An exploration of the links between parasites, trophic ecology, morphology, and immunogenetics in the Lake Tanganyika cichlid radiation

Differences in habitat and diet between species are often associated with morphological differences. Habitat and trophic adaptation have therefore been proposed as important drivers of speciation and adaptive radiation. Importantly, habitat and diet shifts likely impose changes in exposure to different parasites and infection risk. As strong selective agents influencing survival and mate choice, parasites might play an important role in host diversification. We explore this possibility for the adaptive radiation of Lake Tanganyika (LT) cichlids. We first compare metazoan macroparasites infection levels between cichlid tribes. We then describe the cichlids’ genetic diversity at the major histocompatibility complex (MHC), which plays a key role in vertebrate immunity. Finally, we evaluate to what extent trophic ecology and morphology explain variation in infection levels and MHC, accounting for phylogenetic relationships. We show that different cichlid tribes in LT feature partially non-overlapping parasite communities and partially non-overlapping MHC diversity. While morphology explained 15% of the variation in mean parasite abundance, trophic ecology accounted for 16% and 22% of the MHC variation at the nucleotide and at the amino acid level, respectively. Parasitism and immunogenetic adaptation may thus add additional dimensions to the LT cichlid radiation

Molecular characterization of MHC class IIB genes of sympatric Neotropical cichlids

Ministerio de Economía y Competitividad del Gobierno de España, Programa de Formación de Personal Investigador FPI BES-2011-047645 to MJH, Programa Estatal de Fomento de la Investigación Científica y Técnica de Excelencia Proyecto CGL 2010-16103 to MB. This project was further enabled through two German Science Foundation grants to CE (DFG, EI841/4-1 and EI841/6-1) both part of the SPP 1399 priority programme on “host-parasite interactions”

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.Funding was provided by Spanish Ministry of Science and Innovation (MCIN)/Spanish Research Agency (AEI) and European Regional Development Fund (ERDF) “A way to make Europe” through projects CGL2010‑16103, CGL2013‑42462‑P, CGL2017‑82986‑C2‑1‑P to MB. SEB was supported by a Swiss National Science Foundation Early PostDoc.Mobility Fellowship (P2SKP3_191312). MJH was supported by a predoctoral fellowship from the Spanish Ministry of Economy and Competitiveness (FPI‑BES‑2011‑047645), and CLM was supported by a predoctoral fellowship from MCIN/AEI and European Social Funds (FPI‑PRE2018‑085797).Peer reviewe

Alignment of genetic variants of the MHC class II loci of East African cichlids

First, the generated raw reads (11,569 reads) were processed with Roche's demultiplexing and converting tools (sffinfo, sfffile) and sequences of primer annealing sites were removed. For quality filtering we applied a filter for too short reads (≤ 150 bp), we only allowed 1% of ambiguous bases (N) and filtered out low quality sequences (Mean ≥ 15). These sequences were imported into Geneious (6.1.6 Biomatters Ltd, www.geneious.com) and de novo assembled (with custom sensitivity: minimum overlap identity of 95 % and maximum ambiguity 4 using all reads from one species. This resulted in contigs of single individuals with highly identical reads (pairwise identity: median 99.50 %) and contigs of several individuals sharing these reads (pairwise identity: median 99.40 %). The coverage ranged from 2 to 131 for single individual contigs and 2 to 337 reads for contigs originating from multiple individuals. We also kept low coverage contigs as we use our data for measuring genetic diversity among tribes and not for investigating functionality or selection processes (indicated with suffix “low” in the alignment). However, if more than 3 bp of a read were different than the rest of the contig, the read was excluded and also singletons, which differed dramatically (≥ 10 mutations) to other contigs, were removed from the data set (reads N=517). Consensus sequences were generated within Geneious using 50 % strict rule from each contig and for each individual. Most homopolymer regions were correctly called with these settings and ambiguous positions were coded according to IUPAC rules. The obtained variants were aligned using MAFFT (--auto; 200PAM/k=2, 1.53 open penalty/0.123 offset) (Katoh & Standley, 2013) and insertions of ambiguous positions, homopolymers and misalignments were manually checked. This resulted in an alignment of 751 base pairs containing both intronic and exonic regions. A blast search of the alleles led to the exclusion of further sequences (removed contigs N=266). In a next step we shortened the alleles to exon 2 only, in order to (i) reduce our data set to coding nucleotides and (ii) to reduce the amount of missing data and ambiguities. This resulted in a total number of 844 MHC exon 2 variants of 160 bp lengths. Despite our methodological limitations, short reads and relatively low sequence coverage for some contigs, our results are valid as a valuable measurement of immunogenetic diversity that is comparable across all tribes of Lake Tanganyika cichlids as these biases are expected to be similarly distributed across the different tribes. The available alignment was generated as described above and saved as a fasta file. It includes 844 MHC exon 2 variants (160 base pairs). The header of each entry includes information about the scientific species name (e.g. Tropheus moorii) with a specific variant name (e.g. 04_01). This can be followed by the indication "low", which was added if the variant was called with very low coverage (<=2x)

An exploration of the links between parasites, trophic ecology, morphology, and immunogenetics in the Lake Tanganyika cichlid radiation

Differences in habitat and diet between species are often associated with morphological differences. Habitat and trophic adaptation have therefore been proposed as important drivers of speciation and adaptive radiation. Importantly, habitat and diet shifts likely impose changes in exposure to different parasites and infection risk. As strong selective agents influencing survival and mate choice, parasites might play an important role in host diversification. We explore this possibility for the adaptive radiation of Lake Tanganyika (LT) cichlids. We first compare metazoan macroparasites infection levels between cichlid tribes. We then describe the cichlids' genetic diversity at the major histocompatibility complex (MHC), which plays a key role in vertebrate immunity. Finally, we evaluate to what extent trophic ecology and morphology explain variation in infection levels and MHC, accounting for phylogenetic relationships. We show that different cichlid tribes in LT feature partially non-overlapping parasite communities and partially non-overlapping MHC diversity. While morphology explained 15% of the variation in mean parasite abundance, trophic ecology accounted for 16% and 22% of the MHC variation at the nucleotide and at the amino acid level, respectively. Parasitism and immunogenetic adaptation may thus add additional dimensions to the LT cichlid radiation.status: publishe

Parasitological survey and genetic variants of East African cichlids

Differences in habitat and diet between species are often associated with morphological differences. Habitat and trophic adaptation have therefore been proposed as important drivers of speciation and adaptive radiation. Importantly, habitat and diet shifts likely impose changes in exposure to different parasites and infection risk. As strong selective agents influencing survival and mate choice, parasites might play an important role in host diversification. We explore this possibility for the adaptive radiation of Lake Tanganyika (LT) cichlids. We first compare metazoan macroparasites infection levels between cichlid tribes. We then describe the cichlids' genetic diversity at the major histocompatibility complex (MHC), which plays a key role in vertebrate immunity. Finally, we evaluate to what extent trophic ecology and morphology explain variation in infection levels and MHC, accounting for phylogenetic relationships. We show that different cichlid tribes in LT feature partially non-overlapping parasite communities and partially non-overlapping MHC diversity. While morphology explained 15% of the variation in mean parasite abundance, trophic ecology accounted for 16% and 22% of the MHC variation at the nucleotide and at the amino acid level, respectively. Parasitism and immunogenetic adaptation may thus add additional dimensions to the LT cichlid radiation

Parasitological survey of 23 cichlid species for metazoan ecto- and endoparasites

For the parasitological survey, 23 cichlid species were screened for metazoan ecto- and endoparasites (Supplementary Table 1). Sampling was conducted at Toby's place on the Zambian shoreline of Lake Tanganyika. While most fish species were obtained in August 2012, S. diagramma and H. microlepis were captured in August 2011 and July 2013, respectively. One species, A. burtoni, was obtained in July 2013 at Kapata, which is about 20 km southward. About 7 to 18 individuals per species (usually ten) were caught by chasing fish into standing nets (Supplementary Table 1). After capture the fish were kept in tanks of 0.8 m x 0.8 m x 1.2 m depth or 0.8 m x 0.8 m x 2 m depth. Before usage, tanks were cleaned, dried and filled with lake water. All fish were dissected in the field within four days post-capture. The day of dissection (0, 1, 2 or 3 days after capture) was recorded in order to keep track of changes in parasitological parameters while the fish were kept in the tanks. Individual fish were killed with an overdose of MS222. The parasitological survey consisted of three parts. First, the outer surface and the mouth cavity of the fish were inspected for ectoparasitic monogeneans and crustaceans (copepods, branchiurans, isopods), bivalves, and any kind of helminthic cysts. Second, the four gill-branches on the left were dissected and stored on 100% analytical ethanol (EtOH), and later in the lab screened for ectoparasitic monogeneans, crustaceans (copepods and branchiurans), bivalves, and any kind of helminthic cysts. Third, fish were screened for intestinal monogeneans, digeneans, acanthocephalans, nematodes, and any kind of helminthic cysts. To do so, stomach, intestines, gall and urinary bladder were dissected and inspected in a petridish with lake water. Finally, the sex of the fish was determined by visual inspection of the genital papilla and gonad development. The parasitological survey was performed with a stereomicroscope and by multiple observers. Observers were recorded in order to keep track of observer bias. A single observer screened the outer surface and the mouth cavity of the fish. The number of observers varied between years for gills and intestines (gills: two observers in each year; intestines: three, four and one observer(s) in 2011, 2012 and 2013, respectively). All parasites were counted and identified to genus or class level and preserved as follows. Monogeneans were isolated using dissection needles and were either mounted on slides in ammonium picrate glycerine for further morphological characterization, or stored on 100 % EtOH. Acanthocephalans and nematodes were stored on 80 % EtOH, while intestinal monogeneans, branchiurans, copepods, any kind of helminthic cysts, bivalves and unknown groups were stored on 100 % EtOH