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Completing parts of trematode life cycles in the laboratory is a useful way to obtain experimentally infected hosts and identify how specific aspects of parasitism influence host ecology and behavior. However, a lack of knowledge about host specificity and other factors that influence prevalence can hamper those efforts. Echinostoma trivolvis lineage c is a genetically distinct member of the E. trivolvis species complex that is known only from DNA sequences from adult and larval stages recovered from naturally infected muskrats (Ondatra zibethicus) and the marsh pondsnail (Ladislavella elodes), respectively. We determined the effect of host species/morphotype, host size, and miracidial dose on the infection success in potential first intermediate hosts. In the laboratory, we exposed 2 freshwater snail species (L. elodes and Planorbella duryi) and a morphological variant of L. elodes (formerly known as Stagnicola reflexa) to 2 miracidia to determine first intermediate host use. Among these 3 snail groups, we also tested the effect of host size on infection success with 3 size classes (1-5 mm, 5-10 mm, and 10-15 mm). Within 1 host species, L. elodes, we compared the effect of 2 doses (2 and 5 miracidia) and 3 size classes on infection success. At a dose of 2 miracidia, rediae and cercariae developed within 1 host species, L. elodes, as well as the S. reflexa morphotype, although infection success varied according to host size. At a dose of 5 miracidia, infection success increased in small and medium-size L. elodes relative to the low dose group. Our results confirm the first intermediate host species observed in nature but indicate that prevalence is influenced by host species morphotype, host size, and parasite dose. To obtain more infected snails, our experiments suggest exposing small and medium-size L. elodes snails to 5 miracidia. This research encourages further use of E. trivolvis lineage c in the laboratory to explore aspects of host-parasite interactions such as parasite-modified behavior., (© American Society of Parasitologists 2024.)

Turtle acanthocephalans in the genus Neoechinorhynchus have complex life cycles and are thought to involve an ostracod intermediate host and a turtle definitive host. For Neoechinorhynchus emydis, an additional snail paratenic host is part of its life cycle. However, how snails become infected and whether other species of Neoechinorhynchus can be found in snails remains unclear. This study was conducted to understand the survival, development, and host use of 4 species of turtle acanthocephalans (Neoechinorhynchus chrysemydis, N. emydis, Neoechinorhynchus emyditoides, and Neoechinorhynchus pseudemydis) in their intermediate, paratenic, and definitive hosts. Using eggs recovered from adult acanthocephalans from naturally infected turtle hosts in Oklahoma, experimental infections were conducted in 2 species of ostracods and 1 species of freshwater snail. Ionomes also were generated for these acanthocephalans and their turtle definitive hosts to better understand resource competition. Upon ingestion by ostracods, eggs of all 4 species of acanthocephalans hatched in both ostracod species. However, no further acanthocephalan development occurred in Cypridopsis sp. ostracods, whereas all 4 acanthocephalan species grew and developed into infective juveniles over 28-32 days in Physocypria sp. ostracods. Of the 4 species of acanthocephalans, N. emydis grew faster early in ostracods and was the only species to survive and establish in snail hosts. This finding was supported by the observation of proboscis remnants in the feces of snails that ingested ostracods infected with N. chrysemydis, N. emyditoides, and N. pseudemydis. In contrast, when snails were exposed to eggs of all 4 acanthocephalan species, eggs were passed in the feces, resulting in no infections. The overall ionomic composition of adult acanthocephalans and their turtle hosts suggested that the hosts and parasites share host resources. However, specific elemental differences indicated that turtle acanthocephalans can accumulate heavy metals at concentrations higher than those in their hosts. Results of this study support previous field observations and hypotheses that (1) Physocypria sp. ostracods serve as intermediate hosts for N. chrysemydis, N. emydis, N. emyditoides, and N. pseudemydis; (2) N. emydis uses snail paratenic hosts, but snails are a non-viable route of transmission to turtles for N. chrysemydis, N. emyditoides, and N. pseudemydis; and (3) as adults, these 4 acanthocephalan species utilize their turtle definitive hosts similarly in terms of ionomic composition. This work challenges the traditional view of the acanthocephalan life cycle and sets the stage for future hypotheses., (© American Society of Parasitologists 2024.)

Environmental DNA (eDNA) surveys promise to be a sensitive and powerful tool for the detection of trematodes. This can contribute to the limited studies on trematode ecology, specifically in aquatic ecosystems. Here, we developed species-specific primer and probe sets for Moliniella anceps, Opisthioglyphe ranae, and Plagiorchis multiglandularis cercariae and applied a novel eDNA qPCR assay to detect larval trematodes quantitatively. We evaluated the effectiveness of the assays using filtered lake water samples collected from different sites of Lake Fadikha and Kargat River Estuary in Lake Chany, Russia, showing high species specificity and sensitivity in all 3 assays. Further, all 3 assays had high efficiencies ranging from 94.9 to 105.8%. Moliniella anceps, O. ranae, and P. multiglandularis were detected in the environmental water samples through real-time PCR. Thus, we anticipate that our approach will be beneficial for biomonitoring, measuring, and managing ecological systems., (© American Society of Parasitologists 2024.)

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., (© American Society of Parasitologists 2021.)

We investigated the effect of Schistosoma japonicum adenylate kinase 1 (Sjak1) on the growth and development of schistosomula. Quantitative real-time PCR showed that Sjak1 mRNA was expressed in 3-, 10-, 14-, 18-, and 21-day-old schistosomula, and its levels increased gradually with the development of S. japonicum. Using immunohistochemical techniques, ak1 protein was found to be mainly distributed in the tegument and some parenchymal tissues of the schistosomula. Double-stranded RNA-mediated knockdowns of ak1 decreased ak1 mRNA transcripts by more than 90%, and western blot results showed that expression of ak1 protein was decreased by 66%. Scanning electron microscopy following the RNA-mediated ak1 knockdown showed that the sensory papillae did not develop. Transmission electron microscopy showed a lower mean thickness of the tegument in the Sjak1 interference group than in the negative control group. Terminal deoxynucleotidyl transferase dUTP nick-end labeling suggested higher apoptosis in the interference group than the negative control group. These results showed that ak1 may be involved in the growth and development of S. japonicum schistosomula and especially in the development of the integument. Consequently, ak1 may be a potential target in developing prevention methods for schistosomiasis in the future., (© American Society of Parasitologists 2021.)

Snails identified as Triodopsis hopetonensis were collected (n = 18) from the University of Arkansas-Fayetteville campus in December 2018. Additional snails were collected in April 2019 (n = 9) and in September 2019 (n = 9). Kidney tissues were examined using light microscopy, and polysporocystic oocysts were observed. Sporulated oocysts (n = 2) measured 78 ± 3.4 μm × 76 ± 2.9 μm with an irregular oocyst residuum and contained an estimated 44-55 tetrazoic sporocysts. The sporocysts (n = 10) measured 13 ± 0.5 μm × 11 ± 1.5 μm with an indistinct, irregularly placed, sporocyst residuum and usually contained 4 sporozoites, although an octozoic variant was observed. DNA was extracted from the snail kidney tissues and used as a template for polymerase chain reaction (PCR). PCR was used to determine the infection status of the snails; 13 of 36 (36%) specimens were found to be infected with a new Klossia species, and only 3 (8%) of these infections were detected using light microscopy. The complete nuclear 18S ribosomal DNA (1,800 bp) and mitochondrial genomes (6,775 bp) were generated, and they differentiated this parasite from the type species Klossia helicina and support the description of this new Klossia species, Klossia razorbacki n. sp. This is the first Klossia species to be described from any North American snail., (© American Society of Parasitologists 2021.)

To analyze the response of the snail Physella acuta to Echinostoma paraensei, a compatible digenetic trematode, Illumina RNA-seq data were collected from snails with early infection (5 snails at 2 days post-exposure [DPE]) and established infection (4 snails, 8 DPE), and 7 control (unexposed) snails. A reference transcriptome (325,563 transcripts, including 98% of eukaryotic universal single-copy orthologs; BUSCO) and a draft P. acuta genome (employing available genomic Illumina reads; 799,945 scaffolds, includes 88% BUSCO genes) were assembled to guide RNA-seq analyses. Parasite exposure of P. acuta led to 10,195 differentially expressed (DE) genes at 2 DPE and 8,876 DE genes at 8 DPE with only 18% of up-regulated and 22% of down-regulated sequences shared between these time points. Gene ontology (GO) analysis yielded functional annotation of only 1.2% of DE genes but did not indicate major changes in biological activities of P. acuta between 2 and 8 DPE. Increased insights were achieved by analysis of expression profiles of 460 immune-relevant DE transcripts, identified by BLAST and InterProScan. Physella acuta has expanded gene families that encode immune-relevant domains, including CD109/TEP, GTPase IMAP, Limulus agglutination factor (dermatopontin), FReD (≥82 sequences with fibrinogen-related domains), and transcripts that combine C-type lectin (C-LECT) and C1q domains, novel among metazoa. Notably, P. acuta expressed sequences from these immune gene families at all time points, but the assemblages of unique transcripts from particular immune gene families differed between 2 and 8 DPE. The shift in profiles of DE immune genes, from early exposure to parasite establishment, suggests that compatible P. acuta initially respond to infection but switch to express immune genes that likely are less effective against E. paraensei but counter other types of (opportunistic) pathogens and parasites. We propose that the latter expression profile is part of an extended phenotype of E. paraensei, imposed upon P. acuta through parasite manipulation of the host, following successful parasite establishment in the snail after 2 DPE., (© American Society of Parasitologists 2020.)

Some parasite species alter the behavior of intermediate hosts to promote transmission to the next host in the parasite's life cycle. This is the case for Euhaplorchis californiensis , a brain-encysting trematode parasite that causes behavioral changes in the California killifish ( Fundulus parvipinnis ). These manipulations increase predation by the parasite's final host, piscivorous marsh birds. The mechanisms by which E. californiensis achieves this manipulation remain poorly understood. As E. californiensis cysts reside on the surface of the killifish's brain, discerning regional differences in parasite distribution could indicate mechanisms for host control. In this study, we developed a method for repeated experimental infections. In addition, we measured brain-region specific density using a novel methodology to locate and quantify parasite infection. We show that E. californiensis cysts are non-randomly distributed on the fish brain, aggregating on the diencephalon/mesencephalon region (a brain area involved in controlling reproduction and stress coping) and the rhombencephalon (an area involved in controlling locomotion and basal physiology). Determining causal mechanisms behind this pattern of localization will guide future research examining the neurological mechanisms of parasite-induced host manipulation. These findings suggest that parasites are likely targeting the reproductive, monoaminergic, and locomotor systems to achieve host behavioral manipulation.

Truttaedacnitis truttae is a cucullanid nematode of primarily salmonine fishes. Brown trout ( Salmo trutta ) in Europe reportedly become parasitized by ingesting lampreys ( Lampetra planeri ) carrying infective larvae. However, our field and laboratory observations suggested that North American specimens of T. truttae have an alternative life cycle. High abundances and potential impact of T. truttae in rainbow trout, Oncorhynchus mykiss , in the Colorado River drainage in Grand Canyon, where there are no lampreys, prompted a study on the transmission dynamics of this nematode. Eggs of T. truttae , collected from live gravid females, were incubated in the laboratory. Snails, Physa gyrina and Lymnaea sp., were exposed to T. truttae larvae 3-4 wk later. Active larvae of T. truttae were observed penetrating the intestinal wall of exposed snails, and worm larvae were found in the visceral tissues when examined 1 wk after exposure. Larvae in snails showed little growth and development 2 wk later and corresponded to L3 larvae. Infected snails were fed to hatchery-reared juvenile rainbow trout. Developing stages were subsequently found in the mucosal lining and lumen of trout intestines. Adult male and female (gravid) worms were found in the ceca of trout examined 5-6 mo after consuming infected snails. Larvae found in pepsin/trypsin digests and mucosal scrapings from wild, naturally infected, trout corroborate laboratory findings. Screening of Physa sp. and gammarids collected from Colorado River, Grand Canyon, for natural infections with T. truttae using the ITS1 rDNA marker gave positive results. Truttaedacnitis truttae is the second species, after Truttaedacnitis clitellarius of lake sturgeon, capable of using a snail first intermediate/paratenic host and is similar to several other cucullanids in having a histotropic phase of development in the definitive fish host.

Eutrophication of aquatic habitats has become a global problem, with implications for host-parasite dynamics. Blooms of certain cyanobacteria are associated with cyanotoxins, particularly microcystins such as microcystin-LR (MC-LR). These potent toxins have been shown to adversely affect freshwater fauna and can increase host susceptibility to parasite infection. However, to understand how cyanotoxins influence infection outcomes in nature, it is necessary to investigate whether free-living parasite infectious stages, such as that of trematode cercariae, are also affected given their demonstrated sensitivity to various contaminants. Here we examined the effects of environmentally relevant levels of MC-LR representing relatively high (82 μg/L) and low (11 μg/L) concentrations on the activity and survival of four different types of cercariae ( Echinostoma sp., Cephalogonimus sp., Alaria sp., and an unidentified strigeid type) over 24 hr. Exposure to MC-LR did not affect the activity of any cercarial type, nor was survival reduced. In fact, the strigeid-type cercariae had significantly increased longevity if exposed to either MC-LR solution, with the greatest longevity in the highest concentration. Our results indicate that MC-LR may have opposing effects on aquatic parasites and their hosts, potentially increasing host susceptibility but having a neutral or positive effect on motile infectious stages such as cercariae. Cyanobacterial blooms could thus enhance trematode transmission; however, the effects of other cyanotoxins must be studied, as well as a broader range of host and parasite species.

Parasites can affect animal populations and communities in aquatic ecosystems. However, greater understanding is needed for the distributions and drivers of parasite infection levels in many areas. This study focuses on parasite prevalence (percent infected hosts) of an important class of parasites, trematodes, in 2 species of snail first intermediate hosts ( Planorbella trivolvis and Physa sp.) in the Illinois River watershed, which has been impacted by human development. We hypothesized that trematode prevalence depends on local (e.g., water chemistry) and landscape (e.g., proximity to the Illinois River and land cover) factors. To test our hypotheses, we collected at least 20 individuals of 1 or both species of snails from 28 ponds within the watershed, and we made water-quality measurements and recorded habitat characteristics at each site. We then screened the snails for infections in the laboratory and identified the trematode cercariae that emerged based on morphological and molecular techniques. We found 5 cercariae morphotypes, including important parasites of wildlife, such as Echinostoma sp. and Ribeiroia ondatrae . Our results indicate that proximity to the Illinois River and open water or wetlands was positively associated with trematode prevalence in both snail species, whereas water chemistry (higher pH, lower calcium concentration, and lower specific conductance) was associated with increased prevalence, but only in Physa . Our findings offer increased understanding of potential environmental drivers underlying trematode distributions, with implications for wildlife health.

The control of emerging parasites requires a fundamental knowledge of where and when rates of transmission are high. Data on spatiotemporal patterns of infection are challenging to obtain, particularly for complex life cycle parasites that involve transmission into multiple obligate hosts. The lancet liver fluke, Dicrocoelium dendriticum, has a long history of colonization outside its native host and geographical range in continental Europe. Infection patterns involving adult and metacercarial stages have been characterized for this trematode in a region of emergence in western Canada within co-grazing herbivores and ants, but infection patterns in snail intermediate hosts in this region are unknown. We combined spatiotemporal prevalence surveys with sequence analyses of the cytochrome c oxidase subunit 1 ( COI) barcoding gene from samples of sporocyst tissue in infected snails to confirm that D. dendriticum utilizes 3 sympatric species of Oreohelid land snail ( Oreohelix subrudis, Oreohelix sp., and Oreohelix cooperi) as first intermediate host. Mean prevalence within a total sample of 900 adult snails collected over 1 field season from 6 sites was 9.9 ± 2.4%. For each species of snail, prevalence ranged between 5-30% within monthly samples, with peaks in mid-summer followed by declines in fall. Between-site variation in prevalence was low and non-significant, implying that rates of transmission of D. dendriticum miracidia from domestic stock and wildlife into snails are similar within localized sites, despite high variation in local habitat characteristics and in the structure of the definitive host community.

Specimens of the African snail Achatina fulica, collected in Bucaramanga, Colombia, were examined for parasites. Numerous specimens of Caenorhabditis briggsae were collected from the digestive tract of the snails and identified by the structure of male spiculum, caudal bursa, gubernaculum and precloacal lip in males, triangular tooth in metarhabdion, and protandrous hermaphrodites with a female:male ratio of 15:1 and with morphometry. DNA sequences of the ITS2 region of the ribosomal gene array from worms in this study matched with 99% similarity to published sequences of C. briggsae. A redescription of the species is provided. This is the first record of the species in South America.

The human liver fluke Opisthorchis viverrini (Platyhelminthes, Trematoda, Digenea) uses snails of the genus Bithynia as first intermediate host. Peculiarly among trematodes, the eggs of O. viverrini hatch within the digestive tract of its snail host. It remains uncertain whether hatching in this species is mediated through mechanical fracture of the eggshell or by digestion with specific digestive enzymes. This study aimed to characterize enzymes with specific inhibitors and factors involved in the hatching activity of O. viverrini eggs. For measuring egg hatching in vivo, 50 O. viverrini mature eggs were fed to individual Bithynia siamensis goniomphalos snails at various temperature conditions for 24 hr. Ex vivo, mature eggs were incubated with crude snail extract and commercial leucine aminopeptidase (LAP). Egg-hatching of O. viverrini was temperature dependent, with optimal hatching occurring at 24-28 C, with a peak of hatching of 93.54% in vivo and 30.55% ex vivo occurring at these temperatures. Ex vivo hatching rates increased to 45.87% under anaerobic conditions at 28 C. Some 22.70% and 16.21% of heat-killed eggs also hatched within the snail digestive tract and snail extract, respectively, indicating that host molecules are involved in the hatching response. Most eggs hatch in the anterior regions of the digestive tract. Hatching was completely inhibited in the presence of bestatin, an inhibitor of LAP, but not in the presence of phosphatase inhibitors. Bestatin inhibition of hatching was reversible. Finally, egg hatching could be induced by addition of a porcine LAP. The results indicate that this digenean utilizes both LAP of the snail host and movement of miracidia for hatching.

Parasites often use external cues to identify and move toward environments where they are likely to encounter suitable hosts. The trematode parasite Euhaplorchis californiensis produces cercariae that emerge from California horn snails ( Cerithideopsis californica [= Cerithidea californica]) to infect California killifish ( Fundulus parvipinnis) as second intermediate hosts. Based upon work on a congeneric Euhaplorchis species from Florida, and based on the ecology of its killifish host, we hypothesized that E. californiensis cercariae in southern California estuaries are positively phototactic and negatively geotactic, using both sunlight and gravity to guide their movement to the upper water column. To distinguish positive phototaxis from negative geotaxis, we first quantified E. californiensis movement in response to light along a horizontal plane and determined they were positively phototactic. In a second experiment, we quantified E. californiensis movement along a vertical plane in response to an overhead light, a light from below, or no light. We found that E. californiensis exhibit negative geotaxis in the absence of light, but will swim in the direction of gravity to move toward a light source from below. Thus, E. californiensis are both positively phototactic and negatively geotactic, but cercariae prioritize phototactic cues. These results suggest that E. californiensis cercariae aggregate in the open water, indicating that the pelagic zone represents an area of high infection risk for California killifish hosts.

Angiostrongylus cantonensis infection can lead to severe neuropathological damage caused by the development of these nematodes in the central nervous system after penetrating the blood-brain barrier. They commonly cause eosinophilic meningitis or meningoencephalitis in non-permissive hosts (e.g., mice). It has been shown that differences exist in the brains of permissive and non-permissive hosts during the larval development of A. cantonensis; however, the mechanism underlying the difference is not completely understood. This study analyzed and characterized the differentially expressed proteins in the intracranial A. cantonensis larvae in rat (ILR) and mouse (ILM) brains by using proteomics. We found that 29 proteins were differentially expressed: 12 of these proteins were highly expressed in ILR, whereas the remaining 17 proteins were highly expressed in ILM. Three protein spots were homologous to the actin-2, actin-1, and disorganized muscle protein 1 (dim-1) of Caenorhabditis elegans. In addition, proteomic analyses revealed that act-1 and act-2 were up-regulated in ILM compared to ILR, whereas dim-1 was down-regulated in ILM. Annotation using gene ontology revealed that act-1, act-2, and dim-1 were mainly associated with adenosine triphosphate (ATP) catabolic processes and ATP binding. Quantitative real-time polymerase chain reaction analyses of act-1 and dim-1 using the first internal transcribed spacers of A. cantonensis 18S ribosomal RNA (rRNA) was consistent with 2-dimensional gel electrophoresis (2-DE) and the sizes of these parasites; ILR was longer and wider than ILM. These results indicate that the differentially expressed proteins dim-1 and act-1 could be related to the development and pathogenicity of A. cantonensis in different hosts.

The edible land snail Cornu aspersum (Pulmonata: Stylommatophora) acts as a second intermediate host in the terrestrial life cycle of Brachylaima spp. trematodes, harboring unencysted metacercariae in its kidney. The ingestion of undercooked infected snails by humans may allow metacercariae to potentially develop to adult stage in the intestine, causing brachylaimiasis, as already seen in Australia. The prevalence and dynamics of C. aspersum parasitization by Brachylaima spp. metacercariae in specimens intended for human consumption in Spanish marketplaces were studied. In total, 3,710 C. aspersum specimens were analyzed over 5 yr, which were obtained from public marketplaces in the Spanish cities of Barcelona, Bilbao, Madrid, Tudela, Valencia, and Zaragoza. The overall prevalence was 41.97% (95% CI: 40.38-45.56%). The Tudela marketplace had the highest values for both the seasonal prevalence and abundance in all studies during autumn (93.57% and 3.09, respectively). This market also gave the highest individual metacercarial burden recorded, 212 metacercariae in a single specimen. Overall, the highest prevalence of Brachylaima spp. occurred in autumn (58.65%) and the lowest in winter (22.64%). There was a seasonal effect on prevalence, which increased from summer to autumn and then decreased in winter. In total, 96 experimental Brachylaima adults were obtained from the metacercariae parasitizing the analyzed snails. These were identified through morphometric tools (principal component analysis) as Brachylaima mascomai (56 in Barcelona, 1 in Bilbao, 7 in Tudela, and 3 in Valencia), and Brachylaima llobregatensis (17 in Barcelona, 8 in Bilbao, 1 in Valencia, and 3 in Zaragoza). Logistic regression modeling, conducted to predict the probability of purchasing parasitized snails using city and season as predictors showed a correct prediction overall of 79.0%, with a significant (p = 0.001) risk effect in the Barcelona-autumn interaction (2.551-38.442), a significant (p = 0.049) protection effect in the Tudela-spring interaction (0.076-0.997), a significant (p < 0.001) risk effect in the Tudela-autumn interaction (4.330-78.584), and a significant (p = 0.014) protection effect in the Valencia-spring interaction (0.033-0.687). The high overall prevalence of Brachylaima spp. metacercariae should be a matter of concern for public health authorities, mainly in countries where C. aspersum is consumed.

Understanding temporal variation of host-pathogen dynamics can be important for predicting disease risks and anticipating how disease systems may change in response to natural or human disturbances. Seasonal changes in weather, especially those associated with changes in temperature or precipitation, are often a key component of temporal changes in infection risk and can have important impacts on disease systems. However, these patterns can be difficult to track due to interannual variation and the need for longer term, multi-year surveillance efforts. We assessed seasonal and annual changes in the trematode component community of first-intermediate host stream snail Elimia (= Oxytrema = Goniobasis) proxima across 5 streams in the southern Appalachian Mountains. Over 3 yr, we found no evidence of consistent seasonal peaks of trematode infection in E. proxima. There was some across-site consistency in infection prevalence over 4 yr, because high prevalence sites tended to maintain higher prevalence from year to year, relative to lower prevalence sites. In addition, we examined the relationship between prevalence of first-intermediate host infection, weather variables, and site-level factors, including snail density and water quality metrics. Trematode prevalence was negatively related to total precipitation, which may have been due to the movement of infectious parasite stages and hosts downstream during high flows. We found no strong relationships between trematode prevalence and snail density or any of the water quality metrics examined in this study, indicating that snail infection may be driven primarily by definitive host activity.

The expansion of parasite distributions outside of their native host and geographical ranges has occurred repeatedly over evolutionary time. Contemporary examples include emerging infectious diseases (EIDs), many of which pose threats to human, domestic animal, and wildlife populations. Theory predicts that parasites with complex life cycles will be rare as EIDs due to constraints imposed by host specialization at each life-cycle stage. In contrast to predictions of this theory, we report 2 new intermediate hosts in the 3-host life cycle of the liver fluke Dicrocoelium dendriticum in Cypress Hills Provincial Park, Alberta, Canada. Results of sequence analysis of the cytochrome oxidase 1 (cox1) mitochondrial gene identified the terrestrial snail Oreohelix subrudis and the ant Formica aserva as first and second intermediate hosts, respectively, in the region. Neither of these intermediate hosts, nor their suite of domestic and wild mammalian grazers used in the life cycle, occurs within the native range of D. dendriticum in Europe. Our results from host surveys show that the prevalence of D. dendriticum in samples of O. subrudis varied between 4% and 10%, whereas mean metacercariae intensity in F. aserva varied between 33 and 41 (n = 163, mean ± SD = 38 ± 35). These results are the first to describe the complete life cycle of emerging lancet fluke in western North America. The process of multi-level ecological fitting, in which the lancet fluke possesses pre-existing traits to utilize host resources, rather than host species, at each life-cycle stage provides a mechanism for the establishment of this complex life cycle in a novel habitat and in novel hosts.

In the blood fluke family Schistosomatidae, marine snails are well known as intermediate hosts. Eight families of marine snails have thus far been reported to host schistosomes across the world, most of which have been implicated in human cercarial dermatitis (HCD) outbreaks. As part of our larger effort to define the species diversity and biology of schistosomes in Argentina, in particular their role in causing HCD, we searched in the marine pulmonate snail (Siphonaria lessoni) for a schistosome species described previously from S. lessoni from southern Argentina. Additionally, gulls (Larus dominicanus) collected from a different project locality (inland) were examined, because they are known to spend time in the intertidal regions. Schistosome sporocysts were found in S. lessoni, and a small worm fragment was retrieved from a gull. Molecular phylogenies for 28S, ITS1-5.8S-ITS2, and cox1 genes revealed that the specimens from the gull and S. lessoni grouped closely together, suggesting they are conspecifics. Also, ITS1-5.8S-ITS2 sequences suggested one of the schistosomes from S. lessoni and a schistosome from a South African penguin were also conspecifics. Further study is needed to verify if these specimens comprise a distinct marine clade within the larger avian schistosome clade that is comprised mostly of species using freshwater snail hosts. Thus far, it appears this group of marine schistosomes may be more likely found in the southern hemisphere. It is unclear if the observed distribution pattern of schistosomes in Siphonaria is a result of sampling bias and/or indicative of a specific bird-snail-schistosome association. It is clear they are sharply differentiated from the basal marine clade of avian schistosomes that includes Austrobilharzia.

Host specificity of parasites is a basic principle in parasitology; however, it is not easily measured. Previously, host specificity was calculated as the number of species that a parasite infected, but this is not an accurate description of host usage because some species are capable of being infected but do not contribute to the completion of the life cycle. Instead, measures of host specificity should take into consideration interactions between a parasite and a potential host species as well as interactions between current and subsequent hosts in the life cycle. The objectives of this study were to track the development of 2 trematode species, Halipegus eccentricus and Halipegus occidualis , in 3 phylogenetically and ecologically distinct microcrustacean second intermediate hosts, and then evaluate the extent to which each of these hosts contributed to transmission of each Halipegus species to the next odonate host in the life cycle. All 3 microcrustacean species exposed became infected with both species of Halipegus. The patterns of growth of H. eccentricus and H. occidualis were similar, but there were consistent differences in the rates of growth among the microcrustacean species in both Halipegus species. Regardless of host species infected, all individuals of both species were considered to be developmentally infective to the next host in the life cycle by 19 days postexposure (DPE) when they lost their excretory bladder. Worms of varying sizes were capable of surviving without this structure, suggesting that there is not a strong relationship between the rate of growth of the metacercariae and the development of their osmoregulatory system. Although Halipegus species were capable of living without an excretory bladder at 19 DPE, there were differences in their size and rates at which the 3 microcrustaceans contributed to transmission of the parasites to subsequent odonate hosts. Collectively, under controlled laboratory conditions, there was an approximate 2-fold difference in the average percentage of worms that established in odonates from the ostracod, Cypridopsis sp., than from the harpacticoid copepod, Phyllognathopus sp., and the difference was nearly 3-fold between Cypridopsis sp. and the cyclopoid copepod, Thermocyclops sp. Therefore, despite all 3 microcrustacean species becoming infected, not all species were equally suited for transmission and completion of the life cycle. Differences among the 3 microcrustacean species in cercaria ingestion, metacercarial growth and development, and odonate predation rates on infected microcrustacean species were important factors in determining transmission of the 2 Halipegus species to odonate hosts.

The edible land snail Cornu aspersum (Pulmonata: Stylommatophora) acts as second intermediate host in the cycle of Brachylaima sp. trematode, harboring free metacercariae in its kidney. The ingestion of undercooked infected snails by humans allows metacercariae to develop to adult stage in the intestine, causing brachylaimiasis. Praziquantel (PZQ) is the drug of choice to treat trematodiasis and it is effective against Brachylaima sp. metacercariae. The objective of this work was to assess, by transmission electron microscopy (TEM), the ultrastructural changes produced on the tegument and gastrodermis of the Brachylaima metacercariae recovered from C. aspersum treated with PZQ in comparison with untreated ones. Snails naturally infected by Brachylaima sp. metacercariae were treated by PZQ both individually and in groups. Metacercariae recovered from treated and control snails were processed for TEM. The tegument of untreated metacercariae was covered by a regular and thick glycocalyx. The syncytial epithelium contained abundant T2 secretory bodies appearing as membrane-bound biconcave disk-vesicles with high electron-dense and uniform content. The T2 secretory bodies located along the external area of the syncytium were mainly arranged at right angles to the apical plasma membrane. In treated metacercariae, the content of the T2 secretory bodies appeared altered, degenerating from high to low electron density, losing its uniform appearance and forming high electron-dense accumulations scattered around the periphery of the vesicle and separated by low electron-dense spaces. The presence of clusters was detectable in the central area. The characteristic arrangement of the T2 secretory bodies observed in untreated metacercariae was lost in treated ones. Vesicles near the apical area of the tegument no longer maintained their arrangement perpendicular to the apical plasma membrane. The characteristic arrangement of T2 secretory bodies and mitochondria was lost. The T2 secretory bodies were also found altered in the tegumental cell bodies, suggesting that the alterations started at the production stage. Mitochondria were severely degenerated and located in the apical area of the tegument. The digestive system displayed a strong contraction, which included the disappearance of the intracecal lumen.

:  Life history stages of Pleurogonius malaclemys were investigated in wild populations of the eastern mudsnail ( Ilyanassa obsoleta ) and diamondback terrapin ( Malaclemys terrapin ) in New Jersey, New York, and Rhode Island between 2011 and 2015, and laboratory experiments investigating the settling preference of metacercarial cysts of P. malaclemys were conducted. Cysts of P. malaclemys were found on mudsnails on the north and south shores of Long Island, New York and in Rhode Island, approximately 280 km farther north than previously reported. The cysts were found on mudsnails year round, but cyst prevalence increased during the summer months, reaching maximum levels (∼70%) in November. Nearly 58% of Jamaica Bay, New York mudsnails had cysts; mean intensities were 2.63 cysts/mudsnail. Although cyst prevalence was high, only 11 mudsnails (0.28%) were found to have the internal redial stages of P. malaclemys, the stage of infection preceding external cysts. In addition to mudsnails, P. malaclemys could encyst on other biological substrates, including common terrapin prey species. The majority of wild adult terrapins from Stone Harbor, New Jersey were infected with the adult stage of P. malaclemys (80.30%, x¯ = 36.36 trematodes/terrapin, n = 66). Juvenile terrapins were experimentally infected with P. malaclemys and on average 22.5% of the consumed cysts successfully developed into adult trematodes. Studies on the life cycle of P. malaclemys are important because previous research has shown that the frequency of cysts of P. malaclemys on mudsnails can be used as an indirect measure of terrapin abundance.

The microhabitats in which hosts live can potentially influence the ability and success of parasites in finding and infecting these hosts. The infection dynamics of both digenetic trematode parasites and a nematode parasite (Daubaylia potomaca) infecting a pulmonate snail, Helisoma anceps , were observed in a small North Carolina lake using 3 different classifications of substratum type based on percent coverage by leaves and debris. There were no differences in snail site occupancy or density between substratum types, but small-scale differences in microhabitat impacted parasite prevalence and intensity in their gastropod hosts. Snails inhabiting substrata covered in more leaf litter and debris had a lower prevalence and intensity of infection by all of the trematode species and life stages when compared to H. anceps inhabiting other substrata types, while only the intensity of infection was impacted in D. potomaca infections. These findings emphasize the importance of microhabitat, specifically its physical components, in influencing parasite infection in intermediate hosts and that small-scale differences may significantly affect the patterns of infection.

Helminths often demonstrate preferential site selection in which a parasite will only occur in 1 microhabitat or a restricted portion of its fundamental niche within its host. However, factors responsible for helminth site specificity are poorly understood, and very little is known about how these factors vary among multiple host species. Some helminths, such as Halipegus occidualis, have been reported from different habitats (stomach or under the tongue) within multiple anuran host species, suggesting that the site selected varies within anuran species. This study examined the site selection by H. occidualis in 7 definitive anuran host species using experimental infections. Then, the site fidelity of H. occidualis was further tested by transplanting worms from under the tongue to the stomach and vice versa in different anuran host combinations, and the movement of worms was recorded. Halipegus occidualis individuals occupied the habitat under the tongue in 6 of 7 anuran species. However, worms always occupied the stomach of American bullfrogs (Lithobates catesbeianus) and were never found under the tongue or in the mouth of these hosts. More importantly, all worms remained in the original habitat when transplanted from the stomach to the stomach or the buccal cavity to the buccal cavity within another individual of the same amphibian species. However, when worms were transplanted from the stomach to the buccal cavity or vice versa in the same host species, the worms always migrated back to the original habitat. The main contribution of this study is that it experimentally documented the variability in the site fidelity of H. occidualis within multiple definitive host species and determined that site fidelity is not as strongly conserved in this genus as suggested previously. Additionally, this work suggests that the variation in site selection in different host species could lead to speciation of the parasites.

Complex societies where individuals exhibit division of labor with physical polymorphism, behavioral specialization, and caste formation have evolved several times throughout the animal kingdom. Recently, such complex sociality has been recognized in digenean trematodes; evidence is limited to 6 marine species. Hence, the extent to which a soldier caste is present throughout the Trematoda is sparsely documented, and there are no studies detailing the structure of a species lacking such a social structure. Here we examine colony structure for an additional 5 echinostomoid species, 4 of which infect the marine snail Cerithidea californica and 1 (Echinostoma liei) that infects the freshwater snail Biomphalaria glabrata . For all species, we present redia morphology (pharynx and body size) and the distribution of individuals of different castes throughout the snail body. When morphological evidence indicated the presence of a soldier caste, we assessed behavior by measuring attack rates of the different morphs toward heterospecific trematodes. Our findings indicate that each of the 4 species from C. californica have a permanent soldier caste while E. liei does not. The observed intra- and inter-specific variation of caste structure for those species with soldiers, and the documentation of colony structure for a species explicitly lacking permanent soldiers, emphasizes the diverse nature of trematode sociality and the promise of the group to permit comparative investigations of the evolution and ecology of sociality.

Specimens of Transversotrema patialense (sensu lato) ( Soparkar, 1924 ) Crusz and Sathananthan, 1960 (Digenea: Transversotrematidae) infected the skin (epidermal spaces beneath scales near pectoral fins) of 4 of 126 (prevalence 3%; mean intensity 1.8) zebrafish ( Danio rerio (Hamilton, 1822) [Cypriniformes: Cyprinidae]) purchased in 2009 and cultured by a California (USA) fish supplier. These fish were sold as "laboratory-reared" and "specific pathogen free," purportedly raised in a recirculating aquaculture system that included zebrafish only. We herein describe the morphological features of this transversotrematid using light and scanning electron microscopy, provide a comprehensive list of hosts (snails and fishes) and geographic locality records for specimens reported as T. patialense, which is perhaps a species complex, and provide a brief historical synopsis of the taxonomic and life history research that has been conducted on this fluke. No species of Transversotrema previously had been reported from the Americas; however, this discovery is not surprising given that: (1) a suitable intermediate host (red-rimmed melania, Melanoides tuberculata (Müller, 1774) [Cerithioidea: Thiaridae]) has been established in California and elsewhere in North America, (2) the zebrafish is a susceptible definitive host, and (3) T. patialense reportedly matures on a broad ecological and phylogenetic spectrum of freshwater fishes. To our knowledge, this is the northern-most geographic locality record for a species of this genus. We suspect this case study represents an example of a parasite that may now be established in North America by the fortuitous co-occurrence of a susceptible, exotic snail host (the red-rimmed melania) and a susceptible, widely distributed, exotic fish host (the zebrafish).

Our current knowledge of avian schistosomes from South America is scarce in all respects, including species and generic diversity, their life cycles, patterns of host use, potential to cause dermatitis outbreaks, and evolutionary affinities. As a step towards addressing this shortcoming, the goal of this study was to provide discrete reference points relating to snail hosts, locality records, morphological attributes, sequence for nuclear 28S and ITS, and partial mitochondrial cox1 genes, and phylogenetic relationships for schistosome cercariae recovered from different species of Chilina, which are gastropods endemic to South America. In total, 1,308 snails belonging to 6 species of Chilina were collected from 12 localities across Argentina. Thirty-eight snails (2.9%) had schistosome infections. Our data indicate the presence of 3 lineages of Chilina-transmitted schistosomes, all of which group within the major avian schistosome clade. However, none of the lineages grouped within or as sister to other known avian schistosome genera in the tree, indicating they probably represent undescribed genera. The relationships of these schistosomes from Chilina spp. are discussed in relation to their position in the global schistosome phylogenetic tree.

Within Oligochaeta, Chaetogaster limnaei is unusual in exhibiting a parasitic relationship with freshwater pulmonate snails. Taxonomic confusion has been caused by differences in what have been considered 2 subspecies of this worm: Chaetogaster limnaei limnaei is an ectosymbiont and is present inside the mantle cavity of the snail, whereas Chaetogaster limnaei vaghini is parasitic and lives in the kidney of the snail. This study explored the distribution of these annelids in central New York and used mitochondrial DNA sequence data from the COI locus to examine the relationship, evolution, and species status of the ectosymbiotic and parasitic forms of C. limnaei. Snails ( Physa gyrina ) were collected from 6 streams and lakes in central New York, with additional specimens collected from a lake in Massachusetts for comparison. One hundred and forty snails were examined, and at least 1 form of Chaetogaster was present in 88 specimens, a prevalence of 62.9%. COI sequence data from New York and Massachusetts did not reveal separate ectosymbiotic and parasitic lineages. Instead, all parasitic forms were part of a mixed clade that included both ectosymbiotic and parasitic forms. This mixed clade was nested within clades of ectosymbiotic forms only, suggesting that a plastic lineage of C. limnaei, able to be both ectosymbionts and parasites, evolved from ectoparasitic ancestors.

The epidermal mucus covering the surface of a snail represents an important barrier to trematode larvae attempting to penetrate the snail and may play a role in mediating snail-trematode compatibility. In this study, Facioloides magna miracidia were exposed to mucus harvested from a compatible snail host, Lymnaea elodes (palustris), and from an incompatible snail, Helisoma trivolvis . In vitro treatment of freshly hatched miracidia with snail-derived mucus exerted dramatically different effects on larvae depending on snail species. At the lowest dilution of mucus tested (1:3) mean damage rates (tegumental damage and/or larval lysis and death) were as high as 100% for miracidia exposed to H. trivolvis mucus, while none of F. magna miracidia were damaged in L. elodes mucus. A dilution series for each snail species, and treatments with heat and proteinase K were performed to characterize the component(s) of mucus inducing the observed morphological changes. The damaging effects of H. trivolvis mucus were concentration dependent and completely abrogated by heat (65 C, 30 min) and proteinase treatment, strongly implicating a heat-labile protein(s) in mucus as the active cytotoxic agent(s). In contrast to our prediction that miracidial contact with mucus of compatible L. elodes would trigger larval transformation, mucus from either snail species tested exhibited little to no activity. Overall these data demonstrate the presence of a potent cytotoxic protein-like factor in the mucus of F. magna -incompatible H. trivolvis , and its absence in the mucus of the compatible snail, L. elodes . This finding supports the notion that the epidermal mucus layer may be serving as an important determinant of larval trematode-snail compatibility.

Trematodes belonging to the family Stomylotrematidae are intestinal parasites of birds. Despite the worldwide distribution and diversity of host species, the first intermediate host remains unknown. For a survey of parasites of Pomacea maculata , snails were collected from the municipality of São Vicente Férrer, state of Maranhão, northeastern Brazil. In the present study, the xiphidiocercariae shed from these snails were used in the experimental infection of the water bug Belostoma plebejum. The insect mortality was observed 30 days post-infection, and the metacercariae recovered in the body cavity of B. plebejum were identified as Stomylotrema gratiosus. This is the first report of an ampullariid snail as intermediate host of stomylotrematid trematodes.

Angiostrongylus cantonensis , the rat lungworm, is a zoonotic, parasitic nematode that uses the rat as a definitive host and gastropods as intermediate hosts. It is prevalent in parts of Asia, the Pacific islands, and the Caribbean. In the United States, A. cantonensis is established in Hawaii and in recent years has been reported in Alabama, California, Louisiana, and Florida, where it has been found in the reintroduced Lissachatina fulica (also known as Achatina fulica), the giant African snail that was once eradicated from the state. Since 2004, A. cantonensis has been identified as the causative agent for 2 non-human primate deaths in Florida, one attributed to ingestion of the snail Zachrysia provisoria. Our study further supports the presence of A. cantonensis in Z. provisoria in Florida and identifies 2 additional introduced terrestrial snails, Bradybaena similaris and Alcadia striata, that serve as intermediate hosts for A. cantonensis , as well as evidence of rat infection, in southern Florida. The finding of both definitive and intermediate hosts suggests that A. cantonensis may be established in south Florida.

In 1984, work on the parasite population and community ecology in the pulmonate snail, Helisoma anceps , was initiated in Charlie's Pond (North Carolina). Similar research on Physa gyrina was started in 1986. When study in the pond began in 1984, 8 species of larval trematodes were being shed from Hel. anceps. By far, the dominant species was Halipegus occidualis , with prevalences generally ∼60%, except during midsummer, when older snails were dying. For the other 7 trematode species being shed, prevalences were consistently less than 4%. By 2006, 18 species had been identified in Hel. anceps at one time or another. In 1986, Hal. eccentricus was discovered in P. gyrina , with a prevalence of ∼49%. Through 2006, 7 trematodes were found to be shedding cercariae from P. gyrina . Halipegus eccentricus disappeared from the pond in 1998. From March through November of 2012 and 2013, 1,292 Hel. anceps and 716 P. gyrina were collected, using collection protocols that were identical to those used from 1984 through 2006. In 2012, 5 trematode species, including Hal. occidualis, were present in Hel. anceps at one time or another. During the last part of the 2012 collecting season cercariae of just 2 species were being shed from Hel. anceps (and 1 from P. gyrina ). In 2013, only cercariae of Haematoloechus longiplexus and Uvulifer ambloplitis were observed from Hel. anceps. The latter species was lost by 2014, and an echinostome was present (2.1%); a single snail was infected with Haem. longiplexus. Four species were being shed from P. gyrina , i.e., Echinoparyphium sp. (7.9%), Glypthelmins sp. (1.5%), Plagiorchis sp. (4.9%), and Posthodiplostomum sp. (7.4%). Rarefaction curves were generated for Hel. anceps shedding in 1984, 1988, 1989, 2002, 2006, and August of 2014. The data clearly indicate that species diversity was constantly declining over the 31-yr period. We did not include P. gyrina in the analysis since data for this snail species were not acquired until 1991-1992. At present, we have no definitive explanation for the decrease in diversity, although circumstantial evidence suggests that it might be related to periodic declines in water level that negatively affected the colonization and maintenance of emergent vegetation within the pond.

Pond snails ( Physa acuta ) were exposed to a gradient (0, 1, 3, 5, 10, 15, 20, 25, 30, 35, 40, and 50) of trematode (Halipegus eccentricus) eggs and observed to determine egg dose effects on the survival of snails through the prepatent period, snail reproduction, and the production of cercariae. The probability of snail survival through the prepatent period significantly decreased with increasing egg exposures, where the odds of snail survival was 0.94 per trematode egg. Similarly, the probability of snail reproduction significantly decreased with increasing egg exposures, where the odds of snail reproduction was 0.85 per trematode egg. In contrast, the probability of a snail shedding cercariae significantly increased with increasing egg exposures, where the odds of reaching a patent infection were 1.64 per trematode egg. However, snails shedding cercariae that were exposed to higher doses of trematode eggs tended to die sooner. Thus, there appears to be a tradeoff between infecting a snail and killing the host.

Pseudosellacotyla lutzi ( Freitas, 1941), at present included in the Faustulidae, is redescribed, and its life cycle was resolved experimentally. The prosobranch snail Aylacostoma chloroticum Hylton Scott (Thiaridae), collected in the Yacyretá Dam, Province of Misiones, Argentina, was found naturally infected with cercariae that lacked pigmented eyespots, and possessed 7 pairs of penetration glands, 8 pairs of flame cells, and a V-shaped excretory vesicle. The cercariae developed in oval cysts, which were found on fin rays, vertebrae, and spines of poeciliid and tetragonopterid fish species. Adults were obtained experimentally from Hoplias malabaricus (Erythrinidae) infected with metacercariae from albino Gymnocorymbus ternetzi (Tetragonopteridae), which had been exposed to emerging cercariae. Adults were also found in naturally infected H. malabaricus collected in the Yacyretá Dam. The morphology of the cercariae, and the characteristics of the life cycle show that P. lutzi should be included in the Cryptogonimidae.

Cornu aspersum (= syn. Helix aspersa ) snails imported into NE Spain from Algeria and South Africa, and intended for human consumption via sale in public markets, were analyzed to assess their contribution to geographic dissemination of Brachylaima species. Prevalence and viability studies of metacercariae were performed on Algerian and South African Brachylaima adults. Morphometric studies were performed by measuring 16 variables in metacercariae and 18 variables in adults. Principal component analyses (PCA) were performed to assess the contribution of each variable in the separation of the different groups. A MANOVA test performed on morphometric data from Brachylaima llobregatensis and Brachylaima mascomai Algerian and South African adults, revealed a significant multivariate main effect (P < 0.001) and univariate effects in 13 variables (P ≤ 0.001). Pairwise comparisons revealed significant differences in 13 variables between B. mascomai and Algerian adults, as opposed to 3 variables (testis I width, testis II width, and egg width) when compared with B. llobregatensis. South African adults had significant differences in 5 variables (body length, oral sucker length, oral sucker width, egg length, and egg width) when compared with B. llobregatensis, as opposed to 1 variable (testis II width) when compared with B. mascomai. Results from PCA showed 2 different groups: B. llobregatensis/Algerian adults (overlapped) and B. mascomai/South African adults (overlapped). Imported edible C. aspersum specimens can contribute significantly to the geographic spread of Brachylaima species by harboring infective metacercariae which could easily infect animals, given their heteroxenous character. Brachylaima llobregatensis has previously been detected in NE Spain only, and now this species appears to be parasitizing C. aspersum in Algeria (North Africa). This geographic dispersion could be favored by commerce in terrestrial snails such as C. aspersum.

The gill parasite Centrocestus formosanus (Trematoda: Heterophyidae) is an exotic parasite of concern in Texas because it has been shown to infect multiple threatened and endangered fish species. The purpose of this study was to determine if C. formosanus could present a threat to larval anurans, as well as threatened neotenic salamanders endemic to the spring-fed systems of Texas. We exposed adults of the San Marcos salamander Eurycea nana (Caudata: Plethodontidae) and tadpoles of the Rio Grande leopard frog Lithobates berlandieri (Anura: Ranidae) to the cercariae of C. formosanus . The San Marcos salamander showed no signs of metacercarial infection, suggesting that E. nana may be refractory to C. formosanus cercariae. Centrocestus formosanus readily infects the gills of leopard frog tadpoles, but the metacercariae apparently died prior to reaching maturity in our tadpoles.

In this study, we investigated oysters, Crassostrea virginica , from Laguna Madre in South Texas, where a 45-yr old study recorded metacercarial infections of the echinostomatid trematode, Acanthoparyphium spinulosum , an Asian relative of which, Acanthoparyphium tyosenense, has been associated with human infections via the ingestion of raw mollusks. In an effort to examine the base-line infection parameters of Acanthoparyphium sp. in oysters, we examined the effect of distance from the shoreline, which is the habitat of the first intermediate host snail, Cerithidea pliculosa, as well as temporal changes in oyster infection levels, by conducting quarterly collections of oysters during a year. We found that almost all oysters (prevalence = 97.8-100%) were infected regardless of distance to the shoreline and season. However, the abundance of metacercariae was significantly higher close to the shoreline, while no significant temporal changes could be detected. In addition to the echinostomatid, we found a high abundance of the metacestode Tylocephalum sp. and the presence of 4 other metazoan parasites. None of the infections seemed to incur significant tissue damage to the oysters. Our study shows that at least locally, recreational harvesters of oysters may be exposed to Acanthoparyphium sp. Future studies should examine oysters from snail host habitats in the Gulf of Mexico, and the potential zoonotic risk of Acanthoparyphium sp. should be evaluated using experimental infections of animal models.

Daubaylia potomaca is a nematode parasite that exhibits an unusual direct life cycle in planorbid snails in which adult females are the infective stage, after being shed from a definitive host. The present study examined the shedding patterns of this nematode to determine what cues or mechanisms might lead to the parasite leaving its host. A correlation was found between host death and the frequency and number of D. potomaca shed, suggesting that the nematodes can detect that the host is dying and may leave in search of a new host. Furthermore, elevated intensities of D. potomaca in the snail induce shedding earlier, suggesting that competition for space and resources may also play a role in the shedding patterns of the nematode, but not when time to death is controlled. Finally, nematodes shed a longer time before host death were significantly longer and more likely to be gravid than those shed as time to snail death approached, implying that the nematode reaching maturity or being inseminated might also be cues for D. potomaca to leave its snail host. In summary, the shedding patterns of D. potomaca appear to be a complex mix of host death detection, competition, and nematode maturation.

The objectives of this study were to describe (1) the osmotic environment and precise location of the Proterometra macrostoma redia in its snail intermediate host, (2) where retraction of the distome body into the cercarial tail occurs, and (3) the subsequent emergence path of the cercaria out of the snail. Snails, Elimia semicarinata , were collected from North Elkhorn Creek in Scott County, Kentucky and screened daily for patent infections. Live rediae were extracted from infected snails in either artificial pond water (APW) or artificial snail water (ASW) and monitored for changes in morphology and movement every hour over 5 hr at 22 C. Infected and control snails were simultaneously fixed and decalcified in Cal-Ex II, prepared for routine paraffin sectioning, and serial sections subsequently analyzed for rediae and cercariae location. Significantly (χ(2) = 42.45; 1 df; P = 0.0001) more rediae showed movement in ASW than in APW after 5 hr, suggesting a host compartment separate from the mantle cavity. Histological sections clearly showed rediae developing in close association with the snail digestive tract, within the peri-intestinal sinus of the snail, and isolated from the mantle cavity by a mantle membrane. Retraction of the distome body into the cercarial tail follows the emergence of the cercaria from the redia. Cercariae then enter the mantle cavity and emerge into fresh water through a siphon-like structure formed by the mantle collar of the snail.

Infection by trematode parthenitae (larval, asexual trematodes) has severe consequences for molluscan hosts, resulting in cessation of reproduction and early mortality. Here we present evidence that the freshwater snail Planorbella trivolvis can lose infections by trematode parthenitae. Of 8 P. trivolvis infected by reniferin parthenitae, 6 died within 2 wk, whereas the remaining 2 snails lost their infections within 82 days after initial examination. This phenomenon might suggest that molluscs can resist established trematode infections (i.e., "self-cure") or at least out-survive some trematode parthenitae.

Hairworms infect terrestrial arthropods and are 1 of the most understudied groups of parasites. Recently, life cycles of 2 gordiids (Paragordius varius and Paragordius obamai) have been domesticated in the laboratory. We tested the viability of laboratory reared and post-frozen larval and cyst stages of the North American gordiid, P. varius , frozen at -80 C for 7 mo, and the viability of field collected and post-frozen cysts of the African (P. obamai) and North American ( P. varius ) gordiid frozen at -20 C for 2 mo. All snails exposed to post-frozen or control P. varius larvae became infected with cysts, and there was no significant difference in prevalence or mean intensity of cysts among control or experimental snail groups. As with larvae, no significant differences were observed in prevalence or mean intensity of emerging worms from crickets infected with post-frozen or control P. obamai or P. varius cysts. All female P. obamai and P. varius worms from control and post-frozen cyst infections laid eggs and larvae hatched from some of these eggs. Survival and cyst formation of P. varius larvae exposed to different combinations of drying and/or freezing temperatures indicated that gordiid larvae have the ability to survive drying and freezing, but survival significantly increases during freezing at lower temperatures. The major contribution of our study is the demonstration that gordiid larval and cyst stages can survive freezing temperatures to infect and develop in the next host.

Bithynia funiculata is the first intermediate host of the human liver fluke Opisthorchis viverrini in northern Thailand but its identification through morphological analysis is often problematic due to the shortage of gastropod taxonomists. As a consequence, we focused on the development of species-specific primers for use as an identification tool. Our work involved recovery of a 502-base pair (bp) amplicon of unknown function through species-specific primers whose effectiveness was tested by analyzing specimens of B. funiculata from 3 locations in northern Thailand. This primer set did not amplify other species in the Bithyniidae or in other gastropod families. By providing a tool to confirm morphological identifications of B. funiculata, and by enabling the identification of juvenile specimens and those with damaged shells, these primers will improve estimates of the prevalence of parasitic infections in this snail.

Experimental infections and field-collected lizards were used to investigate issues of transmission, host specificity, and seasonal occurrence in the nematode Cyrtosomum penneri (Cosmocercoidea: Atractidae). Anolis sagrei (87 males, 42 females) were captured from the Florida Southern College campus, Polk County, Florida, from October 2010 to September 2011, and 8,803 C. penneri were collected from their intestines. During the breeding season all sexually mature (SVL ≥ 34 mm) A. sagrei were infected, whereas juvenile lizards (SVL <34 mm) were never infected. Experimental infections, using A. sagrei , found that worms were transferred to new hosts venereally, but not during oral exposures. Mating trials confirmed that worms were consistently transferred between hosts during copulation under natural conditions. Experimental exposures found that land snails and crickets do not serve as transport or intermediate hosts, which supports the idea that C. penneri is transferred only during host copulation. Experimental infections to test host specificity in C. penneri successfully infected A. sagrei , Hemidactylus turcicus , and Sceloporus undulatus , but not Anolis carolinensis or Plestiodon inexpectatus. Overall, this is the first study to fully elucidate the life cycle of any atractid nematode, and we suggest a venereal route of transmission for all atractid worms that infect reptilian hosts. Our findings also have implications for the host's reproductive and behavioral biology, e.g., support for covert or satellite males in the A. sagrei mating system.

We elucidate the life cycle of Maritrema orensense for the first time and experimentally confirm that of the sympatric Maritrema bonaerense. In Argentinean estuaries, both species parasitize the cochliopid snail Heleobia australis as first intermediate host, the grapsid crabs Neohelice granulata and Cyrtograpsus angulatus as second intermediate hosts, and gulls as definitive hosts. Here, we describe the daughter sporocysts and cercariae of M. orensense and redescribe these stages for M. bonaerense. Sporocysts of M. orensense are shorter, with fewer developed cercariae than M. bonaerense. The cercariae of M. orensense have longer, larger, and more undulating cephalic glands than M. bonaerense. We redescribe metacercariae and adults of both species and compare them with the previous descriptions. Intestinal ceca length, vitellaria shape and extension, and egg size are the most relevant characteristics in metacercariae and adults for differentiating the species. Hence, the detailed morphological description and comparative analyses of morphometrics obtained from natural and experimental infections permit clear differentiation of M. orensense and M. bonaerense at each life stage.

An unidentified xiphidio-type cercaria, previously thought inconsequential to catfish health, was found to be released from marsh rams-horn snails (Planorbella trivolvis) inhabiting ponds on a commercial catfish operation in the Mississippi Delta. A preliminary challenge of cohabiting channel catfish ( Ictalurus punctatus ) with snails actively shedding the unidentified cercariae resulted in death of some fish. A second cohabitation trial yielded similar results, as did a third challenge of 250 cercariae/fish. Histopathology revealed developing metacercariae concentrated in the cranial region, especially within the branchial chamber, with several metacercariae at the base of the branchial arches within, or adjacent to, blood vessels, possibly the proximate cause of death. Genetic sequence analysis of the 18S small subunit ribosomal DNA (ssDNA), 28S large subunit rDNA (lsDNA), and cytochrome oxidase (Cox1) genes all matched the cercariae to Drepanocephalus spathans (Digenea: Echinostomatidae), a parasite of the double-crested cormorant (Phalacrocorax auritus), a piscivorous bird endemic on most catfish farms. This is the first commentary regarding pathology of D. spathans in juvenile channel catfish as well as the first report of the marsh rams-horn snail as an intermediate host in the D. spathans life cycle. The data presented here suggest this parasite could have limiting effects on catfish production, further supporting the need for adequate snail control programs to reduce trematode prevalence on commercial catfish operations.

Some parasites modify the behavior of intermediate hosts to increase the probability of transmission to the next host in their life cycle. In habitats where this is common, parasites play an important role in predator-prey links and food web dynamics. In this study we used laboratory observations to investigate the behavior of longnose killifish, Fundulus similis, that were naturally infected with metacercariae of the trematode, Euhaplorchis sp. A, from Laguna Madre, south Texas. In particular, we examined whether there was a relationship between the number of metacercariae lodged on the brain of the infected fish and behaviors that made the fish more conspicuous to avian final hosts. We also quantified the abundance and cercariae production of this parasite in its first intermediate snail host, Cerithidea pliculosa , and examined the seasonal variation of Euhaplorchis sp. A in F. similis . Our data demonstrated that Euhaplorchis sp. A affected the surfacing behavior of F. similis in an intensity-dependent manner. Fish with many infections spent longer time at the surface of the water than fish with few infections. Our data also show that Euhaplorchis sp. A is a common parasite in the first intermediate host and produces close to 4,000 cercariae m(-2) day(-1). Consequently 97% of all fish collected and necropsied were infected, with little seasonal variation in the mean abundance of the parasite. Based on our data, Euhaplorchis sp. A is likely important to predator-prey links in Gulf of Mexico estuary food webs, similar to the closely related Euhaplorchis californiensis in southern California. We expect that other closely related species elsewhere may have similar effects on other fish hosts, emphasizing the need for incorporating trophically transmitted parasites in estuarine food web studies.

Metagonimoides oregonensis (Heterophyidae) is a little-known digenetic trematode that uses raccoons and possibly mink as definitive hosts, and stream snails and amphibians as intermediate hosts. Some variation in the life cycle and adult morphology in western and eastern populations has been previously noted. In the southern Appalachians, Pleurocera snails and stream salamanders, e.g., Desmognathus spp., are used as intermediate hosts in the life cycle. We completed a series of studies in this system examining some aspects of larval trematode morphology and first and second intermediate host use. Molecular sequencing of the 28S rDNA of cercariae in our survey placed them clearly within the heterophyid family. However, light and scanning electron microscopy revealed both lateral and dorso-ventral finfolds on the cercariae in our region, whereas original descriptions of M. oregonensis cercariae from the west coast indicate only a dorso-ventral finfold, so further work on the systematics of this group may be warranted. A survey of first intermediate host, Pleurocera proxima, from 7 streams in the region identified only M. oregonensis, virgulate-type cercariae, and cotylomicrocercous-type cercariae in the streams, with M. oregonensis having the highest prevalence, and the only type present that use amphibians as second intermediate hosts. Based on clearing and staining of 6 Desmognathus quadramaculatus salamander larvae, we found that individual salamanders could have over 600 metacercariae, which form between muscle fibers throughout the body. Histological observations suggest that the metacercariae do not cause excessive tissue damage or inflammation, and likely persist through metamorphosis, thereby transmitting potentially large numbers of worms to definitive host raccoons foraging along streams.

Opportunity for parasites to manipulate host behavioral phenotype may be influenced by several factors, including the host ecology and the presence of cohabiting parasites in the same host. Metacercariae of Ornithodiplostomum ptychocheilus and "black spot" Crassiphiala bulboglossa have similar life cycles. Each parasite uses a littoral snail as a first intermediate host, fathead minnows as a second intermediate host, and a piscivorous bird as a final host. Metacercariae of black spot encyst in the dermal and epidermal tissues, while metacercariae of O. ptychocheilus encyst on the brain over a region that coordinates optomotor responses. Because of site differences within the host, we predicted that O. ptychocheilus metacercariae might manipulate the behavioral phenotype of minnows to facilitate transmission to the final host, but metacercariae of black spot would not. In our study population, prevalence was 100% for O. ptychocheilus , with an overall median intensity of 105 metacercariae per minnow. Prevalence of black spot was 60%, with a median abundance and intensity of 12 and 20 metacercariae per minnow for the overall sample and for infected fish, respectively. Minnows accumulated both parasites over time, producing significant correlations between intensity and minnow body length and between intensities of the 2 parasites. Minnows infected with black spot had on average twice as many O. ptychocheilus metacercariae as similar-sized minnows without any black spot cercariae. We found no correlation between body condition of minnows and intensity for either parasite. We measured 2 aspects of anti-predator competence to test for effects linked to parasite intensity. We found no correlation between intensity of either species of parasite and latency to behavioral response to attack from a mechanical model heron, nor was there any effect of parasite intensity on a measure of shoaling affinity. The absence of any detectable effect of metacercariae on anti-predator competence in minnows may reflect selection against parasite pathology from predation by non-hosts of the parasites and overwinter mortality due to low dissolved oxygen.