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Living organisms are vulnerable to thermal stress which causes a diversity of physiological outcomes. Previous work has shown that the snail vectors (

Schistosoma mansoni, a snail-vectored, blood fluke that infects humans, was introduced into the Americas from Africa during the Trans-Atlantic slave trade. As this parasite shows strong specificity to the snail intermediate host, we expected that adaptation to S. American Biomphalaria spp. snails would result in population bottlenecks and strong signatures of selection. We scored 475,081 single nucleotide variants (SNVs) in 143 S. mansoni from the Americas (Brazil, Guadeloupe, and Puerto Rico) and Africa (Cameroon, Niger, Senegal, Tanzania, and Uganda), and used these data to ask: (i) Was there a population bottleneck during colonization? (ii) Can we identify signatures of selection associated with colonization? And (iii) what were the source populations for colonizing parasites? We found a 2.4-2.9-fold reduction in diversity and much slower decay in linkage disequilibrium (LD) in parasites from East to West Africa. However, we observed similar nuclear diversity and LD in West Africa and Brazil, suggesting no strong bottlenecks and limited barriers to colonization. We identified five genome regions showing selection in the Americas, compared with three in West Africa and none in East Africa, which we speculate may reflect adaptation during colonization. Finally, we infer that unsampled African populations from central African regions between Benin and Angola, with contributions from Niger, are likely the major source(s) for Brazilian S. mansoni. The absence of a bottleneck suggests that this is a rare case of a serendipitous invasion, where S. mansoni parasites were preadapted to the Americas and were able to establish with relative ease.

The marble bury test is a commonly applied behavioral test often used to screen pharmaceuticals for treatment of compulsivity or anxiety disorders and to better understand the underlying neurobiology of these conditions using rodent models. We explore the use of the marble bury test in a repeated fashion over longer time intervals to assess how it may be used in a more chronic context. Assuming that marble bury scores represent a neurobiological phenotype of an individual, we hypothesize that the measurement of this phenotype is repeatable over time in a healthy animal. We performed four trials over the course of 12 weeks, using three overlapping scores for marble burying: 100%,50%, and 0% buried. Despite intertrial differences in the mean number of marbles buried, we found significant repeatability scores across 12 weeks for two measurements: marbles buried 100% and marbles buried50%. Exploration of pairwise repeatabilities between the trials revealed highest repeatability during shorter time intervals. Although the effect of time is difficult to disentangle from possible effects of body size/development on the scores, we hypothesize that a combination of these two factors together influence repeatability of scores and should be explored further. Understanding the patterns in repeatability in marble burying scores can inform experimental design to use this test over longer time intervals for chronic conditions or long-term interventions. Additionally, these data indicate that a pre-test, post-test design is possible, which can reduce the number of animals needed for research applications while maintaining statistical power.

Following a 4-year annual praziquantel (PZQ) treatment campaign, the resulting prevalence of Schistosoma mansoni was seen to differ among individual villages along the Kenyan shore of Lake Victoria. We have investigated possible inherent differences in snail-related aspects of transmission among such 10 villages, including six persistent hotspot (PHS) villages (≤ 30% reduction in prevalence following repeated treatments) located along the west-facing shore of the lake and four PZQ-responding (RESP) villages (> 30% prevalence reduction following repeated treatment) along the Winam Gulf. When taking into account all sampling sites, times, and water hyacinth presence/absence, shoreline-associated Biomphalaria sudanica from PHS and RESP villages did not differ in relative abundance or prevalence of S. mansoni infection. Water hyacinth intrusions were associated with increased B. sudanica abundance. The deeper water snail Biomphalaria choanomphala was significantly more abundant in the PHS villages, and prevalence of S. mansoni among villages both before and after control was positively correlated with B. choanomphala abundance. Worm recoveries from sentinel mice did not differ between PHS and RESP villages, and abundance of non-schistosome trematode species was not associated with S. mansoni abundance. Biomphalaria choanomphala provides an alternative, deepwater mode of transmission that may favor greater persistence of S. mansoni in PHS villages. As we found evidence for ongoing S. mansoni transmission in all 10 villages, we conclude that conditions conducive for transmission and reinfection occur ubiquitously. This argues for an integrated, basin-wide plan for schistosomiasis control to counteract rapid reinfections facilitated by large snail populations and movements of infected people around the lake.

Schistosoma mansoni, which causes human intestinal schistosomiasis, continues to be a major public health concern in the Lake Victoria basin in western Kenya, with Biomphalaria sudanica (a shoreline inhabiting snail) and Biomphalaria choanomphala (a deep-water snail) playing roles in transmission. A recent study showed that B. sudanica was abundantly present near all study villages on the lakeshore, but B. choanomphala was significantly more abundant near villages known to be persistent transmission hotspots. The present study investigated the relative compatibility of B. sudanica and B. choanomphala with S. mansoni. A reciprocal cross-infection experiment used young adult F1 generation B. sudanica and B. choanomphala that were exposed to either 1, 5, or 10 sympatric or allopatric human-derived S. mansoni miracidia. Three weeks post-exposure (PE) and weekly thereafter, the snails were counted and screened for schistosome cercariae, and at 7 wk PE, total cercariae shed during a 2 hr period by each infected snail was determined. Pre-patent periods for S. mansoni in both B. sudanica and B. choanomphala were similar, and most snails in all exposure combinations started shedding cercariae 5 wk PE. Prevalences were significantly higher in B. choanomphala (12.2-80.9%) than in B. sudanica (5.2-18.6%) at each dose, regardless of whether miracidia were of an allopatric or a sympatric source (P < 0.0001). Overall, the odds of a snail becoming infected with 5 or 10 miracidia were significantly higher than the odds of being infected with 1 miracidium, (P < 0.0001), and fewer cercariae were produced by snails exposed to single as compared to 5 or 10 miracidia. On average, B. choanomphala produced more cercariae ( = 458, SD = 414) than B. sudanica ( = 238, SD = 208) (P < 0.0001). These results suggest that B. choanomphala is more compatible with S. mansoni than B. sudanica. Though B. choanomphala can be found in shallow shoreline waters, it is, for the most part, a deeper-water taxon. Because dredging is a relatively inefficient means of sampling, B. choanomphala is likely underestimated with respect to its population size, the number of S. mansoni-positive snails, and its role in maintaining transmission.

Oarfish are rarely seen and seldom studied, which makes their parasite fauna even more enigmatic. Necropsy of 12 oarfish, Regalecus russelii (Regalecidae) (Cuvier, 1816), from Japan yielded 2 species of acanthocephalans. One species was found in 2 oarfish and a total of 76 specimens was collected, but only a single, immature specimen of the second species was found. The former represents an undescribed species from the order Echinorhynchida and is described here. Morphological and phylogenetic analyses of the small subunit ( SSU) rDNA place this species in the family Gymnorhadinorhynchidae, and genus Gymnorhadinorhynchus which is characterized by a cylindrical proboscis with longitudinal rows of hooks, basal circle of enlarged hooks, asymmetry of hook shape, 4 cement glands, and a spineless trunk. Diagnostic characters of this species within the genus are the number of longitudinal rows of hooks (14), smaller body size (males: 4.8-6.6 mm and females: 5.3-6.3 mm) and a number of molecular autapomorphies including a number of long insertions in both the SSU and large subunit rDNA (LSU). A single immature female of Bolbosoma sp. (Palaeacanthocephala: Plagiorhynchidae) was also found with its anterior end embedded in the mucosa of the pyloric ceca. The characters of this specimen are not consistent with any other known species of Bolbosoma; however, because only 1 immature specimen with a partially invaginated proboscis was recovered, it was not designated as a new species.

Following a four-year annual praziquantel treatment campaign the resulting prevalence ofS. mansoniwas seen to differ among individual villages along the Kenyan shore of Lake Victoria. We have investigated possible inherent differences in snail-related aspects of transmission among such 10 villages, including six persistent hotspot (PHS) villages (≤30% reduction in prevalence following repeated treatments) located along the west-facing shore of the lake, and four PZQ-responding (RESP) villages (>30% prevalence reduction following repeated treatment) along Winam Gulf. When taking into account all sampling sites and times and water hyacinth presence/absence, shoreline-associatedB. sudanicafrom PHS and RESP villages did not differ in relative abundance or prevalence ofS. mansoniinfection. Water hyacinth intrusions were associated with increasedB. sudanicaabundance. The deeper water snailBiomphalaria choanomphalawas significantly more abundant in the PHS villages and prevalence ofS. mansoniamong villages both before and after control was positively correlated withB. choanomphalaabundance. Worm recoveries from sentinel mice did not differ between PHS and RESP villages, and abundance of non-schistosome trematode species was not associated withS. mansoniabundance.Biomphalaria choanomphalaprovides an alternative, deepwater mode of transmission that may favor greater persistence ofS. mansoniin PHS villages. As we found evidence for ongoingS. mansonitransmission in all 10 villages, we conclude conditions conducive for transmission and reinfection occur ubiquitously. This argues for an integrated, basin-wide plan for schistosomiasis control to counteract rapid reinfections facilitated by large snail populations and movements of infected people around the lake.

Examination of a small portion of the viscera of an oarfish (Regalecus russellii) recovered from Santa Catalina Island, southern California, revealed numerous tetraphyllidean tapeworm plerocercoids, Clistobothrium cf. montaukensis; 2 juvenile nematodes, Contracaecum sp.; and a fragment of an adult acanthocephalan, family Arhythmacanthidae. This suggests that the fish was relatively heavily parasitized. The presence of larval and juvenile worms suggests that oarfish are preyed upon by deep-swimming predators such as the shortfin mako shark, Isurus oxyrinchus, known to be a definitive host for the adult tapeworm, and also by diving mammals such as sperm whales, Physeter catodon L., hosts of Contracaecum spp. nematodes.

A vaccine against schistosomiasis would contribute significantly to reducing the 3–70 million disability-adjusted life years lost annually to the disease. Towards this end, inoculation with the large extracellular loop (EC-2) of Schistosoma mansoni tetraspanin-2 protein (Sm-TSP-2) has proved effective in reducing worm and egg burdens in S. mansoni-infected mice. The EC-2 loop of Schistosoma japonicum TSP-2, however, has been found to be highly polymorphic, perhaps diminishing the likelihood that this antigen can be used for vaccination against this species. Here, we examine polymorphism of the EC-2 of Sm-TSP-2 in genetically unique worms derived from six individuals from Kisumu, Kenya.

Oarfish are rarely seen and seldom studied, which makes their parasite fauna even more enigmatic. Necropsy of 12 oarfish, Regalecus russelii (Regalecidae) (Cuvier, 1816), from Japan yielde...

We review how molecular markers and evolutionary analysis have been applied to the study of schistosome parasites, important pathogens that infect over 200 million people worldwide. Topics reviewed include phylogenetics and biogeography, hybridization, infection within snails, mating systems, and genetic structure. Some interesting generalizations include that schistosome species hybridize frequently and have switched definitive hosts repeatedly in evolutionary time. We show that molecular markers can be used to infer epidemiologically-relevant processes such as spatial variation in transmission, or to reveal complex patterns of mate choice. Analysis of genetic structure data shows that transmission foci can be structured by watershed boundaries, habitat types, and host species. We also discuss sampling and analytical problems that arise when using larvae to estimate genetic parameters of adult schistosome populations. Finally, we review pitfalls in methodologies such as genotyping very small individuals, statistical methods for identifying clonemates or for identifying sibling groups, and estimating allele frequencies from pooled egg samples.

OBJECTIVE To investigate trends in the efficacy of praziquantel (PZQ) suggestive of the emergence of drug resistance against Schistosoma mansoni infection after 12.5 years of intense, repeated use in a small geographic area along the shores of Lake Victoria. METHODS As part of a longitudinal study, 178 men occupationally exposed to schistosomes were repeatedly tested for S. mansoni infection at 4- to 6-week intervals and treated with PZQ at each reinfection. We compared cure rates by year of study and examined factors associated with cure failure in a multivariate logistic regression model. RESULTS Overall, the cure rate after a single dose of PZQ was 66%, ranging annually from 36% to 82%. In multivariate analysis, failure to cure after 1 PZQ dose was significantly associated with high intensity of infection and having fewer previous exposures to dying worms. Even after adjustment for these factors, treatments administered in 2006 were significantly more likely to result in cure failures than treatments administered in 2004, the year in which PZQ efficacy was highest. While cure rates varied over the course of 12 years, there was no consistent downward trend towards decreased efficacy over time. In years for which malacological data were available, periods of low PZQ efficacy coincide with high rates of S. mansoni infection in nearby snail populations. CONCLUSION We did not find a pattern of cure failures consistent with development of clinical resistance to PZQ in our intensely treated cohort.

OBJECTIVES To develop and assess a microsatellite technique to characterize populations of Schistosoma mansoni from humans. METHODS For each of five patients, we calculated the allele count and frequency at 11 loci for several pools of miracidia (50 and 100), and compared these to population values, determined by amplifying microsatellites from 186 to 200 individual miracidia per patient. RESULTS We were able to detect up to 94.5% of alleles in pools. Allele count and frequency strongly and significantly correlated between singles and pools; marginally significant differences (P < 0.05) were detected for one patient (pools of 50) for allele frequencies and for two patients (pools of 100) for allele counts. Kato-Katz egg counts and number of alleles per pool did not co-vary, indicating that further direct comparisons of the results from these two techniques are needed. CONCLUSIONS Allele counts and frequency profiles from pooling provide important information about infection intensity and complexity, beyond that obtained from traditional methods. Although we are not advocating use of pooling to replace individual genotyping studies, it can potentially be useful in certain applications as a rapid and cost effective screening method for studies of S. mansoni population genetics, or as a more informative way to quantify and characterize human worm populations.

Schistosomiasis is one of the major unconquered infectious diseases afflicting people of developing countries, particularly in Africa. A deeper understanding of the epidemiology of schistosomes is complicated by the intravascular location of adult worms which makes them routinely unavailable for study. Their progeny, miracidia, which are hatched from eggs that are passed in feces, are available and can provide valuable insights about human infections, but they are small in size, hindering robust molecular analyses. Here we present a new high-throughput technique to assess the genotypes at 21 previously published microsatellite loci for individual miracidia of S. mansoni. The 21 loci can be amplified in four multiplexed PCR reactions; however, enough template is produced for approximately six PCR reactions, which allows for additional PCR reactions for resampling or obtaining additional data. We validated this technique using a pedigree study employing laboratory crosses of S. mansoni from Kenya to obtain sets of parents and offspring. Of 23 loci examined, 21 loci were found to be reliable: false alleles were rare and missing alleles due to allelic dropout occurred at only two loci in approximately 5% of the offspring. The latter type of error can be further reduced by reamplification which is possible with our method. This technique is amenable to a 96-well format thus facilitating analysis of larger samples of miracidia, allowing more robust molecular epidemiological studies of S. mansoni to infer population size, population structure, gene flow, mating systems, speciation, and host race formation.

An investigation of a parasite species that is broadly host- and habitat-specific and exhibits alternative transmission strategies was undertaken to examine intraspecific variability and if it can be attributed to cryptic speciation or environmentally induced plasticity. Specimens of an acanthocephalan parasite, Leptorhynchoides thecatus, collected throughout North America were analysed phylogenetically using sequences of the cytochrome oxidase I gene and the internal transcribed spacer region. Variation in host use, habitat use, and transmission were examined in a phylogenetic context to determine if they were more likely phylogenetically based or due to environmental influences. Results indicated that most of the variation detected can be explained by the presence of cryptic species. The majority of these species have narrow host and microhabitat specificities although one species, which also may comprise a complex of species, exhibits broad host and habitat specificity. Alternate transmission pathways only occurred in two of the cryptic species and correlate with host use patterns. Taxa that mature in piscivorous piscine hosts use a paratenic fish host to bridge the trophic gap between their amphipod intermediate host and piscivorous definitive host. One potential example of environmentally induced variation was identified in three populations of these parasites, which differ on their abilities to infect different host species.

Parasite fitness is largely influenced by a variation in host response due to the host's genetic background. Here we investigated the impact of host genotype on pathogen success in the snail vector of its castrating parasite, Schistosoma mansoni. We infected five inbred lines of Biomphalaria glabrata with two infection doses and followed their growth, reproductive output and parasite production throughout the course of infection. There was no difference in resistance to infection among inbred lines, but lines varied in their responses to infection and the numbers of parasites produced. Snails did not compensate for castration by increasing their fecundity during the early phase of infection (fecundity compensation). However, some lines were able to delay parasite shedding for up to 30 weeks, thus prolonging reproduction before the onset of castration. Here we propose this strategy as a novel defense against castrating pathogens in snails. Gigantism, a predicted outcome of castration due to energy reallocation, occurred early in infection (

Laboratory-reared cystacanths of Leptorhynchoides thecatus (Acanthocephala: Rhadinorhynchidae) were used to study the effect of cystacanth size on adult success and the factors that influence cystacanth size within the intermediate host. To assess how host size and intensity of infection influence cystacanth size, infected amphipods (Hyalella azteca) were measured, and sex, length, and width of cystacanths were determined. After a subset of cystacanths was measured, small- and large-size classes of cystacanths were designated. To determine how cystacanth size relates to adult size, green sunfish (Lepomis cyanellus) were fed 10 large or small cystacanths. Fish were dissected 6 wk after infection, and worms were removed. After adult worms were permanently mounted on slides, their length and width were measured. Intensity of infection and amphipod size significantly influenced cystacanth size in that large amphipods harbored larger cystacanths than did small amphipods and heavy infections produced smaller cystacanths than did light infections. Adult worms from the small and large cystacanth-size classes showed no significant difference in size; however, large cystacanths had a significantly higher establishment and survival than did small cystacanths: 40% of large worms and 14% of small worms were recovered. The results of this study indicate that host size and host sharing influence cystacanth size and that cystacanth size is an important factor in determining adult success.

Seasonal prevalence and abundance of the helminths of bluegill sunfish in a Louisiana oligohaline bayou were measured by a survey and a field "live-box" experiment. The survey took place from the spring of 1997 to the summer of 1998 and examined fish that were7 cm. Three trematode species, Phagicola nana (Heterophyidae), Ascocotyle tenuicollis (Heterophyidae), and Posthodiplostomum minimum (Diplostomatidae), and 2 nematode species, Camallanus oxycephalus (Camallanidae) and Spinitectus carolini (Cystidicolidae), were examined. Camallanus oxycephalus was the only helminth that showed a distinct seasonal pattern. Abundance and prevalence peaked in summer, which was likely driven by concerted reproductive cycles of females. The survey data indicated that the other helminths fluctuated over time but did not seem to follow a distinct seasonal pattern. The fluctuations could be attributed to the variable nature of the habitat or to the stochastic events that influence transmission dynamics, particularly isolated events such as tropical storms, which cause dramatic changes in salinity. The live-box experiment successfully measured recruitment of A. tenauicollis and suggested a seasonal component of the distribution of this species, which was possibly caused by temperature-dependent emergence of cercariae from the snail intermediate host.

Translating research advances to natural systems using experimental laboratory studies is often difficult because of the variability between the natural environment and experimental conditions. Because environmental conditions have a large effect on an organism's physiology, responses to stressors like nutrient limitation, temperature, oxygen deprivation, predation, and parasite/pathogen infection are likely to be context dependent. Therefore, it is essential to examine the impact the study environment has on the experimental outcome. Here, we explored the effect of light exposure on susceptibility to parasite infection. The Biomphalaria glabrata / Schistosoma mansoni study system is a well-established model for studying schistosomiasis. It has been general practice to maintain the vector, B. glabrata, in dark conditions after exposure to miracidia of the human pathogen S. mansoni. We evaluated susceptibility of B. glabrata to S. mansoni under 3 different light conditions during the prepatent period, light (125 lx) on a 12-12 cycle, dim light (3 lx) on a 12-12 cycle, and no light (24 hr at 0 lx). We hypothesized that stress due to photoperiod disruption (24 hr of darkness) would result in compromised immune function and lead to higher susceptibility to infection. Prevalence of infected snails differed significantly between the light conditions, and higher susceptibility was observed in the full light and complete dark conditions compared with the low light conditions. The dim conditions are representative of current methods for evaluating susceptibility in this system. Our results indicate that light exposure during the prepatent period can affect infection outcomes, and environmental conditions must therefore be considered when assessing fitness and immune response due to interactions between host genotype and environment.

The enteric helminth fauna of the threatened yellow-blotched sawback turtle, Graptemys flavimaculata Cagle, 1954, was examined for the first time. Seven specimens were collected from the Pascagoula River in Jackson, George, and Green counties, Mississippi, U.S.A. The turtles were necropsied for toxicology studies, and their intestinal tracts were salvaged for parasitic analysis. One acanthocephalan species, Neoechinorhynchus stunkardi; 5 trematode species, Telorchis corti, T. angustus, T. singularis, an unidentified pronocephalid, and Cotylaspsis cokeri; and 2 nematode species, Falcaustra procera, and Spiroxys sp., were found. No new species were encountered; however, G. flavimaculata is a new host record, and the Pascagoula River is a new location record for all 8 helminth species. Transmission of at least 3 of the 8 helminth species involves ingestion of a mollusc intermediate host by the turtle host, which is not unexpected given the molluscivorous diet of adult G. flavimaculata.

Schistosomes are trematode parasites of global importance, causing infections in millions of people, livestock, and wildlife. Most studies on schistosomiasis, involve human subjects; as such, there is a paucity of longitudinal studies investigating parasite dynamics in the absence of intervention. As a consequence, despite decades of research on schistosomiasis, our understanding of its ecology in natural host populations is centered around how environmental exposure and acquired immunity influence acquisition of parasites, while very little is known about the influence of host physiology, coinfection and clearance in the absence of drug treatment. We used a 4-year study in free-ranging African buffalo to investigate natural schistosome dynamics. We asked (i) what are the spatial and temporal patterns of schistosome infections; (ii) how do parasite burdens vary over time within individual hosts; and (iii) what host factors (immunological, physiological, co-infection) and environmental factors (season, location) explain patterns of schistosome acquisition and loss in buffalo? Schistosome infections were common among buffalo. Microgeographic structure explained some variation in parasite burdens among hosts, indicating transmission hotspots. Overall, parasite burdens ratcheted up over time; however, gains in schistosome abundance in the dry season were partially offset by losses in the wet season, with some hosts demonstrating complete clearance of infection. Variation among buffalo in schistosome loss was associated with immunologic and nutritional factors, as well as co-infection by the gastrointestinal helminth Cooperia fuelleborni. Our results demonstrate that schistosome infections are surprisingly dynamic in a free-living mammalian host population, and point to a role for host factors in driving variation in parasite clearance, but not parasite acquisition which is driven by seasonal changes and spatial habitat utilization. Our study illustrates the power of longitudinal studies for discovering mechanisms underlying parasite dynamics in individual animals and populations., Author summary Schistosomes are a parasite of global importance, affecting over 200 million people worldwide, while also infecting livestock and wildlife. Despite decades of research on schistosomiasis in humans, little is known about what drives patterns of infection in untreated naturally occurring populations. We took advantage of a study in African buffalo to understand how geography, nutrition, immunity and coinfection drive schistosome acquisition and loss. The most striking outcome of our study was that schistosome burden varied seasonally within an individual, with some hosts able to completely clear infection, and others unable to do so. The ability of a buffalo to clear infection was affected by immune response and co-infection with other gastrointestinal parasites while host immunity and coinfection were not important in determining whether a buffalo became infected. These outcomes should be taken into consideration when designing control programs for human schistosomiasis.

Six new species of Leptorhynchoides from the southeastern United States are described. These new species were once part of the Leptorhynchoides thecatus complex of species that was previously recognized on the basis of DNA sequence data. Multivariate morphometric analysis including discriminant function analysis and decision tree analysis indicated that each of the species is morphologically distinct. Both analyses classified more than 90% of specimens correctly and most misclassifications occurred between members of 2 pairs of species that are morphologically similar. The most discriminating continuous characters were: trunk length, number of longitudinal rows of hooks, length of the longest hook, and testes width. Hook asymmetry and missing hooks on the proboscis were also important taxonomic characters. The discriminant function and the decision tree generated from the data were used to classify new specimens, yielding a 96% and 84% correct classification rate, respectively. The new taxonomic designations account for much of the previously recognized variability in host use, habitat use, and development as determined by survey data. With the addition of these 6 new taxa, 10 species currently are recognized within the genus.

Background Schistosomiasis is a debilitating neglected tropical disease that infects over 200 million people worldwide. To combat this disease, in 2012, the World Health Organization announced a goal of reducing and eliminating transmission of schistosomes. Current control focuses primarily on mass drug administration (MDA). Therefore, we monitored transmission of Schistosoma mansoni via fecal egg counts and genetic markers in a typical school based MDA setting to ascertain the actual impacts of MDA on the targeted schistosome population. Methods For 4 years, we followed 67 children enrolled in a MDA program in Kenya. Infection status and egg counts were measured each year prior to treatment. For 15 of these children, for which there was no evidence of acquired resistance, meaning they became re-infected following each treatment, we collected microsatellite genotype data from schistosomes passed in fecal samples as a representation of the force of transmission between drug treatments. We genotyped a total of 4938 parasites from these children, with an average of 329.2 parasites per child for the entire study, and an average of 82.3 parasites per child per annual examination. We compared prevalence, egg counts, and genetic measures including allelic richness, gene diversity (expected heterozygosity), adult worm burdens and effective number of breeders among time points to search for evidence for a change in transmission or schistosome populations during the MDA program. Findings We found no evidence of reduced transmission or schistosome population decline over the course of the program. Although prevalence declined in the 67 children as it did in the overall program, reinfection rates were high, and for the 15 children studied in detail, schistosome egg counts and estimated adult worm burdens did not decline between years 1 and 4, and genetic diversity increased over the course of drug treatment. Interpretation School based control programs undoubtedly improve the health of individuals; however, our data show that in an endemic area, such a program has had no obvious effect on reducing transmission or of significantly impacting the schistosome population as sampled by the children we studied in depth. Results like these, in combination with other sources of information, suggest more integrated approaches for interrupting transmission and significantly diminishing schistosome populations will be required to achieve sustainable control., Author Summary Schistosomiasis is a chronic and debilitating disease. Current control focuses primarily on mass drug administration (MDA). We monitored schistosome transmission via fecal egg counts and genetic markers in a school based MDA setting to learn how the intervention was influencing transmission or the targeted schistosome population. For 4 years, we followed 67 children enrolled in a MDA program in Kenya, and for 15 of these, we repeatedly acquired in depth genetic data regarding the schistosome populations they harbored. Although prevalence declined in the 67 children, we found no evidence of reduced egg counts/worm burdens in those that reacquired infections and genetic diversity of schistosomes increased over the course of treatment. Our data indicate that this school based MDA program had a strong benefit to individual health as fewer children were infected over time. However, this decline does not appear to be due to schistosome population reduction, and may be caused by either acquired resistance or behavioral changes of the children. In conclusion, control programs based on chemotherapy alone or based on only a subset of the population will need to be supplemented with additional approaches if we are to achieve the WHO goal of eliminating human schistosomiasis by 2025.

For ethical and logistical reasons, population-genetic studies of parasites often rely on the non-invasive sampling of offspring shed from their definitive hosts. However, if the sampled offspring are naturally derived from a small number of parents, then the strong family structure can result in biased population-level estimates of genetic parameters, particularly if reproductive output is skewed. Here, we document and correct for the strong family structure present within schistosome offspring (miracidia) that were collected non-invasively from humans in western Kenya. By genotyping 2,424 miracidia from 12 patients at 12 microsatellite loci and using a sibship clustering program, we found that the samples contained large numbers of siblings. Furthermore, reproductive success of the breeding schistosomes was skewed, creating differential representation of each family in the offspring pool. After removing the family structure with an iterative jacknifing procedure, we demonstrated that the presence of relatives led to inflated estimates of genetic differentiation and linkage disequilibrium, and downwardly-biased estimates of inbreeding coefficients (FIS). For example, correcting for family structure yielded estimates of FST among patients that were 27 times lower than estimates from the uncorrected samples. These biased estimates would cause one to draw false conclusions regarding these parameters in the adult population. We also found from our analyses that estimates of the number of full sibling families and other genetic parameters of samples of miracidia were highly intercorrelated but are not correlated with estimates of worm burden obtained via egg counting (Kato-Katz). Whether genetic methods or the traditional Kato-Katz estimator provide a better estimate of actual number of adult worms remains to be seen. This study illustrates that family structure must be explicitly accounted for when using offspring samples to estimate the genetic parameters of adult parasite populations., Author Summary Genetic epidemiology uses genetic data to uncover patterns of disease processes. To acquire data for these analyses, individual pathogens are collected and scored at genetic markers, and the resultant data are analyzed to infer biological patterns about the pathogen populations. In lieu of invasive sampling of adult pathogens in humans, researchers have relied on non-invasive sampling of parasite offspring (often shed in fecal samples). One potential problem with this approach is that analyses using the offspring data will be biased because many of the offspring are related and family sizes are likely to be unequal. We show that this sampling issue is relevant in a natural transmission zone in western Kenya and that it yields biases in three important parameters: genetic differentiation, inbreeding coefficients, and estimates of the amount of non-random association between loci (linkage disequilibrium). We also develop a method to remove these biases by removing the sibling structure present in the dataset. Finally, we suggest that our measure of family number, as well as other genetic measures, may be useful measures of the worm burdens in patients.

Metacercariae of an unidentified species of Apophallus Luhe, 1909 are associated with overwinter mortality in coho salmon, Oncorhynchus kisutch (Walbaum, 1792), in the West Fork Smith River, Oregon. We infected chicks with these metacercariae in order to identify the species. The average size of adult worms was 197 × 57 μm, which was 2 to 11 times smaller than other described Apophallus species. Eggs were also smaller, but larger in proportion to body size, than in other species of Apophallus. Based on these morphological differences, we describe Apophallus microsoma n. sp. In addition, sequences from the cytochrome c oxidase 1 gene from Apophallus sp. cercariae collected in the study area, which are likely conspecific with experimentally cultivated A. microsoma, differ by >12% from those we obtained from Apophallus donicus (Skrjabin and Lindtrop, 1919) and from Apophallus brevis Ransom, 1920. The taxonomy and pathology of Apophallus species is reviewed.

SUMMARYSchistosoma kisumuensisn. sp. is described based on 6 adult males and 2 adult females collected from the circulatory system of 3 murid rodent species,Pelomys isseli, Mastomys natalensis, andDasymys incomtus. Specimens were collected from a single location, Nyabera Swamp, in Kisumu, Kenya in the Lake Victoria Basin. This new species is morphologically similar to members of theS. haematobiumgroup, currently represented by 8 species parasitizing artiodactyls and primates, including humans.Schistosoma kisumuensisdiffers from these species by producing relatively smallSchistosoma intercalatum-like eggs (135·2×52·9 μm) with a relatively small length to width ratio (2·55). Comparison of approximately 3000-base-pair sequences of nuclear rDNA (partial 28S) and mtDNA (partialcox1,nad6, 12S) strongly supports the status ofS. kisumuensisas a new species and as a sister species ofS. intercalatum. Thecox1 genetic distance between these two species (6·3%) is comparable to other pairwise comparisons within theS. haematobiumgroup. Separation of the Congo River and Lake Victoria drainage basins is discussed as a possible factor favoring the origin of this species.

A recently developed high-throughput technique that allows multi-locus microsatellite analysis of individual miracidia of Schistosoma mansoni was used to assess the levels of genetic diversity and population structure in 12 infrapopulations of the parasite, each infrapopulation derived from an infected school child from the Mwea area, central Kenya. The mean number of alleles per locus was in the range 8.22-10.22, expected heterozygosity in Hardy-Weinberg equilibrium was 0.68-0.70, and pairwise F(ST) values ranged from 0.16% to 3.98% for the 12 infrapopulations. Although the genetic diversity within each infrapopulation of S. mansoni in this area was generally high, low levels of genetic structure were observed, suggestive of high levels of gene flow among infrapopulations. Private alleles were found in 8 of the 12 infrapopulation, the highest number of private alleles recorded per infrapopulation was 3. Our data suggest that the level of gene flow among infrapopulations of S. mansoni in Mwea is extremely high, thus providing opportunity for spread of rare alleles, including those that may confer character traits such as drug resistance and virulence.

The mating systems of internal parasites are inherently difficult to investigate although they have important implications for the evolutionary biology of the species, disease epidemiology, and are important considerations for control measures. Using parentage analyses, three topics concerning the mating biology of Schistosoma mansoni were investigated: the number of mates per adult male and female, variance in reproductive success among individuals, and the potential role for sexual selection on male body size and also mate choice for genetically dissimilar individuals. Results indicated that schistosomes were mostly monogamous, and evidence of only one mate change occurred over a period of 5-6 weeks. One male was polygynous and contained two females in its gynecophoral canal although offspring were only detected for one of the females. Even though they were primarily monogamous and the sex ratio near even, reproductive success was highly variable, indicating a potential role for sexual selection. Male body size was positively related to reproductive success, consistent with sexual selection via male-male competition and female choice for large males. However, relatedness of pairs was not associated with their reproductive success. Finally, genetically identical individuals differed significantly in their reproductive output and identical males in their body size, indicating important partner and environmental effects on these traits.

Hybridization and introgression can have important consequences for the evolution, ecology and epidemiology of pathogenic organisms. We examined the dynamics of hybridization between a trematode parasite of humans, Schistosoma mansoni, and its sister species, S. rodhaini, a rodent parasite, in a natural hybrid zone in western Kenya. Using microsatellite markers, rDNA and mtDNA, we showed that hybrids between the two species occur in nature, are fertile and produce viable offspring through backcrosses with S. mansoni. Averaged across collection sites, individuals of hybrid ancestry comprised 7.2% of all schistosomes collected, which is a large proportion given that one of the parental species, S. rodhaini, comprised only 9.1% of the specimens. No F1 individuals were collected and all hybrids represented backcrosses with S. mansoni that were of the first or successive generations. The direction of introgression appears highly asymmetric, causing unidirectional gene flow from the rodent parasite, S. rodhaini, to the human parasite, S. mansoni. Hybrid occurrence was seasonal and most hybrids were collected during the month of September over a 2-year period, a time when S. rodhaini was also abundant. We also examined the sex ratios and phenotypic differences between the hybrids and parental species, including the number of infective stages produced in the snail host and the time of day the infective stages emerge. No statistical differences were found in any of these characteristics, and most of the hybrids showed an emergence pattern similar to that of S. mansoni. One individual, however, showed a bimodal emergence pattern that was characteristic of both parental species. In conclusion, these species maintain their identity despite hybridization, although introgression may cause important alterations of the biology and epidemiology of schistosomiasis in this region.

The complete sequence of the mitochondrial genome of Leptorhynchoides thecatus (Acanthocephala) was determined, and a phylogenetic analysis was carried out to determine its placement within Metazoa. The genome is circular, 13,888 bp, and contains at least 36 of the 37 genes typically found in animal mitochondrial genomes. The genes for the large and small ribosomal RNA subunits are shorter than those of most metazoans, and the structures of most of the tRNA genes are atypical. There are two significant noncoding regions (377 and 294 bp), which are the best candidates for a control region; however, these regions do not appear similar to any of the control regions of other animals studied to date. The amino acid and nucleotide sequences of the protein coding genes of L. thecatus and 25 other metazoan taxa were used in both maximum likelihood and maximum parsimony phylogenetic analyses. Results indicate that among taxa with available mitochondrial genome sequences, Platyhelminthes is the closest relative to L. thecatus, which together are the sister taxon of Nematoda; however, long branches and/or base composition bias could be responsible for this result. The monophyly of Ecdysozoa, molting organisms, was not supported by any of the analyses. This study represents the first mitochondrial genome of an acanthocephalan to be sequenced and will allow further studies of systematics, population genetics, and genome evolution.

Being his former graduate students, we are thrilled and honored to present the 2012 Clark P. Read Mentor Award to Dr. William (Bill) F. Font. Bill is a native of New Orleans, where he obtained a B.S. from Tulane University in 1966. He served honorably in the U.S. Navy from 1966–1970 and is a Vietnam Veteran. In 1970, he began his academic career at Louisiana State University under Dr. Kenneth Corkum. He earned an M.S. in 1972 and then a Ph.D. in 1975. His first appointment was as an Assistant Professor in 1975 at the University of Wisconsin–Eau Claire (UW–Eau Claire) and became an Associate Professor in 1983. In 1985, he moved to Southeastern Louisiana University (SELU) where he obtained the rank of Professor in 1988. In 2010, Bill retired from classroom teaching but remains a Scholar in Residence and Professor Emeritus at SELU. Presenting ASP’s mentor award to Bill speaks volumes to Bill’s mentoring impact in the field of parasitology. You see, Bill is the first recipient of this award to have been at institutions where there were no Ph.D. programs. At both UW–Eau Claire and SELU, there is a greater emphasis on classroom teaching and fewer resources available to establish and maintain a large-scale research program. Nevertheless, Bill’s mentoring has reached far and wide to many students and colleagues at all levels. He has mentored 14 students in an M.S. degree, served as an external committee member for 10 students in a Ph.D. degree, 3 students for an Undergraduate Honors Thesis, and 46 undergraduate students in scientific research. Bill is a natural, thoughtful, and dedicated mentor and, today, we recognize and celebrate his mentoring contributions. Bill has stated on several occasions that the best part of his career was working with students. He is a gracious and giving person and outwardly expresses his gratitude for the opportunity to work with students. Upon his recent retirement, he wrote a heartfelt letter to his students stating, ‘‘I am writing to let you know that I have retired. I think that this is a good time to thank you all for what you have meant to me. Both teaching and research have been enjoyable parts of my career, but I would have to say that the very best part of the past 25 years at Southeastern has been getting to know, and doing research with, my graduate students. Working with you has been absolutely great!’’ When we requested letters for Bill’s nomination for this award, there was overwhelming enthusiasm by his past students to support his recommendation. We would like to share several themes that are common throughout the support letters. Bill has had an enormous impact on each of our professional and personal developments. His role has been integral in helping us determine our career paths, and for many this guidance came at pivotal times in their lives. Bill is a truly caring and supportive mentor for both professional and personal matters, and this support has been extended far past our graduation days. Also, Bill held up the highest expectations for each of us and truly believed that we could meet them. He expected us to put in the same long hours and dedication as he did. Bill is also very talented at inspiring students and he could easily maintain his faith, support, and encouragement to live up to his high standards even through what felt like trying times for his students. Part of what made him such a great mentor was that he had an ‘‘open-door policy’’ and, thus, was never too busy to mentor. We could walk into his office at any time and he would put down what he was doing to talk with us. This unique mentoring quality is one we took for granted then, but now, as overbooked faculty members ourselves, we realize the time sacrifices he made to help us. Another insight gained from reading these letters is that Bill was able to tailor his mentoring style to be what each of us needed. This is the hallmark of a good mentor: that one can understand and appreciate the differences among students and how to best guide them. For Sarah Brock, it was encouraging her that she could do more; for Amanda Vincent it was challenging her to think critically and guiding her to discover links between biology and policy; for Chrystal Kain it was his faith in her to succeed as a graduate student; for Ashleigh Smythe it was patience and faith in her ability; for Ricky Fiorillo it was ‘‘tough’’ love, honesty, and continued support. Speaking from a personal experience (CDC), Bill possessed several traits that made him a great research mentor. He was patient, enthusiastic, knowledgeable, supportive, and inquisitive. He often led by example rather than dictating how one should conduct research. Indeed, it is said that imitation is the greatest form of flattery. I must say that Bill’s methodological (and often ‘‘anal’’) attention to detail in his research is probably one of the most important skills I have taken with me in my own career. I cannot state with more sincerity that Bill’s mentorship has greatly influenced my career and he deserves much of the credit for where I am today. From the very beginning, he was responsible for my current academic path. I recall when I applied to SELU, I wanted to work with a herpetologist who ended up not taking students. Bill called me out of the blue and offered me a chance to work with him. I said yes, and ever since am indebted to the opportunity he provided me. As with all his students, Bill continued to look out for our future interests. Indeed, I remember when he came back from a meeting in England, where he saw a talk by Dr. Mike Blouin. Bill made it a point to direct me to Mike’s papers. A year later, I was starting my Ph.D. in Mike’s laboratory. Even through my Ph.D., I often relied upon Bill for research advice. Mike was a population geneticist, but not a parasitologist. Thus, I would often call Bill to get his guidance on parasitological methods. Bill always provided me with the time, even though I was no longer his responsibility. Today, Bill’s research continues to inspire me and we continue to work * Department of Biology, Texas A&M University, 3258 TAMU, College Station, Texas 77843-3258. DOI: 10.1645/12-79.1

Background The near exclusive use of praziquantel (PZQ) for treatment of human schistosomiasis has raised concerns about the possible emergence of drug-resistant schistosomes. Methodology/Principal Findings We measured susceptibility to PZQ of isolates of Schistosoma mansoni obtained from patients from Kisumu, Kenya continuously exposed to infection as a consequence of their occupations as car washers or sand harvesters. We used a) an in vitro assay with miracidia, b) an in vivo assay targeting adult worms in mice and c) an in vitro assay targeting adult schistosomes perfused from mice. In the miracidia assay, in which miracidia from human patients were exposed to PZQ in vitro, reduced susceptibility was associated with previous treatment of the patient with PZQ. One isolate (“KCW”) that was less susceptible to PZQ and had been derived from a patient who had never fully cured despite multiple treatments was studied further. In an in vivo assay of adult worms, the KCW isolate was significantly less susceptible to PZQ than two other isolates from natural infections in Kenya and two lab-reared strains of S. mansoni. The in vitro adult assay, based on measuring length changes of adults following exposure to and recovery from PZQ, confirmed that the KCW isolate was less susceptible to PZQ than the other isolates tested. A sub-isolate of KCW maintained separately and tested after three years was susceptible to PZQ, indicative that the trait of reduced sensitivity could be lost if selection was not maintained. Conclusions/Significance Isolates of S. mansoni from some patients in Kisumu have lower susceptibility to PZQ, including one from a patient who was never fully cured after repeated rounds of treatment administered over several years. As use of PZQ continues, continued selection for worms with diminished susceptibility is possible, and the probability of emergence of resistance will increase as large reservoirs of untreated worms diminish. The potential for rapid emergence of resistance should be an important consideration of treatment programs., Author Summary The emergence of drug resistant pathogens is a great challenge to the control of infectious diseases. Schistosomiasis is one of the world's greatest neglected tropical diseases, and it is primarily controlled with the drug praziquantel. This drug is often used by repeatedly treating patients to maintain reduced worm burdens, an ideal situation to encourage the evolution of resistant worms. Although drug based control programs are increasing, monitoring efforts for drug resistance remain rare. We measured drug susceptibility of schistosomes from a cohort of patients in Kenya who are enrolled in a longitudinal study in which they are repeatedly treated with praziquantel. We found that schistosomes from previously treated patients were significantly less susceptible than those that were not. Also, schistosomes derived from a single patient who had been treated with praziquantel 18 times showed marked resistance. Although the findings of this study indicated that reduced drug susceptibility occurs in this population of schistosomes, this trait does not seem to be spreading widely or creating clinical levels of resistance. We hypothesize that the trait remains at low frequency because of the large population of schistosomes that are not exposed to the drug and/or potential fitness costs associated with reduced susceptibility.

Background Schistosoma mansoni exists in a complex environmental milieu that may select for significant evolutionary changes in this species. In Kenya, the sympatric distribution of S. mansoni with S. rodhaini potentially influences the epidemiology, ecology, and evolutionary biology of both species, because they infect the same species of snail and mammalian hosts and are capable of hybridization. Methodology/Principal Findings Over a 2-year period, using a molecular epidemiological approach, we examined spatial and temporal distributions, and the overlap of these schistosomes within snails, in natural settings in Kenya. Both species had spatially and temporally patchy distributions, although S. mansoni was eight times more common than S. rodhaini. Both species were overdispersed within snails, and most snails (85.2% for S. mansoni and 91.7% for S. rodhaini) only harbored one schistosome genotype. Over time, half of snails infected with multiple genotypes showed a replacement pattern in which an initially dominant genotype was less represented in later replicates. The other half showed a consistent pattern over time; however, the ratio of each genotype was skewed. Profiles of circadian emergence of cercariae revealed that S. rodhaini emerges throughout the 24-hour cycle, with peak emergence before sunrise and sometimes immediately after sunset, which differs from previous reports of a single nocturnal peak immediately after sunset. Peak emergence for S. mansoni cercariae occurred as light became most intense and overlapped temporally with S. rodhaini. Comparison of schistosome communities within snails against a null model indicated that the community was structured and that coinfections were more common than expected by chance. In mixed infections, cercarial emergence over 24 hours remained similar to single species infections, again with S. rodhaini and S. mansoni cercarial emergence profiles overlapping substantially. Conclusions/Significance The data from this study indicate a lack of obvious spatial or temporal isolating mechanisms to prevent hybridization, raising the intriguing question of how the two species retain their separate identities., Author Summary One of the world's most prevalent neglected diseases is schistosomiasis, which infects approximately 200 million people worldwide. Schistosoma mansoni is transmitted to humans by skin penetration by free-living larvae that develop in freshwater snails. The origin of this species is East Africa, where it coexists with its sister species, S. rodhaini. Interactions between these species potentially influence their epidemiology, ecology, and evolutionary biology, because they infect the same species of hosts and can hybridize. Over two years, we examined their distribution in Kenya to determine their degree of overlap geographically, within snail hosts, and in the water column as infective stages. Both species were spatially and temporally patchy, although S. mansoni was eight times more common than S. rodhaini. Both species overlap in the time of day they were present in the water column, which increases the potential for the species to coinfect the same host and interbreed. Peak infective time for S. mansoni was midday and dawn and dusk for S. rodhaini. Three snails were coinfected, which was more common than expected by chance. These findings indicate a lack of obvious isolating mechanisms to prevent hybridization, raising the intriguing question of how the two species retain separate identities.