 and 2.7).
Phylogenetic analyses using both maximum likelihood (ML, Figure 2.6) and Bayesian
analysis (Figure 2.7) showed similar topologies, forming two distinct and well-
supported clades (with 100% support value) for P. australiana and P. xylostella.
Reference sequences from Genbank were well resolved between the two clades (see
Appendix D and E). All outgroups were also well resolved.
While interspecific variation between the two Australian taxa was 9% the intraspecific
variation within both Australian taxa was 1.3%.
Both taxa were found sympatrically in most locations, including Tasmania (Figure
2.5). Four collections (Samford 2014, Theresa Park 2014, Gatton 2016 and Laidley
2016) contained only P. xylostella. All New Zealand collections were P. xylostella (not
shown in Figure 2.5).
40
Figure 2.5 Distribution of DBM adults caught in light traps at Samford (QLD), Hobart
(TAS), Theresa Park (NSW), Werombi (NSW), Mowbray Park (NSW), Birkdale
(QLD), Gatton (QLD) and Laidley (QLD). Numbers within bars represent the
individuals identified and assigned to relevant taxa (see Table 2.1. for total number of
individuals).
313
89
1111
2
3
5 4
31
1811
11
2
0%10%20%30%40%50%60%70%80%90%
100%
Per
cen
tag
e o
f ta
xa
in
ea
ch c
oll
ecti
on
P. xylostella P. australiana
41
Figure 2.6 Bipartition maximum likelihood (ML) tree with bootstrap values. The tree
was collapsed to remove low supported nodes (≥75%) and the nodes were further
collapsed (shape of the clade) because of the large number of specimens assigned to
each taxon. See Appendix D for the original phylogenetic tree.
42
Figure 2.7 Bayesian analysis with posterior probabilities. The tree was collapsed to
remove low supported nodes (≥75%) and the nodes were further collapsed (shape of
the clade) because of the large number of specimens assigned to each taxon. See
Appendix E for the original phylogenetic tree.
43
2.3.2 Morphology
The majority of key features observed were not consistently different between the two
taxa examined in the current study. The only consistently diagnostic feature was found
in females, in which the form of the tubular projection was consistently curved in P.
australiana, and in P. xylostella the raised pair of folds forming surrounding the
antrum consistently formed two conical projections bracing the tubular projection of
the antrum. In females, these forms matched the identification by CO1 sequence in
100% of female specimens from all three populations.
In males, species identification using differences in the appearance of the vinculum
saccus (which had been reported to be slender in P. australiana) were found to be
unreliable diagnostic features. The appearance of the vinculum saccus showed
variation across the taxa and included intermediate characteristics. Similarly, the slight
sinuation in the ventral margin and the rounded ventro-distal margin of the valva
showed variation across the two taxa (see Appendix F). Two individuals were found
to have opposing results when comparing morphology to the CO1 data: in one,
sequence data confirmed the species as P. xylostella but the vinculum saccus was
slender; in the other, CO1 data identified P. australiana but the vinculum saccus was
broad (Table 2.4). The CO1 sequences from these two individuals were re-examined;
DNA from each specimen was re-extracted and PCR and sequencing was repeated.
The morphology was also re-examined, which confirmed the observation. CO1
identification of the taxa were used for these two individuals when conducting the
subsequent statistical analyses of morphometric data.
44
Table 2.4 Images show the appearance of the vinculum saccus in two males where
one had the characteristic morphology of P. australiana but were identified as P.
xylostella from CO1 sequence data and the other had the characteristic morphology of
P. xylostella but were identified as P. australiana from CO1 sequence data. These
specimens were collected from Hobart and Theresa Park respectively.
Sample ID Morphology Molecular Genitalia image (4x)
HOB3_3 P. australiana P. xylostella
SYD15T_22m_Oct P. xylostella P. australiana
Statistical analysis of female genitalia
One-way ANOVA results showed that the mean length of the TPL and S7L were
significantly different between the groups (Table 2.5). TukeyHSD results showed that
the mean length of TPL was shorter in P. australiana (P < 0.001) than in either the
New Zealand or Australian P. xylostella populations but the tubular projection length
was not significantly different in the two P. xylostella populations. The mean length
of S7L was shorter in P. australiana (P < 0.001) than in Australian P. xylostella
population and shorter in New Zealand P. xylostella population than in Australian P.
xylostella population. However, the sternite 7 length was not significantly different
between the New Zealand P. xylostella population and the Australian P. australiana
population.
However, density plots of both TPL and S7L for all three groups showed significant
overlap of 94% and 82% respectively with values within the variance of the opposite
45
taxon (Figure 2.8). There was no significant difference in the whole length, whole
width and upper part length among the three populations.
Table 2.5 Mean values ± SD of parameters for each female population are shown in
micrometers (μm). P-value of the one-way ANOVA (at 0.95 confidence intervals) are
presented which showed a significance difference for the TPL (tubular projection
length) parameter and the S7L (sternite 7 length) parameter. Statistically significant
codes: ***P < 0.001, **P < 0.01.
Parameter
Populations
P-value
F-value
Australian
P. australiana
(AA) (n=14)
Australian
P. xylostella
(AX) (n=27)
New Zealand
P. xylostella
(NX) (n=25)
WL 747.4 ± 86 760 ± 98.5 739.2 ± 74.5 0.702 0.356 WW 468.6 ± 59.9 464.9 ± 54.2 465 ± 38.8 0.895 0.111 UPL 351.3 ± 75.9 303.8 ± 87.2 321.4 ± 81.4 0.244 1.442 TPL 177 ± 20.6 210.1 ± 24.7 218.9 ± 24.2 7.59e-06 *** 14.34 S7L 396.2 ± 71.2 463.7 ± 58.6 417.8 ± 36.7 0.00103 ** 7.729
46
a)
b)
Figure 2.8 Density plots showing the distribution of measurements taken for a) tubular
projection length (TPL) and b) sternite 7 length (S7L) parameters of females showing
an overlap between Australian P. australiana (AA), Australian P. xylostella (AX) and
New Zealand P. xylostella (NX).
47
Statistical analysis of male genitalia
One-way ANOVA results showed that the mean length of the PL and VSL were
significantly different between the groups (Table 2.6). TukeyHSD results showed that
the mean length of PL was longer in P. australiana (P < 0.001) than in either the New
Zealand or Australian P. xylostella populations, but the PL was not significantly
different in the two P. xylostella populations. The mean length of VSL was longer in
P. australiana (P < 0.05) than in New Zealand P. xylostella population. Vinculum
saccus length of Australian P. xylostella was not significantly different from both
Australian P. australiana and New Zealand P. xylostella. However, density plots of
both PL and VSL for all three groups showed a significant overlap of 85% with values
within the variance of the opposite taxon (Figure 2.9). There was no significant
difference in the whole length, valva width and valva length among the three
populations.
Table 2.6 Mean values ± SD of parameters for each male population are shown in
micrometers (μm). P-value of the one-way ANOVA (at 0.95 confidence interval) is
presented which showed a significance difference for the PL (phallus length)
parameter and VSL (vinculum saccus length) parameter. Statistically significant
codes: *P < 0.05, ***P < 0.001.
Parameter Populations P-value F-value
Australian
P. australiana
(AA) (n=12)
Australian
P. xylostella
(AX) (n=13)
New Zealand
P. xylostella
(NX) (n=29)
WL 1006.7 ± 38.9 989.6 ± 36.5 973.5 ± 54.5 0.148 1.986
VW 321.4 ± 24.1 307.4 ± 21.7 313.7 ± 24.5 0.342 1.096
VL 607.9 ± 26.4 616.7 ± 25.8 611.1 ± 38.7 0.798 0.227
PL 565.9 ± 30.6 498 ± 31.2 493.9 ± 22.2 1.8e-08 *** 26.46
VSL 396.9 ± 34 368 ± 45 362.4 ± 29.8 0.0235 * 4.061
48
a)
b)
Figure 2.9 Density plots showing the distribution of measurements taken for (a)
phallus length (PL) and (b) vinculum saccus length (VSL) parameters of males
showing an overlap between Australian P. australiana (AA), Australian P. xylostella
(AX) and New Zealand P. xylostella (NX).
49
2.4 Discussion
The results overall confirm the separation of the two taxa based on mitochondrial
(CO1) sequence data. Both maximum likelihood and Bayesian analyses showed
similar topologies by forming two distinct well supported clades (100%) for P.
australiana and P. xylostella confirming them as two separate taxa. Moreover, an
interspecific divergence of 9% and intraspecific divergence of 1.3% were found,
further confirming the separation of the two taxa. These results are consistent with the
description in Hebert et al. (2003) that the value closer to 3% in the CO1 sequence
divergence suggests the presence of a separate taxon (Hebert et al., 2003).
Both P. xylostella and P. australiana were found sympatrically in most locations,
including Tasmania. Two collections in 2014 (Samford and Theresa Park) and two
collections (Gatton and Laidley) in 2016 consisted only of P. xylostella adults. The
presence of P. australiana in Tasmania has been suggested previously based on an
observation of morphology (personal communication from Lionel Hill, Senior
Entomologist, in Tasmania) and the presence of P. australiana in Tasmania was
confirmed by CO1 sequence data and morphological comparison in this study. A
single female specimen with pronounced curvature of the tubular projection (which
was described as P. xylostella) may indicate the presence P. australiana in New
Zealand (Dugdale, 1973). Further supporting it another two female specimens, one
collected from Ivercargill, Boulder Bank, Nelson in New Zealand in 1907 and another
from Fiordland collected before 1910 were observed to have morphological
characteristics of P. australiana based on the S7 oval sclerotisation with no raised pair
of folds and the antrum projection in the side view (curvature and tubular projection
projecting for less than 1/3 its length beyond 7S) as described by Landry and Hebert
(2013) (personal communication from John Dugdale, Senior Entomologist, New
Zealand). However, specimens examined in this study did not support the presence of
P. australiana in New Zealand.
The morphology of genitalia in P. xylostella and P. australiana have been previously
described (Baraniak, 2007; Dugdale, 1973; Landry & Hebert, 2013; Moriuti, 1986;
Robinson & Sattler, 2001) but comparative analysis of measurements have not been
previously recorded. This is the first study to address this gap.
50
There were no significant differences in the measures or features in the New Zealand
or Australian P. xylostella populations except the sternite 7 length which showed a
significant difference. There were significant differences between the two taxa except
for the vinculum saccus length in males which showed no significant difference.
The length of the tubular projection in females was significantly longer in P. xylostella
(from both Australia and New Zealand) than in P. australiana. The length of the
sternite 7 was shorter in P. australiana than in Australian P. xylostella and in New
Zealand P. xylostella than in Australian P. xylostella. However, the extensive overlap
in the variance does not support those as diagnostic features. Similarly in males, the
presence of individuals with intermediate and opposing characteristics of the vinculum
saccus does not support the use of morphological characteristics in general as a
diagnostic feature in males. Examinations of the vinculum saccus feature to be slender,
the sinuation at the ventral margin of the valva and the rounded ventro-distal margin
in P. australiana as described in Landry and Hebert (2013) cannot be used in the
identification of the two taxa.
The examination of the curvature of the tubular projection in P. australiana and the
raised folds surrounding the antrum in P. xylostella were reliable diagnostic features
in females, further supporting the separation of the two taxa. The observations of the
three female specimens reported by Dugdale (personal communication) suggests that
further examination of New Zealand populations should be conducted to identify if P.
australiana is present.
Whole length, whole width, and upper part length in females and whole length, valva
width, and valva length in males were found not to be statistically different in the two
taxa. Examination of genitalia measurements in their ventral position may show a
different outcome, but it is difficult to use the ventral position, especially when
handling a large number of specimens.
An allozyme study on P. xylostella populations from 14 locations worldwide,
including specimens from five different locations from Australia (Adelaide, Brisbane,
Mareeba, Melbourne and Sydney), found that the Australian and Japanese populations
were different from all other populations (Pichon et al., 2006). Using six microsatellite
51
loci, Endersby et al. (2006) found no genetic variation within Australian populations
or between Australian and New Zealand P. xylostella populations. Similarly, Juric et
al. (2017) found that the Australian population clustered with the New Zealand
haplotypes. These results are further supported by the results of this study, which show
very little mitochondrial divergence and no significant morphological variation in the
structure of genitalia between Australian and New Zealand populations. These results
suggest that Australian and New Zealand populations of P. xylostella represent one
population, and is potentially a consequence of a single, rapid invasion to both
countries (Chu, 1986; Saw et al., 2006; Talekar & Shelton, 1993). Furthermore, the
results are consistent with previous observations on strong migration capacity of P.
xylostella (Saw et al., 2006) and of a possible recent introduction of DBM to Australia,
New Zealand and North America from a highly variable population from Europe (Juric
et al., 2017).
This study has confirmed that P. xylostella and P. australiana can be separated into
two taxa based on both mitochondrial DNA and some morphological data
(examination of female morphological characteristics). However, the species status of
P. australiana has not been confirmed. The use of mitochondrial DNA alone to
identify or describe new species has been questioned (Rubinoff et al., 2006; Will et al.,
2005; Will & Rubinoff, 2004). Examination of only a single, maternally-inherited gene
(mtDNA/CO1) may be insufficient, and ideally bi-parentally inherited nuclear markers
should be identified and compared in order to identify species boundaries (see chapter
4 for further discussion). In addition, a combination of multiple sources of data in an
integrative taxonomic framework using specimens from a wide geographic range
(Dayrat, 2005; Schlick-Steiner et al., 2010; Springer et al., 2001) and interbreeding
studies are required to determine if P. australiana is a new species.
52
53
Chapter 3: Host plants and distribution of
Plutella species in Australia
3.1 Introduction
The diamondback moth (DBM), Plutella xylostella L. (Lepidoptera: Plutellidae), is
one of the most destructive insect pests of brassica crops worldwide, attacking cole
crops (cabbage, cauliflower), oilseeds (canola, mustard) and root vegetables (radish,
turnip) (Sarfraz et al 2005). Crop damage is caused by larvae that feed on the leaves
or the head of crops like in cabbage and broccoli (Figure 3.1) or on the flowers and
young pods of canola.
Since the introduction of P. xylostella into Australia, it has been reported to attack
brassica vegetable crops and now considered to cause severe damages to the canola
production (Endersby et al., 2004). Damage in canola, can be severe and is increasing
as the area planted increases in South Australia (SA), Western Australia (WA),
Victoria (VIC) and New South Wales (NSW) (Furlong et al., 2008; Gu et al., 2007;
Perry et al., 2015).
Plutella xylostella has developed resistance to almost all insecticides including Bt (the
toxin from Bacillus thuringensis) and was the first agricultural pest to develop
resistance against DDT (Ankersmit, 1953; Atumurirava et al., 2011; Sun et al., 1986;
Talekar & Shelton, 1993; Zhou et al., 2011). The rapid and widespread evolution of
resistance highlights the need for an effective integrated resistance management
strategy including both biological controls and biopesticides. However, the release of
biological controls against the exotic P. xylostella in Australia has been made more
challenging with the description by Landry and Hebert (2013) of a new, closely related
taxon, Plutella australiana. Little is known about this new taxon, particularly its
potential economic importance as a pest along with its distribution. If it is an endemic
species, it could be at risk from the biological control agents released against P.
xylostella. Moreover, rigorous risk assessment on host specificity and impacts on non-
54
target organisms must be taken prior to the release of any new biopesticides based on
microorganisms and insecticides against P. xylostella (BiosecurityAct, 2015).
Figure 3.1 DBM larvae damaging A) cabbage leaf, B) head formation of red cabbage.
The availability of brassica host plants and the abundance of P. xylostella on them are
critical determinants of the economic importance of this pest (Ahuja et al., 2010).
Plutella xylostella occurs on brassica crops but also occurs on weedy brassicas such as
wild mustard and wild radish (Sarfraz et al., 2006; Sarfraz et al., 2011). Both P.
xylostella and associated parasitoids use them as host plants and as refugia when
cultivated crops are damaged or prior to their cultivation (Kahuthia-Gathu et al., 2009;
Sarfraz et al., 2006; Talekar & Shelton, 1993). The incidence of P. xylostella on native
Australian brassicas is not well known but there are many native species of brassicas
in Australia, including caper berries, Capparis spp. P. xylostella have been
successfully reared on caperbush (Capparis sandwichiana: Brassicales, capparacea) in
Hawaii (Robinson & Sattler, 2001). Reports of P. xylostella on crops other than
brassicas are very rare, but with few incidences recorded on; okra (Abelmoschus
esculentus: Malvales, Malvaceae) in 1971 in Ghana (FAO, 1971), chickpea (Cicer
arietinum: Fabales, Fabaceae) and prickly Russian thistle (Salsola kali:
Caryophyllales, Chenopodiaceae) in northern Russia (Reichart, 1919; Talekar et al.,
1985). Reappearance of DBM on those crops were not reported ever since (Löhr &
Gathu, 2002). But recent records on a strain of P. xylostella has been reported to feed
on sugar snap peas (Pisum sativum: Fabales, Fabaceae) (Löhr & Rossbach, 2001;
Rossbach et al., 2006). The reason for this host shift was due to the high P. xylostella
damage caused in the cabbage field causing the P. xylostella to shift to the only
55
available nearby crop, the sugar pea (Henniges-Janssen et al., 2014). However, the
alleles of the newly evolved DBM population surviving on sugar peas were not fixed,
showing genetic variation in adaptation for suitable environments (Henniges-Janssen
et al., 2011).
There is limited data on the larval host plants of P. australiana, and its potential pest
status is only now being explored. Only three larvae of P. australiana have been
previously reported on Lincoln weed (Diplotaxis tenufolia: Brassicales, Brassicaceae)
in South Australia (Perry et al., 2015). There are now large scale studies on its pest
status in canola in South Australia (Perry et al., 2015; Perry et al., 2017), but data on
its incidence in vegetable brassicas have not yet been reported and nothing is known
of the host plants in eastern Australia.
This chapter investigates the larval host plants of the Plutella species, especially P.
australiana on crops and weeds by collecting the larvae from both brassica crops and
weeds in the fields. Identification of larvae collected from the field used both
cytochrome oxidase 1 ‘barcode’ (CO1) sequence analysis and rearing of larvae
through to adult emergence in the laboratory, followed by dissection, examination and
morphometric analysis of the genitalia. Moreover, the genitalia morphological
measurements between adults reared from field collected larvae and the light trapped
adults (in chapter 2) were examined to look at any morphological variations caused by
rearing individuals under laboratory conditions.
56
3.2 Materials and Methods
3.2.1 Sampling
Larvae were collected by plant inspection and hand collection, by sweep netting and
using beat sheets at the same locations and same dates in Queensland, New South
Wales and Tasmania as those from which adults were collected by light trapping (see
chapter 2, section 2.2.1).
Thirty larvae were collected from each crop or from weeds on the field margin on the
same day as light trapping was conducted. Larvae were identified from their pale
brown head capsule and green and segmented body. Host plants were recorded, and
extra leaves were collected from the field to feed the larvae when under laboratory
conditions. Larvae were transported in take-away containers lined with paper tissue,
along with host plant material, and closed with a ventilated lid sealed using a nappy
liner to prevent larvae from escaping. Thirty larvae collected from Nelson, New
Zealand in January 2016 were received from New Zealand as 40% ethanol preserved
larvae. The majority of larvae were then reared on the extra leaves collected from the
field accordingly and if the diet was not enough organic cabbage was used to feed the
larvae. The larvae were reared in temperature controlled cabinets at 20-23 °C and
humidity at 65-70% under 12 h light: 12 h dark until pupation and adult emergence.
Adults were then frozen and stored for molecular and morphological analyses. Larvae
collected from Queensland in 2016 were used for molecular analysis without rearing
to their adult stage as well as the larvae shipped in ethanol from New Zealand.
3.2.2 Molecular Analysis
DNA extraction, PCR amplification and CO1 ‘barcode’ Sanger sequencing were
conducted following the same procedure described in section 2.2.2 (chapter 2) to
establish the identity of the taxa. Two hundred and four (n=204) larvae were included
(Table 3.1) in the phylogenetic analyses described in section 2.2.2. (chapter 2).
57
Table 3.1 Larvae collection details including location, crop type and number of
individuals taken for genetic and morphological analyses. Specimens not taken for
morphological analyses are marked as ‘/’ and unknown GPS coordinates are marked
as ‘-‘.
3.2.3 Morphology
Detailed morphological analysis including measurements of adult genitalia
characteristics to determine the statistical variance across taxa and populations
(chapter 2, Figure 2.4); and observations of morphological characteristics to identify
reliable diagnostic features (chapter 2, Table 2.2 and 2.3) were performed. A subset of
both male and female larvae (n=47) from the same individuals taken for the
phylogenetic tree construction were included in the statistical analyses (represents only
Hobart, Theresa Park and Werombi). A comparison was made of the measured features
of the genitalia of the adults reared from larvae within and between the taxa and with
adults of both taxa from light traps (chapter 2) to determine any effect of laboratory
conditions and diets, on the morphological features of the laboratory reared
individuals. A total of 19 adult males and 28 adult females reared from larvae collected
in the field were measured (see Appendix G and H for measurement data).
Sample
code
Date
(year)GPS Elevation Location Crop type
Number of sequences
included in the
phylogenetic analyses
Females Males
HOB4 2014
33.98295° S,
150.63965° E 31m Hobart, Tasmania cauliflower, swedes 13 8 5
TL 2015
33.98295° S,
150.63965° E 58m Theresa Park, NSW cabbage 28 9 5
WL15 2015
34.00338 °S,
150.56716 °E 286m Werombi, NSW kale 22 5 5
WW15 2015
34.00338 °S,
150.56716 °E 286m Werombi, NSW weed (field mustard) 28 6 4
GL 2016
27.32139°S,
152.20061°E 98m Gatton, QLD cauliflower 15 / /
LL 2016
27.45113°S,
152.22041°E 151m Laidley, QLD broccoli 29 / /
CL 2016
28.13129 °S,
153.23248°E 244m Currumbin, QLD broccoli , kale 11 / /
BL 2016
27.493653 °S,
153.204733 °E 10m Birkdale, QLD broccoli 31 / /
NL 2016 - - Nelson, New Zealand kale 27 / /
Number of specimens
used for morphological
identification
58
Statistical analyses
Field collected larvae
All data were tested for normality and homogeneity of variance using a Shapiro Wilk
normality test and Levene’s test for equality of variances before being analysed. All
of the data for both females and males were normally distributed except the vinculum
saccus length (VSL) in males, which was transformed by raising to the power of 3 (^3)
transformation to obtain a normal distribution before analysis. One-way ANOVA was
conducted to examine the differences of the mean of each parameter between the
Australian P. xylostella and Australian P. australiana populations. Statistically
significant parameters were visualized using density plots. All statistical analyses were
conducted using the statistical software R (R Development Core Team, 2013).
Statistical analysis between adults caught in light traps and adults reared from
field collected larvae
P. xylostella female individuals
Measurements of all parameters were normally distributed. A one-way ANOVA was
conducted to examine the differences of the mean of each parameter across the three
populations (Australian P. xylostella adults, New Zealand P. xylostella adults and P.
xylostella larvae). When the ANOVA results showed significant differences for any
parameter, a post hoc Tukey Honest Significant Differences (TukeyHSD) test was
used to assess the significant differences between the three populations. Statistically
significant parameters were visualized using density plots.
P. australiana female individuals
Measurements of whole length (WL), upper part length (UPL), tubular projection
length (TPL) and the sternite 7 length (S7L) data were normally distributed, but whole
width (WW) was not. Although transformation of the WW data to obtain a normal
distribution was performed using a log10, log, raised to the power of 3 (^3), took the
ninth root (^1/9), raises the constant e to the power of WW (exp), found the absolute
value (abs), the sine of WW (sin), square root of WW (sqrt), it did not show a normal
distribution. A non-parametric Kruskal Wallis test was conducted to examine the
differences of the mean of each parameter across the two populations; Australian P.
59
australiana adults and P. australiana larvae. Statistically significant parameters were
visualized using density plots.
P. xylostella male individuals
While measurements of whole length (WL) and phallus length (PL) were not normally
distributed other parameters were normally distributed. Normal distribution of WL and
PL data were obtained using a power of 3 (^3) transformation and log10 transformation,
respectively before analysis. A one-way ANOVA was conducted to examine the
differences of the mean of each parameter across the three populations (Australian P.
xylostella adults, New Zealand P. xylostella adults and P. xylostella larvae).
P. australiana male individuals
Measurements of all parameters were normally distributed. A one-way ANOVA was
conducted to examine the differences of the mean of each parameter across the two
populations; Australian P. australiana adults and P. australiana larvae. Statistically
significant parameters were visualized using density plots.
3.3 Results
3.3.1 CO1 sequence Data
The sequences of all larvae could be aligned completely with the two taxa described
from light trapped adult sequences (chapter 2), and all the samples (n=204) were fully
resolved into either of the two taxa.
While the average interspecific variation between the two Australian taxa was 8.3%
the intraspecific variation within P. xylostella and P. australiana were 1.3% and 0.3%
respectively.
3.3.2 Host plants of P. xylostella and P. australiana
The majority of the larvae collected (n=160) were P. xylostella: 44 were larvae of P.
australiana. The majority of collections were entirely composed of P. xylostella
60
(Figure 3.2). All New Zealand collections were P. xylostella (not included in Figure
3.2).
Figure 3.2 Distribution of larvae from field collections in Hobart (TAS), Theresa Park
(NSW), Werombi (NSW), Gatton (QLD), Laidley (QLD), Currumbin (QLD) and
Birkdale (QLD). Numbers within bars represent the individuals identified and assigned
to relevant taxa (for total numbers see Table 3.1.).
Larvae of P. australiana were present in only two collections: on cabbage in Theresa
Park, NSW in 2015 and on field mustard weeds (Brassica rapa) amongst a kale crop
in Werombi NSW in 2015 (Figure 3.2). Larvae collected from all other brassica crops
were entirely composed of P. xylostella, including the New Zealand larvae. In the two
collections in which P. australiana was present, they were sympatric with P. xylostella
larvae, but the majority of larvae analysed from those two collections were P.
australiana (22 out of 28 in both collections).
The larvae collected from the weed (B. rapa) on the margin of the kale crop at
Werombi were predominantly P. australiana. Both larvae and pupae were found on
the plants (Figure 3.3). However, larvae collected from the crop itself were entirely P.
xylostella.
13
6
22
6
15 29 11 3122 22
0%10%20%30%40%50%60%70%80%90%
100%
Per
cen
tag
e o
f ta
xa
in
th
e co
llec
tio
n
P. xylostella P. australiana
61
Figure 3.3 Field mustard (B. rapa) (above) and the cabbage field (below) that P.
australiana larvae were collected. In order from left to right are the field mustard plant,
its flower, larvae feeding on the leaf, pupae found on the stem and the cabbage field
where P. australiana larvae were collected.
62
3.3.3 Morphology
As in chapter 2, the majority of observed morphological features of the genitalia used
to differentiate the two taxa were found not to be consistently reliable in adults reared
from larvae collected in the field. The two consistent diagnostic features were again
found in females, in which the form of the tubular projection was consistently curved
in P. australiana, and in P. xylostella the raised pair of folds surrounding the antrum
consistently formed two conical projections bracing the tubular projection of the
antrum. As for the adults caught in light traps (chapter 2), identification of the taxon
using these two features matched the identification by CO1 sequence in 100% of
female specimens.
In males, species identification using differences in the appearance of the vinculum
saccus, which had been reported to be slender in P. australiana, the sinuation at the
ventral margin of the valva and the rounded ventro-distal margin in P. australiana
(Landry & Hebert 2013) were found to be unreliable diagnostic features. In one male
adult reared from larvae, the morphological appearance of the vinculum saccus directly
contradicted the CO1 sequence data. The individual was confirmed as P. xylostella by
sequence data but the appearance of the vinculum saccus was slender (Table 3.2). The
CO1 sequence from this individual was re-examined, with DNA re-extracted and
subjected to PCR and Sanger sequencing, and the morphology was also re-examined.
This re-confirmed the observation. The individual was categorised as P. xylostella in
the statistical analysis of morphometric data.
63
Table 3.2 The image shows the appearance of the vinculum saccus of a male that had
the characteristic morphology of P. australiana but was identified as P. xylostella from
CO1 sequence data. The larva was collected from B. rapa weeds in Werombi NSW in
2015.
Sample ID Morphology Molecular Genitalia image (4x)
WW15_18
P. australiana
P. xylostella
Statistical analysis of field collected larvae
Statistical analysis of female genitalia
One-way ANOVA results showed that the mean of whole length (WL), whole width
(WW), tubular projection length (TPL) and sternite 7 length (S7L) were significantly
different between the taxa. The mean of WL in P. xylostella was longer (P < 0.01) than
in P. australiana, WW in P. xylostella was wider (P < 0.05) than in P. australiana,
TPL in P. xylostella was longer (P < 0.01) than in P. australiana and S7L in P.
xylostella was (P < 0.01) longer than in P. australiana (Table 3.3). However, density
plots of the WL, WW, TPL and S7L showed significant overlap of 64%, 68%, 54%
and 61% respectively with values within the variance of the opposite taxon (Figure
3.4). The upper part length was not significantly different between the taxa.
64
Table 3.3 Mean ± SD values of parameters for each female population are shown in
micrometres (μm). P-value of the one-way ANOVA (at 0.95 confidence intervals) is
presented which showed a significance difference for the whole length (WL), whole
width (WW) and tubular projection length (TPL) parameters. Statistically significant
codes: *P < 0.05, **P < 0.01.
Parameter Populations P-value F-value
P. xylostella (n=15) P. australiana (n=13)
WL 814.6 ± 80 747.2 ± 35.9 0.00958 ** 7.822
WW 485.6 ± 46.3 448.5 ± 16.9 0.0113 * 7.427
UPL 383.4 ± 68.4 385.4 ± 41.1 0.927 0.009
TPL 204.6 ± 42.1 159.7 ± 13.3 0.00105 ** 13.59
S7L 431.2 ± 81.5 361.8 ± 32.5 0.00799 ** 8.256
65
a) b)
c) d)
Figure 3.4 Density plots showing the distribution of measurements taken for a) whole
length (WL), b) whole width (WW), c) tubular projection length (TPL) and d) sternite
7 length (S7L) parameters of females showing an overlap between P. xylostella and
P. australiana. However, mean values are significantly different.
66
Statistical analysis of male genitalia
One-way ANOVA results showed that the mean phallus length (PL) (P < 0.001) was
significantly longer in P. xylostella than in P. australiana (Table 3.4). A density plot
of PL showed an overlap of 11% with values within the variance of the opposite taxon
(Figure 3.5). There was no significant difference in the whole length, valva width,
valva length and vinculum saccus length between the two taxa.
Table 3.4 Mean ± SD values of parameters for each male population are shown in
micrometres (μm). P-value of the one-way ANOVA (at 0.95 confidence intervals) is
presented which showed a significance difference for the phallus length (PL)
parameter. Statistically significant codes: ***P < 0.001.
Parameter Populations P-value F-value
P. xylostella (n=12) P. australiana (n=7)
WL 959.8 ± 39.5 961.5 ± 53.2 0.942 0.005
VW 316.2 ± 14.9 308.3 ± 18.5 0.348 0.931
VL 569.3 ± 27.2 606.2 ± 63.6 0.166 2.092
PL 552.2 ± 13.1 479.5 ± 17.3 1.12e-07 *** 81.48
VSL 390.5 ± 29.6 355.3 ± 54.6 0.133 2.485
67
Figure 3.5 Density plot showing the distribution of measurements taken for phallus
length (PL) parameter of males showing an overlap between P. xylostella and P.
australiana. However, mean values are significantly different.
Statistical analysis between adults caught in light traps and adults reared from
field collected larvae
Female P. xylostella individuals
One-way ANOVA results showed that the mean of whole length (WL), upper part
length (UPL) and sternite 7 length (S7L) were weakly significantly different (P < 0.05)
(Table 3.5). TukeyHSD results showed that the mean length of WL was shorter in New
Zealand P. xylostella than in larvae. There was no significant difference between the
WL of Australian P. xylostella and larvae; and New Zealand and Australian P.
xylostella populations. The mean length of UPL was shorter in Australian P. xylostella
than in larvae. There was no significant difference between New Zealand P. xylostella
and larvae; and New Zealand P. xylostella and Australian P. xylostella. The mean
length of S7L was shorter in New Zealand P. xylostella than in Australian P. xylostella.
There was no significant difference between Australian P. xylostella and larvae; and
New Zealand P. xylostella and larvae. However, density plots of the WL, UPL and
S7L showed significant overlap of 94%, 91% and 93% respectively with values within
68
the variance of the opposite taxon (Figure 3.6). There was no significant difference in
the whole width and tubular projection length among the three populations.
Table 3.5 Mean ± SD values of parameters for each female population are shown in
micrometres (μm). One-way ANOVA (at 0.95 confidence intervals) is presented
which showed a significance difference for the whole length (WL) and upper part
length (UPL) parameters. Statistically significant codes: *P < 0.05.
Parameter Populations P-value F-value
Australian
adults (n=27)
New Zealand
adults (n=25)
Larvae
(n=15)
WL 760.0 ± 98.5 739.2 ± 74.5 814.6 ± 80 0.0307 * 3.687
WW 464.9 ± 54.2 465 ± 38.8 485.6 ± 46.3 0.335 1.112
UPL 303.8 ± 87.2 321.4 ± 81.4 383.4 ± 68.4 0.013 * 4.664
TPL 210.1 ± 24.7 218.9 ± 24.2 204.6 ± 42.1 0.305 1.212
S7L 463.7 ± 58.6 417.8 ± 36.7 431.2 ± 81.5 0.0235 * 3.989
69
a) b)
c)
Figure 3.6 Density plots showing the distribution of measurements taken for a) whole
length (WL), b) upper part length (UPL) and c) sternite 7 length (S7L) parameters
showing an overlap between Australian P. xylostella adults (AUS adults), New
Zealand P. xylostella adults (NZ adults) and P. xylostella larvae. However, mean
values are significantly different.
70
Female P. australiana individuals
One-way ANOVA and Kruskal Wallis test results (Table 3.6) showed that the mean
whole width (WW) (P < 0.01) and the mean tubular projection length (TPL) (P < 0.05)
were significantly different between P. australiana adults from the field and P.
australiana adults reared from larvae. Both WW and TPL were shorter in adults from
larvae than in adults from the field. However, both density plots of WW and TPL
showed an overlap of 78% with values within the variance of the opposite taxon
(Figure 3.7). There was no significant difference in the whole length, upper part length
and sternite 7 length among the two populations.
Table 3.6 Mean ± SD values of parameters for each female P. australiana population
are shown in micrometres (μm). One-way ANOVA (at 0.95 confidence intervals) and
Kruskal Wallis test are presented which showed a significance difference for the whole
width (WW) and tubular projection length (TPL) parameters. Statistically significant
codes: *P < 0.05.
Parameter Populations P-value F-value
Adults from light
traps (n=14)
Larvae
(n=13)
WL 747.4 ± 86 747.2 ± 35.9 0.994 0
WW 468.6 ± 59.9 448.5 ± 16.9 0.01152* -
UPL 351.3 ± 75.9 385.4 ± 41.1 0.176 1.943
TPL 177 ± 20.6 159.7 ± 13.3 0.0191 * 6.281
S7L 396.2 ± 71.2 361.8 ± 32.5 0.136 2.374
71
Figure 3.7 Density plots showing the distribution of measurements taken for whole
width (WW) and tubular projection length (TPL) parameters of females, showing an
overlap between Australian P. australiana adults from light traps and P. australiana
adults reared from larvae. However, mean values are significantly different.
72
Male P. xylostella individuals
One-way ANOVA results showed that the mean phallus length (PL) (P < 0.1) was
weakly significantly different between adults caught in light traps including the New
Zealand specimens, and adults reared from field collected larvae (Table 3.7). The mean
of PL was longer in adults from larvae than in adults from the field. A density plot of
PL showed an overlap of 93% with values within the variance of the opposite taxon
(Figure 3.8). There was no significant difference in the whole length, valva width,
valva length and vinculum saccus length among the three populations.
Table 3.7 Mean ± SD values of parameters for each male P. xylostella population are
shown in micrometres (μm). One-way ANOVA (at 0.95 confidence intervals) is
presented which showed a slight difference for the phallus length (PL) parameter.
Statistically significant codes: ‘.’0.1.
Parameter Populations P-value F-value
Australian
adults (n=13)
New Zealand
adults
(NX) (n=29)
Larvae
(n=12)
WL 989.6 ± 36.5 973.5 ± 54.5 959.8 ± 39.5 0.159 2.135
VW 307.4 ± 21.7 313.7 ± 24.5 316.2 ± 14.9 0.913 0.012
VL 616.7 ± 25.8 611.1 ± 38.7 569.3 ± 27.2 0.588 0.302
PL 498 ± 31.2 493.9 ± 22.2 552.2 ± 13.1 0.084 . 3.263
VSL 368 ± 45 362.4 ± 29.8 390.5 ± 29.6 0.552 0.365
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Figure 3.8 Density plot showing the distribution of measurements taken for phallus
length (PL) parameter of males, showing an overlap between P. xylostella adults from
light traps and P. xylostella adults reared from larvae.
Male P. australiana individuals
One-way ANOVA results showed that the mean whole length (WL) (P < 0.05), valva
length (VL) (P < 0.01) and the phallus length (PL) (P < 0.001) were significantly
different between P. australiana adults from the field and P. australiana adults reared
from larvae (Table 3.8). The mean of WL, VL and PL were shorter in adults from
larvae than in adults from the field. However, density plots of WL, VL and PL showed
an overlap of 58%, 58% and 42% respectively with values within the variance of the
opposite taxon (Figure 3.9). There was no significant difference in the valva width and
vinculum saccus length among the two populations.
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Table 3.8 Mean ± SD values of parameters for each male P. australiana population
are shown in micrometres (μm). One-way ANOVA (at 0.95 confidence intervals) is
presented which showed a significance difference for the whole length (WL), valva
length (VL) and phallus length (PL) parameters. Statistically significant codes: *P <
0.05, **P < 0.01,***P < 0.001.
Parameter Populations P-value F-value
Adults from light
traps (n=12) Larvae (n=7)
WL 1006.7 ± 38.9 961.5 ± 53.2 0.0245 * 6.167
VW 321.4 ± 24.1 308.3 ± 18.5 0.612 0.267
VL 607.9 ± 26.4 606.2 ± 63.6 0.0073 ** 9.278
PL 565.9 ± 30.6 479.5 ± 17.3 0.000841 *** 17.29
VSL 396.9 ± 34 355.3 ± 54.6 0.691 0.164
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Figure 3.9 Density plots showing the distribution of measurements taken for whole
length (WL), valva length (VL) and phallus length (PL) parameters of males, showing
an overlap between P. australiana adults from light traps and P. australiana adults
reared from larvae. However, mean values are significantly different.
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3.4 Discussion
The results of the larval collections confirm the separation of the two taxa based on
mitochondrial (CO1) sequence as in chapter 2. The CO1 sequences were well resolved
into P. australiana and P. xylostella. Moreover, an interspecific divergence of 8.3%
and intraspecific divergence of 1.3% and 0.3% within P. xylostella and P. australiana
respectively were found further confirming the separation of the two taxa as in chapter
2. The number of specimens of P. australiana considered in the analysis was not as
high as for P. xylostella.
Since P. australiana was first described in 2013, very few larvae have been reported
in the field, and those mostly on weeds. Three larvae were found on Lincoln weed
(Diplotaxis tenufolia, family Brassicaceae) in South Australia (Perry et al., 2015). This
study is the first report of wild field mustard, B. rapa, as a host for P. australiana
larvae. P. australiana larvae are reported to attack canola, a cultivar of B. rapa or B.
napus in industry reports (personal communication from Michael Keller, Professor,
Adelaide) but the data has not yet been published.
This study has provided the first documentation of vegetable cabbage as a host for
larvae of P. australiana. Plutella australiana appears to have little association with
crop plants other than canola (as reported above). Larvae collected from cabbage, kale,
broccoli, swede and cauliflower crops in Australia and analysed in this study were
entirely composed of P. xylostella with the notable exception of the cabbage crop at
Theresa Park NSW in 2015, in which both P. xylostella and P. australiana larvae were
identified. The larvae collected from a kale crop in Nelson, New Zealand, were all
found to be P. xylostella.
It was also noteworthy that while both P. australiana and P. xylostella larvae were
identified in the B. rapa weeds on the margin of a kale crop at Werombi NSW, all the
larvae analysed from the kale crop itself were P. xylostella. Furthermore, the results in
chapter 2 show that at both these locations and sampling dates, a significant proportion
of the adults caught in light traps were P. australiana. However, while the majority of
larvae recovered from the cabbage crop at Theresa Park were P. australiana, at
Werombi the larvae from the kale crop were only P. xylostella, despite the presence of
77
P. australiana as larvae on adjacent weeds and as adults in light traps. Similarly, the
larval samples from cauliflower and swedes in Hobart, Tasmania were 100% P.
xylostella but light trap samples from the same location were shown (in chapter 2) to
have a small proportion of P. australiana (Figure 2.5).
Overall, these findings suggest that P. australiana may be an occasional or emerging
pest of brassica crops, present only in some crops (canola, cabbage) but not yet widely
attacking vegetable brassicas and possibly primarily found on weedy brassica species.
Further investigation on a wider range of Brassicaceae plants is required which may
reveal more host plants used by P. australiana.
The presence of P. australiana larvae on B. rapa, but not on kale in the same field,
indicates a potentially significant host preference. Studies have found that P. xylostella
choose their host according to the nutrient levels of host plants (Sarfraz, Dosdall, &
Keddie, 2010) or even that they select host plants with less leaf wax (Stoner, 1990).
The implications of potential host preference will be further discussed in chapter 4.
As in chapter 2, examination of the curvature of the tubular projection in P. australiana
and the raised folds surrounding the antrum in P. xylostella were the only reliable
diagnostic features in females. The other features measured were found to be
statistically significantly different between the two taxa, but the overlap in the variance
indicates that these features cannot be used as a diagnostic characteristic. In males, the
presence of individuals with intermediate and contradictory appearance of the
vinculum saccus further confirmed it as an unreliable feature, though both phallus
length and vinculum saccus length were found to be significantly longer in P.
australiana than in P. xylostella. As in chapter 2 observation of the sinuation of the
ventral margin and the round ventro-distal margin of the valva were found not to be
diagnostic due to their variation between the taxa.
Field collected larvae that were reared under laboratory conditions exhibited
morphological differences in both females and males. Between light caught adults and
larval females of P. xylostella, whole length and upper part length were found to be
longer in larvae than in adults from both Australia and New Zealand. In P. australiana
the whole length and tubular projection length were found to be shorter in larvae than
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in adults. Between adult and larval males of P. xylostella, phallus length was found to
be longer in larvae than in adults. In P. australiana, whole length, valva length and
phallus length were found to be shorter in larvae than in adults. However, phallus
length was found to be different between P. xylostella larvae and P. australiana larvae
as mentioned above. These findings suggest that the length of some morphological
features vary depending on rearing conditions.
In conclusion, the molecular results presented here further support the classification of
P. australiana and P. xylostella into two taxa. However, morphological characters
were found to be largely unreliable as a diagnostic tool, particularly for laboratory
reared specimens except the diagnostic morphological features in females. The results
of the field collections suggest that P. australiana may be an emerging pest of crops
in Australia, but further studies are required to determine the full range of host plants
used by this species and potential host plant preferences.
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Chapter 4: General Discussion
This study investigated the taxonomy, distribution, host plants, pest status and the pest
management implications of Plutella species in Australia including New Zealand. The
main aim of this study was to clarify the validity of Plutella australiana as a distinct
species by sampling individuals from different locations in Australia and a larger
sample size by using a mitochondrial gene (CO1) and by using a detailed genitalia
morphological analysis of the two Plutella taxa. Moreover, the distribution, pest status
and management implications of these taxa were examined.
The key findings of this study were;
• Plutella xylostella and P. australiana are two distinct taxa based on CO1 data.
• The measurement of key morphological features, while significantly different,
overlap and cannot be used as diagnostic tools in differentiating the two taxa.
The examinations of the morphological features revealed only two reliable
diagnostic features in females (the form of the tubular projection was
consistently curved in P. australiana, and in P. xylostella the raised pair of
folds surrounding the antrum consistently formed two conical projections
bracing the tubular projection of the antrum).
• Both taxa are found sympatrically in most locations in Australia and share
similar host plants, but P. australiana are rarely found on vegetable brassicas.
Preference of P. australiana for B. rapa suggests that canola crops widely
grown in South Australia and Western Australia may be at risk.
• Plutella xylostella was found in New Zealand but P. australiana was not. No
significant differences in morphology between Australian and New Zealand P.
xylostella were identified except the sternite 7 length which was longer in
Australian P. xylostella than in New Zealand P. xylostella. Together, with the
low genetic divergence found between the P. xylostella populations and strong
migration capacity of P. xylostella, these data suggest a single widespread
invasion to both countries or an invasion from New Zealand to Australia or
vice versa. (Saw et al., 2006).
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• The origin of P. australiana remains undetermined and this has implications
for pest management strategies in Australia. Based on the reported estimated
rate of divergence at the CO1 locus in insects (Brower, 1994; Juan et al., 1995)
and the results of this study, P. xylostella and P. australiana diverged about
4.5 million years ago before even the introduction of P. xylostella into
Australia.
• No records have been found in the region, but the sequence diversity is low,
suggesting a recent incursion. It is not clear if P. australiana evolved in
Australia or elsewhere, or if it is a native or introduced taxon.
These findings are discussed below in relation to the existing knowledge on Plutella
taxa in Australia including New Zealand.
4.1 CO1 ‘barcode’ analysis of Australian and New Zealand Plutella taxa
This study confirmed the separation of P. xylostella and P. australiana into two taxa
using CO1 ‘barcode’ sequence data from both adults and larvae. Both maximum
likelihood and Bayesian analyses showed similar topologies, forming two distinct well
supported clades (100%) for P. australiana and P. xylostella, and confirming them as
two separate taxa. Among adults the interspecific divergence of the CO1 ‘barcode’
sequence data between the two taxa was on average 9% while intraspecific variation
within both taxa was 1.3%. Among larvae, the interspecific divergence of the CO1
‘barcode’ sequence data between the two taxa was of average 8.3% while intraspecific
variation within P. xylostella and P. australiana were 1.3% and 0.3% respectively,
though the number of specimens of P. australiana was not as high as for P. xylostella.
Hebert et al. (2003) suggested that in lepidopterans a value closer to 3% in the CO1
sequence divergence represents a new species. The Landry and Hebert (2013) supports
this, so the interspecific divergence of the current study (9% and 8.3%) shows P.
xylostella and P. australiana as two distinct taxa. Similarly, Perry et al. (2017) showed
a high genetic divergence between P. australiana and P. xylostella. These findings
were consistent with other studies on mosquitoes (Ruiz-Lopez et al., 2012) and flies
(Renaud et al., 2012) which have reported the existence of new taxa using CO1
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barcode sequence divergence. However, the identity of P. xylostella and P. australiana
as separate species, and their capacity to interbreed remains unresolved, requiring
further molecular studies using nuclear markers and mating compatibility studies.
Many insect studies have used the mitochondrial cytochrome oxidase 1 marker to
examine population genetics, taxonomy and evolution (Chang et al., 1997; Landry et
al., 1999; Miller et al., 2015; Schmidt et al., 2015) due to its high genetic variability
(Simon et al., 1994). However, there are arguments against using mitochondrial DNA
alone in identifying or describing new species because it is haploid and maternally
inherited with its rate of evolution inconsistent within and between species (Rubinoff
et al., 2006; Will et al., 2005; Will & Rubinoff, 2004). A study of Phyciodes butterflies
demonstrated that using mitochondrial DNA alone resulted in failure to correctly
identify species (Wahlberg et al., 2003). Nuclear (RAD seq) markers have recently
been used to identify different taxa (Perry et al., 2017) and may be a potential nuclear
marker for future studies on Plutella taxa. However, mating compatibilities between
P. australiana and P. xylostella also needs to be examined to confirm the species status
of P. australiana.
The status of P. australiana as originating in and endemic to Australia is not clear. The
estimated rate of divergence at the CO1 locus in insects was reported as 2% per million
years (Brower, 1994; Juan et al., 1995). Based on this estimate and the results of this
study (interspecific variation of 9% between the two taxa), P. australiana and P.
xylostella diverged about 4.5 million years ago, prior to the introduction of P.
xylostella approximately 120 years ago. The low level of variation of P. australiana
CO1 sequences also suggests very limited gene flow among populations in Australia,
and possibly recent introduction from elsewhere. Most Plutella spp. collections
examined by Landry and Hebert (2013) were collected between 2004 and 2012.
However, one museum pinned specimen examined, collected from NSW in 1971 by
V. J. Robinson, was identified as P. australiana and therefore suggests the occurrence
of P. australiana in Australia from at least that time.
A study using six microsatellite loci did not show any variation among Australian
populations of P. xylostella across 17 locations (Endersby et al., 2006), and concluded
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that the species had been recently introduced into Australia. However, Pichon et al.
(2006) conducted a regional study of allozyme variation and found significant genetic
differentiation among Australian populations of P. xylostella. Saw et al. (2006)
examined haplotype frequencies and suggested that Australian populations may be the
result of migration from northern regions of south-eastern Asia. Similarly, Talekar and
Shelton (1993) suggested a possible migration of P. xylostella to the Southern
Hemisphere including Australia. However, Juric et al. (2017) suggested a recent
introduction of P. xylostella to Australia, New Zealand and North America from a
highly variable European population and found that Australian population clustered
with New Zealand haplotypes, and that two independent lineages were found to have
arisen from Australia and New Zealand groups, one of them an entirely Australian
haplotype.
Given the low levels of mitochondrial DNA divergence among populations of P.
australiana found in the current study, it is unlikely that it is an Australian native
species, but rather a recent introduction. Further examination of the phylogeography
of P. australiana is required to determine if a migration pathway into Australia, similar
to that of P. xylostella, has occurred or if it may have evolved in Australia.
4.2 Diagnostic morphological features
While Landry and Hebert (2013) proposed that a number of morphological
characteristics could be used to distinguish the two Australian taxa, the majority of
these features were found not to be reliable in differentiating adults of the two taxa.
Two reliable diagnostic features to differentiate the taxa were found in females:
examination of the curvature of the tubular projection in P. australiana and raised
folds surrounding the antrum in P. xylostella. In males, species identification using
differences in the appearance of the vinculum saccus (slender in P. australiana) was
found to be an unreliable diagnostic feature. Similarly, the shape of the ventro-distal
margin (rounded in P. australiana) and the slight sinuation in the ventral margin of P.
australiana were found to be unreliable diagnostic features. Other features described
by Landry and Hebert (2013) such as teguminal processes (ventral view), the zone of
spiniform setae of the valva in males and the extension of the antrum projection beyond
83
the posterior margin of S7 (ventral view), ductus bursae in females still require further
examination. While all the genitalia measurements in this study were taken in their
lateral position, except the phallus in males, measurements of the features such as
vinculum saccus width in males and whole width of sternite 7 in females in their
ventral position would be interesting to examine if they give different results.
In males, the presence of individuals with intermediate and opposing characteristics of
the vinculum saccus does not support the use of it as a diagnostic feature. This study
found three male individuals (two field collected adults and one individual reared from
larvae collected on field mustard) which showed opposing results for molecular and
morphological identifications. These results seen only in males may suggest the
possibility of mating compatibilities between the two taxa where P. australiana males
mate with P. xylostella females and progeny produced have P. australiana genitalia
morphology but P. xylostella mitochondrial DNA or vice versa.
Descriptions of morphological characteristics of Plutella species such as genitalia
characteristics, wing venation, larval characteristics, pupal characteristics and adult
external morphology of male and females (Clarke, 1971; Dugdale, 1973; Moriuti,
1986; Robinson & Sattler, 2001) have been used to identify Plutella species in other
countries such as Hawaii and New Zealand. The observation on a curved tubular
projection described by Dugdale (1973) in a single female collected from New Zealand
suggests that P. australiana may be present in New Zealand but this requires further
examination. Furthermore, Dugdale reported that the dark brown or blackish antennal
integument used to distinguish P. xylostella from P. antiphona in New Zealand, and
that the wing venation characters were variable in Australian populations, suggesting
the presence of variants in Australia at the time. Interestingly, there were no significant
differences in morphology between Australian and New Zealand P. xylostella except
a slight difference in the sternite 7 length (longer in Australian P. xylostella). This may
indicate that a single widespread invasion occurred to both countries (see Juric et al.
(2017)).
Measurements of morphological features between adults caught in light traps and
adults reared from field collected larvae and reared under laboratory conditions,
exhibited morphological differences in both females and males. These findings suggest
84
that some morphological features vary depending on rearing conditions. Similarly,
progeny of adults of one crop may differ from the adults from different crops. These
findings suggest that adults collected from the field are suitable for taxonomic studies.
The current study provides the first comprehensive, comparative analysis of genitalia
measurements in P. australiana and P. xylostella. Overall, the findings indicate that
most morphological characters examined here have limited capacity to differentiate
both taxa in the absence of molecular data.
4.3 Summary and conclusion on the taxonomy of P. xylostella and P. australiana
Like Landry and Hebert (2013), the current study found that the CO1 barcode can be
used to identify P. australiana. and P. xylostella. However, analysis of morphological
features in the current study were not reliable in identifying the two taxa, except for
the two morphological diagnostic features in females. These two features were
consistent with the CO1 data and can therefore be used to identify female P.
australiana from P. xylostella in future studies. This study has shown that three male
characteristics (appearance of the vinculum saccus, valva structure at the ventro-distal
margin and the ventral margin) and all male characteristics tested in this study and
used by Robinson and Sattler (2001) and Landry and Hebert (2013) for species
identification were not diagnostic. The ventro-distal margins described in Dugdale
1973 requires further examination.
Plutella xylostella taxonomy has been largely based on morphological characteristics
such as genitalia features, wing venation, larval characteristics, pupal characteristics
and adult external morphology of male and females (Baraniak, 2007; Clarke, 1971;
Dugdale, 1973; Moriuti, 1986) and one study that examined the measurements of
external morphology of Plutella populations in India (Chacko & Narayanasamy,
2004). However, there is evidence that sole reliance on morphological characters may
be misleading, as has been found in the current study. For example, environmental
conditions (such as temperature, humidity, light conditions and diets) can affect
morphological development and lead to variation in morphological characters. Several
studies have confirmed that diet influences the developmental and reproductive
parameters in Plutella (Begum et al., 1996; Muhamad et al., 1994; Sarfraz, Dosdall, &
Keddie, 2010; Shelton & Nault, 2004; Talekar & Shelton, 1993; Yamada, 1983). In
85
the current study, it has been shown that laboratory conditions have impacted the
development of larvae leading to morphological differences in both females and males.
Therefore, it is necessary that an integrated morphological and molecular framework
is used to re-evaluate and resolve the systematic relationships of Plutella species
worldwide.
4.4 Distribution, host range and pest status of the two taxa
4.4.1 Distribution
This study has shown that P. australiana occurs sympatrically with P. xylostella in
most locations in Queensland, New South Wales and Tasmania. While the presence of
P. australiana in QLD and NSW further confirms the findings of Landry and Hebert
(2013), the presence of P. australiana in Tasmania has been confirmed in this study.
In the current study, P. australiana was not found to occur in New Zealand but a more
extensive examination is required because of a number of records which suggest its
possible presence. For example, Dugdale (1973) observed a curved tubular projection
in a single female specimen and three female specimens collected from Ivercargill,
Boulder Bank, Nelson in New Zealand in 1907 and one from Fiordland collected
before 1910 were observed to have morphological characteristics of P. australiana.
This was based on the S7 oval sclerotisation with no raised pair of folds and the antrum
projection in the side view (curvature and tubular projection projecting for less than
1/3 its length beyond S7) (personal communication from John Dugdale, Senior
Entomologist, New Zealand). However, specimens used in this study collected from
New Zealand between 2008 and 2016 were entirely P. xylostella. As this study has
shown, several of the morphological features identified by Landry and Hebert (2013)
cannot be used to differentiate the two taxa, but the curvature of the female tubular
projection is a consistent diagnostic feature, supporting the suggestion that P.
australiana may also be present in New Zealand (Dugdale, 1973). These findings
suggest that further examination of New Zealand populations should be conducted to
identify diversity within Plutella spp. Similarly, investigation of Australian specimens
from the Australian National Insect Collection (ANIC) may provide a good indication
of any early presence of P. australiana in Australia.
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The origins and distribution of P. australiana in the wider region are not known.
Plutella australiana has not been found in New Caledonia (2015) (personal
communication from Christian Mille, researcher in entomology, New Caledonia) and
further examination on other islands is required. All current available CO1 barcode
sequence data (NCBI, BOLD) for P. australiana are from Australian specimens.
Further studies on the regional distribution and relationships to other Plutellidae are
required.
4.4.2 Host preference and pest status of P. australiana
In the current study, host plants of P. xylostella were found to be cabbage, kale,
broccoli, swedes, cauliflower and the weed field mustard (Brassica rapa). Moreover,
they are known to damage canola crops and are considered to be a significant and
sporadic pest in canola (Furlong et al., 2008), recolonising winter canola crops in
southern and western regions of Australia (Perry et al., 2015).
This study is the first to describe the occurrence of P. australiana larvae on cabbage
(Brassica oleracea) and field mustard (B. rapa). Plutella australiana is known to occur
on canola, the cultivar of B. rapa, in South Australia (personal communication from
Michael Keller, Professor, Adelaide). Three P. australiana larvae were previously
found on Lincoln weed (Diplotaxis tenufolia, family Brassicaceae) in South Australia
(Perry et al., 2015; Perry et al., 2017). The occurrence of P. australiana larvae on
weedy field mustard in a kale field in Werombi (NSW) but not on kale itself, and the
frequent co-occurrence of both taxa in light traps but only detection of P. xylostella
larvae in the crop, indicate a host preference in P. australiana. Similarly, the detection
of P. australiana on Lincoln weed but not on nearby canola crops suggests that P.
australiana is not widely adapted to brassica crops (Perry et al., 2015). No P.
australiana larvae were found on cauliflower and swedes in Hobart (TAS) from the
same location where they were present in light traps, further supporting their host
preference for weeds. However, weeds in Tasmania were not explored in this study
and remain to be examined. Both taxa were found to co-occur on host plants found in
this study (cabbage and field mustard) with similar results reported from South
Australia where both taxa were found to co-occur on Lincoln weed (Perry et al., 2017).
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Plutella xylostella are also known to use wild crucifer hosts between brassica cropping
seasons (Harcourt, 1986; Talekar & Shelton, 1993; Yamada, 1983) but have clearly
adapted to use brassica crops as a host. Host preference and oviposition preference in
P. xylostella is known to be influenced by the nutrient value of host plants (Sarfraz,
Dosdall, Blake, et al., 2010) and on the amount of glossy leaf wax in the host plant,
which is a defense mechanism (Stoner, 1990). As an example, sulfur fertilizers and
nitrogen are known to promote egg laying in P. xylostella (Badenes‐Perez et al., 2010;
Furlong et al., 2013; Staley et al., 2010). Similarly, green leaf volatiles,
isothiocyanates, nitriles, dimethyl trisulfide, and terpenes emitted by cabbage and
canola (Girling et al., 2011; Kugimiya et al., 2010) may impact P. ausraliana host
preference. However, this need further examination and may help to identify their
target host plants.
Existence of P. xylostella on wild brassica crops as a refuge during the seasons which
are not suitable for cultivation has been reported earlier (Furlong et al., 2013; Harcourt,
1986; Sarfraz et al., 2006; Talekar & Shelton, 1993). As a solution, Sarfraz et al. (2011)
suggested that monitoring and controlling P. xylostella in weed species even before
crops are cultivated may help control this pest in the fields, and may also be the case
with P. australiana. Previous studies have reported the presence of DBM on different
host plants (Begum et al., 1996; Löhr & Rossbach, 2001; Robinson & Sattler, 2001)
so including host and native plants in Australia needs to be investigated to determine
whether P. australiana is a native species.
Overall, these findings suggest that P. australiana is an occasional or emerging pest
of brassica crops.
4.4.3 Summary and conclusions on the distribution, host range and pest status
of the two taxa.
Overall, these findings suggest that P. australiana is an occasional or emerging pest
of brassica cabbage crops. Preference of P. australiana for B. rapa suggests that canola
crops widely grown in South Australia and Western Australia are at risk. Reports of
P. australiana on canola, Lincoln weed and in light traps in South Australia already
show a threat to the canola industry in Australia.
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4.5 Pest management
Controlling P. xylostella through extensive usage of insecticides had led to a build-up
of insecticide resistance to almost all the available insecticides (APRD, 2012; Ridland
& Endersby, 2011; Sarfraz et al., 2005). All chemical insecticides have high mutagenic
power and their extensive use can increase the number of mutations and create
bottlenecks in DBM populations (Roux et al., 2007). For these reasons, integrated pest
management strategies which include chemical, biological and cultural control tactics
(Sarfraz et al., 2006) are best for effective control of P. xylostella. In particular, an
emphasis on maintaining natural enemies has been advocated with common biological
control agents including parasitoids, pathogenic fungi, bacteria and viruses. The
existence of P. australiana in Australia has made these applications more challenging
as their resistance to any of these is unknown. However, a recent study on the
frequency of mutations associated with pyrethroid resistance in Australian populations
showed that P. australiana were susceptible to insecticides (Perry et al., 2017).
As there is mounting evidence that P. australiana poses a possible risk to the
Australian canola industry, it is critical that the status of P. australiana as a native or
exotic species in Australia is clarified to avoid any delay in the release or registration
of new biological controls. As an example, PlxyGV isolates which are being
investigated as potential biological controls by QUT (Spence et al., 2016) will need to
undergo extensive risk assessments on non-target organisms and host specificity prior
to their release (BiosecurityAct, 2015).
4.6 Limitations and recommendations
This study adds to the existing knowledge of molecular, morphology, distribution and
host plant use of Plutella species in Australia and New Zealand. However, there is still
a significant knowledge gap regarding the life history of P. australiana and it presents
issues for biological control, pest management and potentially, market access for
Australian production.
A range of limitations were identified in this study. Firstly, to enable confirmation that
the two taxa are in fact different species, fixed differences at nuclear DNA loci need
89
to be demonstrated. This was not possible in the current study due to time constraints.
Secondly, while this study provides the most extensive examination of morphological
characters in Plutella species undertaken to date, morphological identification used
only the lateral view. This was due to the difficulty in examining the genitalia
structures in their ventral position when handling large numbers of specimens. It is
possible that characters which are diagnostic in identifying the two taxa may have been
revealed if the ventral position had been examined. Finally, the number of male and
female individuals used for morphological measurements varied because sex was not
known when the initial collections were made. This lead to a biased sample number
for males and females.
To expand the knowledge of P. australiana in Australia and other Plutella species in
other countries, future research must use a combination of both mitochondrial and
nuclear markers to examine the taxonomy of this species. Furthermore, morphological
features should not be used in isolation to identify new species because environmental
factors can influence morphology. A combined approach based on morphology and
molecular data will decrease the potential for error. Additionally, adult specimens
collected from the field are recommended for morphological examinations. Future
studies should sample DBM from a wider geographical range across Australia
(including WA and northern Australia). It is recommended that a broad range of both
crop and weed varieties are examined to further clarify the host range of P. australiana.
Such an investigation should also include a wide range of Australian native
Brassicaceae plants. Further sampling should also be undertaken in New Zealand and
other islands where it has been anecdotally recorded (example; Macquarie Island,
Christian Mille pers comm). A wider systematic analysis across multiple locations is
required to establish the origin of P. australiana.
90
91
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102
103
104
Appendices
Appendix A
Genbank accession numbers of the individuals taken for the phylogenetic analyses.
Accession number Species
KF370582 P. xylostella
KF370599 P. xylostella
KF370638.1 P. xylostella
KF370642 P. xylostella
KF370657 P. xylostella
KF370673 P. xylostella
KF370770.1 P. xylostella
KF370856 P. xylostella
KF370743 P. xylostella
KF370633 P. xylostella
KF370794 P. xylostella
KF370724 P. xylostella
KF370749 P. australiana
KF370728 P. australiana
KF370780 P. australiana
KF370791 P. australiana
KF370824 P. australiana
KF370833 P. australiana
KF370844 P. australiana
KF370849 P. australiana
KF370853 P. australiana
KF370854 P. australiana
KF370862 P. australiana
KF370868 P. australiana
HQ923231.1 Hyperxena scierana
KF808771.1 Plutella hyperboreella
KT140166.1 Plutella porrectella
105
Appendix B
Summary of female genitalia morphological measurement data of adults collected from light traps taken for the analyses.
Year of
collection Location Sample ID Molecular (CO1) Female measurements (μm)
Whole
length
(WL)
Whole
width
(WW)
Upper
part
length
(UPL)
Tubular
projection
length
(TPL)
Sternite 7
Length
(S7L)
2015 Werombi, NSW SYDW15_3 P. australiana 840.5 503.7 489.6 155.0 350.9
SYDW15_8 P. australiana 934.3 564.9 434.5 193.8 499.8
SYDW15_13 P. australiana 712.0 285.6 489.5 206.0 222.5
2015 Mowbray Park, 2015 M15_2_Nov P. australiana 837.8 469.2 390.3 158.1 447.6
M15_3_Nov P. australiana 829.6 520.2 338.3 176.8 491.3
M15_4_Nov P. australiana 736.1 462.4 253.3 195.5 482.8
M15_5_Nov P. australiana 758.2 455.6 343.3 212.5 414.9
M15_6_Nov P. australiana 719.1 464.1 307.7 161.5 411.4
M15_8_Nov P. australiana 654.5 464.1 334.9 180.2 319.6
M15_9_Nov P. australiana 695.3 448.8 334.9 197.2 360.4
M15_10_Nov P. australiana 717.4 498.1 316.2 173.4 401.2
M15_12_Nov P. australiana 661.3 445.4 289.0 170.0 372.3
M15_13_Nov P. australiana 770.1 508.3 372.3 156.4 397.8
M15_11_Nov P. xylostella 642.6 408.0 236.3 171.7 406.3
106
Appendix B Continued
2014 Birkdale, QLD BIR1 P. xylostella 668.1 496.4 273.7 231.2 394.4
2014 Hobart, QLD HOB3_12 P. xylostella 749.7 445.4 368.9 236.3 380.8
HOB3_13 P. xylostella 683.4 436.9 187.0 209.1 496.4
HOB3_14 P. xylostella 705.5 554.2 263.5 181.9 442.0
HOB3_18 P. xylostella 751.4 465.8 360.4 202.3 391.0
2014 Theresa Park, NSW SYDT1 P. xylostella 867.9 445.0 382.0 210.1 485.9
SYDT4 P. xylostella 799.6 368.6 376.6 188.3 423.0
SYDT6 P. xylostella 646.8 406.8 177.4 202.0 469.4
SYDT8 P. xylostella 968.8 513.3 431.2 245.6 537.6
SYDT11 P. xylostella 780.5 466.9 390.0 199.2 390.5
SYDT20 P. xylostella 674.9 481.0 236.3 251.6 438.6
2014 Werombi, NSW SYDW1 P. xylostella 734.1 431.4 215.6 188.3 518.5
SYDW2 P. xylostella 679.6 415.0 354.8 212.9 324.8
SYDW3 P. australiana 597.7 469.6 223.8 141.9 373.9
SYDW4 P. xylostella 780.5 466.9 311.1 232.0 469.4
SYDW5 P. xylostella 742.3 488.7 204.7 229.2 537.6
SYDW6 P. xylostella 698.7 447.7 155.6 234.7 543.1
SYDW7 P. xylostella 581.5 NA NA 199.2 NA
SYDW8 P. xylostella 698.7 538.0 199.2 240.2 499.4
SYDW9 P. xylostella NA 488.7 NA 191.0 NA
SYDW10 P. xylostella 780.5 431.4 267.5 199.2 513.1
107
Appendix B Continued
2015 Werombi, NSW SYDW15_6 P. xylostella NA 471.1 NA 151.0 NA
SYDW15_7 P. xylostella 854.8 540.4 381.5 220.3 473.3
SYDW15_9 P. xylostella 899.6 526.2 397.8 189.7 501.8
SYDW15_10 P. xylostella 956.8 505.8 438.6 NA 518.2
SYDW15_11 P. xylostella 818.0 328.4 322.3 236.6 495.7
SYDW15_12 P. xylostella 836.4 520.0 359.0 210.1 477.4
2016 New Zealand DBM1_5 P. xylostella 836.4 485.4 385.6 244.8 450.8
DBM1_6 P. xylostella 838.4 526.2 363.1 222.4 475.3
DBM1_9 P. xylostella 767.0 499.6 357.0 236.6 410.0
DBM1_11 P. xylostella 844.6 489.5 375.4 238.7 469.2
DBM1_12 P. xylostella 799.6 573.8 425.7 259.3 373.9
2013 New Zealand DBM4_1 P. xylostella 664.7 404.6 300.9 207.4 363.8
DBM4_6 P. xylostella 776.9 440.3 370.6 232.9 406.3
DBM4_11 P. xylostella 674.9 440.3 256.7 175.1 418.2
DBM4_17 P. xylostella 654.5 404.6 229.5 209.1 425.0
2013 New Zealand DBM5_11 P. xylostella 809.2 467.5 346.8 258.4 462.4
DBM5_16 P. xylostella 705.5 469.2 311.1 219.3 394.4
DBM5_17 P. xylostella 639.2 447.1 166.6 197.2 472.6
DBM5_21 P. xylostella 834.7 493.0 396.1 243.1 438.6
2008 New Zealand DBM9_1 P. xylostella 727.6 460.7 294.1 207.4 433.5
DBM9_3 P. xylostella 722.5 486.2 282.2 222.7 440.3
DBM9_4 P. xylostella 617.1 464.1 170.0 214.2 447.1
DBM9_5 P. xylostella 669.8 404.6 312.8 226.1 357.0
108
Appendix B Continued
DBM9_7 P. xylostella 639.2 452.2 193.8 173.4 445.4
DBM10_1 P. xylostella 822.8 472.6 470.9 212.5 351.9
DBM10_2 P. xylostella 710.6 430.1 283.9 178.5 426.7
DBM10_3 P. xylostella 674.9 474.3 272.0 210.8 402.9
DBM10_4 P. xylostella 765.0 413.1 375.7 192.1 389.3
DBM10_5 P. xylostella 678.3 481.1 272.0 205.7 406.3
DBM10_6 P. xylostella 790.5 467.5 374.0 234.6 416.5
DBM10_12 P. xylostella 816.0 477.7 448.8 249.9 367.2
109
Appendix C
Summary of male genitalia morphological measurement data of adults collected from light traps taken for the analyses.
Year of
collection Location Sample ID
Molecular
(CO1) Male measurements (μm)
Whole
length
(WL)
Valva
width
(VW)
Valva
length
(VL)
Phallus
length
(PL)
Vinculum
saccus
length
(VSL)
2014 Birkdale, QLD BIR2 P. australiana 1074.4 370.6 656.2 598.4 418.2
BIR4 P. australiana 1064.2 328.1 598.4 NA 465.8
BIR3 P. xylostella 989.4 294.1 625.6 503.2 363.8
BIR5 P. xylostella 1008.1 299.2 632.4 510 375.7
2014 Hobart, QLD HOB3_6 P. australiana 999.6 340 623.9 578 375.7
HOB3_10 P. australiana 972.4 329.8 584.8 540.6 387.6
HOB3_17 P. australiana NA 290.7 586.5 NA NA
HOB3_2 P. xylostella 980.9 280.5 678.3 484.5 302.6
HOB3_3 P. xylostella 972.4 312.8 591.6 562.7 380.8
HOB3_4 P. xylostella NA 300 588.2 489.6 NA
HOB3_5 P. xylostella NA 306 591.6 494.7 NA
2015 Theresa Park, NSW SYD15T_3_Oct P. australiana 982.6 341.7 635.8 593.3 346.8
SYD15T_21_Oct P. australiana 1020 316.2 642.6 617.1 377.4
SYD15T_22m_Oct P. australiana 1037 300.9 598.4 521.9 438.6
SYD15T_29_Oct P. australiana 952 292.4 571.2 532.1 380.8
110
Appendix C Continued
SYD15T_16m_Oct P. australiana 972.4 302.6 603.5 559.3 368.9
SYD15T_17m_Oct P. australiana 1004.7 338.3 608.6 557.6 396.1
SYD15T_30_Oct P. australiana 994.5 306 584.8 561 409.7
SYD15T_18m_Oct P. xylostella 972.4 324.7 598.4 494.7 374
SYD15T_19m_Oct P. xylostella 999.6 323 615.4 482.8 384.2
SYD15T_20m_Oct P. xylostella 1018.3 299.2 608.6 532.1 409.7
2014 Theresa Park, NSW SYDT17 P. xylostella 899.3 309.4 615.4 476 283.9
2014 Samford, QLD SAM1 P.xylostella 1033.6 360.4 612 515.1 421.6
SAM7 P.xylostella 986 273.7 652.8 428.4 333.2
SAM9 P.xylostella 1025.1 312.8 606.9 499.8 418.2
2016 New Zealand DBM1_1 P. xylostella 948.6 302.6 600.1 452.2 348.5
DBM1_2 P. xylostella 1054.35 312.12 632.2 493 422.15
DBM1_3 P. xylostella 1008.1 326.4 596.7 521.9 411.4
DBM1_4 P. xylostella 1004.7 333.2 627.3 489.6 377.4
DBM1_7 P. xylostella 1047.2 324.7 668.1 511.7 379.1
2016 New Zealand DBM3_1 P. xylostella 1030.2 368.9 666.4 518.5 363.8
DBM3_2 P. xylostella 989.4 326.4 613.7 511.7 375.7
DBM3_3 P. xylostella 1031.9 319.6 656.2 501.5 375.7
DBM3_4 P. xylostella 1048.9 312.8 661.3 496.4 387.6
DBM3_5 P. xylostella 962.2 309.4 603.5 479.4 358.7
DBM3_6 P. xylostella 1025.1 326.4 688.5 513.4 336.6
DBM3_7 P. xylostella 965.6 324.7 629 476 336.6
2013 New Zealand DBM4_3 P. xylostella 1052.3 321.3 632.4 503.2 419.9
111
Appendix C Continued
DBM4_4 P. xylostella 970.7 324.7 634.1 501.5 336.6
DBM4_5 P. xylostella 991.1 345.1 622.2 NA 368.9
DBM4_8 P. xylostella 906.1 297.5 552.5 487.9 353.6
DBM4_9 P. xylostella 955.4 346.8 586.5 498.1 368.9
2013 New Zealand DBM5_12 P. xylostella 977.5 345.1 630.7 520.2 346.8
DBM5_15 P. xylostella 936.7 299.2 601.8 499.8 334.9
DBM5_18 P. xylostella 967.3 278.8 555.9 527 411.4
DBM5_19 P. xylostella 974.1 326.4 608.6 510 365.5
DBM5_22 P. xylostella 941.8 312.8 612 484.5 329.8
DBM5_26 P. xylostella 963.9 316.2 617.1 498.1 346.8
2008 New Zealand DBM9_2 P. xylostella 882.3 292.4 562.7 443.7 319.6
DBM9_6 P. xylostella 863.6 300.9 544 481.1 319.6
DBM9_8 P. xylostella 938.4 268.6 562.7 457.3 375.7
DBM9_10 P. xylostella 841.5 256.7 535.5 450.5 306
DBM9_13 P. xylostella 975.8 278.8 613.7 491.3 362.1
DBM10_19 P. xylostella 975.8 299.2 605.2 508.3 370.6
113
Appendix D
Bipartition maximum likelihood (ML) tree with bootstrap values. The tree was
collapsed to remove low supported nodes (≥75%). For the full tree view go to:
https://1drv.ms/b/s!AjKrDFNSXOJphq1UQfDEnH0kiVYWgw
114
Appendix E
Bayesian analysis with posterior probabilities. The tree was collapsed to remove low
supported nodes (≥75%). For the full tree view go to:
https://1drv.ms/b/s!AjKrDFNSXOJphroJo2cRme38DE6gug
115
Appendix F
Male genitalia specimens showing sinuation in the ventral margin of the valva in (A) P. xylostella (specimen HOB4_1) and no sinuation in (B) P.
australiana (specimen TL15_4) which contradict the description in Landry and Hebert (2013).
A) B)
116
Appendix G
Summary of female genitalia morphological measurement data of adults reared from field collected larvae taken for the analyses.
Year of
collection Location Crop type Sample ID
Molecular
(CO1) Female measurements (μm)
Whole
length
(WL)
Whole
width
(WW)
Upper
part
length
(UPL)
Tubular
projection
length
(TPL)
Sternite
7 Length
(S7L)
2015 Theresa Park, NSW cabbage TL15_3 P. australiana 768.4 464.1 442.0 173.4 326.4
TL15_6 P. australiana 739.5 433.5 326.4 144.5 413.1
TL15_7 P. australiana 739.5 457.3 408.0 180.2 331.5
TL15_8 P. australiana 734.4 436.9 392.7 144.5 341.7
TL15_9 P. australiana 732.7 435.2 377.4 154.7 355.3
TL15_10 P. australiana 765.0 459.0 351.9 176.9 413.1
TL15_12 P. xylostella 661.3 375.7 367.2 147.9 294.1
TL15_19 P. xylostella 656.2 431.8 368.9 202.3 287.3
TL15_11 P. australiana 736.1 484.5 379.1 178.5 357.0
2015 Werombi, NSW weed (field mustard) WW15_3 P. australiana 727.6 421.6 416.5 147.9 311.1
WW15_4 P. australiana 751.4 453.9 402.9 147.9 348.5
WW15_6 P. australiana 659.6 455.6 306.0 158.1 353.6
WW15_8 P. australiana 812.6 442.0 445.4 158.1 367.2
WW15_2 P. australiana 759.9 455.6 363.8 149.6 396.1
117
Appendix G Continued
WW15_9 P. australiana 787.1 431.8 397.8 161.5 389.3
2014 Hobart, TAS cauliflower, swedes HOB4_3 P. xylostella 829.6 453.9 392.7 107.2 436.9
HOB4_4 P. xylostella 803.8 495.6 316.2 213.4 487.6
HOB4_5 P. xylostella 792.2 498.1 285.6 200.6 506.6
HOB4_6 P. xylostella 840.5 471.1 348.8 193.8 491.6
HOB4_7 P. xylostella 854.8 571.0 320.3 193.8 534.5
HOB4_8 P. xylostella 830.3 505.8 471.2 206.0 359.0
HOB4_9 P. xylostella 789.5 475.2 438.6 212.2 350.9
HOB4_10 P. xylostella 852.7 460.9 438.6 175.4 414.1
2015 Werombi, NSW kale WL15_6 P. xylostella 775.2 508.3 275.4 268.6 499.8
WL15_7 P. xylostella 963.4 546.1 423.0 272.9 540.4
WL15_8 P. xylostella 831.3 511.7 430.1 244.8 401.2
WL15_9 P. xylostella 922.5 500.9 510.0 226.7 412.5
WL15_10 P. xylostella 816.0 477.7 363.8 204.0 452.2
118
Appendix H
Summary of male genitalia morphology measurement data of adults reared from field collected larvae taken for the analyses.
Year of
collection Location Crop type Sample ID
Molecular
(CO1) Male measurements (μm)
Whole
length
(WL)
Valva
width
(VW)
Valva
length
(VL)
Phallus
length
(PL)
Vinculum
saccus
length
(VSL)
2015 Theresa Park, NSW cabbage TL15_4 P. australiana 935 290.7 557.6 545.7 377.4
TL15_5 P. australiana 940.1 331.5 527 533.8 413.1
TL15_13 P. australiana 984.3 304.3 593.3 543.7 391
TL15_14 P. australiana 960.5 317.9 552.5 NA 408
2014 Hobart, TAS cauliflower, swedes HOB4_1 P. xylostella 980.9 312.8 559.3 472.6 421.6
HOB4_2 P. xylostella 1033.6 317.9 639.2 481.1 394.4
HOB4_11 P. xylostella 1004.7 300.9 642.6 489.6 362.1
HOB4_12 P. xylostella 979.2 324.7 646 499.8 333.2
HOB4_13 P. xylostella 936.7 328.1 574.6 470.9 362.1
TL15_28 P. xylostella 846.6 307.7 525.3 450.5 321.3
2015 Werombi, NSW kale WL15_21 P. xylostella 938.4 309.4 705.5 460.7 232.9
WL15_22 P. xylostella 999.6 312.8 693.6 477.7 306
WL15_23 P. xylostella 952 299.2 627.3 482.8 324.7
WL15_24 P. xylostella 1011.5 294.1 598.4 479.4 413.1
WL15_25 P. xylostella 963.9 329.8 559.3 472.6 404.6
119
Appendix H Continued
2015 Werombi, NSW weed (field mustard) WW15_16 P. australiana 895.9 317.9 566.1 562.7 329.8
WW15_18 P. xylostella 890.8 261.8 503.2 516.8 387.6
WW15_17 P. australiana 1011.5 333.2 608.6 567.8 402.9
WW15_19 P. australiana 991.1 317.9 579.7 559.3 411.4
121
Supplementary Materials
Conference: Australian Entomological Society annual conference, 2015, Cairns,
Queensland.
122
Conference: International Congress of Entomology, 2016, Orlando, Florida.
123
Grants received
1) Australian Government’s National Taxonomy Student Travel Grant 2014-15 of the
Research Grant Programme (NTRGP) - Australian Biological Resources Study
(ABRS) – The amount of AUD 825 was used to travel to the AES conference and
the registration of the same conference.
Membership of professional societies
1. Australian Entomological Society.
2. Entomological society of America.
