Distinct evolutionary lineages of Schistocephalus parasites infecting co-occurring sculpin and stickleback fishes in Alaska.

Sculpins (coastrange and slimy) and sticklebacks (ninespine and threespine) are widely distributed fishes cohabiting 2 south-central Alaskan lakes (Aleknagik and Iliamna), and all these species are parasitized by cryptic diphyllobothriidean cestodes in the genus Schistocephalus. The goal of this investigation was to test for host-specific parasitic relationships between sculpins and sticklebacks based upon morphological traits (segment counts) and sequence variation across the NADH1 gene. A total of 446 plerocercoids was examined. Large, significant differences in mean segment counts were found between cestodes in sculpin (mean = 112; standard deviation [s.d.] = 15) and stickleback (mean = 86; s.d. = 9) hosts within and between lakes. Nucleotide sequence divergence between parasites from sculpin and stickleback hosts was 20.5%, and Bayesian phylogenetic analysis recovered 2 well-supported clades of cestodes reflecting intermediate host family (i.e. sculpin, Cottidae vs stickleback, Gasterosteidae). Our findings point to the presence of a distinct lineage of cryptic Schistocephalus in sculpins from Aleknagik and Iliamna lakes that warrants further investigation to determine appropriate evolutionary and taxonomic recognition.

Freshwater Colonization, Adaptation, and Genomic Divergence in Threespine Stickleback.

The Threespine Stickleback is ancestrally a marine fish, but many marine populations breed in fresh water (i.e., are anadromous), facilitating their colonization of isolated freshwater habitats a few years after they form. Repeated adaptation to fresh water during at least 10 My and continuing today has led to Threespine Stickleback becoming a premier system to study rapid adaptation. Anadromous and freshwater stickleback breed in sympatry and may hybridize, resulting in introgression of freshwater-adaptive alleles into anadromous populations, where they are maintained at low frequencies as ancient standing genetic variation. Anadromous stickleback have accumulated hundreds of freshwater-adaptive alleles that are disbursed as few loci per marine individual and provide the basis for adaptation when they colonize fresh water. Recent whole-lake experiments in lakes around Cook Inlet, Alaska have revealed how astonishingly rapid and repeatable this process is, with the frequency of 40% of the identified freshwater-adaptive alleles increasing from negligible (∼1%) in the marine founder to ≥50% within ten generations in fresh water, and freshwater phenotypes evolving accordingly. These high rates of genomic and phenotypic evolution imply very intense directional selection on phenotypes of heterozygotes. Sexual recombination rapidly assembles freshwater-adaptive alleles that originated in different founders into multilocus freshwater haplotypes, and regions important for adaptation to freshwater have suppressed recombination that keeps advantageous alleles linked within large haploblocks. These large haploblocks are also older and appear to have accumulated linked advantageous mutations. The contemporary evolution of Threespine Stickleback has provided broadly applicable insights into the mechanisms that facilitate rapid adaptation.

Cross-continental experimental infections reveal distinct defence mechanisms in populations of the three-spined stickleback Gasterosteus aculeatus.

Epidemiological traits of host-parasite associations depend on the effects of the host, the parasite and their interaction. Parasites evolve mechanisms to infect and exploit their hosts, whereas hosts evolve mechanisms to prevent infection and limit detrimental effects. The reasons why and how these traits differ across populations still remain unclear. Using experimental cross-infection of three-spined stickleback Gasterosteus aculeatus and their species-specific cestode parasites Schistocephalus solidus from Alaskan and European populations, we disentangled host, parasite and interaction effects on epidemiological traits at different geographical scales. We hypothesized that host and parasite main effects would dominate both within and across continents, although interaction effects would show geographical variation of natural selection within and across continents. We found that mechanisms preventing infection (qualitative resistance) occurred only in a combination of hosts and parasites from different continents, while mechanisms limiting parasite burden (quantitative resistance) and reducing detrimental effects of infection (tolerance) were host-population specific. We conclude that evolution favours distinct defence mechanisms on different geographical scales and that it is important to distinguish concepts of qualitative resistance, quantitative resistance and tolerance in studies of macroparasite infections.

Predicting future from past: The genomic basis of recurrent and rapid stickleback evolution.

Similar forms often evolve repeatedly in nature, raising long-standing questions about the underlying mechanisms. Here, we use repeated evolution in stickleback to identify a large set of genomic loci that change recurrently during colonization of freshwater habitats by marine fish. The same loci used repeatedly in extant populations also show rapid allele frequency changes when new freshwater populations are experimentally established from marine ancestors. Marked genotypic and phenotypic changes arise within 5 years, facilitated by standing genetic variation and linkage between adaptive regions. Both the speed and location of changes can be predicted using empirical observations of recurrence in natural populations or fundamental genomic features like allelic age, recombination rates, density of divergent loci, and overlap with mapped traits. A composite model trained on these stickleback features can also predict the location of key evolutionary loci in Darwin's finches, suggesting that similar features are important for evolution across diverse taxa.

Estimating effective population size for a cestode parasite infecting three-spined sticklebacks.

Remarkably few attempts have been made to estimate contemporary effective population size (Ne) for parasitic species, despite the valuable perspectives it can offer on the tempo and pace of parasite evolution as well as coevolutionary dynamics of host-parasite interactions. In this study, we utilized multi-locus microsatellite data to derive single-sample and temporal estimates of contemporary Ne for a cestode parasite (Schistocephalus solidus) as well as three-spined stickleback hosts (Gasterosteus aculeatus) in lakes across Alaska. Consistent with prior studies, both approaches recovered small and highly variable estimates of parasite and host Ne. We also found that estimates of host Ne and parasite Ne were sensitive to assumptions about population genetic structure and connectivity. And, while prior work on the stickleback-cestode system indicates that physiographic factors external to stickleback hosts largely govern genetic variation in S. solidus, our findings indicate that stickleback host attributes and factors internal to the host - namely body length, genetic diversity and infection - shape contemporary Ne of cestode parasites.

Are solo infections of the diphyllobothriidean cestode Schistocephalus solidus more virulent than multiple infections?

We performed a long-term natural experiment investigating the impact of the diphyllobotriidean cestode Schistocephalus solidus on the body condition and clutch size (CS) of threespine stickleback Gasterosteus aculeatus, its second intermediate host, and the growth of larval parasites in host fish. We tested the hypothesis that single S. solidus infections were more virulent than multiple infections. We also asked whether the metrics of mean and total parasite mass (proxies for individual and total volume, respectively) were consistent with predictions of the resource constraints or the life history strategy (LHS) hypothesis for the growth of, hence exploitation by, larval helminths in intermediate hosts. The samples were drawn from Walby Lake, Alaska in eight of 11 years. Host body condition and CS (egg number per spawning bout) decreased significantly with intensity after adjustments for host size and parasite index. Thus, infections have an increasingly negative impact on measures of host fitness with greater intensity, in contrast to the hypothesis that single infections are more harmful than multiple infections. We also found that mean parasite mass decreased with intensity while total parasite mass increased with intensity as predicted by the LHS hypothesis.

The cestode parasite Schistocephalus pungitii: castrator or nutrient thief of ninespine stickleback fish?

In this investigation, the host-parasite relationship of ninespine stickleback fish Pungitius pungitius and the cestode parasite Schistocephalus pungitii was studied using samples from Dog Bone Lake, Kenai Peninsula, Alaska, to test the hypothesis that S. pungitii is a castrator of ninespine stickleback. Infected, adult females of all sizes (ages) were capable of producing clutches of eggs. S. pungitii had a negative effect on the ability of host females to produce a clutch, which was related to increasing parasite:host mass ratio (parasite index, PI). Among infected females with egg clutches, both clutch size and egg size were reduced; and the reduction increased with greater PI. The results of this study are consistent with the hypothesis that S. pungitii causes host sterility as a result of simple nutrient theft and is not a true castrator as hypothesized in earlier reports. The degree of parasite-induced sterility appears to vary among populations of the ninespine stickleback, perhaps reflecting differences in resource availability. Populations of ninespine stickleback appear to show a greater reduction in host reproductive capacity with PI than populations of the threespine stickleback infected by Schistocephalus solidus, possibly owing, in part, to the length-adjusted somatic mass of the threespine stickleback being greater.

Mutual dilution of infection by an introduced parasite in native and invasive stream fishes across Hawaii.

The presence of introduced hosts can increase or decrease infections of co-introduced parasites in native species of conservation concern. In this study, we compared parasite abundance, intensity, and prevalence between native Awaous stamineus and introduced poeciliid fishes by a co-introduced nematode parasite (Camallanus cotti) in 42 watersheds across the Hawaiian Islands. We found that parasite abundance, intensity and prevalence were greater in native than introduced hosts. Parasite abundance, intensity and prevalence within A. stamineus varied between years, which largely reflected a transient spike in infection in three remote watersheds on Molokai. At each site we measured host factors (length, density of native host, density of introduced host) and environmental factors (per cent agricultural and urban land use, water chemistry, watershed area and precipitation) hypothesized to influence C. cotti abundance, intensity and prevalence. Factors associated with parasitism differed between native and introduced hosts. Notably, parasitism of native hosts was higher in streams with lower water quality, whereas parasitism of introduced hosts was lower in streams with lower water quality. We also found that parasite burdens were lower in both native and introduced hosts when coincident. Evidence of a mutual dilution effect indicates that introduced hosts can ameliorate parasitism of native fishes by co-introduced parasites, which raises questions about the value of remediation actions, such as the removal of introduced hosts, in stemming the rise of infectious disease in species of conservation concern.

Landscape genetics of Schistocephalus solidus parasites in threespine stickleback (Gasterosteus aculeatus) from Alaska.

The nature of gene flow in parasites with complex life cycles is poorly understood, particularly when intermediate and definitive hosts have contrasting movement potential. We examined whether the fine-scale population genetic structure of the diphyllobothriidean cestode Schistocephalus solidus reflects the habits of intermediate threespine stickleback hosts or those of its definitive hosts, semi-aquatic piscivorous birds, to better understand complex host-parasite interactions. Seventeen lakes in the Cook Inlet region of south-central Alaska were sampled, including ten in the Matanuska-Susitna Valley, five on the Kenai Peninsula, and two in the Bristol Bay drainage. We analyzed sequence variation across a 759 bp region of the mitochondrial DNA (mtDNA) cytochrome oxidase I region for 1,026 S. solidus individuals sampled from 2009-2012. We also analyzed allelic variation at 8 microsatellite loci for 1,243 individuals. Analysis of mtDNA haplotype and microsatellite genotype variation recovered evidence of significant population genetic structure within S. solidus. Host, location, and year were factors in structuring observed genetic variation. Pairwise measures revealed significant differentiation among lakes, including a pattern of isolation-by-distance. Bayesian analysis identified three distinct genotypic clusters in the study region, little admixture within hosts and lakes, and a shift in genotype frequencies over time. Evidence of fine-scale population structure in S. solidus indicates that movement of its vagile, definitive avian hosts has less influence on gene flow than expected based solely on movement potential. Observed patterns of genetic variation may reflect genetic drift, behaviors of definitive hosts that constrain dispersal, life history of intermediate hosts, and adaptive specificity of S. solidus to intermediate host genotype.

Fecundity compensation and fecundity reduction among populations of the three-spined stickleback infected by Schistocephalus solidus in Alaska.

We surveyed nine populations of the three-spined stickleback infected by the diphyllobothriidean cestode Schistocephalus solidus from south-central Alaska for two apparent forms of tolerance to infection in females capable of producing egg clutches notwithstanding large parasite burdens. Seven populations exhibited fecundity reduction, whereas two populations showed fecundity compensation. Our data suggest that fecundity reduction, a side effect resulting from nutrient theft, occurs in two phases of host response influenced by the parasite : host body mass (BM) ratio. The first is significantly reduced ovum mass without significant reduction in clutch size, and the second one involves significant reductions in both ovum mass and clutch size. Thus, ovum mass of host females who are functionally being starved through nutrient theft seems to be more readily influenced by parasitism and, therefore, decreased before clutch size is reduced. This inference is consistent with expectations based on the biology of and effect of feeding ration on reproduction in stickleback females. Fecundity compensation appears to be uncommon among populations of three-spined stickleback in Alaska and rare among populations throughout the northern hemisphere. Fecundity reduction seems to be common, at least among stickleback populations in Alaska.

The rise and fall of an epizootic of the diphyllobothriidean cestode Schistocephalus pungitii infecting the ninespine stickleback.

Epizootics of diphyllobothriidean cestodes appear to be simple, but deceptive similarity conceals the myriad ways in which these events are shaped by complex abiotic and biotic interactions. In Dog Bone Lake, Alaska, an epizootic of Schistocephalus pungitii infecting the ninespine stickleback (Pungitius pungitius) was short-lived. Its duration, with a peak that lasted only 1 yr, was shorter than for previously documented epizootics in Schistocephalus solidus . The ability of the ninespine stickleback to sustain infections, which appears to be related to species-specific characteristics of the host, may have played an important role in shaping the epizootic. Moreover, the epizootic of S. pungitii was not coincident with those observed for S. solidus in earlier studies within this region, supporting the hypothesis that processes involved in epizootics largely reflect local (lake-level) influences on population dynamics of the parasite. The outbreak occurred at a time when the host population was not relatively dense, which is inconsistent with epidemiological theory and may be a consequence of the parasite's indirect life cycle. The variability of the unregulated and unstable epizootic events of diphyllobothriidean cestodes presents a challenge to understand the ecological and evolutionary factors influencing the prevalence of infections in host populations.

Distinct lineages of Schistocephalus parasites in threespine and ninespine stickleback hosts revealed by DNA sequence analysis.

Parasitic interactions are often part of complex networks of interspecific relationships that have evolved in biological communities. Despite many years of work on the evolution of parasitism, the likelihood that sister taxa of parasites can co-evolve with their hosts to specifically infect two related lineages, even when those hosts occur sympatrically, is still unclear. Furthermore, when these specific interactions occur, the molecular and physiological basis of this specificity is still largely unknown. The presence of these specific parasitic relationships can now be tested using molecular markers such as DNA sequence variation. Here we test for specific parasitic relationships in an emerging host-parasite model, the stickleback-Schistocephalus system. Threespine and ninespine stickleback fish are intermediate hosts for Schistocephalus cestode parasites that are phenotypically very similar and have nearly identical life cycles through plankton, stickleback, and avian hosts. We analyzed over 2000 base pairs of COX1 and NADH1 mitochondrial DNA sequences in 48 Schistocephalus individuals collected from threespine and ninespine stickleback hosts from disparate geographic regions distributed across the Northern Hemisphere. Our data strongly support the presence of two distinct clades of Schistocephalus, each of which exclusively infects either threespine or ninespine stickleback. These clades most likely represent different species that diverged soon after the speciation of their stickleback hosts. In addition, genetic structuring exists among Schistocephalus taken from threespine stickleback hosts from Alaska, Oregon and Wales, although it is much less than the divergence between hosts. Our findings emphasize that biological communities may be even more complex than they first appear, and beg the question of what are the ecological, physiological, and genetic factors that maintain the specificity of the Schistocephalus parasites and their stickleback hosts.

Processes influencing the duration and decline of epizootics in Schistocephalus solidus.

The interplay of intermediate host fish and plerocercoids of diphyllobothriidean cestodes results in epizootics that are deceptively simple, but conceal complex biotic and abiotic interactions shaping each event independently. Although general descriptions of epizootics and some details of biotic interactions between enemies are known, much remains to be discovered about the abiotic and biotic forces and their interactions driving epizootics. This study shows that the duration of an epizootic of Schistocephalus solidus was sustained by high prevalence, mean intensity, and PI (parasite index-parasite : host biomass ratio) levels among young-of-the-year and 1-yr-old threespine sticklebacks. Many infections and most parasite growth in young-of-the-year fish apparently occurred under the ice during the winter. Few new infections appear to have occurred among 1-yr-old fish, which may live 2 yr and sometimes 3 yr. The decline of the epizootic occurred as the recruitment of 1 to 2-yr-old hosts decreased significantly, followed by reduced infections of young-of-the-year fish. Thus, a major factor influencing parasite population dynamics was reduced transmission (probability of infection) as a result of overwinter host mortality among 1-yr-old fish. Mega-epizootics, named and described herein, appear to represent a "perfect storm" phenomenon dependent on a particular and rare combination of circumstances. Less extreme and more gradual epizootics may be more common and play out in myriad ways, because of complex abiotic and biotic factors influencing both parasite and host populations. The interplay of parasite and host resulting in reciprocal effects upon one another occurs during both the emergence and decline phases of an epizootic.

Do heavy burdens of Schistocephalus solidus in juvenile threespine stickleback result in disaster for the parasite?

The diphyllobothriidean cestode Schistocephalus solidus typically infects threespine sticklebacks that are too small to allow the parasite to reach a mature size. As a result, the parasite must allow further growth of its host to reach the size at which it becomes competent to infect and reproduce in the definitive host. At times, however, intensity of infection can be high, leading to crowding among parasites and to heavy burdens causing mortality among hosts. Our data show that, during a previously observed epizootic, large percentages of plerocercoids (average 75% per host, 82% among all parasites pooled) did not grow to become massive enough in 1-yr-old threespine sticklebacks to be capable of establishment and maturation in the definitive host. Massive deaths of 1-yr-old sticklebacks due to infection during the epizootic resulted in the great misfortune of a disaster for a large number of parasites, resulting in dramatically reduced transmission of S. solidus.

Morphological responses of a stream fish to water impoundment.

Water impoundment imposes fundamental changes on natural landscapes by transforming rivers into reservoirs. The dramatic shift in physical conditions accompanying the loss of flow creates novel ecological and evolutionary challenges for native species. In this study, we compared the body shape of Cyprinella venusta collected from eight pairs of river and reservoir sites across the Mobile River Basin (USA). Geometric morphometric analysis of the body shape showed that river populations differ from reservoir populations. Individuals inhabiting reservoirs are deep-bodied and have a smaller head, a more anterior dorsal fin, a shorter dorsal fin base and a more ventral position of the eye than C. venusta in streams. The direction of shape divergence within reservoir-river pairs was consistent among pairs of sites, and the shape of C. venusta in reservoirs is strongly correlated with reservoir size. These findings show that physical characteristics of reservoirs drive changes in the morphological attributes of native fish populations, indicating that water impoundment may be an important, yet largely unrecognized, evolutionary driver acting on aquatic biodiversity.

Castration of female ninespine stickleback by the pseudophyllidean cestode Schistocephalus pungitii: evolutionary significance and underlying mechanism.

Parasitic castration may result from manipulation of host energy allocation away from reproduction, which should result in castration of lightly infected hosts as well as heavily infected ones. Castration also may result from nutrient theft alone, which incidentally influences host energy allocation to reproduction and should cause reproduction to end in heavily infected hosts. Although the pseudophyllidean cestode Schistocephalus pungitii is a castrator of ninespine stickleback fish (Pungitius pungitius), the cause and significance of castration remain unknown. We used predictions about reproductive capacity in lightly and heavily infected hosts and host size (age) at which castration occurs to address these questions. In Airolo Lake, Alaska, we found that inhibition of reproduction in ninespine stickleback occurs in small fish before the size (age) of sexual maturity and is not instantaneous. Castration was observed in lightly and heavily infected adult-size hosts. Infected fish do not reach the same size as uninfected fish, possibly because of growth inhibition or selective predation. Castration in infected adult-size female ninespine stickleback appears to be the result of adaptive manipulation of host energy allocation by the parasite, but it could result from an energy budget readily influenced by parasitism.

The effect of phylogeny on interspecific body shape variation in darters (Pisces: Percidae).

We conducted a geometric morphometric analysis of interspecific body shape variation among representatives of 31 species of darters (Pisces: Percidae) to determine whether there is evidence of a phylogenetic effect in body shape variation. Cartesian transformation grids representing relative shape differences of individual species and subspecies revealed qualitative similarities within most traditionally recognized taxonomic groups (genera and subgenera). Canonical variates analysis and a UPGMA cluster analysis were conducted to explore further the relationships among body shapes of species; both analyses revealed patterns of variation consistent with the interpretation that shape is associated with taxonomic affinities. Normalized Mantel statistics revealed a significant positive association between body shape differences and phylogenetic interrelatedness for each of four recent phylogenetic hypotheses, providing evidence of a phylogenetic effect. This result is somewhat surprising, however, given the largely incompatible nature of these four phylogenies. We provide evidence that this result may be due to (1) the inclusion of multiple sets of closely related species to represent the traditionally recognized genera and subgenera within each phylogeny and/or (2) the inclusion of several species with relatively divergent shapes and their particular positions within the phylogenies relative to one another or to the other species of darters.

Reduction of egg size in natural populations of threespine stickleback infected with a cestode macroparasite.

Manifestations of infectious disease may represent host adaptations to avoid or reduce the effects of infection on host fitness, parasite manipulations that benefit the pathogen's fitness, or nonadaptive side effects of parasitism. Threespine stickleback fish (Gasterosteus aculeatus) from Alaska and the cestode macroparasite Schistocephalus solidus provide an excellent system for study of the effects of parasitism on host egg size because females in populations there are capable of producing clutches of eggs in the face of substantial infection, contrary to the inhibition of reproduction that has been observed in other stickleback populations or other species of fish. A side effect resulting in reduction of mean ovummass among infected females was predicted based on the egg production process in female stickleback, the considerable energy and resource demands of S. solidus, and the chronic and progressive nature of the effects the macroparasite should have on the host fish. In each of 9 populations of G. aculeatus representing replicate natural experiments in lakes scattered across the Matanuska-Susitna Valley and the Kenai Peninsula of south-central Alaska and among all populations combined, the mean ovum mass of infected female fish is significantly reduced in comparison with that of uninfected females taken from the same population at the same time. Reduction in mean female egg mass ranged from 8 to 32% across all populations. To examine whether reduction in mean female ovum mass was a nonadaptive side effect or an adaptation, relatively large data sets from 2 of the populations were used. Mean ovum mass of infected females was predicted to decrease directly in relation to parasite index (PI) if the diminution in mean egg mass were the result of a nonadaptive side effect resulting from host nutrient loss. Alternatively, the absence of a relationship between PI and reduction in ovum mass is predicted if decreases in mean female ovum mass result from host or parasite adaptation (or both) because lightly infected hosts should show a response similar to that of heavily infected ones. In each of the 2 populations, there is a significant, negative relationship between mean female ovum mass and PI, demonstrating a correlation between the decrease in ovum mass and the level of infection. Thus, the results are consistent with the hypothesis that the reductions in mean female egg mass represent side effects of parasitism involving nutrient theft. Moreover, the proportional decline in egg mass with increasing PI apparently differed between the 2 populations, and there was no significant relationship between mean percent decrease in mean female ovum mass and mean PI across populations. These observations suggest that unknown ecological and evolutionary factors influence the degree of reduction in mean ovum mass in a population-specific manner.

The "crowding effect" in the cestode Schistocephalus solidus: density-dependent effects on plerocercoid size and infectivity.

The occurrence of the crowding effect was demonstrated in plerocercoids of the cestode Schistocephalus solidus infecting threespine stickleback Gasterosteus aculeatus from Walby and Scout lakes, Alaska. Contrary to an earlier report, relatively large numbers of parasites (>3-4 plerocercoids) were observed to grow large enough in an intermediate host fish to become competent to infect and to mature in the definitive host under any of 3 assumed threshold values and 1 scenario of graded sizes for parasite competency. In Walby Lake, intensity and host body mass were significant predictors of mean plerocercoid mass per host, whereas intensity, host body mass, and combined parasite index were significant predictors in Scout Lake. Slopes of equations expressing the relationship between mean parasite mass and intensity for both lakes were less than 1, implying that processes other than or in combination with simple resource limitation might be producing the observed crowding effect. The causal mechanism for the crowding effect could include exploitative competition, interference competition, and host immune response. There were significant differences in infection between the two lakes, including different distributions of parasite intensities among hosts and different expressions of the crowding effect; however, an explanation of the differences awaits further investigation.