Host phenotype and microbiome vary with infection status, parasite genotype, and parasite microbiome composition.

A growing literature demonstrates the impact of helminths on their host gut microbiome. We investigated whether the stickleback host microbiome depends on ecoevolutionary variables by testing the impact of exposure to the cestode parasite Schistocephalus solidus with respect to infection success, host genotype, parasite genotype, and parasite microbiome composition. We observed constitutive differences in the microbiome of sticklebacks of different origin, and those differences increased when sticklebacks exposed to the parasite resisted infection. In contrast, the microbiome of successfully infected sticklebacks varied with parasite genotype. More specifically, we revealed that the association between microbiome and immune gene expression increased in infected individuals and varied with parasite genotype. In addition, we showed that S. solidus hosts a complex endomicrobiome and that bacterial abundance in the parasite correlates with expression of host immune genes. Within this comprehensive analysis we demonstrated that (i) parasites contribute to modulating the host microbiome through both successful and unsuccessful infection, (ii) when infection is successful, the host microbiome varies with parasite genotype due to genotype-dependent variation in parasite immunomodulation, and (iii) the parasite-associated microbiome is distinct from its host and impacts the host immune response to infection.

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.

Repeatability of Adaptive Radiation Depends on Spatial Scale: Regional Versus Global Replicates of Stickleback in Lake Versus Stream Habitats.

The repeatability of adaptive radiation is expected to be scale-dependent, with determinism decreasing as greater spatial separation among "replicates" leads to their increased genetic and ecological independence. Threespine stickleback (Gasterosteus aculeatus) provide an opportunity to test whether this expectation holds for the early stages of adaptive radiation-their diversification in freshwater ecosystems has been replicated many times. To better understand the repeatability of that adaptive radiation, we examined the influence of geographic scale on levels of parallel evolution by quantifying phenotypic and genetic divergence between lake and stream stickleback pairs sampled at regional (Vancouver Island) and global (North America and Europe) scales. We measured phenotypes known to show lake-stream divergence and used reduced representation genome-wide sequencing to estimate genetic divergence. We assessed the scale dependence of parallel evolution by comparing effect sizes from multivariate models and also the direction and magnitude of lake-stream divergence vectors. At the phenotypic level, parallelism was greater at the regional than the global scale. At the genetic level, putative selected loci showed greater lake-stream parallelism at the regional than the global scale. Generally, the level of parallel evolution was low at both scales, except for some key univariate traits. Divergence vectors were often orthogonal, highlighting possible ecological and genetic constraints on parallel evolution at both scales. Overall, our results confirm that the repeatability of adaptive radiation decreases at increasing spatial scales. We suggest that greater environmental heterogeneity at larger scales imposes different selection regimes, thus generating lower repeatability of adaptive radiation at larger spatial scales.

Specificity of resistance and geographic patterns of virulence in a vertebrate host-parasite system.

BACKGROUND: Host genotype - parasite genotype co-evolutionary dynamics are influenced by local biotic and abiotic environmental conditions. This results in spatially heterogeneous selection among host populations. How such heterogeneous selection influences host resistance, parasite infectivity and virulence remains largely unknown. We hypothesized that different co-evolutionary trajectories of a vertebrate host-parasite association result in specific virulence patterns when assessed on a large geographic scale. We used two reference host populations of three-spined sticklebacks and nine strains of their specific cestode parasite Schistocephalus solidus from across the Northern Hemisphere for controlled infection experiments. Host and parasite effects on infection phenotypes including host immune gene expression were determined. RESULTS: S. solidus strains grew generally larger in hosts coming from a population with high parasite diversity and low S. solidus prevalence (DE hosts). Hosts from a population with low parasite diversity and high S. solidus prevalence (NO hosts) were better able to control the parasite's growth, regardless of the origin of the parasite. Host condition and immunological parameters converged upon infection and parasite growth showed the same geographic pattern in both host types. CONCLUSION: Our results suggest that NO sticklebacks evolved resistance against a variety of S. solidus strains, whereas DE sticklebacks are less resistant against S. solidus. Our data provide evidence that differences in parasite prevalence can cause immunological heterogeneity and that parasite size, a proxy for virulence and resistance, is, on a geographic scale, determined by main effects of the host and the parasite and less by an interaction of both genotypes.

Correction to: Specificity of resistance and geographic patterns of virulence in a vertebrate hostparasite system.

After publication of the original article [1], the authors have notified us that the incorrect version of Fig. 4 was used. Below you can find the both incorrect and correct versions of the figure.

The right response at the right time: Exploring helminth immune modulation in sticklebacks by experimental coinfection.

Parasites are one of the strongest selective agents in nature. They select for hosts that evolve counter-adaptive strategies to cope with infection. Helminth parasites are special because they can modulate their hosts' immune responses. This phenomenon is important in epidemiological contexts, where coinfections may be affected. How different types of hosts and helminths interact with each other is insufficiently investigated. We used the three-spined stickleback (Gasterosteus aculeatus) - Schistocephalus solidus model to study mechanisms and temporal components of helminth immune modulation. Sticklebacks from two contrasting populations with either high resistance (HR) or low resistance (LR) against S. solidus, were individually exposed to S. solidus strains with characteristically high growth (HG) or low growth (LG) in G. aculeatus. We determined the susceptibility to another parasite, the eye fluke Diplostomum pseudospathaceum, and the expression of 23 key immune genes at three time points after S. solidus infection. D. pseudospathaceum infection rates and the gene expression responses depended on host and S. solidus type and changed over time. Whereas the effect of S. solidus type was not significant after three weeks, T regulatory responses and complement components were upregulated at later time points if hosts were infected with HG S. solidus. HR hosts showed a well orchestrated immune response, which was absent in LR hosts. Our results emphasize the role of regulatory T cells and the timing of specific immune responses during helminth infections. This study elucidates the importance to consider different coevolutionary trajectories and ecologies when studying host-parasite interactions.

Examining the role of parasites in limiting unidirectional gene flow between lake and river sticklebacks.

Parasites are important selective agents with the potential to limit gene flow between host populations by shaping local host immunocompetence. We report on a contact zone between lake and river three-spined sticklebacks (Gasterosteus aculeatus) that offers the ideal biogeographic setting to explore the role of parasite-mediated selection on reproductive isolation. A waterfall acts as a natural barrier and enforces unidirectional migration from the upstream river stickleback population to the downstream river and lake populations. We assessed population genetic structure and parasite communities over four years. In a set of controlled experimental infections, we compared parasite susceptibility of upstream and downstream fish by exposing laboratory-bred upstream river and lake fish, as well as hybrids, to two common lake parasite species: a generalist trematode parasite, Diplostomum pseudospathaceum, and a host-specific cestode, Schistocephalus solidus. We found consistent genetic differentiation between upstream and downstream populations across four sampling years, even though the downstream river consisted of ~10% first-generation migrants from the upstream population as detected by parentage analysis. Fish in the upstream population had lower genetic diversity and were strikingly devoid of macroparasites. Through experimental infections, we demonstrated that upstream fish and their hybrids had higher susceptibility to parasite infections than downstream fish. Despite this, naturally sampled upstream migrants were less infected than downstream residents. Thus, migrants coming from a parasite-free environment may enjoy an initial fitness advantage, but their descendants seem likely to suffer from higher parasite loads. Our results suggest that adaptation to distinct parasite communities can influence stickleback invasion success and may represent a barrier to gene flow, even between close and connected populations.

Genomics of divergence along a continuum of parapatric population differentiation.

The patterns of genomic divergence during ecological speciation are shaped by a combination of evolutionary forces. Processes such as genetic drift, local reduction of gene flow around genes causing reproductive isolation, hitchhiking around selected variants, variation in recombination and mutation rates are all factors that can contribute to the heterogeneity of genomic divergence. On the basis of 60 fully sequenced three-spined stickleback genomes, we explore these different mechanisms explaining the heterogeneity of genomic divergence across five parapatric lake and river population pairs varying in their degree of genetic differentiation. We find that divergent regions of the genome are mostly specific for each population pair, while their size and abundance are not correlated with the extent of genome-wide population differentiation. In each pair-wise comparison, an analysis of allele frequency spectra reveals that 25-55% of the divergent regions are consistent with a local restriction of gene flow. Another large proportion of divergent regions (38-75%) appears to be mainly shaped by hitchhiking effects around positively selected variants. We provide empirical evidence that alternative mechanisms determining the evolution of genomic patterns of divergence are not mutually exclusive, but rather act in concert to shape the genome during population differentiation, a first necessary step towards ecological speciation.

Resist Globally, Infect Locally: A Transcontinental Test of Adaptation by Stickleback and Their Tapeworm Parasite.

Parasite infections are a product of both ecological processes affecting host-parasite encounter rates and evolutionary dynamics affecting host susceptibility. However, few studies examine natural infection variation from both ecological and evolutionary perspectives. Here, we describe the ecological and evolutionary factors generating variation in infection rates by a tapeworm (Schistocephalus solidus) in a vertebrate host, the threespine stickleback (Gasterosteus aculeatus). To explore ecological aspects of infection, we measured tapeworm prevalence in Canadian stickleback inhabiting two distinct environments: marine and freshwater. Consistent with ecological control of infection, the tapeworm is very rare in marine environments, even though marine fish are highly susceptible. Conversely, commonly infected freshwater stickleback exhibit substantial resistance in controlled laboratory trials, suggesting that high exposure risk overwhelms their recently evolved resistance. We also tested for parasite adaptation to its host by performing transcontinental reciprocal infections, using stickleback and tapeworm populations from Europe and western Canada. More infections occurred in same-continent host-parasite combinations, indicating parasite "local" adaptation, at least on the scale of continents. However, the recently evolved immunity of freshwater hosts applies to both local and foreign parasites. The pattern of adaptation described here is not wholly compatible with either of the common models of host-parasite coevolution (i.e., matching infection or targeted recognition). Instead, we propose a hybrid, eco-evolutionary model to explain the remarkable pattern of global host resistance and local parasite infectivity.

Virulence in the three-spined stickleback specific parasite Schistocephalus solidus is inherited additively.

Parasite virulence is a key trait in host-parasite interactions and plays a crucial role in infection dynamics. Our study system offers the rare opportunity to study the virulence of an individual macroparasite (Schistocephalus solidus) in its vertebrate fish host (Gasterosteus aculeatus). The size of the tapeworm in the fish can be regarded as a good proxy for individual parasite virulence, as parasite size correlates negatively with fitness traits of the stickleback host (i.e. the bigger the parasite, the lower the host's reproductive success) as well as directly with the number of parasite offspring to be expected. To investigate how virulence is inherited, laboratory bred, parasite-naïve stickleback were infected with a cross of two S. solidus populations of either high or low virulence, as well as one hybrid cross between the two. The relative weight of the parasite as expressed in the parasite index served as a measure of virulence. Furthermore, we measured several condition and immune related traits in the fish host to assess parasite impact on the stickleback. We hypothesized that parasite virulence is to a large extent genetically determined and correlated with several fitness traits in the stickleback host. We found that virulence is inherited additively in S. solidus, with hybrids of high and low virulence parasites displaying intermediate levels. However, contrary to expectation, infection rate of S. solidus in three-spined stickleback is not related to virulence. Even though the presence of the parasite caused differences in host condition, these were indistinguishable between the different levels of virulence in this experiment. Fish immune traits also showed a response to infection but had no correlation with level of parasite virulence. With this experiment we have taken the first step towards understanding how virulence is inherited and how it is driven in the Schistocephalus-stickleback system, even though virulence, as measured here, does not directly translate into cost for the host. A better understanding of the costs inflicted on the host by S. solidus infection is needed to understand this interaction in greater detail.

Extensive copy-number variation of young genes across stickleback populations.

Duplicate genes emerge as copy-number variations (CNVs) at the population level, and remain copy-number polymorphic until they are fixed or lost. The successful establishment of such structural polymorphisms in the genome plays an important role in evolution by promoting genetic diversity, complexity and innovation. To characterize the early evolutionary stages of duplicate genes and their potential adaptive benefits, we combine comparative genomics with population genomics analyses to evaluate the distribution and impact of CNVs across natural populations of an eco-genomic model, the three-spined stickleback. With whole genome sequences of 66 individuals from populations inhabiting three distinct habitats, we find that CNVs generally occur at low frequencies and are often only found in one of the 11 populations surveyed. A subset of CNVs, however, displays copy-number differentiation between populations, showing elevated within-population frequencies consistent with local adaptation. By comparing teleost genomes to identify lineage-specific genes and duplications in sticklebacks, we highlight rampant gene content differences among individuals in which over 30% of young duplicate genes are CNVs. These CNV genes are evolving rapidly at the molecular level and are enriched with functional categories associated with environmental interactions, depicting the dynamic early copy-number polymorphic stage of genes during population differentiation.

The role of prezygotic isolation mechanisms in the divergence of two parasite species.

BACKGROUND: The formation of reproductive barriers in diverging lineages is a prerequisite to complete speciation according to the biological species concept. In parasites with complex life cycles, speciation may be driven by adaptation to different intermediate hosts, yet diverging lineages can still share the same definitive host where reproduction takes place. In these cases, prezygotic isolation mechanisms should evolve very early and be particularly strong, preventing costly unfavourable matings. In this study, we investigated the importance of prezygotic barriers to reproduction in two cestode species that diverged 20-25mya and show an extraordinary degree of specificity to different intermediate hosts. Both species share the same definitive hosts and hybridize in the laboratory. Yet, natural hybrids have so far not been detected. METHODS: We used a combination of different experiments to investigate the role of prezygotic barriers to reproduction in the speciation of these parasites. First, we investigated whether hybridization is possible under natural conditions by exposing lab-reared herring gulls (Larus argentatus, the definitive hosts) to both parasites of either sympatric or allopatric combinations. In a second experiment, we tested whether the parasites prefer conspecifics over parasites from a different species in dichotomous mate choice trials. RESULTS: Our results show that the two species hybridize under natural conditions with parasites originating either from sympatric or allopatric populations producing hybrid offspring. Surprisingly, the mate choice experiment indicated that both parasite species prefer mates of the different species to conspecifics. CONCLUSIONS: Neither fundamental constraints against hybridization in a natural host nor assortative mate choice sufficiently explain the persistent segregation of the two tapeworm species in nature. Hence, postzygotic ecological selection against hybrids is presumably the more important driving force limiting gene flow between the two parasite sister species.

Transcriptome profiling of immune tissues reveals habitat-specific gene expression between lake and river sticklebacks.

The observation of habitat-specific phenotypes suggests the action of natural selection. The three-spined stickleback (Gasterosteus aculeatus) has repeatedly colonized and adapted to diverse freshwater habitats across the northern hemisphere since the last glaciation, while giving rise to recurring phenotypes associated with specific habitats. Parapatric lake and river populations of sticklebacks harbour distinct parasite communities, a factor proposed to contribute to adaptive differentiation between these ecotypes. However, little is known about the transcriptional response to the distinct parasite pressure of those fish in a natural setting. Here, we sampled wild-caught sticklebacks across four geographical locations from lake and river habitats differing in their parasite load. We compared gene expression profiles between lake and river populations using 77 whole-transcriptome libraries from two immune-relevant tissues, the head kidney and the spleen. Differential expression analyses revealed 139 genes with habitat-specific expression patterns across the sampled population pairs. Among the 139 differentially expressed genes, eight are annotated with an immune function and 42 have been identified as differentially expressed in previous experimental studies in which fish have been immune challenged. Together, these findings reinforce the hypothesis that parasites contribute to adaptation of sticklebacks in lake and river habitats.

Experimental parasite community ecology: intraspecific variation in a large tapeworm affects community assembly.

Non-random species associations occur in naturally sampled parasite communities. The processes resulting in predictable community structure (e.g. particular host behaviours, cross-immunity, interspecific competition) could be affected by traits that vary within a parasite species, like growth or antigenicity. We experimentally infected three-spined sticklebacks with a large tapeworm (Schistocephalus solidus) that impacts the energy needs, foraging behaviour and immune reactions of its host. The tapeworms came from two populations, characterized by high or low growth in sticklebacks. Our goal was to evaluate how this parasite, and variation in its growth, affects the acquisition of other parasites. Fish infected with S. solidus were placed into cages in a lake to expose them to the natural parasite community. We also performed a laboratory experiment in which infected fish were exposed to a fixed dose of a common trematode parasite. In the field experiment, infection with S. solidus affected the abundance of four parasite species, relative to controls. For two of the four species, changes occurred only in fish harbouring the high-growth S. solidus; one species increased in abundance and the other decreased. These changes did not appear to be directly linked to S. solidus growth though. The parasite exhibiting elevated abundance was the same trematode used in the laboratory infection. In that experiment, we found a similar infection pattern, suggesting that S. solidus affects the physiological susceptibility of fish to this trematode. Associations between S. solidus and other parasites occur and vary in direction. However, some of these associations were contingent on the S. solidus population, suggesting that intraspecific variability can affect the assembly of parasite communities.

In vitro leukocyte response of three-spined sticklebacks (Gasterosteus aculeatus) to helminth parasite antigens.

Helminth parasites of teleost fish have evolved strategies to evade and manipulate the immune responses of their hosts. Responsiveness of fish host immunity to helminth antigens may therefore vary depending on the degree of host-parasite counter-adaptation. Generalist parasites, infective for a number of host species, might be unable to adapt optimally to the immune system of a certain host species, while specialist parasites might display high levels of adaptation to a particular host species. The degree of adaptations may further differ between sympatric and allopatric host-parasite combinations. Here, we test these hypotheses by in vitro exposure of head kidney leukocytes from three-spined sticklebacks (Gasterosteus aculeatus) to antigens from parasites with a broad fish host range (Diplostomum pseudospathaceum, Triaenophorus nodulosus), a specific fish parasite of cyprinids (Ligula intestinalis) and parasites highly specific only to a single fish species as second intermediate host (Schistocephalus pungitii, which does not infect G. aculeatus, and Schistocephalus solidus, infecting G. aculeatus). In vitro responses of stickleback leukocytes to S. solidus antigens from six European populations, with S. solidus prevalence from <1% to 66% were tested in a fully crossed experimental design. Leukocyte cultures were analysed by means of flow cytometry and a chemiluminescence assay to quantify respiratory burst activity. We detected decreasing magnitudes of in vitro responses to antigens from generalist to specialist parasites and among specialists, from parasites that do not infect G. aculeatus to a G. aculeatus-infecting species. Generalist parasites seem to maintain their ability to infect different host species at the costs of relatively higher immunogenicity compared to specialist parasites. In a comparison of sympatric and allopatric combinations of stickleback leukocytes and antigens from S. solidus, magnitudes of in vitro responses were dependent on the prevalence of the parasite in the population of origin, rather than on sympatry. Antigens from Norwegian (prevalence 30-50%) and Spanish (40-66%) S. solidus induced generally higher in vitro responses compared to S. solidus from two German (<1%) populations. Likewise, leukocytes from stickleback populations with a high S. solidus prevalence showed higher in vitro responses to S. solidus antigens compared to populations with low S. solidus prevalence. This suggests a rather low degree of local adaptation in S. solidus populations, which might be due to high gene flow among populations because of their extremely mobile final hosts, fish-eating birds.

Making the in vitro breeding of Schistocephalus solidus more flexible.

Schistocephalus solidus is one of the few cestodes that can be bred in vitro. Worms have typically been bred in pairs, so the parents of each offspring can clearly be assigned. From a genetic perspective, it would be useful to be able to mate an individual worm to multiple partners while still being able to distinguish among different parents. As each adult S. solidus possesses numerous reproductive complexes, cutting worms and breeding the pieces separately would facilitate such breeding designs. We halved worms before in vitro breeding and evaluated whether this affected outcrossing rates and reproductive output. Cutting did not influence clutch mass, i.e. egg number and size, or outcrossing rates, but eggs from cut worms had a lower hatching rate than eggs from uncut worms. We found that when two anterior worm halves were bred together, they produced fewer, smaller eggs with higher hatching rates, compared to two posterior halves. Moreover, once we controlled for this effect of 'worm half', the two halves of an individual worm tended to reproduce similarly under comparable circumstances. We conclude that cutting plerocercoids increases the flexibility with which this tapeworm can be experimentally bred without dramatically affecting the production of viable, outcrossed eggs.

Morphological and molecular data for larval stages of four species of Petasiger Dietz, 1909 (Digenea: Echinostomatidae) with an updated key to the known cercariae from the Palaearctic.

Large-tailed echinostomatid cercariae of the genus Petasiger Dietz, 1909 (Digenea: Echinostomatidae) from the planorbid snails Gyraulus albus (Müller) and Planorbis planorbis (L.) collected in Germany and the Czech Republic and metacercariae from Gasterosteus aculeatus L. (Gasterosteiformes: Gasterosteidae) collected in Canada are characterised morphologically and molecularly. The rediae, cercariae and metacercariae are described in detail and compared with the existing data on the larval stages of Petasiger spp. Comparative molecular analyses using 28S rDNA and nad1 mitochondrial sequences supported the distinct status of four species of Petasiger. Molecular and morphological evidence for their distinction and an updated key to the known large-tailed cercariae of Petasiger from the Palaearctic are provided.

Exploring local immunological adaptation of two stickleback ecotypes by experimental infection and transcriptome-wide digital gene expression analysis.

Understanding the extent of local adaptation in natural populations and the mechanisms that allow individuals to adapt to their native environment is a major avenue in molecular ecology research. Evidence for the frequent occurrence of diverging ecotypes in species that inhabit multiple ecological habitats is accumulating, but experimental approaches to understanding the biological pathways as well as the underlying genetic mechanisms are still rare. Parasites are invoked as one of the major selective forces driving evolution and are themselves dependent on the ecological conditions in a given habitat. Immunological adaptation to local parasite communities is therefore expected to be a key component of local adaptation in natural populations. Here, we use next-generation sequencing technology to compare the transcriptome-wide response of experimentally infected three-spined sticklebacks from a lake and a river population, which are known to evolve under selection by distinct parasite communities. By comparing overall gene expression levels as well as the activation of functional pathways in response to parasite exposure, we identified potential differences between the two stickleback populations at several levels. Our results suggest locally adapted patterns of gene regulation in response to parasite exposure, which may reflect different local optima in the trade-off between the benefits and the disadvantages of mounting an immune response because of quantitative differences of the local parasite communities.

Genome-wide patterns of standing genetic variation in a marine population of three-spined sticklebacks.

Since the end of the Pleistocene, the three-spined stickleback (Gasterosteus aculeatus) has repeatedly colonized and adapted to various freshwater habitats probably originating from ancestral marine populations. Standing genetic variation and the underlying genomic architecture both have been speculated to contribute to recent adaptive radiations of sticklebacks. Here, we expand on the current genomic resources of this fish by providing extensive genome-wide variation data from six individuals from a marine (North Sea) stickleback population. Using next-generation sequencing and a combination of paired-end and mate-pair libraries, we detected a wide size range of genetic variation. Among the six individuals, we found more than 7% of the genome is polymorphic, consisting of 2599111 SNPs, 233464 indels and structural variation (SV) (>50 bp) such as 1054 copy-number variable regions (deletions and duplications) and 48 inversions. Many of these polymorphisms affect gene and coding sequences. Based on SNP diversity, we determined outlier regions concordant with signatures expected under adaptive evolution. As some of these outliers overlap with pronounced regions of copy-number variation, we propose the consideration of such SV when analysing SNP data from re-sequencing approaches. We further discuss the value of this resource on genome-wide variation for further investigation upon the relative contribution of standing variation on the parallel evolution of sticklebacks and the importance of the genomic architecture in adaptive radiation.

Absence of major histocompatibility complex class II mediated immunity in pipefish, Syngnathus typhle: evidence from deep transcriptome sequencing.

The major histocompatibility complex (MHC)-mediated adaptive immune system is the hallmark of gnathostome immune defence. Recent work suggests that cod-like fishes (Gadidae) lack important components of the MHC class II mediated immunity. Here, we report a putative independent loss of functionality of this pathway in another species, the pipefish Syngnathus typhle, that belongs to a distantly related fish family (Syngnathidae). In a deep transcriptome sequencing approach comprising several independent normalized and non-normalized expressed sequence tag (EST) libraries with approximately 7.5 × 10(8) reads, sequenced with two next generation platforms (454 and Illumina), we were unable to identify MHC class IIα/ß genes as well as genes encoding associated receptors. Along with the recent findings in cod, our results suggest that immune systems of the Euteleosts may be more variable than previously assumed.