ARTICLE

Helminth Parasites of the Wood Frog,

Lithobates sylvaticus, in Prairie Pothole Wetlands

of the Northern Great Plains

Eric E. Pulis & Vasyl V. Tkach & Robert A. Newman

Received: 8 September 2010 /Accepted: 6 May 2011# Society of Wetland Scientists 2011

Abstract Parasites are ubiquitous members of biotic

communities. Because their persistence and abundance is

closely tied to other taxa and numerous environmental

factors, information about parasite diversity may provide

unique insights into ecosystem health. Parasites may also

impact host health and population dynamics. Because they

are relatively inconspicuous, however, little is known about

parasite diversity and prevalence in specific host species in

most geographic regions. We sampled parasites from wood

frogs in the ecologically unique Prairie Pothole Region of

North Dakota, USA.Most frogs were infected with helminths.

We found a minimum of 7 species of trematodes, 3 of

nematodes, and 1 cestode. Two species had prevalence >50%:

the lung nematode Rhabdias bakeri and the trematode

Echinoparyphium rubrum. Helminth species richness ranged

from 0 to 6 taxa per host, with a median of 2. Total helminth

abundance within infected frogs ranged from 1 to 503

worms. Males caught during the spring breeding season

were infected with more taxa of helminths than either recent

metamorphs or frogs captured later in the summer. The total

abundance of helminths was also greater in breeding

frogs. These data provide the foundation for further

analyses of the ecology of amphibian-parasite interactions on

the Northern Plains.

Keywords Amphibian . Host-parasite interactions .

Larval-adult DNA matching . Prairie Pothole region

Introduction

Amphibians have been the focal hosts of numerous studies

of helminth parasites (Aho 1990; Sutherland 2005). Yet, Aho

(1990) noted that the parasite fauna of most amphibians

remains poorly known and our understanding of their

ecology is limited. Despite worldwide concerns about

amphibian declines (Stuart et al. 2004) and the potential

role of parasites (Johnson and Lunde 2005; Rohr et al.

2008a, b; Szuroczki and Richardson 2009), we still know

little about parasite spatial distribution, transmission, and

influence on amphibian population dynamics in most

ecosystems. Most of what we know about amphibian-

helminth interactions comes from a small but growing

number of experimental studies involving a few parasite

taxa and primarily larval amphibians (Szuroczki and

Richardson 2009), although a few studies focused on

recently metamorphosed amphibians (e.g., Goater 1994).

These studies have demonstrated some effects of parasitic

infection on some aspects of host fitness, with impacts often

dependent on interactions with other environmental factors

such as larval density and aspects of water quality (Goater

1994; Kiesecker and Skelly 2001; Belden 2006; Johnson et

al. 2007; Rohr et al. 2008a; Holland 2010; Koprivnikar

2010). There is thus reason to think that parasites might play

a role in amphibian population dynamics, at least under

some conditions.

Identifying effects of helminths on wild amphibian

populations is more difficult. Johnson et al. (2002) provided

a compelling case that one helminth species is associated with

limb malformations in nature, with effects varying among

E. E. Pulis :V. V. Tkach : R. A. Newman (*)

Department of Biology, University of North Dakota,

10 Cornell St. STOP 9019,

Grand Forks, ND 58202–9019, USA

e-mail: robert.newman@und.edu

Present Address:

E. E. Pulis

Gulf Coast Research Lab,

703 East Beach Dr.,

Ocean Springs, MS 39564, USA

Wetlands

DOI 10.1007/s13157-011-0183-6

host species. Most field studies of natural populations do not

directly document parasite impacts on host populations, but

rather test for relationships between occurrence of parasites

(prevalence, species richness, abundance) and ecological

context. Rohr et al. (2008b), for example, found that

exposure to agrochemicals was positively correlated with

the abundance of larval trematodes in leopard frogs

(Lithobates pipiens (Schreber 1782)) in Minnesota. Ecolog-

ical relationships are usually complex and depend on the

host and parasite species, the host community, and the

specific range and combination of environmental conditions

encompassed in the study (King et al. 2007; McKenzie

2007). In northern leopard frogs in Quebec, for example,

King et al. (2007) found the lowest parasite species richness

in agricultural wetlands, which they attributed to disruption

of transmission because of effects of land use on the

community of bird and mammal hosts. This finding is

consistent with the hypothesis that parasite diversity will be

greatest in healthy, intact ecosystems, where higher host

diversity supports a more robust helminth community,

leading to the notion that parasite community structure may

provide a useful indicator of ecosystem condition

(Hudson et al. 2006). With regard to host population

dynamics, these observational studies provide a necessary

assessment of the potential relevance of parasites in

amphibian ecology, if not direct evidence of an impact.

However, because natural systems are highly variable,

identifying general patterns and refining our knowledge of

the circumstances in which parasites are more abundant or

diverse requires field studies of additional host species,

geographic regions, and ecological conditions.

The Prairie Pothole Region of the north central plains of

North America (Stewart and Kantrud 1971) encompasses a

vast array of wetlands embedded in a landscape dominated

by large-scale agriculture (Kantrud 1983; Leitch 1989;

USDA 1996). It is a critically important region both

ecologically (e.g., waterfowl production, Batt et al. 1989)

and economically (e.g., crop production, USDA 1996;

North Dakota Agriculture 2008). The frequent juxtaposition

of intensive agriculture with breeding habitat for aquatic

species, including amphibians, creates the potential for

anthropogenic alteration of host-parasite interactions.

However, the region is heterogeneous in land use and

wetland characteristics, and thus provides a useful stage to

investigate the ecology of amphibian-parasite interactions in

an important natural system.

Several amphibian species are common in the eastern

portion of the Prairie Pothole Region in north-central North

Dakota. We focus here on the wood frog (Lithobates

sylvaticus = Rana sylvatica (Le Conte 1825), Anura:

Ranidae). Wood frogs are broadly distributed in North

America (Georgia to Alaska) and occur in a variety of

habitats (woodlands to tundra, Conant and Collins 1998),

making them a good candidate for comparative studies of

ecology and species interactions. Although the parasitic

worms of wood frogs have been documented in many

studies (e.g., Baker 1979; Muzzall and Peebles 1991;

McAllister et al. 1995), most surveys have been limited to

wooded habitats in the eastern portion of the range and

based on relatively small sample sizes. To our knowledge

no studies of wood frog parasites have been conducted in

the northern plains, including the Prairie Pothole Region of

North Dakota.

Wood frogs have been the subject of many other

ecological studies (Redmer and Trauth 2005). They are

medium-sized frogs that tend to breed in vernal ponds and

are terrestrial after metamorphosis, except during the early

spring breeding season. They are explosive breeders,

completing reproductive activity in a 1–3 week period on

the northern plains (Newman unpublished data). Distribu-

tion of adults during the summer depends on food

availability, adequate cover, and moisture (Redmer 2002).

Adults are carnivorous and prey primarily on insects,

although worms, snails, crustaceans, and spiders may also

be consumed (Davis and Folkerts 1986). They, in turn, are

preyed on by a variety of birds, reptiles, and mammals.

Like many amphibians, wood frogs are positioned centrally

in food webs and consequently may play an important role

in parasite transmission.

Perhaps the most important reason parasite ecology is

poorly documented in most systems is that parasites are

small, inconspicuous, and difficult to identify (Szuroczki

and Richardson 2009). Some, particularly trematodes

(flukes), have complex life cycles, requiring multiple hosts

at different points in the life cycle, thus compounding the

challenge of identification and understanding natural

history. Elucidating the role of parasites in amphibian, or

any host’s, ecology requires, at a minimum, precise

identification of parasite species and a detailed understanding

of life cycles. Without this foundation, it is impossible to

estimate accurately parasite prevalence, community structure,

habitat relations, or transmission dynamics. With limited prior

knowledge, the first step in understanding host-parasite

interactions, the impact of parasites on host populations, and

the ecological context of such impacts, is a careful and

accurate account of the parasite community. Our objectives in

the present study were (1) to conduct a comprehensive survey

of the helminth parasites of wood frogs in one portion of the

Prairie Pothole Region, and (2) to examine the temporal

dynamics of infection throughout the active season and across

host life stages to gain insight into the nature of host-parasite

interactions in this region. We also demonstrate a novel use of

molecular markers to identify larval parasite stages found in

amphibians by genetically matching them to morphologically

more readily identifiable adult stages found in other vertebrate

taxa that serve as definitive hosts. Through this approach, we

Wetlands

can elucidate the life cycle of the parasites and mechanisms of

transmission. Our work, then, will not only serve as the basis

for further investigations of amphibian – parasite interaction

in the Prairie Pothole Region, but also provide generally

useful information for helminth identification in other species

or regions and a greater understanding of parasite ecology in

northern prairie wetlands.

Methods

Frog and Parasite Sampling

The study was conducted in the glacial drift plain ecoregion

(Bryce et al. 1998) of northeastern Nelson Co., North

Dakota, USA. We collected 705 wood frogs by hand or dip

net from 84 different wetlands between April 7, 2004 and

April 18, 2006 (2004: N=303 frogs from 67 wetlands,

2005: n=265 from 57 wetlands, 2006: n=137 from 33

wetlands). Wetlands were contained within an area of

approximately 19 km2 and were included if calling males

were judged sufficiently numerous that year. Across the

3 year period, 34 wetlands were sampled in a single year,

27 wetlands were sampled in 2 years, and 23 wetlands were

sampled all 3 years. Sample sizes from included wetlands

ranged from one to 15 frogs per wetland per year

ðx ¼ 4:49! 2:89 SDÞ, yielding a 3 year combined total of

1–29 frogs per wetland ðx ¼ 8:39! 5:88 SDÞ.

During the spring breeding season (mid-April) only

males were captured. Through the summer months (May–

August) of 2004 and 2005 wood frogs, including females

and metamorphs, were captured opportunistically. After

capture the frogs were transported to the laboratory, where

mass and snout-vent length were measured. Frogs were

then euthanized, following an IACUC-approved protocol,

by topical application of benzocaine and immediately

necropsied. The gastrointestinal tract, lungs, kidneys, fat

bodies, urinary bladder, and leg muscles were carefully

searched for helminths using a dissecting microscope. Any

helminths found were identified and counted. Flatworms

were heat killed in water and nematodes were heat killed in

saline and fixed in 70% ethanol suitable for both morphological

and molecular studies. Whenever numbers allowed, some

specimens were placed in 95% ethanol for molecular analysis.

In order to identify larval stages of digeneans (flukes) found in

frogs by matching their sequences with sequences of adult

worms, we also collected adult digeneans from numerous

species of potential definitive hosts in the study area, using

sampling methods appropriate for each taxon, and under

necessary permits from North Dakota Game and Fish.

In addition, some animals were obtained from hunters.

Potential hosts included a garter snake (Thamnophis

sirtalis (Linnaeus, 1766)), tiger salamander (Ambystoma

mavortium (Baird, 1850)), raccoon (Procyon lotor (Linnaeus,

1758)), American badger (Taxidea taxus (Schreber, 1777)),

American mink (Neovison vison (Schreber, 1777)), long-tailed

weasel (Mustela frenata Lichtenstein, 1831), striped skunk

(Mephitis mephitis (Schreber, 1776)), and several species of

birds, including a ring-necked pheasant (Phasianus colchicus

(Linnaeus, 1758)), black-crowned night heron (Nycticorax

nycticorax (Linnaeus, 1758)), hooded merganser (Lophodytes

cucullatus (Linnaeus, 1758)) and various waterfowl or wading

birds that are known to prey on amphibians.

To examine possible effects of life stage, sex, and

phenology, the frog sample was divided into three groups.

The category Breeding Male includes those males caught in

April at breeding choruses. Metamorphs were young of the

year individuals still small enough to distinguish from

yearlings or older frogs based on size (in this area,

metamorphs often grow to a size similar to small breeding

males by the end of the summer, Newman unpublished

data). Metamorphs were not necessarily caught immediately

upon leaving their natal ponds, however. Summer Frogs

include any frog caught after the breeding season, including

both adults and possibly juveniles that were indistinguishable

from adults based on size (Schneider 2004). Summer Frogs

were separated by sex for some analyses. We pooled frogs

across all 3 years because helminth community structure and

species prevalence varied little among years, in general

(Pulis 2007).

We followed existing terminology in describing patterns

of parasitic infection whenever possible (Bush et al. 1997).

Prevalence refers to the percentage of frogs infected with a

given helminth. Mean intensity is mean number (i.e.,

abundance) of parasites of a particular species per infected

frogs. Total infection intensity is the total number of all

helminths infecting an individual frog. Species richness is

the total number of helminth taxa inhabiting an individual

frog. We tested for differences between sexes in parasite

species richness and total infection intensity with Mann-

Whitney tests (Zar 1999). Correlation analyses (Spearman’s

rank, rs) were used to test for relationships between frog

length or mass and helminth infection (total intensity and

species richness). To test for differences among categories

of frogs in helminth species richness and infection intensity

we used Kruskal-Wallis tests with post hoc Tukey’s HSD

comparisons among groups if overall group differences

were significant (Zar 1999). All statistical analyses were

conducted using Statistica version 6.0 software (Statsoft,

Inc. 2004).

Molecular Data

For larval digeneans, we attempted to match sequences of

metacercariae with those obtained from adult worms from

locally collected definitive hosts (snakes, birds and mammals).

Wetlands

Metacercariae are usually morphologically indistinguishable,

hence not identifiable to species. Lung nematodes Rhabdias

were identified using both morphological and molecular

characters (Tkach et al. 2006). Genomic DNA was extracted

from single metacercariae or single adult digeneans and

nematodes according to Tkach and Pawlowski (1999). DNA

fragments of approximately 1,350 base pairs at the 5′ end of

the nuclear 28S ribosomal DNA gene (including variable

domains D1-D3), and in some cases ITS region (ITS1 +

5.8S + ITS2) were amplified by PCR in an Eppendorf

Mastercycler thermocycler according to protocol described by

Tkach et al. (2003). Forward primers ITSf (5′- CGC

CCGTCGCTACTACCGATTG -3′), digl2 (5′-AAGCATAT

CACTAAGCGG-3′), and reverse primer 1500R (5′- GCTAT

CCTGAGGGAAACTTCG -3′) were used for amplification.

PCR products were cleaned-up using Qiagen QiaQuick kit

according to manufacturer instructions. PCR primers and

several internal primers were used for sequencing. Sequencing

reactions were prepared using BigDye chemistry and run on an

automated capillary sequencer ABI Prism 3100. Contiguous

sequences were assembled using Sequencher™ software

(GeneCodes Corp., ver. 4.1.4) and compared with our

sequences and sequences available in the GenBank using

BioEdit alignment software, version 7.0.1 (Hall 1999). Newly

obtained sequences of adult and larval digeneans were

submitted to GenBank: Telorchis bonnerensis Waitz, 1960

(JF820590–JF820593), Echinoparyphium rubrum (Cort,

1914) (JF820594–JF820596), Apharyngostrigea pipientis

(Faust, 1918) (JF820597–JF820598), Lechriorchis tygarti

Talbot, 1933, (JF820599–JF820604), and Alaria taxideae

Swanson et Erickson, 1946 (JF820605–JF820609).

Results

A total of 14,396 parasites belonging to at least 11 species

were recovered from 629 (89.2%) of the 705 wood frogs.

The majority of frogs harbored 1–3 taxa, with a maximum

parasite community within an individual frog of 6 taxa

(Fig. 1). For all frogs, the overall species richness of

parasites was 1.6±1.0 (SD) taxa. For just the infected frogs,

the mean parasite species richness was 1.8±0.9 taxa with a

mean infection intensity of 22.9±38.1.

Parasite Identification

Metacercariae of five digenean species belonging to five

different families were precisely identified by matching

their ribosomal DNA sequences with those of adults worms

collected from potential definitive hosts. Metacercariae of

E. rubrum (Echinostomatidae) matched sequences of adults

obtained from a ring-necked pheasant, as well as adults

obtained from chickens and ducklings experimentally

infected with metacercariae from frog kidneys. Metacercariae

of L. tygarti (Reniferidae) matched sequences of adult

digeneans of this species obtained from a common garter

snake in the study area. Metacercariae of T. bonnerensis

(Telorchiidae) matched sequences of adult digeneans

obtained from tiger salamanders in the study area. Meso-

cercariae of Alaria spp. (Diplostomidae) found in wood

frogs were a mix of at least two species. Due to problematic

morphological differentiation among mesocercariae belonging

to different species, we pooled them for statistical analysis.

Some of several sequenced specimens of mesocercariae from

frogs matched exactly the sequences from adult A. taxideae

obtained from a badger, a striped skunk, and a long-tailed

weasel in the study area. It should be noted, however, that at

least two additional species of Alaria can be found in

carnivore mammals in the study area. We did not have a

chance, however, to dissect any wild canids or other potential

hosts. In any case, Alaria is represented in wood frogs by at

least two species. Metacercariae of strigeids potentially might

also be a mix of more than one species. One of the sequenced

strigeid metacercariae matched the sequence of adult

Apharyngostrigea pipientis (Faust, 1918) (Strigeidae) obtained

from a black-crowned night-heron in the study area.

Structure of the Helminth Community

Parasite species differed substantially in their representation

in the sample. The most commonly encountered parasites

among all frogs were metacercariae of the trematode, E.

rubrum, and the lung nematode, R. bakeri, which were the

only taxa with prevalence greater than 50% (Table 1).

Fibricola sp. was the only other taxon found in more than

10% of frog hosts. Abundances of particular parasite

species found in a single host varied widely among hosts

and parasite taxa, with mean intensity of infection greatest

Fig. 1 Helminth species richness in individual wood frog hosts

collected from Prairie Pothole wetlands of northeastern North Dakota,

USA

Wetlands

Table

1Prevalance

(P)andmeanintensity

(MI)ofhelminthsfoundin

Lithobatessylvaticusfrom

fivedem

ographiccategories

from

2004to

2006in

NorthDakota

Helminth

Metam

orphs(72)

Summer

Frogs(163)

Summer

Fem

ales

(89)

Summer

Males

(70)

BreedingMales

(470)

AllFrogs(705)

P%

MI

P%

MI

P%

MI

P%

MI

P%

MI

P%

MI

N±SD

N±SD

N±SD

N±SD

N±SD

N±SD

Trematoda

Echinoparyphium

rubrum

51.4

28.8

41.7

18.3

36.0

16.0

47.1

20.9

53.6

21.9

50.6

21.9

37

±44.4

68

±31.5

32

±35.9

33

±28.5

252

±44.2

367

±42.1

Fibricola

sp.

0.0

0.0

11.7

8.8

13.5

9.3

10.0

8.0

21.3

14.5

16.8

13.6

0–

19

±8.5

12

±8.0

7±10.0

100

±29.2

119

±27.0

Alariaspp.

2.8

12.0

5.5

6.1

4.5

4.0

7.1

6.5

9.6

13.1

7.9

11.9

2±15.6

9±7.7

4±3.6

5±9.5

45

±26.1

56

±23.8

Apharyngostrigea

pipientis

1.4

1.0

1.8

9.0

1.1

9.0

2.9

9.0

5.7

3.0

4.4

3.5

1–

3±1

1–

2±1.4

27

±4.4

31

±4.5

Telorchisbonnerensis

9.7

3.0

1.2

1.5

2.3

1.5

0.0

0.0

4.5

12.5

4.3

9.6

7±2.4

2±0.7

2±0.7

0–

21

±17.5

30

±15.3

Lechriorchistygarti

1.4

1.0

1.8

3.0

2.3

4.0

1.4

1.0

1.7

1.8

1.8

1.9

2±0

3±3.5

2±4.2

1–

8±1.0

13

±1.8

Haem

atoloechuscomplexus

0.0

0.0

0.6

1.0

0.0

0.0

1.4

1.0

1.9

1.6

1.4

1.5

0–

1–

0–

1–

9±1.0

10

±1

Nem

atoda

Rhabdiasbakeri

36.1

2.1

63.2

4.3

69.7

4.0

52.9

5.5

83.2

8.1

73.8

7.1

26

±1.4

103

±4.4

62

±4.5

37

±4.2

391

±8.9

520

±8.2

Spiruridaesp.

0.0

0.0

1.2

4.5

2.3

4.5

0.0

0.0

3.2

1.7

2.4

2.0

0–

2±3.5

2±3.5

0–

15

±1.0

17

±1.6

Cosm

ocercoides

dukae

1.4

51.2

35

1.1

3.0

1.4

67

0.0

0.0

0.4

25.0

1–

2±45.3

1–

1–

0–

3±36.4

Cestoda

Mesocestoides

sp.

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.4

40

0.3

40.0

0–

0–

0–

0–

2±42.4

242.4

Total

81.4

23.0

80.4

15.6

83.1

12.4

75.7

20.3

95.1

25

89.2

22.9

51

±40.2

131

±27.6

74

±26.7

53

±28.1

447

±40.3

629

±38.1

N=totalnumber

ofinfected

frogsforeach

parasitetaxon,SD=1standarddeviation

Wetlands

for the trematodes E. rubrum, Fibricola sp., and Alaria spp.,

and two otherwise rare taxa, the nematode Cosmocercoides

dukae (Holl, 1928) and the cestode Mesocestoides sp.

(Table 1). Accordingly, the parasite community composition

within all wood frogs was dominated by trematodes, with E.

rubrum and Fibricola sp. accounting for the majority of

individual parasites (Table 2). The lung nematode R. bakeri

accounted for most of the remaining individual worms, with

other nematodes and cestodes making up only a tiny fraction

of the assemblage. Rhabdias bakeri larvae found in the

intestinal tract were not counted, because they are just

passing through. Only two taxa (25.6% of the community),

R. bakeri and Haematolechus complexus (Seely, 1905), were

represented by adult stages using frogs as definitive hosts.

For the other taxa, frogs act as intermediate or paratenic

hosts.

Frog characteristics influenced both the composition of

parasite communities and infection intensity. We found a

weak, but significant positive correlation between frog

length and helminth species richness, (rs=0.30, p=0.049,

df=705), but not frog length and total infection intensity,

(rs=0.136, p>0.05, df=705), frog mass and species

richness (rs=0.29, p>0.05, df=705), or frog mass and

total infection intensity (rs=0.166 P>0.05, df=705). The

parasite community was generally similar among frogs of

different categories, although there were some differences

in both community composition and parasite load which

were consistent with differences associated with size

(Tables 1, 2 and 3, Figs. 2 and 3). Over all frogs within

a category, we found 10 helminth taxa in breeding males,

the same number in summer frogs, and 7 taxa in

metamorphs (Table 3). Breeding males had a higher

percentage of infection by at least one parasite taxon

(95.1%), followed by summer frogs (80.4%) and then

metamorphs (70.8%). Host categories differed in parasite

community composition because Mesocestoides sp. was

found only in breeding males, C. dukae was found in all

groups other than breeding males, and several taxa,

principally Fibricola sp., were absent in metamorphs

(Tables 1 and 2).

Within individual frogs, helminth richness was greater in

breeding males than metamorphs and summer caught frogs

(Kruskal-Wallis H=27.7, p<0.001, Table 3, Fig. 2). Total

infection intensity was greater in breeding males than in

summer frogs but did not differ between metamorphs and

either group (Kruskal-Wallis H=55.1, p<0.001, Fig. 3).

Breeding males were infected by significantly more R.

bakeri (Kruskal-Wallis H=114.0, p<0.001, Fig. 3) and

Fibricola sp. (Kruskal-Wallis H=24.0, p<0.001, Fig. 3)

than either summer frogs or metamorphs. Summer frogs had

greater numbers of both of these parasites than metamorphs

(Fig. 3). There was no difference between male and female

Table 2 Contribution of parasite taxa to helminth community of frog groups as percentage of individuals of a taxa out of the total helminth

population

Helminth Parasite

Life State

Metamorphs

(72)

Summer

Frogs (163)

Summer

Females (89)

Summer

Males (70)

Breeding

Males (470)

All Frogs

(705)

% % % % % %

Community Community Community Community Community Community

Trematoda 96.4 73.7 71.9 74.8 70.9 73.2

Echinoparyphium

rubrum

Larval 90.8 61.0 55.8 64.1 49.3 54.3

Fibricola sp. Larval * 8.2 12.2 5.2 13.0 11.2

Alaria spp. Larval 2.0 2.7 1.7 3.6 5.3 4.6

Apharyngostrigea

pipientis

Larval 0.1 1.3 1.0 1.7 0.7 0.8

Telorchis

bonnerensis

Larval 1.8 0.1 0.3 * 2.4 2.0

Lechriorchis tygarti Larval 1.7 0.4 0.9 0.1 0.1 0.2

Haematoloechus

complexus

Adult * <0.1 * 0.1 0.1 0.1

Nematoda 4.8 26.1 28.0 25.8 28.4 26.2

Rhabdias bakeri Adult 4.7 22.3 26.7 18.9 28.2 25.5

Spiruridae sp. Larval * 0.4 1.0 * 0.2 0.2

Cosmocercoides

dukae

Adult 0.1 3.4 0.3 6.2 * 0.5

Cestoda * * * * 0.7 0.6

Mesocestoides sp. Larval * * * * 0.7 0.6

Total Helminths 1,173 2,045 916 1,073 11,178 14,396

Wetlands

frogs caught during the summer in helminth richness (Mann-

Whitney U=2784.5, p=0.23, Table 3) or total infection

intensity (Mann-Whitney U=2743.5, p=0.20). Male (n=24)

and female (n=43) metamorphs also did not differ in

helminth richness (Mann-Whitney U=439.0, p=0.30) or

total infection intensity (Mann-Whitney U=449.0, p=0.37).

Discussion

Overall Diversity of Wood Frog Helminth Communities

Our results provide a strong foundation for assessment of

parasitism in wood frogs of the Prairie Pothole Region of

northeastern North Dakota. The 12 parasite taxa we found

is the largest number recorded to date for wood frogs

(Bouchard 1951; Muzzall and Peebles 1991; McAllister et al.

1995; Yoder and Coggins 1996). Other studies, however,

typically involved much smaller samples collected at fewer

sites. For example, Muzzall and Peebles (1991), obtained 7

helminth taxa from 100 wood frogs in Michigan, Bouchard

(1951) recorded one helminth species from 46 wood

frogs in Maine, McAllister et al. (1995) found only 5

species of helminths in 13 wood frogs from Arkansas, and

Yoder and Coggins (1996) found 10 helminth species in

20 wood frogs from southeastern Wisconsin. The small

sample sizes of many studies make estimation and

comparison of levels of parasitism and parasite community

composition problematic. In addition, the majority of the

above studies overlooked most larval stages, which are

difficult to detect and identify. No more than three species

of larvae were reported in any of the previous studies.

More importantly, inadequate accounting of larval forms

leads to an underestimate of the role of amphibians in the

circulation of parasites of other animals that prey on frogs.

The only other survey of Lithobates parasites in the

northern plains involved northern leopard frogs in North

and South Dakota (Goldberg et al. 2001). Their sample is

most comparable to our summer frogs, because they do not

say if they included metamorphs in their work. Fewer

(74%) leopard frogs were infected with helminths com-

pared to our Summer Frogs (80.4%), although species

richness and mean intensity per infected frog were slightly

higher in the leopard frogs. Ten of the 17 helminth taxa

found in their leopard frogs were adults, compared to only

3 in our wood frogs. Most notably missing from Goldberg

et al.’s leopard frogs were echinostome metacercaria,

which were found in 41.7% of wood frogs and made up

61% of the total helminths. We have also found E.

rubrum in the leopard frogs at our study site (pers. obs.);

hence, this parasite is capable of infecting leopard frogs.

However, Goldberg et al. (2001) examined fixed frogs,

and it is more difficult to see tiny metacercariae in fixed

tissue. Overall, it seems that the trophic webs involving

frogs in the Northern Plains are similar enough among

locations to support ecologically similar communities

across individual sites and species of frogs. It is difficult,

however, to compare patterns across studies when parasite

Table 3 Frequency (%) distributions of helminth species richness within frogs of each category, and mean number of parasite taxa for each

category. The number in parentheses refers to the sample size for each frog category

# Taxa per frog Metamorphs (72) Summer Frogs (163) Summer Females (89) Summer Males (70) Breeding Males (470)

0 29.2 19.6 16.9 24.3 4.9

1 45.8 43.6 43.8 44.3 35.7

2 15.3 27.0 30.3 20.0 37.0

3 9.7 7.4 7.9 7.1 15.7

4 0 1.8 1.1 2.9 5.1

5 0 0.6 0 1.4 1.3

6 0 1 0 0 0.2

Mean ± SD 1.1±0.9 1.3±1.0 1.3±0.9 1.2±1.1 1.9±1.0

Total taxa 7 10 9 8 10

Fig. 2 Mean species (±1 SE) richness per frog group. B = breeding

males, M = recent metamorphs, S = adult-sized frogs caught during

the summer

Wetlands

inventories are incomplete and do not include all life

stages.

Helminth Communities, Host Life Stage, and Seasonal

Patterns

We divided our frog sample into demographic categories

partly to refine our understanding of patterns of parasitism,

but also because of unavoidable sampling biases associated

with these groups. Sampling was heavily skewed towards

breeding males because sex-ratio at breeding choruses is

strongly skewed toward males (Howard and Kluge 1985;

Crouch and Paton 2000); removing males also has much

less impact on populations. We did not sample females

during the breeding season. Summer frogs should provide a

less sex-biased view of the helminth component community

since all frogs were retained during this time.

Among all demographic groups, breeding males (indi-

vidually and collectively) had the highest helminth richness

and intensities of infection. One possible explanation for

this pattern is accumulation of parasites in older frogs,

assuming breeding males are older, on average, than

summer frogs and metamorphs. This also assumes parasites

can survive over winter in their freeze-tolerant hosts (Layne

et al. 1998), which has been confirmed for at least some

helminths (Woodhams et al. 2000). Breeding males and

summer frogs are obviously older than metamorphs, and

although breeding males were more heavily parasitized than

metamorphs, summer frogs were not. This phenomenon

requires further studies.

Greater richness of the component community (the

combined helminth species richness across all frogs in the

sample) in breeding males is most likely an artifact of their

much larger sample size resulting in recovery of rare

parasites. However, further explanation is required for the

higher parasite infracommunity richness (the helminth

species richness in individual frogs), overall prevalence,

and total infection intensity in breeding males than in the

other two groups, particularly in light of the observation

that summer males did not differ significantly from summer

females. The greater total infection intensity in breeding

males is primarily due to Alaria spp., Fibricola sp. and R.

bakeri. Rhabdias bakeri has a terrestrial stage in its life

cycle and begins to appear in the lungs about 20 days after

the metamorphs emerge. The trematode Fibricola sp.,

despite aquatic transmission, also was not detected until

after frog metamorphosis. A small sample of tadpoles and

recently emerged metamorphs yielded no Fibricola sp.,

although we found other larval trematodes. These observations

suggest a simple explanation for the higher parasite numbers in

adult frogs compared to metamorphs. In the case of Fibricola

(and other larval trematodes) transmission occurs in the water,

strongly suggesting that adult wood frogs are infected during

the breeding period, because they are terrestrial at other times.

This also explains the higher prevalence and intensity of

infection in breeding males, which remain in the water much

longer than females. Lower parasitism in summer males

versus breeding males might result from mortality of heavily

infected frogs, or loss of infections due to host immune

response. Our data do not allow us to discriminate between

these alternatives.

Studies of other amphibian species have also found

increases in parasitism following metamorphosis. Gillilland

and Muzzall (1999) showed that leopard frog metamorphs

accumulated helminths after they left the water through the

first year. King et al. (2008) found an increase in abundance

Fig 3 Mean (±1 SE) helminth

infection intensity per frog

group. Open bars are total

infection intensity, lightly

shaded bars are number of

Rhabdias bakeri per infected

host, and darker shaded bars are

number of Fibricola sp. per

infected host. B = breeding

males, M = recent metamorphs,

S = adult-sized frogs caught

during the summer

Wetlands

of nematodes in northern leopard frogs in Quebec from

shortly after metamorphosis until later in the season, but a

decrease in abundance of some trematodes, which they

suggested was due to mortality of more heavily infected

froglets. Because at least some of the parasites (Rhabdias,

Fibricola) only appear in young frogs after metamorphosis,

and species richness was higher in both adult groups

compared to metamorphs, helminth species richness clearly

increases in young frogs in the period between metamor-

phosis and attainment of adult size.

Potential Impacts of Helminths on Amphibian Demography

There is little direct evidence for impacts of most parasitic

infections on amphibian health in wild populations

(Sutherland 2005). However, several laboratory experiments

have found that echinostome infection can impair renal

function, increase mortality, or decrease growth, at least

in tadpoles (Martin and Conn 1990; Fried et al. 1997;

Holland et al. 2007) and under some environmental

conditions (Belden 2006; Holland 2010; Koprivnikar

2010). Echinoparyphium rubrum dominated the helminth

community of wood frog metamorphs in our study,

accounting for 90.8% of all worms in those frogs. Their

prevalence in metamorphs was 51.4%, similar in breeding

adult males (53.6%), but lower (41.7%) in summer caught

frogs. The reduction in prevalence from metamorphs or

breeding males to terrestrial summer-caught frogs suggests

that either the hosts (metamorphs and breeding males) are

able to eliminate the metacercariae through an immune

response or there is higher mortality of echinostome-

infected frogs, as seemed to be the case for Fibricola sp.

Rhabdias bakeri, the most common wood frog parasite

in our sample, has not been shown to cause adverse effects

in wood frogs metamorphs (Goater and Vandenbos 1997).

In Bufo bufo, Rhabdias bufonis lungworm infection has

been shown to reduce growth, locomotory performance,

and survival of post metamorphic toads under stress

(Goater and Ward 1992; Goater et al. 1993; Goater 1994).

In Bufo marinus in Australian, R. pseudosphaerocephala

infection reduces growth rates in the field and lab (Kelehear

et al. 2011). Because R. bakeri in natural conditions is

specific to wood frogs (Tkach et al. 2006) its pathogenicity

may be lower in comparison to more generalist Rhabdias

species.

Wood frogs in this area appear to serve primarily as

intermediate hosts of helminths. Based on the dominance of

larval forms of worms in the wood frog, and presence of the

adult worms in animals known to include frogs in their

diets (i.e. striped skunks, raccoons, tiger salamanders) the

wood frog appears to be an important carrier of parasite

larvae. Our observations support the point made by Esch

et al. (1990) that amphibians are important agents of

parasite transmission in ecological communities. However,

more research is required to determine the impacts of

parasites on wood frog health and population dynamics in

the Prairie Pothole Region.

Parasite Identification and Novel Ecological Insights

Accurate species identification is an essential first step in

estimating biodiversity of any ecosystem, comparing

locations or habitats, or understanding interactions among

species. In studies of host-parasite dynamics, species

identification and host associations are fundamental to

correct identification of transmission pathways. For exam-

ple, if we did not know that wood frogs and leopard frogs

host different species of Rhabdias (Tkach et al. 2006), we

might assume that the presence of one frog species would

influence parasite transmission to the other. Yet, accurate

identification of helminths and their host associations is a

challenging task, particularly for trematode larvae, which

provide few morphological characteristics on which to base

identification.

Matching DNA sequences of metacercariae found in

frogs to those of adult worms collected from potential

definitive hosts proved to be a useful method for accurately

identifying parasites infecting frogs in this area and

elucidating transmission pathways. One of the matches in

our study has provided a particularly interesting new insight

into the life cycle of the parasite. Telorchis bonnerensis

known from salamanders was synonymized by Macdonald

and Brooks (1989) with Telorchis corti Stunkard, 1915

parasitic in turtles, mainly based on experimental evidence

from Watertor (1967) that T. bonnerensis can reach maturity

in chelonians, which are specific hosts of T. corti.

Macdonald and Brooks (1989) neglected the fact that T.

corti reported from turtles are always fully developed,

while T. bonnerensis experimentally grown in turtles by

Watertor (1967) were small, contained very few eggs and

needed a long time to reach maturity. In contrast, T.

bonnerensis readily matures in tiger salamanders, reaching

larger size and producing numerous eggs. We have

experimentally infected snapping turtles Chelydra serpentina

(Linnaeus, 1758) hatched from eggs in the laboratory, with

metacercariae of T. bonnerensis from naturally infected

mollusks Physa. The mollusks were collected in the study

area from a pond where salamanders were heavily infected

with T. bonnerensis. Turtles are completely absent from the

study area, but tiger salamanders are common. Two to

4 week old turtles were successfully infected and digeneans

developed to normal size and contained numerous eggs.

However, in 1 year old turtles the digeneans developed

extremely slowly, were very small and only a few of them

were able to develop a few eggs. DNA sequences of T.

bonnerensis obtained from naturally infected tiger salaman-

Wetlands

ders and experimentally infected turtles matched each other,

but showed differences compared to sequences of adult fully

developed T. corti obtained from turtles in south-eastern

North Dakota. All these facts support the status of T.

bonnerensis as a separate species specific to salamanders,

but to a lesser extent capable of infecting chelonians. We

consider all Telorchis metacercariae found in wood frogs in

our study to be T. bonnerensis, especially taking into account

the lack of turtles in the study area. Only by matching DNA

sequences of metacercariae to adult worms, could we

determine the helminth life cycle and the transmission route

to wood frogs in this location. This example clearly

illustrates the importance of accurate identification both for

improved accounting of biological diversity as well as

elucidation of ecological interactions and highlights the

value of collaborations among ecologists and taxonomic

specialists.

Acknowledgments We are grateful to North Dakota EPSCoR (NSF

grant #EPS-0132289) and the Department of Biology for financial

support for this project. LeAnne Froese helped with frog necropsies

and Eugene Katsman assisted in processing specimens for molecular

analysis. We thank the editors and reviewers for feedback which we

hope resulted in greater clarity.

References

Aho JM (1990) Helminth communities of amphibians and reptiles:

Comparative approaches to understanding patterns and processes.

In: Esch GW, Bush AO, Aho JM (eds) Parasite communities:

patterns and processes. Chapman and Hall, New York, pp 175–

195

Baker MR (1979) Seasonal population changes in Rhabdias ranae

Walton, 1929 (Nematoda: Rhabdiasidae) from Rana sylvatica of

Ontario. Canadian Journal of Zoology 57:179–183

Batt BDJ, Anderson MG, Anderson CD, Caswell FD (1989) The use

of prairie potholes by North American ducks. In: van der Valk A

(ed) Northern prairie wetlands. Iowa State Universtity Press,

Ames, pp 204–227

Belden LK (2006) Impact of eutrophication on wood frog, Rana

sylvatica, tadpoles infected with Echinostoma trivolvis cercaria.

Canadian Journal of Zoology 84:1315–1321

Bouchard JL (1951) The platyhelminthes parasitizing some northern

Maine amphibia. Transactions of the American Helminthological

Society 70:245–250

Bryce S,Omernik JM, Pater DE, Ulmer M, Schaar J, Freeouf J, Johnson

R, Kuck P, Azevedo SH (1998) Ecoregions of North Dakota and

South Dakota. Northern Prairie Wildlife Research Center Online:

Jamestown, NDAvailable via http://www.npwrc.usgs.gov/resource/

habitat/ndsdeco/index.htm. (Version 30NOV1998)

Bush AO, Lafferty KD, Lotz JM, Shostak AW (1997) Parasitology

meets ecology on its own terms: Margolis et al. revisited. The

Journal of Parasitology 83:575–583

Conant R, Collins JT (1998) A field guide to reptiles and amphibians

of eastern and central North America, 3rd edn. Houghton Mifflin,

Boston

Crouch WB, Paton PWC (2000) Using egg mass counts to monitor

wood frog populations. Wildlife Society Bulletin 28:895–901

Davis MS, Folkerts GW (1986) Life History of the wood frog, Rana

sylvatica LeConte (Amphibia: Ranidae), in Alabama. Brimleyana

12:29–50

Esch GW, Shostak AW, Marcogliese DJ, Goater TM (1990) Pattern

and process in helminth parasite communities: an overview. In:

Esch GW, Bush AO, Aho JM (eds) Parasite communities:

patterns and processes. Chapman and Hall, New York, pp 1–19

Fried B, Pane PL, Reddy A (1997) Experimental infection of Rana

pipiens tadpoles with Echinostoma trivolvis cercaria. Parasitology

Research 83:666–669

Gillilland MG, Muzzall PM (1999) Helminthes infecting froglets of

the northern leopard frog (Rana pipiens) from Foggy Bottom

marsh, Michigan. Journal of the Helminthological Society of

Washington 66:73–77

Goater CP (1994) Growth and survival of postmetamorphic toads:

Interaction among larval history, density, and parasitism. Ecology

75:2264–2274

Goater CP, Vandenbos RE (1997) Effects of larval history and

lungworm infection on the growth and survival of wood frogs

(Rana sylvatica). Herpetologica 53:331–338

Goater CP, Ward PI (1992) Negative effects of Rhabdias bufonis

(Nematoda) on the growth and survival of toads (Bufo Bufo).

Oecologia 89:161–165

Goater CP, Semlitsch RD, Bernasconi MV (1993) Effects of body size

and parasite infection on the locomotory performance of juvenile

toads, Bufo bufo. Oikos 66:129–136

Goldberg SR, Bursey CR, McKinnell RG, Tan IS (2001) Helminths of

northern leopard frogs, Rana pipiens (Ranidae), from North

Dakota and South Dakota. Western North American Naturalist

61:248–251

Hall TA (1999) BioEdit: a user-friendly biological sequence alignment

editor and analysis program for Windows 95/98/NT. Nucleic

Acids Symposium Series 41:95–98

Holland MP (2010) Echinostome-induced mortality varies across

amphibian species in the field. The Journal of Parasitology

96:851–855

Holland MP, Skelly DK, Kashgarian M, Bolden SR, Harrison LM,

Cappello M (2007) Echinostome infection in green frogs (Rana

clamitans) is stage and age dependant. Journal of Zoology

271:455–462

Howard RD, Kluge AG (1985) Proximate mechanisms of sexual

selection in wood frogs. Evolution 39:260–277

Hudson PJ, Dobson AP, Lafferty KD (2006) Is a healthy ecosystem

one that is rich in parasites? Trends in Ecology & Evolution

21:381–385

Johnson PTJ, Lunde KB (2005) Parasite infection and limbmalformations:

a growing problem in amphibian conservation. In: Lannoo M (ed)

Amphibian declines: the conservation status of United States Species.

University of California Press, Berkeley, pp 124–138

Johnson PTJ, Lunde KB, Thurman EM, Ritchie EG, Wray SN,

Sutherland DR, Kapfer JM, Frest TJ, Bowerman J, Blaustein AR

(2002) Parasite (Ribeiroia ondatrae) infection linked to amphibian

malformations in the western United States. Ecological Mono-

graphs 72:151–168

Johnson PTJ, Chase JM, Dosch KL, Hartson RB, Gross JA, Larson

DJ, Sutherland DR, Carpenter SR (2007) Aquatic eutrophication

promotes pathogenic infection in amphibians. Proceedings of the

National Academy of Sciences 104:15781–15786

Kantrud HA (1983) An environmental overview of North Dakota: past

and present. Jamestown, ND: Northern Prairie Wildlife Research

Center Online. Available via http://www.npwrc.usgs.gov/resource/

habitat/envovrvw/envovrvw.htm. (Version 16JUL97)

Kelehear C, Brown GP, Shine R (2011) Influence of lung parasites on

the growth rates of free-ranging and captive adult cane toads.

Oecologia 165:585–592

Wetlands

Kiesecker JM, Skelly DK (2001) Effects of disease and pond drying

on gray tree frog growth, development, and survival. Ecology

82:1956–1963

King KC, McLaughlin JD, Gendron AD, Pauli BD, Giroux I,

Rondeau B, Boily M, Juneau P, Marcogliese DJ (2007) Impacts

of agriculture on the parasite communities of northern leopard

frogs (Rana pipiens) in southern Quebec, Canada. Parasitology

134:2063–2080

King KC, Gendron AD, McLaughlin JD, Giroux I, Brousseau P, Cyr

D, Ruby SM, Fournier M, Marcogliese DJ (2008) Short-term

seasonal changes in parasite community structure in northern

leopard froglets (Rana pipiens) inhabiting agricultural wetlands.

The Journal of Parasitology 94:12–22

Koprivnikar J (2010) Interactions of environmental stressors impact

survival and development of parasitized larval amphibians.

Ecological Applications 20:2263–2272

Layne JR Jr, Costanzo JP, Lee RE Jr (1998) Freeze duration influences

postfreeze survival in the frog Rana sylvatica. Journal Experi-

mental Zoolology 280:197–201

Leitch JA (1989) Politicoeconomic overview of prairie potholes. In:

Van der Valk AG (ed) Northern Prairie Wetlands. Iowa State

University Press, Ames, pp 2–14

MacDonald CA, Brooks DR (1989) Revision and phylogenetic

analysis of the North American species of Telorchis Luehe,

1899 (Cercomeria: Trematoda: Digenea: Telorchiidae). Canadian

Journal of Zoology 67:2301–2320

Martin TR, Conn DB (1990) The pathogenicity, localization and cyst

structure of echinostomatid metacercaria (trematoda) infecting

the kidneys of the frogs Rana clamitans and Rana pipiens. The

Journal of Parasitology 76:414–419

McAllister CT, Upton SJ, Trauth SE, Bursey CR (1995) Parasites of

wood frogs, Rana sylvatica (Ranidae), from Arkansas, with a

description of a new species of Eimeria (apicomplexa: eimeriidae).

Journal of the Helminthological Society of Washington 62:143–

149

McKenzie VJ (2007) Human land use and patterns of parasitism in

tropical amphibian hosts. Biological Conservation 137:102–116

Muzzall PM, Peebles CR (1991) Helminths of the wood frog, Rana

sylvatica, and Spring Peeper, Pseudacris c. crucifer, from

Southern Michigan. Journal of the Helminthological Society of

Washington 58:263–265

North Dakota Agriculture (2008) North Dakota Agriculture 2008. Available

via http://www.agdepartment.com/PDFFiles/agbrochure2008.pdf.

Accessed 25 Nov 2008

Pulis EE (2007) Ecology of wood frog (Rana sylvatica) parasites on

the northern plains. M.S. Thesis. University of North Dakota

Redmer M (2002) Natural history of the wood frog (Rana sylvatica) in

the Shawnee National Forest, Southern Illinois. Illinois Natural

History Survey Bulletin 36:163–194

Redmer M, Trauth SE (2005) Rana sylvatica LeConte, 1825. In:

Lannoo M (ed) Amphibian declines: the conservation status of

United States Species. University of California Press, Berkeley,

pp 590–593

Rohr JR, Raffel TR, Sessions SK, Hudson PJ (2008a) Understanding

the net effects of pesticides on amphibian trematodes infections.

Ecological Applications 18:1743–1753

Rohr JR, Schotthoefer AM, Raffel TR, Carrick HJ, Halstead N,

Hoverman JT, Johnson CM, Johnson LB, Lieske C, Piwoni MD,

Schoff PK, Beasley VR (2008b)Agrochemicals increase trematodes

infections in a declining amphibian species. Nature 455:1235–1240

Schneider KK (2004) Age structure of a northern prairie population of

wood frogs (Rana sylvatica). M.S. Thesis, University of North

Dakota

Statsoft, Inc (2004) STATISTICA version 6. Statsoft inc. Tulsa,

Oklahoma, USA

Stewart RE, Kantrud HA (1971) Classification of natural ponds and

lakes in the glaciated prairie region. Resource Publication 92,

Bureau of Sport Fisheries and Wildlife, U.S. Fish and Wildlife

Service, Washington D.C. 57pp

Stuart SN, Chanson JS, Cox NA, Young BE, Rodrigues ASL,

Fischman DL, Waller RW (2004) Status and trends of amphibian

declines and extinctions worldwide. Science 306:1783–1786

Sutherland D (2005) Parasites of North American frogs. In: Lannoo M

(ed) Amphibian declines: the conservation status of United States

species. University of California Press, Berkeley, pp 109–123

Szuroczki D, Richardson JML (2009) The role of trematode parasites

in larval anuran communities: an aquatic ecologist’s guide to the

major players. Oecologia 161:371–385

Tkach V, Pawlowski J (1999) A new method of DNA extraction from

the ethanol-fixed parasitic worms. Acta Parasitololgica 44:147–

148

Tkach VV, Littlewood DTJ, Olson PD, Kinsella MJ, Swiderski Z

(2003) Molecular phylogenetic analysis of the Microphalloidea

Ward, 1901 (Trematoda: Digenea). Systematic Parasitology 56:1–

15

Tkach VV, Kuzmin Y, Pulis EE (2006) A new species of Rhabdias

from lungs of the wood frog, Rana sylvatica: the last sibling of

Rhabdias ranae? The Journal of Parasitology 92:631–636

USDA (1996) America’s Northern Plains: An Overview and Assess-

ment of Natural Resources. U.S. Dept. of Agriculture, Natural

Resources Conservation Service. 16pp. Jamestown, ND: USA.

Northern Prairie Wildlife Research Center Online. Available via

http://www.npwrc.usgs.gov/resource/habitat/amnorpln/index.

htm. (Version 15AUG97)

Watertor JL (1967) Intraspecific variation of adult Telorchis bonnerensis

(Trematoda: Telorchiidae) in amphibian and reptilian hosts. The

Journal of Parasitology 53:962–968

Woodhams DC, Costanzo JP, Kelty JD, Lee RE Jr (2000) Cold

hardiness in two helminth parasites of the freeze-tolerant wood frog,

Rana sylvatica. Canadian Journal of Zoology 78:1085–1091

Yoder RH, Coggins JR (1996) Helminth communities in the northern

spring peeper, Pseudacris c. crucifer Weid, and wood frog, Rana

sylvatica Le Conte, from southeastern Wisconsin. Journal of the

helminthological Society of Washington 63:211–213

Zar JH (1999) Biostatistical Analysis, 4th edn. Prentice-Hall, Upper

Saddle River

Wetlands