Evolution of MHC IIB Diversity Across Cichlid Fish Radiations.

The genes of the major histocompatibility complex (MHC) are among the most polymorphic genes in vertebrates and crucial for their adaptive immune response. These genes frequently show inconsistencies between allelic genealogies and species phylogenies. This phenomenon is thought to be the result of parasite-mediated balancing selection maintaining ancient alleles through speciation events (trans-species polymorphism [TSP]). However, allele similarities may also arise from postspeciation mechanisms, such as convergence or introgression. Here, we investigated the evolution of MHC class IIB diversity in the cichlid fish radiations across Africa and the Neotropics by a comprehensive review of available MHC IIB DNA sequence information. We explored what mechanism explains the MHC allele similarities found among cichlid radiations. Our results showed extensive allele similarity among cichlid fish across continents, likely due to TSP. Functionality at MHC was also shared among species of the different continents. The maintenance of MHC alleles for long evolutionary times and their shared functionality may imply that certain MHC variants are essential in immune adaptation, even in species that diverged millions of years ago and occupy different environments.

Divergent and non-parallel evolution of MHC IIB in the Neotropical Midas cichlid species complex.

BACKGROUND: Ecological diversification is the result of divergent natural selection by contrasting habitat characteristics that favours the evolution of distinct phenotypes. This process can happen in sympatry and in allopatry. Habitat-specific parasite communities have the potential to drive diversification among host populations by imposing selective pressures on their host's immune system. In particular, the hyperdiverse genes of the major histocompatibility complex (MHC) are implicated in parasite-mediated host divergence. Here, we studied the extent of divergence at MHC, and discuss how it may have contributed to the Nicaraguan Midas cichlid species complex diversification, one of the most convincing examples of rapid sympatric parallel speciation. RESULTS: We genotyped the MHC IIB for individuals from six sympatric Midas cichlid assemblages, each containing species that have adapted to exploit similar habitats. We recovered large allelic and functional diversity within the species complex. While most alleles were rare, functional groups of alleles (supertypes) were common, suggesting that they are key to survival and that they were maintained during colonization and subsequent radiations. We identified lake-specific and habitat-specific signatures for both allelic and functional diversity, but no clear pattern of parallel divergence among ecomorphologically similar phenotypes. CONCLUSIONS: Colonization and demographic effects of the fish could have contributed to MHC evolution in the Midas cichlid in conjunction with habitat-specific selective pressures, such as parasites associated to alternative preys or environmental features. Additional ecological data will help evaluating the role of host-parasite interactions in the Midas cichlid radiations and aid in elucidating the potential role of non-parallel features differentiating crater lake species assemblages.

Encapsulation of Anguillicola crassus reduces the abundance of adult parasite stages in the European eel (Anguilla anguilla).

Encapsulation of the parasitic nematode Anguillicola crassus Kuwahara, Niimi & Hagaki is commonly observed in its native host, the Japanese eel (Anguilla japonica Temminck & Schlegel). Encapsulation has also been described in a novel host, the European eel (A. anguilla L.), and there is evidence that encapsulation frequency has increased since the introduction of A. crassus. We examined whether encapsulation of A. crassus provides an advantage to its novel host in Lake Müggelsee, NE Germany. We provide the first evidence that encapsulation was associated with reduced abundance of adult A. crassus. This pattern was consistent in samples taken 3 months apart. There was no influence of infection on the expression of the two metabolic genes studied, but the number of capsules was negatively correlated with the expression of two mhc II genes of the adaptive immune response, suggesting a reduced activation. Interestingly, eels that encapsulated A. crassus had higher abundances of two native parasites compared with non-encapsulating eels. We propose that the response of A. anguilla to infection by A. crassus may interfere with its reaction to other co-occurring parasites.

Gene expression response to a nematode parasite in novel and native eel hosts.

Invasive parasites are involved in population declines of new host species worldwide. The high susceptibilities observed in many novel hosts have been attributed to the lack of protective immunity to the parasites which native hosts acquired during their shared evolution. We experimentally infected Japanese eels (Anguilla japonica) and European eels (Anguilla anguilla) with Anguillicola crassus, a nematode parasite that is native to the Japanese eel and invasive in the European eel. We inferred gene expression changes in head kidney tissue from both species, using RNA-seq data to determine the responses at two time points during the early stages of infection (3 and 23 days postinfection). At both time points, the novel host modified the expression of a larger and functionally more diverse set of genes than the native host. Strikingly, the native host regulated immune gene expression only at the earlier time point and to a small extent while the novel host regulated these genes at both time points. A low number of differentially expressed immune genes, especially in the native host, suggest that a systemic immune response was of minor importance during the early stages of infection. Transcript abundance of genes involved in cell respiration was reduced in the novel host which may affect its ability to cope with harsh conditions and energetically demanding activities. The observed gene expression changes in response to a novel parasite that we observed in a fish follow a general pattern observed in amphibians and mammals, and suggest that the disruption of physiological processes, rather than the absence of an immediate immune response, is responsible for the higher susceptibility of the novel host.

Experimental infection with Anguillicola crassus alters immune gene expression in both spleen and head kidney of the European eel (Anguilla anguilla).

Invasive parasites have been implicated in the declines of several freshwater species. The swim bladder nematode Anguillicola crassus was introduced into Europe in the 1980s and is considered a threat to the European eel (Anguilla anguilla). Infection affects stress resistance and swimming behaviour. European eels produce an immune response against the parasite during the late stages of infection and after repeated infections. We used RNA-seq to examine the molecular response to infection during the poorly understood early stage and identify expression of genes and associated processes that are modified in two immune organs of European eels 3 days post infection with A. crassus. In the spleen, 67 genes were differentially expressed, 32 of which were annotated. Most of these were involved in immune processes and their regulation. Other differentially expressed genes in the spleen were important for heme metabolism and heme turn-over. In the head kidney, 257 genes (134 annotated) were differentially expressed. Several of these were associated with immune functions. Other differentially expressed genes in the head kidney were related to renal function, in particular osmoregulation and paracellular flow. We conclude that the early response of European eels to A. crassus is complex and involves various processes aside from the immune system. We identified molecular changes occurring early during the infection and identified candidate genes and processes which will facilitate future studies aimed at determining the factors affecting European eel viability in the face of this invasive parasite.

Molecular characterization of MHC class IIB genes of sympatric Neotropical cichlids.

BACKGROUND: The Major Histocompatibility Complex (MHC) is a key component of the adaptive immune system of all vertebrates and consists of the most polymorphic genes known to date. Due to this complexity, however, MHC remains to be characterized in many species including any Neotropical cichlid fish. Neotropical crater lake cichlids are ideal models to study evolutionary processes as they display one of the most convincing examples of sympatric and repeated parallel radiation events within and among isolated crater lakes. RESULTS: Here, we characterized the genes of MHC class IIB chain of the Midas cichlid species complex (Amphilophus cf. citrinellus) including fish from five lakes in Nicaragua. We designed 19 new specific primers anchored in a stepwise fashion in order to detect all alleles present. We obtained 866 genomic DNA (gDNA) sequences from thirteen individuals and 756 additional sequences from complementary DNA (cDNA) of seven of those individuals. We identified 69 distinct alleles with up to 25 alleles per individual. We also found considerable intron length variation and mismatches of alleles detected in cDNA and gDNA suggesting that some loci have undergone pseudogenization. Lastly, we created a model of protein structure homology for each allele and identified their key structural components. CONCLUSIONS: Overall, the Midas cichlid has one of the most diverse repertoires of MHC class IIB genes known, which could serve as a powerful tool to elucidate the process of divergent radiations, colonization and speciation in sympatry.

Evaluating the adaptive potential of the European eel: is the immunogenetic status recovering?

The recent increased integration of evolutionary theory into conservation programs has greatly improved our ability to protect endangered species. A common application of such theory links population dynamics and indices of genetic diversity, usually estimated from neutrally evolving markers. However, some studies have suggested that highly polymorphic adaptive genes, such as the immune genes of the Major Histocompatibility Complex (MHC), might be more sensitive to fluctuations in population dynamics. As such, the combination of neutrally- and adaptively-evolving genes may be informative in populations where reductions in abundance have been documented. The European eel (Anguilla anguilla) underwent a drastic and well-reported decline in abundance in the late 20th century and still displays low recruitment. Here we compared genetic diversity indices estimated from neutral (mitochondrial DNA and microsatellites) and adaptive markers (MHC) between two distinct generations of European eels. Our results revealed a clear discrepancy between signatures obtained for each class of markers. Although mtDNA and microsatellites showed no changes in diversity between the older and the younger generations, MHC diversity revealed a contemporary drop followed by a recent increase. Our results suggest ongoing gain of MHC genetic diversity resulting from the interplay between drift and selection and ultimately increasing the adaptive potential of the species.

Characterization of MHC class IIB for four endangered Australian freshwater fishes obtained from ecologically divergent populations.

Genetic diversity is an essential aspect of species viability, and assessments of neutral genetic diversity are regularly implemented in captive breeding and conservation programs. Despite their importance, information from adaptive markers is rarely included in such programs. A promising marker of significance in fitness and adaptive potential is the major histocompatibility complex (MHC), a key component of the adaptive immune system. Populations of Australian freshwater fishes are generally declining in numbers due to human impacts and the introduction of exotic species, a scenario of particular concern for members of the family Percichthyidae, several of which are listed as nationally vulnerable or endangered, and hence subject to management plans, captive breeding, and restoration plans. We used a next-generation sequencing approach to characterize the MHC IIB locus and provide a conservative description of its levels of diversity in four endangered percichthyids: Gadopsis marmoratus, Macquaria australasica, Nannoperca australis, and Nannoperca obscura. Evidence is presented for a duplicated MHC IIB locus, positively selected sites and recombination of MHC alleles. Relatively moderate levels of diversity were detected in the four species, as well as in different ecotypes within each species. Phylogenetic analyses revealed genus specific clustering of alleles and no allele sharing among species. There were also no shared alleles observed between two ecotypes within G. marmoratus and within M. australasica, which might be indicative of ecologically-driven divergence and/or long divergence times. This represents the first characterization and assessment of MHC diversity for Percichthyidae, and also for Australian freshwater fishes in general, providing key genetic resources for a vertebrate group of increasing conservation concern.