No Compensatory Relationship between the Innate and Adaptive Immune System in Wild-Living European Badgers.

The innate immune system provides the primary vertebrate defence system against pathogen invasion, but it is energetically costly and can have immune pathological effects. A previous study in sticklebacks found that intermediate major histocompatibility complex (MHC) diversity correlated with a lower leukocyte coping capacity (LCC), compared to individuals with fewer, or many, MHC alleles. The organization of the MHC genes in mammals, however, differs to the highly duplicated MHC genes in sticklebacks by having far fewer loci. Using European badgers (Meles meles), we therefore investigated whether innate immune activity, estimated functionally as the ability of an individual's leukocytes to produce a respiratory burst, was influenced by MHC diversity. We also investigated whether LCC was influenced by factors such as age-class, sex, body condition, season, year, neutrophil and lymphocyte counts, and intensity of infection with five different pathogens. We found that LCC was not associated with specific MHC haplotypes, MHC alleles, or MHC diversity, indicating that the innate immune system did not compensate for the adaptive immune system even when there were susceptible MHC alleles/haplotypes, or when the MHC diversity was low. We also identified a seasonal and annual variation of LCC. This temporal variation of innate immunity was potentially due to physiological trade-offs or temporal variation in pathogen infections. The innate immunity, estimated as LCC, does not compensate for MHC diversity suggests that the immune system may function differently between vertebrates with different MHC organizations, with implications for the evolution of immune systems in different taxa.

MHC class II-assortative mate choice in European badgers (Meles meles).

The major histocompatibility complex (MHC) plays a crucial role in the immune system, and in some species, it is a target by which individuals choose mates to optimize the fitness of their offspring, potentially mediated by olfactory cues. Under the genetic compatibility hypothesis, individuals are predicted to choose mates with compatible MHC alleles, to increase the fitness of their offspring. Studies of MHC-based mate choice in wild mammals are under-represented currently, and few investigate more than one class of MHC genes. We investigated mate choice based on the compatibility of MHC class I and II genes in a wild population of European badgers (Meles meles). We also investigated mate choice based on microsatellite-derived pairwise relatedness, to attempt to distinguish MHC-specific effects from genomewide effects. We found MHC-assortative mating, based on MHC class II, but not class I genes. Parent pairs had smaller MHC class II DRB amino acid distances and smaller functional distances than expected from random pairings. When we separated the analyses into within-group and neighbouring-group parent pairs, only neighbouring-group pairs showed MHC-assortative mating, due to similarity at MHC class II loci. Our randomizations showed no evidence of genomewide-based inbreeding, based on 35 microsatellite loci; MHC class II similarity was therefore the apparent target of mate choice. We propose that MHC-assortative mate choice may be a local adaptation to endemic pathogens, and this assortative mate choice may have contributed to the low MHC genetic diversity in this population.

The phylogenetic utility and functional constraint of microRNA flanking sequences.

MicroRNAs (miRNAs) have recently risen to prominence as novel factors responsible for post-transcriptional regulation of gene expression. miRNA genes have been posited as highly conserved in the clades in which they exist. Consequently, miRNAs have been used as rare genome change characters to estimate phylogeny by tracking their gain and loss. However, their short length (21-23 bp) has limited their perceived utility in sequenced-based phylogenetic inference. Here, using reference taxa with established phylogenetic relationships, we demonstrate that miRNA sequences are of high utility in quantitative, rather than in qualitative, phylogenetic analysis. The clear orthology among miRNA genes from different species makes it straightforward to identify and align these sequences from even fragmentary datasets. We also identify significant sequence conservation in the regions directly flanking miRNA genes, and show that this too is of utility in phylogenetic analysis, as well as highlighting conserved regions that will be of interest to other fields. Employing miRNA sequences from 12 sequenced drosophilid genomes, together with a Tribolium castaneum outgroup, we demonstrate that this approach is robust using Bayesian and maximum-likelihood methods. The utility of these characters is further demonstrated in the rhabditid nematodes and primates. As next-generation sequencing makes it more cost-effective to sequence genomes and small RNA libraries, this methodology provides an alternative data source for phylogenetic analysis. The approach allows rapid resolution of relationships between both closely related and rapidly evolving species, and provides an additional tool for investigation of relationships within the tree of life.

Identification of putative ecdysteroid and juvenile hormone pathway genes in the shrimp Neocaridina denticulata.

Although the sesquiterpenoid juvenile hormone (JH) and the steroidal ecdysteroids are of vital importance to the development and reproduction of insects, our understanding of the evolution of these crucial hormonal regulators in other arthropods is limited. To better understand arthropod hormone evolution and regulation, here we describe the hormonal pathway genes (e.g. those involved in hormone biosynthesis, degradation, regulation and signal transduction) of a new decapod model, the shrimp Neocaridina denticulata. The majority of known insect sesquiterpenoid and ecdysteroid pathway genes and their regulators are contained in the N. denticulata genome. In the sesquiterpenoid pathway, these include biosynthetic pathway components: juvenile hormone acid methyltransferase (JHAMT); hormone binding protein: juvenile hormone binding protein (JHBP); and degradation pathway components: juvenile hormone esterase (JHE), juvenile hormone esterase binding protein (JHEBP) and juvenile hormone epoxide hydrolase (JHEH), with the JHBP, JHEBP and JHEH genes being discovered in a crustacean for the first time here. Ecdysteroid biosynthetic pathway genes identified include spook, phantom, disembodied, shadow and CYP18. Potential hormonal regulators and signal transducers such as allatostatins (ASTs), Methoprene-tolerant (Met), Retinoid X receptor (RXR), Ecdysone receptor (EcR), calponin-like protein Chd64, FK509-binding protein (FKBP39), Broad-complex (Br-c), and crustacean hyperglycemic hormone/molt-inhibiting hormone/gonad-inhibiting hormone (CHH/MIH/GIH) genes are all present in the shrimp N. denticulata. To our knowledge, this is the first report of these hormonal pathways and their regulatory genes together in a single decapod, providing a vital resource for further research into development, reproduction, endocrinology and evolution of crustaceans, and arthropods in general.

Pathogen burden, co-infection and major histocompatibility complex variability in the European badger (Meles meles).

Pathogen-mediated selection is thought to maintain the extreme diversity in the major histocompatibility complex (MHC) genes, operating through the heterozygote advantage, rare-allele advantage and fluctuating selection mechanisms. Heterozygote advantage (i.e. recognizing and binding a wider range of antigens than homozygotes) is expected to be more detectable when multiple pathogens are considered simultaneously. Here, we test whether MHC diversity in a wild population of European badgers (Meles meles) is driven by pathogen-mediated selection. We examined individual prevalence (infected or not), infection intensity and co-infection of 13 pathogens from a range of taxa and examined their relationships with MHC class I and class II variability. This population has a variable, but relatively low, number of MHC alleles and is infected by a variety of naturally occurring pathogens, making it very suitable for the investigation of MHC-pathogen relationships. We found associations between pathogen infections and specific MHC haplotypes and alleles. Co-infection status was not correlated with MHC heterozygosity, but there was evidence of heterozygote advantage against individual pathogen infections. This suggests that rare-allele advantages and/or fluctuating selection, and heterozygote advantage are probably the selective forces shaping MHC diversity in this species. We show stronger evidence for MHC associations with infection intensity than for prevalence and conclude that examining both pathogen prevalence and infection intensity is important. Moreover, examination of a large number and diversity of pathogens, and both MHC class I and II genes (which have different functions), provide an improved understanding of the mechanisms driving MHC diversity.

Genomic sequence and experimental tractability of a new decapod shrimp model, Neocaridina denticulata.

The speciose Crustacea is the largest subphylum of arthropods on the planet after the Insecta. To date, however, the only publically available sequenced crustacean genome is that of the water flea, Daphnia pulex, a member of the Branchiopoda. While Daphnia is a well-established ecotoxicological model, previous study showed that one-third of genes contained in its genome are lineage-specific and could not be identified in any other metazoan genomes. To better understand the genomic evolution of crustaceans and arthropods, we have sequenced the genome of a novel shrimp model, Neocaridina denticulata, and tested its experimental malleability. A library of 170-bp nominal fragment size was constructed from DNA of a starved single adult and sequenced using the Illumina HiSeq2000 platform. Core eukaryotic genes, the mitochondrial genome, developmental patterning genes (such as Hox) and microRNA processing pathway genes are all present in this animal, suggesting it has not undergone massive genomic loss. Comparison with the published genome of Daphnia pulex has allowed us to reveal 3750 genes that are indeed specific to the lineage containing malacostracans and branchiopods, rather than Daphnia-specific (E-value: 10⁻6). We also show the experimental tractability of N. denticulata, which, together with the genomic resources presented here, make it an ideal model for a wide range of further aquacultural, developmental, ecotoxicological, food safety, genetic, hormonal, physiological and reproductive research, allowing better understanding of the evolution of crustaceans and other arthropods.

Evolution of MHC class I genes in the European badger (Meles meles).

The major histocompatibility complex (MHC) plays a central role in the adaptive immune system and provides a good model with which to understand the evolutionary processes underlying functional genes. Trans-species polymorphism and orthology are both commonly found in MHC genes; however, mammalian MHC class I genes tend to cluster by species. Concerted evolution has the potential to homogenize different loci, whereas birth-and-death evolution can lead to the loss of orthologs; both processes result in monophyletic groups within species. Studies investigating the evolution of MHC class I genes have been biased toward a few particular taxa and model species. We present the first study of MHC class I genes in a species from the superfamily Musteloidea. The European badger (Meles meles) exhibits moderate variation in MHC class I sequences when compared to other carnivores. We identified seven putatively functional sequences and nine pseudogenes from genomic (gDNA) and complementary (cDNA) DNA, signifying at least two functional class I loci. We found evidence for separate evolutionary histories of the α1 and α2/α3 domains. In the α1 domain, several sequences from different species were more closely related to each other than to sequences from the same species, resembling orthology or trans-species polymorphism. Balancing selection and probable recombination maintain genetic diversity in the α1 domain, evidenced by the detection of positive selection and a recombination event. By comparison, two recombination breakpoints indicate that the α2/α3 domains have most likely undergone concerted evolution, where recombination has homogenized the α2/α3 domains between genes, leading to species-specific clusters of sequences. Our findings highlight the importance of analyzing MHC domains separately.

Molecular characterization of the microbial communities in the subcaudal gland secretion of the European badger (Meles meles).

Many mammals possess specialized scent glands, which convey information about the marking individual. As the chemical profile of scent marks is likely to be affected by bacteria metabolizing the primary gland products, the variation in bacterial communities between different individuals has been proposed to underpin olfactory communication. However, few studies have investigated the dependency of microbiota residing in the scent organs on the host's individual-specific parameters. Here, we used terminal restriction fragment length polymorphism analysis of the PCR-amplified 16S rRNA gene and clone library construction to investigate the microbial communities in the subcaudal gland secretion of the European badger (Meles meles). As the secretion has been shown to encode individual-specific information, we investigated the correlation of the microbiota with different individual-specific parameters (age, sex, body condition, reproductive status, and season). We discovered a high number of bacterial species (56 operational taxonomic units from four phyla: Actinobacteria, Firmicutes, Proteobacteria, and Bacteroidetes), dominated by Actinobacteria (76.0%). The bacterial communities of cubs and adults differed significantly. Cubs possessed considerably more diverse communities dominated by Firmicutes, while in adults the communities were less diverse and dominated by Actinobacteria, suggesting that the acquisition of a 'mature bacterial community' is an ontogenetic process related to physiological changes during maturation.

MHC class II genes in the European badger (Meles meles): characterization, patterns of variation, and transcription analysis.

The major histocompatibility complex (MHC) comprises many genes, some of which are polymorphic with numerous alleles. Sequence variation among alleles is most pronounced in exon 2 of the class II genes, which encodes the α1 and ß1 domains that form the antigen-binding site (ABS) for the presentation of peptides. The MHC thus plays an important role in pathogen defense. European badgers (Meles meles) are a good species in which to study the MHC, as they harbor a variety of pathogens. We present the first characterization of MHC class II genes, isolated from genomic DNA (gDNA) and complementary DNA (cDNA), in the European badger. Examination of seven individuals revealed four DRB, two DQB, two DQA, and two DRA putatively functional gDNA sequences. All of these sequences, except DRA, exhibited high variability in exon 2; DRB had the highest variability. The ABS codons demonstrated high variability, due potentially to balancing selection, while non-ABS codons had lower variability. Positively selected sites were detected in DRB and DQA. Phylogenetic analysis demonstrated trans-species polymorphism of class II genes. Comparison with cDNA from whole blood revealed that only DRB had a transcription pattern reflecting the alleles that were present in the gDNA, while the other three genes had disparities between gDNA and cDNA. Only one sequence was transcribed, even though two gDNA sequences were present, from each of both DQB and DRA. Our characterization of badger MHC sequences forms a basis for further studies of MHC variability, mate choice, and pathogen resistance in this, and other, species.

Evidence for a role of the host-specific flea (Paraceras melis) in the transmission of Trypanosoma (Megatrypanum) pestanai to the European badger.

We investigated the epidemiology of Trypanosoma pestanai infection in European badgers (Meles meles) from Wytham Woods (Oxfordshire, UK) to determine prevalence rates and to identify the arthropod vector responsible for transmission. A total of 245 badger blood samples was collected during September and November 2009 and examined by PCR using primers derived from the 18S rRNA of T. pestanai. The parasite was detected in blood from 31% of individuals tested. T. pestanai was isolated from primary cultures of Wytham badger peripheral blood mononuclear cells and propagated continually in vitro. This population was compared with cultures of two geographically distinct isolates of the parasite by amplified fragment length polymorphism (AFLP) and PCR analysis of 18S rDNA and ITS1 sequences. High levels of genotypic polymorphism were observed between the isolates. PCR analysis of badger fleas (Paraceras melis) collected from infected individuals at Wytham indicated the presence of T. pestanai and this was confirmed by examination of dissected specimens. Wet smears and Giemsa-stained preparations from dissected fleas revealed large numbers of trypanosome-like forms in the hindgut, some of which were undergoing binary fission. We conclude that P. melis is the primary vector of T. pestanai in European badgers.