Parasite effects on receivers in animal communication: Hidden impacts on behavior, ecology, and evolution.

Parasites exert a profound effect on biological processes. In animal communication, parasite effects on signalers are well-known drivers of the evolution of communication systems. Receiver behavior is also likely to be altered when they are parasitized or at risk of parasitism, but these effects have received much less attention. Here, we present a broad framework for understanding the consequences of parasitism on receivers for behavioral, ecological, and evolutionary processes. First, we outline the different kinds of effects parasites can have on receivers, including effects on signal processing from the many parasites that inhabit, occlude, or damage the sensory periphery and the central nervous system or that affect physiological processes that support these organs, and effects on receiver response strategies. We then demonstrate how understanding parasite effects on receivers could answer important questions about the mechanistic causes and functional consequences of variation in animal communication systems. Variation in parasitism levels is a likely source of among-individual differences in response to signals, which can affect receiver fitness and, through effects on signaler fitness, impact population levels of signal variability. The prevalence of parasitic effects on specific sensory organs may be an important selective force for the evolution of elaborate and multimodal signals. Finally, host-parasite coevolution across heterogeneous landscapes will generate geographic variation in communication systems, which could ultimately lead to evolutionary divergence. We discuss applications of experimental techniques to manipulate parasitism levels and point the way forward by calling for integrative research collaborations between parasitologists, neurobiologists, and behavioral and evolutionary ecologists.

Is there life after parasitism? Survival, longevity, and oogenesis in Acheta domesticus (Orthoptera: Gryllidae) infected with the hairworm, Paragordius varius (Phylum: Nematomorpha).

The costs parasites impose on hosts can lead to reductions in survival and fecundity, but few studies have evaluated the impacts after infection. Hairworms are parasites of terrestrial arthropods that are free-living in aquatic systems as adults. As parasitic juveniles, hairworms acquire nutrients from their definitive hosts, shifting resources away from host development to parasite growth. However, until now, only one study has examined survivorship of naturally infected hosts with hairworms. Using a different hairworm and host system, we conducted experimental infections to examine growth, survivorship, and egg production in virgin female Acheta domesticus infected with the hairworm, Paragordius varius. We found that infected crickets grew significantly less during hairworm development compared to sham-infected control crickets. After releasing their worms, infected crickets survived for 73 ± 32 days but had significantly shorter life spans by an average of 13 days compared to sham-infected control crickets. However, we found that 50% of previously infected crickets produced eggs after releasing their worms. Taken together, these observations suggest that female crickets infected with hairworms may experience less mortality than previous anecdotal evidence suggests. Finally, we discuss the definition of parasitoid and how it relates to nematomorphs, and we suggest that more field and laboratory research is required before suggesting hairworms are parasitoids.

Selected Wildlife Trematodes.

The trematodes are a species-rich group of parasites, with some estimates suggesting that there are more than 24,000 species. However, recent interests on the biology of trematodes of wildlife indicate that the taxonomic status and nomenclature of many of the previous species descriptions of wildlife trematodes throughout the world are confusing and difficult to decipher. In this chapter, we review work on selected trematodes of amphibians, birds, mammals, and their snail intermediate hosts, in the hope of providing a tool kit on how to study trematodes of wildlife. We provide a brief introduction to each group of wildlife trematodes, followed by some examples of the challenges each group of trematodes has relative to the goal of their identification and understanding of their biology and interactions with their wildlife hosts.

Distribution and reproductive plasticity of Gyrinicola batrachiensis (Oxyuroidea: Pharyngodonidae) in tadpoles of five anuran species.

Previous studies on Gyrinicola batrachiensis indicate that these pinworms have distinct reproductive strategies dependent on the development time to metamorphosis of their anuran tadpole hosts. In tadpoles of amphibian species with short developmental periods (a few weeks), female nematodes reproduce parthenogenetically, and only produce thick-shelled eggs used as transmission agents from tadpole to tadpole. In contrast, nematodes in tadpoles with longer larval developmental periods (months to years) reproduce by haplodiploidy, and females produce thick-shelled as well as autoinfective thin-shelled eggs. However, recent investigations on the haplodiploidy strain of G. batrachiensis indicate that plasticity exists in the ability of these nematodes to produce thin-shelled autoinfective eggs when these nematodes infect tadpoles of co-occurring amphibian species. Yet, little information is available on the potential mechanism for this reproductive plasticity because few co-occurring amphibian species have been examined for the reproductive strategies of these nematodes. Therefore, our goals were to document field host specificity and reproductive strategies of nematode populations in tadpoles of five co-occurring amphibian species that varied in their larval developmental periods. Additionally, we evaluated adult worm morphology from each infected amphibian species to assess any differences in worm development and reproductive strategy of pinworm populations in different amphibian species. Of the five amphibian species examined, four were infected with the haplodiploid strain of G. batrachiensis. Prevalence of G. batrachiensis ranged from a high of 83% in Acris blandchardi to a low of 15% in Pseudacris clarkii; whereas mean intensity was highest for Rana sphenocephala (10 ± 10.36) and lowest for Hyla chrysoscelis (3.23 ± 3.35). Prevalence appeared to be controlled by tadpole ecology and life history, while mean intensity appeared to be controlled by tadpole physiology and worm reproductive strategy, but not necessarily the developmental period of each anuran species. G. batrachiensis observed in long developing tadpoles of R. sphenocephala had high mean intensities and conformed to the haplodiploidy reproductive strategy with both male and female worms being present, and females produced thick-shelled and thin-shelled eggs. In contrast, tadpoles of A. blanchardi, H. chrysoscelis, and P. clarkii, which varied in their developmental times from long to short, had relatively low mean intensities and contained both male and female G. batrachiensis. However, female worms only produced thick-shelled eggs in these hosts. Importantly, morphological differences existed among female worms recovered from R. sphenocephala and female worms recovered from A. blanchardi tadpoles with long developmental periods. These data strongly suggest that when the haplodiploidy strain of G. batrachiensis is shared by tadpoles of different amphibian species, species-specific differences in interactions between these nematodes and their development in different amphibian host species have a strong influence on the reproductive plasticity of these nematodes.

Atrazine reduces the transmission of an amphibian trematode by altering snail and ostracod host-parasite interactions.

Trematodes are ubiquitous members of aquatic environments, have many functional roles in ecosystems, and can cause diseases in humans, livestock, and wild animals. Despite their importance and reports of parasite population declines, few studies have concurrently assessed the effects of aquatic contaminants on multiple hosts, multiple parasite life cycle stages, and on transmission-related host-parasite interactions. Here, we test the effects of environmentally relevant concentrations of the herbicide atrazine (0, 3, 30 µg/L) on the establishment and development of an amphibian trematode (Halipegus eccentricus) in a first-intermediate snail host (Physa acuta) and in a second-intermediate ostracod host (Cypridopsis sp.). Additionally, we test the interactive effects of atrazine and parasitism on snail and ostracod survival. Our results indicate that atrazine negatively affects trematode transmission by altering snail and ostracod host-parasite interactions. Although atrazine did not affect the survival of uninfected snails alone, atrazine acted synergistically with parasitism to reduce the longevity of infected snails. As a result, the number of cercariae (i.e., larval trematodes) produced by snails was 50.7 % (3 µg/L) and 14.9 % (30 µg/L) relative to controls. Atrazine exhibited direct negative effects on ostracod survival at 30 µg/L. However, when ostracods were also exposed to trematodes, the negative effects of atrazine on survival were diminished. Although infected ostracod survival remained high, trematode development was significantly reduced, resulting in reduced infectivity of metacercariae (i.e., nongravid adult cysts infective to definite host) to 32.2 % (3 µg/L) and 28.6 % (30 µg/L) relative to the controls. The combination of reduced cercaria production and reduced metacercarial infectivity in the 3 and 30 µg/L atrazine treatment groups reduced the net number of infective worms produced to 16.4 and 4.3 % (respectively) relative to the control. These results demonstrate the complex nature of pesticide effects on trematode infections and indicate that trematodes can affect their first- and second-intermediate hosts differently under different pesticide concentrations. Our work has broad implications for parasite transmission and conservation and provides a testable mechanism for understanding trematode population declines in contaminated wetlands.

Cryptic species of hairworm parasites revealed by molecular data and crowdsourcing of specimen collections.

Recognizing cryptic species promotes a better understanding of biodiversity, systematics, evolutionary biology, and biogeography. When cryptic species are disease-causing organisms, such as parasites, their correct recognition has important implications for the study of epidemiology, disease ecology, and host-parasite relationships. Freshwater nematomorphs (Nematomorpha: Gordiida) or hairworms, are an enigmatic yet fascinating group of parasites that are known to manipulate host behavior to aid transition from the parasitic phase, within terrestrial insects, to the free-living aquatic stage. Hairworm taxonomy has been hampered by a paucity of informative diagnostic characters and it has long been suspected that this group contains numerous cryptic species. Study of single hairworm species over large geographical areas has been difficult due to extremely rare encounters and unreliable methods of collecting adult worms. Here we report that by using crowdsourcing, citizen scientists have collected and submitted samples of Gordius cf. robustus from throughout its range in North America making its genetic study possible. Combined with our own collections, we examined samples from 28 localities within the USA; despite the collection of numerous hairworms from Canada and Mexico, G. cf. robustus were not collected outside of the contiguous United States. Mitochondrial CO1 genetic distances revealed that specimens grouped into 8 clades separated by 8-24.3%. In addition, molecular evidence from mitochondrial (CO1 and cytB) and nuclear (partial 28S, ITS1, 5.8S and ITS2) DNA suggests that these 8 clades are distinct species and that this group of species is paraphyletic, since the North American species G. attoni and the European species G. aquaticus and G. balticus group among the G. robustus lineages. Furthermore, there was a significant correlation between genetic (CO1) and geographic distance between the 8 Gordius species. This study demonstrates the value of involving the general public in biodiversity studies and highlights the feasibility of using the mitochondrial CO1 gene as a taxonomic marker for genetic barcoding and species identification within the phylum Nematomorpha.

Helminth community structure in tadpoles of northern leopard frogs (Rana pipiens) and Woodhouse's toads (Bufo woodhousii) from Nebraska.

Currently, little information is available on helminth community structure in tadpoles, the ephemeral, feeding, non-reproductive larva stages of anurans. We examined the helminth community structure in tadpoles of northern leopard frogs, Rana pipiens, and Woodhouse's toads, Bufo woodhousii, from a first-order stream in Western Nebraska. Specifically, we were interested in how species-specific difference in size, time to metamorphosis, and habitat partitioning among larval anurans affect their helminth community structure. From June-July 2009, we collected 30 tadpoles of northern leopard frogs, R. pipiens, and 50 tadpoles of Woodhouse's toads, B. woodhousii, from Cedar Creek, Keith County, NE, USA. The compound helminth community was dominated by trematode metacercarial stages (echinostomatids and plagiorchids) and the nematode Gyrinicola batrachiensis with only a single non-gravid adult trematode, Gorgoderina attenuata, present in low numbers. The helminth component communities were depauperate and isolationist in nature, with a maximum of four helminth species/types per component community. Although no species-specific helminth infections were observed, clear differences were found in overall abundance of nematode versus trematode infections among tadpoles of these two anuran species. The component community of tadpoles of R. pipiens was dominated by nematodes, whereas the component community of tadpoles of B. woodhousii was dominated by trematodes. Although differences in tadpole susceptibility of these two anuran species could not be ruled out, differences in the reproductive strategy of G. batrachiensis in tadpoles of R. pipiens and B. woodhousii and anuran species-specific habitats partitioning among tadpoles of these two anuran species were important factors in structuring their helminth communities. Our study indicates that time to metamorphosis (older tadpoles) play a significant role in structuring helminth communities of tadpoles within species at the infracommunity level with older tadpoles having higher helminth species richness and/or abundances. However, species-specific differences in anuran tadpole habitat partitioning outweighed the effects of differences in time to metamorphosis at the component community level. Finally, our study is the first to report deformities in hind limb development in a tadpole from Nebraska. Of the 30 northern leopard frog tadpoles collected, a single individual contained an extra hind limb and was infected with two plagiorchid metacercariae located in the musculature above the hind limbs.

Temporal occurrence and community structure of helminth parasites in southern leopard frogs, Rana sphenocephala, from north central Oklahoma.

Currently, little information is available about the temporal recruitment of helminth communities in amphibian hosts. We examined the helminth community structure and temporal recruitment of helminth parasites in southern leopard frogs, Rana sphenocephala. Specifically, we were interested in how host life history such as habitat, age and/or size, diet, sex, and temporal variation in abiotic factors (precipitation and temperature) were important in determining monthly infection patterns of helminth populations and communities in southern leopard frogs. From May to September 2011, 74 southern leopard frogs were collected from Teal Ridge in Stillwater Payne County, OK, USA. Sixty-nine (93 %) of 74 frogs were infected with 1 or more helminth species. During our collecting period, the average monthly temperature was lowest in May and highest in July, and monthly precipitation was highest in May and lowest during the first week of September. The component community consisted of 11 species of helminth, including 1 larval and 1 adult cestode, 2 larval and 3 adult trematodes, and 1 juvenile and 3 adult nematodes. Of the 1790 helminths recovered, 51 % (911) were nematodes, 47 % (842) were cestodes, and 2 % (37) were trematodes. There were significant differences in the total abundance and mean species richness of helminths acquired by skin contact or through frog diet in monthly component communities of southern leopard frogs. A positive correlation existed for percentage of all helminths acquired by skin contact and monthly precipitation (r = 0.94, P < 0.01). Conversely, a negative correlation existed for monthly precipitation and percentage of helminths acquired by diet (r = -0.94, P < 0.01). Our results indicate that abiotic conditions such as precipitation have a major influence on the avenues for and constraints on the transmission of helminths with life cycles associated with water/moisture or terrestrial intermediate/paratenic hosts and are important in structuring helminth communities of amphibian hosts.

The effects of the herbicide atrazine on freshwater snails.

Atrazine has been shown to affect freshwater snails from the subcellular to community level. However, most studies have used different snail species, methods, endpoints, and atrazine exposure concentrations, resulting in some conflicting results and limiting our understanding. The goal of this study was to address these concerns by (1) investigating the acute and chronic effects of atrazine on four species of freshwater snails (Biomphalaria glabrata, Helisoma trivolvis, Physa acuta, and Stagnicola elodes) using the same methods, endpoints, and concentrations, and (2) summarizing the current literature pertaining to the effects of atrazine on freshwater snails. We conducted a 48 h acute toxicity test with an atrazine concentration higher than what typically occurs in aquatic environments (1000 µg/L). Additionally, we exposed snails to environmentally relevant atrazine concentrations (0, 0.3, 3, and 30 µg/L) for 28 days and assessed snail survival, growth, and reproduction. We also summarized all known literature pertaining to atrazine effects on freshwater snails. The literature summary suggests snails are often affected by environmentally relevant atrazine concentrations at the subcellular and cellular levels. These effects are typically not transitive to effects on survival, growth, or reproduction at the same concentrations. Our acute exposures corroborate the general trend of no direct effect on snail populations as atrazine did not directly affect the survival of any of the four snail species. Similarly, environmentally relevant concentrations did not significantly affect the survival, growth, or reproduction of any snail species. These results indicate that, in the absence of other possible stressors, the direct effects of environmentally relevant atrazine concentrations may not be realized at the snail population level.

Damselflies (Zygoptera) as paratenic hosts for Serpinema trispinosum and its report from turtle hosts from Oklahoma, USA.

Third-stage larvae of the nematode Serpinema trispinosum (Leidy, 1852) were collected from the midgut of four of five species of adult damselflies (Zygoptera) from a non-irrigated restored semipermanent wetland located in Stillwater, Oklahoma, USA. Of the four infected damselfly species, prevalence and mean abundance was highest for the southern spreadwing, Lestes disjunctus australis Walker (10%, 0.2 ± 0.8) and lowest for the familiar bluet, Enallagma civile (Hagen) (2.5%, 0.04 ± 0.3); whereas mean intensities were lowest for the citrine forktail, Ischnura hastata (Say) (1.5 ± 0.5) and the eastern forktail, Ischnura verticalis (Say) (1.0 ± 0). This is the first record of larvae of S. trispinosum from damselflies. Serpinema trispinosum adults have been reported from 18 species of North and Central American freshwater turtles, whereas microcrustaceans such as copepods serve as intermediate hosts and snails, fish and amphibians serve as paratenic hosts in this nematode's life cycle. However, dietary studies of the 18 species of freshwater turtles reported as definitive hosts for S. trispinosum indicate that aquatic insects including damselflies are more commonly reported in turtle diets than are fish or amphibians. Additionally, unlike snails and amphibians, larval damselflies predominantly feed on microcrustaceans, and our observation of S. trispinosum infecting damselflies may reflect the importance of these insects as paratenic hosts. In the present study, we provide new host information and measurements for third-stage larvae of S. trispinosum from damselfly hosts along with measurements for adult male and female S. trispinosum from turtle hosts from Oklahoma, USA.

Comparative descriptions of non-adult stages of four genera of Gordiids (Phylum: Nematomorpha).

Freshwater hairworms infect terrestrial arthropods as larvae but are free-living in aquatic habitats as adults. Estimates suggest that only 18% of hairworm species have been described globally and biodiversity studies on this group have been hindered by unreliable ways of collecting adult free living worms over large geographical areas. However, recent work indicates that non-adult cyst stages of hairworms may be the most commonly encountered stages of gordiids in the environment, and can be used for discovering the hidden diversity of this group. Unfortunately, little information is available on the morphological characteristics of non-adult stages of hairworms. To address this problem, we describe and compare morphological characteristics of non-adult stages for nine species of African and North American gordiids from four genera (Chordodes, Gordius, Paragordius, and Neochordodes). Observations were made on the oviposition behavior of adult worms and morphological characteristics were recorded for egg strings, larvae and cysts using light and differential interference contrast microscopy and/or scanning electron microscopy. Our study indicates that three distinct types of oviposition behaviors and three distinct morphological types of egg string, larva, and cysts were present among the four genera of gordiids. Although species identification based on cyst characteristics was not always possible among different species of gordiids, cyst morphology was conserved among some genera and all clades of gordiids. More importantly, our work indicates that gordiid larval morphology can be used for predicting cyst morphology among other gordiid genera. The capability to identify and predict gordiid genera and/or clades based on cyst morphology will be useful for culturing gordiids in the laboratory from field collected cysts and these new techniques will undoubtedly allow others to discover new species of gordiids from around the world.

Novel techniques for biodiversity studies of gordiids and description of a new species of Chordodes (Gordiida, Nematomorpha) from Kenya, Africa.

We review recent advances in the use of non-adult gordiid cyst stages to locate gordiids over large geographical regions and new culturing techniques which can help overcome current difficulties in nematomorph biodiversity studies. Using these techniques, we collected a new species of gordiid as cysts in aquatic snails (Biomphalaria pfeifferi) from the Lake Victoria Basin, western Kenya, Africa and cultured them in the laboratory. We describe the adult free-living male and female worms using morphological (light and scanning electron microscopy) and molecular data as well as the life cycle, mating and oviposition behavior, egg strings, eggs, larvae, and cysts of this new species. Chordodes kenyaensis n. sp. belongs to a large group of African Chordodes in which simple areoles are smooth or superficially structured less so than "blackberry" areoles but contain filamentous projections. Present among the simple areoles are clusters of bulging areoles, crowned and circurmcluster areoles along with thorn and tubercle areoles. In the laboratory, worms developed and emerged within 53-78 days from three, species of laboratory-reared crickets exposed to cysts of this species. Adult male and female C. kenyaensis n. sp. initiated typical Gordian knots within hours to days of being placed together and males deposited masses of sperm on the cloacal region of females. Females began oviposition within a week of copulating and attached egg strings in a continuous zigzag pattern on small branches or air-hoses but never free in the water column. Larvae hatched within two to three weeks, and cysts developed in laboratory-reared and exposed snails within 14-24 days. Morphological characteristics of egg strings, eggs, larvae and cysts of C. kenyaensis were most similar to other gordiids in the genus Chordodes but differed morphologically from other gordiid genera for which similar information is available.

A METHOD FOR ISOLATING AND SEQUENCING TRYPANOSOME CELLS TO INVESTIGATE SPECIES ASSOCIATIONS IN MULTIPLE MORPHOTYPE INFECTIONS.

Trypanosome infections containing multiple morphologies have been described from all classes of vertebrates, including mammals, birds, non-avian reptiles, amphibians, and fish. These mixed infections make it challenging to evaluate trypanosome diversity, as it is not immediately clear whether the forms present in the bloodstream represent different species or a single pleomorphic species. Amphibians are common hosts for trypanosomes and are often infected by multiple trypanosome morphologies in the bloodstream. Based on morphological observations and life cycle studies, many authors have considered multiple trypanosome morphotypes found infecting the same frogs to be a single pleomorphic species. However, molecular evidence supporting pleomorphic trypanosome species in amphibians is lacking, primarily because linking sequence data to bloodstream trypanosome morphology in mixed infections is extremely challenging. Here we present methods to isolate individual trypanosome cells of 6 morphotypes from frog blood for nested PCR of the 18S rRNA and gGAPDH genes. Single trypanosome cells were isolated by dilution and 3 DNA extraction methods, and 5 nested PCR primer regimes were utilized to optimize amplification from very low starting concentrations. The success rates of extraction methods ranged from 29 to 50% with the use of a Direct PCR kit having the highest success rate. Although the success rate varied in the different combinations of extraction methods and primer regimes, multiple individuals of all 6 trypanosome morphotypes were sequenced for both genes in a novel way that links sequence data to cell morphology by observing isolated cells with a microscope before PCR amplification. All 6 morphologically distinguishable morphotypes coinfecting a frog were genetically distinct. The only other recent molecular study on amphibian trypanosomes also found genetic differences between morphotypes in multiple infections. Together these studies suggest that the occurrence of pleomorphism may be overestimated in amphibian trypanosomes. The methods presented here offer a promising solution to characterize trypanosome diversity within multiple morphotype infections.

MOLECULAR PHYLOGENETIC ANALYSIS OF GYRINICOLA BATRACHIENSIS (NEMATODA: OXYUROIDEA) LENDS SUPPORT TO THE MONOPHYLY OF THE GENUS AND THE RESURRECTION OF GYRINICOLINAE.

Gyrinicola Yamaguti, 1938, includes 6 species of oxyurid found within the intestinal tract of numerous, larval, anuran species in Europe, Asia, South America, and North America. The systematic placement and hierarchical treatment of the genus has shifted at least 5 times since its discovery; the group was first considered as its own family (Gyrinicolidae), then treated as a subfamily (Gyrinicolinae) of Cosmocercidae, then as a member of the Pharyngodonidae, followed by movement back to the Cosmocercidae, and finally a recent proposal suggested the resurrection of the Gyrinicolidae. Species in the genus vary widely in the morphology of the uterine tract, a characteristic often used to indicate membership in the genus, as it is tied to the reproductive mode. However, until recently very few genetic data were available to aid in the placement of this unique group of worms, and before this study to the best of our knowledge none existed for the North American species. To examine the monophyly and placement of the Gyrinicola we sampled populations of Gyrinicola batrachiensis across North America and screened them for genetic diversity using nuclear markers 18S and 28S. Phylogenies suggest at least 3 clades exist among the nematodes from North America and that these clades, alongside Gyrinicola japonica, form a well-supported group within Oxyuroidea. Further representation of Pharyngodonidae from other vertebrate classes may help clarify the relationship of this historical grouping to other members of the Oxyuroidea.

SEASONAL OCCURRENCE OF NEOECHINORHYNCHUS EMYDIS (PHYLUM: ACANTHOCEPHALA) IN THE FRESHWATER SNAIL, PLANORBELLA CF. P. TRIVOLVIS, FROM OKLAHOMA.

The acanthocephalan Neoechinorhynchus emydis has a complex life cycle and infects turtle, ostracod, and snail hosts. However, little information is available on the seasonal distribution or the effects of N. emydis on freshwater snail hosts. To address this, we examined the seasonal distribution and melanization of acanthocephalans in Planorbella cf. Planorbella trivolvis snails from a single location in north-central Oklahoma. Seasonally, prevalence of N. emydis was 0% during the winter, increased to 50% during the summer, and declined to 17% in the fall. Mean abundance exhibited more variation but generally followed a similar pattern as prevalence. More important, all acanthocephalans located within the head/foot region of snail hosts contained melaninlike pigment surrounding each worm, suggesting that snails were mounting an immunological reaction to infections with N. emydis. Snail shell diameter was greatest during the fall and decreased during the winter, indicating that larger or older snails were dying during the winter. However, because field-collected snails were commonly infected with trematodes, and snail size varied significantly with season, it was unclear whether the observed seasonal dynamics of acanthocephalan infections were a result of snail mortality resulting from snail age, parasitic infections, or a combination of factors. To control for these factors, we exposed laboratory-reared Planorbella cf. P. trivolvis snails to naturally infected ostracods in field cages for 5-wk intervals during the winter, spring, and summer. Data from snail-cage infections were consistent with the seasonal field survey such that N. emydis infections were highest in the summer (20%) and lowest (0%) in the winter, suggesting that snails were not ingesting infected ostracods during the winter. However, fewer of our laboratory-reared snails survived in field cages during winter than during spring and summer, suggesting that snails may die more often during harsh winter conditions. Finally, we conducted a laboratory survival experiment by testing the life span and egg production of field-collected snails of various sizes that were naturally infected with acanthocephalans or trematodes or both. Our snail-survival experiment indicated that snail size but not infection status with acanthocephalans or trematodes affected snail survival, with larger snails surviving a shorter amount of time than smaller snails. In addition, snails infected with trematodes laid significantly fewer eggs compared with uninfected snails or snails infected with acanthocephalans. However, we found no significant difference in the number of eggs laid by acanthocephalan-infected and uninfected snails. Although other abiotic factors still need evaluation, we suggest that the occurrence of acanthocephalans in snails throughout the year may be partially influenced by the abundance of infected ostracods that snails may be ingesting and snail population fluctuations during the year.

Field and Laboratory Observations on the Life History of Gordius terrestris (Phylum Nematomorpha), A Terrestrial Nematomorph.

To date, all free-living adult hairworms have been reported from aquatic habitats. However, in Oklahoma, a recently described gordiid, Gordius terrestris, is consistently encountered in terrestrial habitats. We found this gordiid species has a unique egg morphology, unlike that of any other hairworm species, with an outer shell separated by distinct space from a thick inner membrane surrounding the developing larva. Because of this unique egg morphology and the occurrence of free-living hairworms in terrestrial habitats, it was hypothesized that G. terrestris represents the first report of a hairworm species with a terrestrial life cycle. In this study, we observed thousands of free-living adult worms in terrestrial habitats such as wet lawns and underneath wet sod during the winter. We found evidence of worms mating in these terrestrial habitats, followed by female worms burrowing and ovipositing in the soil. In the laboratory, significantly more females burrowed in the soil than males, providing a plausible explanation for the extreme male-biased sex ratio observed for free-living worms found on wet lawns. Finally, we collected terrestrial earthworms infected with the cyst stage of this gordiid species in the field and confirmed those observations by infecting earthworms with G. terrestris larvae in the laboratory. Taken together, these observations strongly support the hypothesis that G. terrestris has a terrestrial life cycle.

Morphological and molecular characterization of adult hairworms (Phylum Nematomorpha) from Iceland and the Faroe Islands, and documentation of their non-adult stages and hosts.

No species of freshwater Nematomorpha have been described from Iceland, but they have been identified anecdotally. Recent surveys in Iceland using freshwater gastropods as biodiversity indicators resulted in the collection of adult free-living hairworms and their non-adult stages including cysts in snail paratenic hosts and juvenile worms in ground beetle definitive hosts. Additionally, specimens acquired from the Icelandic Institute of Natural History indicate nematomorphs are common in Iceland. A single specimen from the Faroe Islands National Museum represents a new species record. Our morphological and molecular characterization indicated all the samples belong to the species Gordionus wolterstorffii, a common nematomorph found throughout Europe. Also, we provide the first descriptions of the cyst stage for the genus Gordionus. Molecular phylogenetic analysis based on 10 species of Gordionus and one species of the closely related genus Parachordodes indicates that Gordionus is not monophyletic. Combining our morphological and phylogenetic investigations, we discuss the lack of clarity in diagnostic morphological characters and the need for additional global collections to clarify the taxonomy of Gordionus.

Lucilia bufonivora, Not Lucilia silvarum (Diptera: Calliphoridae), Causes Myiasis in Anurans in North America With Notes About Lucilia elongata and Lucilia thatuna.

In North America, until recently, all cases of anuran myiasis were attributed to Lucilia silvarum (Meigen) or Lucilia elongata Shannon. The latter species is exceedingly rare and its life history is unknown, but L. silvarum is common and was thought to be capable of being either parasitic or saprophytic in North America. Until recently, the anuran parasite Lucilia bufonivora Moniez was thought to be strictly Palearctic, but a study in 2014 has determined this species is established throughout southern Canada. In 2019, a study demonstrated, with molecular and morphological evidence, that two adult flies formerly identified as L. silvarum and reared from amphibian myiasis cases from Canada, are actually L. bufonivora. Although the mentioned study detected relatively high genetic distances with European L. bufonivora, the lack of evident morphological differentiation suggest that they are the same species. The current study examined 12 adult males and eleven adult females morphologically from three additional North American studies. Specimens were examined which had been identified as L. silvarum or L. elongata, and they all proved to be L. bufonivora. We now suspect L. silvarum is strictly saprophagous in North America like they are in the Palearctic Region. We also provide evidence that the pattern of myiasis differs between European and North American specimens.

MOLECULAR IDENTIFICATION OF JUVENILE NEOECHINORHYNCHUS SPP. (PHYLUM: ACANTHOCEPHALA) INFECTING OSTRACOD AND SNAIL HOSTS PROVIDES INSIGHT INTO ACANTHOCEPHALAN HOST USE.

The role of invertebrates in some acanthocephalan life cycles is unclear because juvenile acanthocephalans are difficult to identify to species using morphology. Most reports suggest acanthocephalans from turtle definitive hosts use ostracods as intermediate hosts and snails as paratenic hosts. However, laboratory studies of the life cycle suggest that ostracods and snails are both required hosts in the life cycle. To elucidate the role of ostracods and snails in acanthocephalan life cycles better, we collected 558 freshwater snails of 2 species, including Planorbella cf. Planorbella trivolvis and Physa acuta, from 23 wetlands in Oklahoma, U.S.A., and examined them for acanthocephalan infections. Additionally, we examined 37,208 ostracods of 4 species, Physocypria sp. (morphotype 1), Cypridopsis sp., Stenocypris sp., and Physocypria sp. (morphotype 2) for juvenile acanthocephalans from 2 wetlands in Oklahoma. Juvenile acanthocephalans were morphologically characterized, and the complete internal transcribed spacer (ITS) region of nuclear rDNA was sequenced from acanthocephalans infecting 11 ostracod and 13 snail hosts. We also sampled 10 red-eared slider turtles, Trachemys scripta elegans, and 1 common map turtle, Graptemys geographica, collected from Oklahoma, Arkansas, and Texas and recovered 1,854 adult acanthocephalans of 4 species. The ITS of 17 adult acanthocephalans of 4 species from turtle hosts were sequenced and compared to juvenile acanthocephalan sequences from ostracod and snail hosts from this study and GenBank to determine conspecificity. Of the 23 locations sampled for snails, 7 (30%) were positive for juvenile acanthocephalans in the genus Neoechinorhynchus. The overall prevalence and mean intensity of acanthocephalans in Planorbella cf. P. trivolvis and P. acuta were 20% and 2 (1-6) and 2% and 1 (1), respectively. In contrast, only 1 of 4 species of ostracods, Physocypria sp. (morphotype 1), was infected with larval/juvenile Neoechinorhynchus spp. with an overall prevalence of 0.1% and a mean intensity of 1 (1-2). Although 4 species of acanthocephalans infected turtle definitive hosts, including Neoechinorhynchus chrysemydis, Neoechinorhynchus emydis, Neoechinorhynchus emyditoides, and Neoechinorhynchus pseudemydis, all the ITS sequences from cystacanths infecting snail hosts were conspecific with N. emydis. In contrast, the ITS sequences from larval/juvenile acanthocephalans from ostracods were conspecific with 2 species of acanthocephalans from turtles (N. emydis and N. pseudemydis) and 1 species of acanthocephalan from fish (Neoechinorhynchus cylindratus). These results indicate that N. emydis infects freshwater snails, whereas other species of Neoechinorhynchus appear not to infect snail hosts. We document new ostracod and snail hosts for Neoechinorhynchus species, including the first report of an ostracod host for N. pseudemydis, and we provide novel molecular barcodes that can be used to determine larva, juvenile, and adult conspecificity of Neoechinorhynchus species.

A New Species of Gordionus (Nematomorpha: Gordiida) from the Rocky Mountains of New Mexico.

The 19 genera within the phylum Nematomorpha contain approximately 350 species. The cosmopolitan genus Gordionus Müller, 1926 contains about 58 species, 6 of which occur in the contiguous United States of America. Recently, 2 new Gordionus species were described from high-altitude streams within the southern Rocky Mountains, near Santa Fe, New Mexico. Here we describe another new Gordionus species, from a high-altitude stream in the southern Rocky Mountains, from near Taos, New Mexico. The sites consisted of temporary puddles and a small human-made stream at 3,175-3,250-m altitude in aspen/pine woodland. Gordionus lokeri n. sp. has 1 areole type, which varies in shape and size between and within body regions. Midbody areoles are elongated, polygonal, or triangular, shingled, with the raised side of the areole serrated. The interareolar space is narrow, containing few bristles. The male cloacal opening is surrounded inside and outside by narrow branching bristles that bifurcate or trifurcate deeply; the furcae then subdivide several times terminally. Adhesive warts are lacking. Genetic data, consisting of partial cytochrome c oxidase subunit I (COI) gene sequences, clearly separated G. lokeri n. sp. from other Nearctic species. This is the third Gordionus species described from high-elevation streams in the Rocky Mountains. It appears that this high-altitude habitat represents the preferential niche for numerous species of this genus, and thus future work should focus on describing gordiid diversity in other parts of the Rocky Mountains.