Species-specific oxylipins and the effects of ontogeny and predation on their emission from freshwater snails.

Chemical cues play important roles in mediating ecological interactions. Oxylipins, oxygenated metabolites of fatty acids, are one signalling molecule type that influences the physiology and function of species, suggesting their broader significance in chemical communication within aquatic systems. Yet, our current understanding of their function is restricted taxonomically and contextually making it difficult to infer their ecological significance. Snails and leeches are ubiquitous in freshwater ecosystems worldwide, yet little is known about their oxylipin profiles and the factors that cause their profiles to change. As snails and leeches differ taxonomically and represent different trophic groups, we postulated oxylipin profile differences. For snails, we hypothesized that ontogeny (non-reproductive vs reproductive) and predation (non-infested vs leech-infested) would affect oxylipin profiles. Oxylipins were characterized from water conditioned with the snail Planorbella duryi and leech Helobdella lineata, and included three treatment types (snails, leeches, and leech-infested snails) with the snails consisting of three size classes: small (5-6 mm, non-reproductive) and medium and large (13-14 and 19-20 mm, reproductive). The two species differed in the composition of their oxylipin profiles both in diversity and amounts. Further, ontogeny and predation affected the diversity of oxylipins emitted by snails. Our experimental profiles of oxylipins show that chemical cues within freshwater systems vary depending upon the species emitting the signals, the developmental stage of the species, as well as from ecological interactions such as predation. We also identified some candidates, like 9-HETE and PGE2, that could be explored more directly for their physiological and ecological roles in freshwater systems.

DNA sequencing confirms meningeal worm (Parelaphostrongylus tenuis) and muscle worm (Parelaphostrongylus andersoni) in white-tailed deer (Odocoileus virginianus): Implications for moose (Alces alces) management.

In North America, some moose populations are declining, and meningeal worm (Parelaphostrongylus tenuis) infections may be contributing. Moose are aberrant hosts for meningeal worm and develop severe pathology whereas white-tailed deer (WTD) are definitive hosts that experience minimal pathology and spread parasite larvae into the environment. Analyses of harvested WTD heads confirmed meningeal worm in Western Manitoba, Canada including in areas where moose have experienced population declines and are currently of management concern. The prevalence of larval meningeal worm from WTD feces in these areas are unknown, particularly because the dorsal-spined larvae (DSL) are morphologically indistinguishable from muscle worm (Parelaphostrongylus andersoni). To assess transmission risk of DSL, we investigated the spatial and temporal variation of prevalence in WTD feces from four areas (two with historical moose population declines and two without) sampled across two summers. We predicted higher prevalence of DSL in areas where moose are of management concern and surveys have shown higher meningeal worm prevalence in WTD heads. Further, we expected to only recover meningeal worm, as muscle worm has only been reported from caribou in more northern areas of Manitoba. We collected WTD feces by transect sampling, used the Baermann technique to obtain larvae, and sequenced partial cytochrome oxidase 1 and internal transcribed spacer 2 genes to confirm species identity. Zero-inflated models revealed that DSL prevalence did not differ temporally but was higher in areas where moose are of management concern. Genetic analyses revealed that meningeal worm and muscle worm were both present in Western Manitoba and co-occurred in three areas. Our results reveal novel insights into the geographic distribution of muscle worm and emphasize the importance of DNA sequencing for DSL identification. We suggest that concern for moose populations is warranted given the increased risk of parasite infection in some management areas.

Changes in snail chemical profiles through host-parasite interactions.

Host behavior may be modified by their parasites to increase the likelihood of transmission, but mechanisms underlying these interactions are not well understood. Hosts and parasites release chemical signaling molecules, like oxylipins, that may affect transmission. Oxylipins are oxygenated metabolites of fatty acids that function as signaling molecules and have essential physiological and functional roles. Yet, the limited taxonomic and contextual scope of these studies constrains our ability to understand their role in parasite-modified behavior. We characterized oxylipins in field-collected File Ramshorn snails, Planorbella pilsbryi. We tested for differences in oxylipin profiles based on infection status (infected with the trematode Echinostoma trivolvis lineage a and uninfected) and parasite activity (high and low). Snail-conditioned water samples were produced by placing five snails into artificial spring water for four hours. Oxylipins were extracted from snail-conditioned water samples and quantified using high performance liquid chromatography-tandem mass spectrometry. Infected snails emitted 69 oxylipins in higher amounts, with 37 only released by this group. Within infected snails, 18 oxylipins were emitted in higher amounts in snails with increased parasite activity. Oxylipins emitted in higher amounts by infected snails with increased parasite activity were predominantly derived from the cytochrome P450 pathway. As infected snails emit different oxylipin profiles than uninfected snails, their production may play a role in altering transmission success. By characterizing the oxylipins produced by snails, and how they are altered by infection, we can test their physiological and ecological roles in freshwater systems.

Density-Dependent Prophylaxis in Freshwater Snails Driven by Oxylipin Chemical Cues.

While animal aggregations can benefit the fitness of group members, the behaviour may also lead to higher risks of parasite infection as group density increases. Some animals are known to moderate their investment in immunity relative to the risk of infection. These animals exhibit density-dependent prophylaxis (DDP) by increasing their immune investment as group density increases. Despite being documented in many taxa, the mechanisms of DDP remain largely unexplored. Snails are known to aggregate and experience large fluctuations in density and serve as required hosts for many parasites. Further, they are known to use chemical cues to aggregate. To test whether freshwater snails exhibit DDP and investigate the role that chemical signaling compounds may play in triggering this phenomenon, we performed four experiments on the freshwater snail Stagnicola elodes, which is a common host for many trematode parasite species. First, we tested if DDP occurred in snails in laboratory-controlled conditions (control vs snail-conditioned water) and whether differences in exposure to chemical cues affected immune function. Second, we used gas chromatography to characterize fatty acids expressed in snail-conditioned water to determine if precursors for particular signaling molecules, such as oxylipins, were being produced by snails. Third, we characterized the oxylipins released by infected and uninfected field-collected snails, to better understand how differences in oxylipin cocktails may play a role in inducing DDP. Finally, we tested the immune response of snails exposed to four oxylipins to test the ability of specific oxylipins to affect DDP. We found that snails exposed to water with higher densities of snails and raised in snail-conditioned water had higher counts of haemocytes. Additionally, lipid analysis demonstrated that fatty acid molecules that are also precursors for oxylipins were present in snail-conditioned water. Trematode-infected snails emitted 50 oxylipins in higher amounts, with 24 of these oxylipins only detected in this group. Finally, oxylipins that were higher in infected snails induced naïve snails to increase their immune responses compared to sham-exposed snails. Our results provide evidence that snails exhibit DDP, and the changes in oxylipins emitted by infected hosts may be one of the molecular mechanisms driving this phenomenon.

The complete mitochondrial genome of the file ramshorn snail Planorbella pilsbryi (Mollusca: Gastropoda: Hygrophila: Planorbidae).

The file ramshorn snail Planorbella pilsbryi Baker, 1926 (Gastropoda: Hygrophila: Planorbidae) is a widespread herbivorous North American freshwater snail found in diverse habitats, including standing and moving water bodies. Genome skimming by Illumina sequencing allowed the assembly of a complete nuclear rRNA repeat sequence and a complete circular mitogenome of 13,720 bp from P. pilsbryi consisting of 75.3% AT nucleotides, 22 tRNAs, 13 protein-coding genes, 2 rRNAs and a control region in the typical order found in panpulmonate snails. Planorbella pilsbryi COXI features a rare TTG start codon while COXII, CYTB, ND2, ND3, and ND5 exhibit incomplete stop codons completed by the addition of 3' A residues to the mRNA. Phylogenetic reconstruction of mitochondrial protein-coding gene and rRNA sequences places P. pilsbryi as sister taxon to Planorbella duryi (Planorbidae) within family Planorbidae, which is consistent with previous phylogenetic hypotheses.

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

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

Morphological and molecular characterization of Quinqueserialis (Digenea: Notocotylidae) species diversity in North America.

Estimates of trematode diversity are inaccurate due to unrecognized cryptic species and phenotypic plasticity within species. Integrative taxonomy (genetics, morphology and host use) increases the clarity of species delineation and improves knowledge of parasite biology. In this study, we used this approach to resolve taxonomic issues and test hypotheses of cryptic species in a genus of trematode, Quinqueserialis. Specimens from throughout North America were field collected from hosts and obtained from museums. We found three morphologically distinct groups and successfully sequenced specimens from two of these groups. DNA sequencing at the 28S and CO1 gene regions revealed that two of the three groups were genetically distinct. One genetic group included two morphological clusters demonstrating host-induced phenotypic plasticity within Quinqueserialis quinqueserialis. The other unique genetic group is a novel species, Quinqueserialis kinsellai n. sp., which is described herein. Our study illustrates the importance of integrating multiple sources of evidence when investigating trematode diversity to account for the influence of cryptic species or phenotypic plasticity. However, further sampling is needed to understand Quinqueserialis spp. diversity as some species have no genetic information associated with them.

Spatial scale and structure of complex life cycle trematode parasite communities in streams.

By considering the role of site-level factors and dispersal, metacommunity concepts have advanced our understanding of the processes that structure ecological communities. In dendritic systems, like streams and rivers, these processes may be impacted by network connectivity and unidirectional current. Streams and rivers are central to the dispersal of many pathogens, including parasites with complex, multi-host life cycles. Patterns in parasite distribution and diversity are often driven by host dispersal. We conducted two studies at different spatial scales (within and across stream networks) to investigate the importance of local and regional processes that structure trematode (parasitic flatworms) communities in streams. First, we examined trematode communities in first-intermediate host snails (Elimia proxima) in a survey of Appalachian headwater streams within the Upper New River Basin to assess regional turnover in community structure. We analyzed trematode communities based on both morphotype (visual identification) and haplotype (molecular identification), as cryptic diversity in larval trematodes could mask important community-level variation. Second, we examined communities at multiple sites (headwaters and main stem) within a stream network to assess potential roles of network position and downstream drift. Across stream networks, we found a broad scale spatial pattern in morphotype- and haplotype-defined communities due to regional turnover in the dominant parasite type. This pattern was correlated with elevation, but not with any other environmental factors. Additionally, we found evidence of multiple species within morphotypes, and greater genetic diversity in parasites with hosts limited to in-stream dispersal. Within network parasite prevalence, for at least some parasite taxa, was related to several site-level factors (elevation, snail density and stream depth), and total prevalence decreased from headwaters to main stem. Variation in the distribution and diversity of parasites at the regional scale may reflect differences in the abilities of hosts to disperse across the landscape. Within a stream network, species-environment relationships may counter the effects of downstream dispersal on community structure.

Selected Wildlife Trematodes.

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

Recognizing the Causes of Parasite Morphological Variation to Resolve the Status of a Cryptogenic Pentastome.

Exotic species can threaten biodiversity by introducing parasites to native hosts. Thus, it is critical to identify if the same parasite species infects both native and exotic hosts. However, developmental- or environmental-induced morphological variation may render species identification ambiguous. Our study reports a range expansion in the southern United States of the pentastome Raillietiella indica from the Mediterranean gecko, Hemidactylus turcicus, as well as a host expansion into the green anole, Anolis carolinensis, in the anole's native range. Species identification was based on sequence data and male spicule shape. In agreement with a study from Australia, we found that much of the morphological variation in hook measurements, the primary diagnostic traits of raillietiellid pentastomes, was due to development. Here, we explicitly link this developmental variation to instar stage by incorporating experimental infection data obtained from the literature. We also show that the various hook traits are themselves highly correlated and, thus, likely not independent. Taking instar stage and correlated hook variables into account, we directly controlled for development on a composite hook size measurement. Using a large sample size from H. turcicus, we did not find any consistent effects of potential factors (host sex, host snout-vent-length, or parasite intensity) that may result in environmental-induced variation in relative hook size (corrected for body length). However, relative male spicule size tended to be negatively correlated with parasite intensity. In contrast, both pentastome body length and relative hook size significantly varied among host species whereas relative male spicule size was not significantly different among host species. Our study independently supports the conclusions that developmental- and host-induced morphological variations need to be accounted for to accurately identify pentastome species.

Isotopic turnover rates and diet-tissue discrimination depend on feeding habits of freshwater snails.

Estimates of animal diets and trophic structure using stable isotope analysis are strongly affected by diet-tissue discrimination and tissue turnover rates, yet these factors are often unknown for consumers because they must be measured using controlled-feeding studies. Furthermore, these parameters may be influenced by diet quality, growth, and other factors. We measured the effect of dietary protein content on diet-tissue discrimination and tissue turnover in three freshwater snail species. We fed lettuce to individually housed snails (n = 450 per species) for ten weeks, then half were switched to a high-protein diet. Isotopic values of muscle and gonad tissue were assessed at 48 and 80 days post-diet change. Snail discrimination factors varied by diet (low-protein > high-protein) and usually differed among species for both N and C, although species had similar carbon discrimination when fed the low-protein diet. Carbon turnover rates were similar among species for a given tissue type, but nitrogen turnover varied more among species. In addition, diet affected growth of species differently; some species grew larger on high-protein (H. trivolvis) while others grew larger on low-protein diet (Lymnaea spp.). These differences among species in growth influenced turnover rates, which were faster in the species with the highest growth rate following the diet switch from low to high-protein. Thus, growth is one of the main processes that affects tissue turnover, but growth and feeding preference did not affect diet-tissue discrimination, which was greater on low-protein than high-protein diets for all species regardless of growth performance. These results suggest that diet might influence two key parameters of stable isotope analysis differently.

Role of parasite transmission in promoting inbreeding: I. Infection intensities drive individual parasite selfing rates.

Among parasitic organisms, inbreeding has been implicated as a potential driver of host-parasite co-evolution, drug-resistance evolution and parasite diversification. Yet, fundamental topics about how parasite life histories impact inbreeding remain to be addressed. In particular, there are no direct selfing-rate estimates for hermaphroditic parasites in nature. Our objectives were to elucidate the mating system of a parasitic flatworm in nature and to understand how aspects of parasite transmission could influence the selfing rates of individual parasites. If there is random mating within hosts, the selfing rates of individual parasites would be an inverse power function of their infection intensities. We tested whether selfing rates deviated from within-host random mating expectations with the tapeworm Oochoristica javaensis. In doing so, we generated, for the first time in nature, individual selfing-rate estimates of a hermaphroditic flatworm parasite. There was a mixed-mating system where tapeworms self-mated more than expected with random mating. Nevertheless, individual selfing rates still had a significant inverse power relationship to infection intensities. The significance of this finding is that the distribution of parasite infection intensities among hosts, an emergent property of the transmission process, can be a key driver in shaping the primary mating system, and hence the level of inbreeding in the parasite population. Moreover, we demonstrated how potential population selfing rates can be estimated using the predicted relationship of individual selfing rates to intensities and showed how the distribution of parasites among hosts can indirectly influence the primary mating system when there is density-dependent fecundity.

Role of parasite transmission in promoting inbreeding: II. Pedigree reconstruction reveals sib-transmission and consequent kin-mating.

Even though parasitic flatworms are one of the most species-rich groups of hermaphroditic organisms, we know virtually nothing of their mating systems (selfing or kin-mating rates) in nature. Hence, we lack an understanding of the role of inbreeding in parasite evolution. The natural mating systems of parasitic flatworms have remained elusive due to the inherent difficulty in generating progeny-array data in many parasite systems. New developments in pedigree reconstruction allow direct inference of realized selfing rates in nature by simply using a sample of genotyped individuals. We built upon this advancement by utilizing the closed mating systems, that is, individual hosts, of endoparasites. In particular, we created a novel means to use pedigree reconstruction data to estimate potential kin-mating rates. With data from natural populations of a tapeworm, we demonstrated how our newly developed methods can be used to test for cosibling transmission and inbreeding depression. We then showed how independent estimates of the two mating system components, selfing and kin-mating rates, account for the observed levels of inbreeding in the populations. Thus, our results suggest that these natural parasite populations are in inbreeding equilibrium. Pedigree reconstruction analyses along with the new companion methods we developed will be broadly applicable across a myriad of parasite species. As such, we foresee that a new frontier will emerge wherein the diverse life histories of flatworm parasites could be utilized in comparative evolutionary studies to broadly address ecological factors or life history traits that drive mating systems and hence inbreeding in natural populations.

Physiological Status Drives Metabolic Rate in Mediterranean Geckos Infected with Pentastomes.

Negative effects of parasites on their hosts are well documented, but the proximate mechanisms by which parasites reduce their host's fitness are poorly understood. For example, it has been suggested that parasites might be energetically demanding. However, a recent meta-analysis suggests that they have statistically insignificant effects on host resting metabolic rate (RMR). It is possible, though, that energetic costs associated with parasites are only manifested during and/or following periods of activity. Here, we measured CO2 production (a surrogate for metabolism) in Mediterranean geckos (Hemidactylus turcicus) infected with a lung parasite, the pentastome Raillietiella indica, under two physiological conditions: rested and recently active. In rested geckos, there was a negative, but non-significant association between the number of pentastomes (i.e., infection intensity) and CO2 production. In recently active geckos (chased for 3 minutes), we recorded CO2 production from its maximum value until it declined to a stationary phase. We analyzed this decline as a 3 phase function (initial decline, secondary decline, stationary). Geckos that were recently active showed, in the secondary phase, a significant decrease in CO2 production as pentastome intensity increased. Moreover, duration of the secondary phase showed a significant positive association with the number of pentastomes. These results suggest that the intensity of pentastome load exerts a weak effect on the metabolism of resting geckos, but a strong physiological effect on geckos that have recently been active; we speculate this occurs via mechanical constraints on breathing. Our results provide a potential mechanism by which pentastomes can reduce gecko fitness.

Revealing cryptic parasite diversity in a definitive host: echinostomes in muskrats.

Many trematode groups have a long history of systematic revision, which can make parasite identification a difficult task. The trematode parasites of muskrats are no exception. Here, we highlight the systematic issues associated with trematodes of muskrats (Ondatra zibethicus). Then, we demonstrate the utility of using both morphological and molecular tools to identify these parasites. Morphological examinations of specimens from muskrats (n = 63) first suggested that at least 4 genera were present including Echinostoma, Wardius, Quinqueserialis, and Notocotylus. For the latter 3 groups, the 28S region verified this assessment. For echinostomes, ND1 sequences revealed at least 5 genetic lineages. A particular lineage, Echinostoma trivolvis lineage b, predominated in both prevalence and intensity of infection. Molecular sequences provided a more accurate estimate of echinostome diversity in the muskrats and further support the idea that E. trivolvis is a species complex. Future studies will focus on whether there are differences in host specificity among the E. trivolvis lineages. In addition, this study has provided initial sequences that will help verify the life cycles of Wardius, Quinqueserialis, and especially, Notocotylus. By linking molecular, morphological, and life history information, we can better understand parasite diversity.

Testing local-scale panmixia provides insights into the cryptic ecology, evolution, and epidemiology of metazoan animal parasites.

When every individual has an equal chance of mating with other individuals, the population is classified as panmictic. Amongst metazoan parasites of animals, local-scale panmixia can be disrupted due to not only non-random mating, but also non-random transmission among individual hosts of a single host population or non-random transmission among sympatric host species. Population genetics theory and analyses can be used to test the null hypothesis of panmixia and thus, allow one to draw inferences about parasite population dynamics that are difficult to observe directly. We provide an outline that addresses 3 tiered questions when testing parasite panmixia on local scales: is there greater than 1 parasite population/species, is there genetic subdivision amongst infrapopulations within a host population, and is there asexual reproduction or a non-random mating system? In this review, we highlight the evolutionary significance of non-panmixia on local scales and the genetic patterns that have been used to identify the different factors that may cause or explain deviations from panmixia on a local scale. We also discuss how tests of local-scale panmixia can provide a means to infer parasite population dynamics and epidemiology of medically relevant parasites.

Testing Mendelian inheritance from field-collected parasites: Revealing duplicated loci enables correct inference of reproductive mode and mating system.

Cryptic aspects of parasite population biology, e.g., mating systems, are increasingly being inferred from polymorphic and co-dominant genetic markers such as microsatellite loci. Underlying the use of such co-dominant markers is the assumption of Mendelian inheritance. The failure to meet this assumption can lead to artifactual statistics and erroneous population inferences. Here, we illustrate the importance of testing the Mendelian segregation and assortment of genetic markers and demonstrate how field-collected samples can be utilised for this purpose. To examine the reproductive mode and mating system of hermaphroditic parasites, we developed microsatellites for the cestode, Oochoristica javaensis. Among loci, we found a bimodal distribution of F(IS) (a fixation index that quantifies the deviation from Hardy-Weinberg equilibrium within subpopulations) values where loci were either highly negative (close to -1) or highly positive (∼0.8). By conducting tests of Mendelian segregation from natural crosses, we determined that loci with negative F(IS) values were in fact duplicated loci that were amplified by a single primer pair. Genetic crosses also provided linkage data and indicated that the duplicated loci most likely arose via tandem duplications rather than whole genome/chromosome duplications. By correcting for the duplicated loci, we were able to correctly infer that O. javaensis has sexual reproduction, but the mating system is highly inbred. To assist others in testing Mendelian segregation and independent assortment from natural samples, we discuss the benefits and limitations, and provide guidelines for particular parasite systems amenable to the methods employed here.