Winter is coming-The role of seasonality through the lens of the rodent virome.

Rodent virus communities (viromes) are overrepresented with zoonotic viruses, and as such are a key host system for the study of zoonotic viruses. However, the extent of viral diversity beyond characterized zoonotic viruses, and the factors that modulate the viromes of rodents remain opaque. In this issue of Molecular Ecology, Raghwani et al. (2023) use rodents as a model to understand the role of seasonality in dictating virome abundance and composition-a factor known to play an important role in most animal one-host, one-pathogen systems. These data are not only highly relevant to rodents, but have broad applications across understanding and disentangling animal virome ecology.

Distinct life history strategies underpin clear patterns of succession in microparasite communities infecting a wild mammalian host.

Individual animals in natural populations tend to host diverse parasite species concurrently over their lifetimes. In free-living ecological communities, organismal life histories shape interactions with their environment, which ultimately forms the basis of ecological succession. However, the structure and dynamics of mammalian parasite communities have not been contextualized in terms of primary ecological succession, in part because few datasets track occupancy and abundance of multiple parasites in wild hosts starting at birth. Here, we studied community dynamics of 12 subtypes of protozoan microparasites (Theileria spp.) in a herd of African buffalo. We show that Theileria communities followed predictable patterns of succession underpinned by four different parasite life history strategies. However, in contrast to many free-living communities, network complexity decreased with host age. Examining parasite communities through the lens of succession may better inform the effect of complex within host eco-evolutionary dynamics on infection outcomes, including parasite co-existence through the lifetime of the host.

Predicting fungal infection rate and severity with skin-associated microbial communities on amphibians.

Pathogen success (risk and severity) is influenced by host-associated microbiota, but the degree to which variation in microbial community traits predict future infection presence/absence (risk) and load (severity) for the host is unknown. We conducted a time-series experiment by sampling the skin-associated bacterial communities of five amphibian species before and after exposure to the fungal pathogen, Batrachochytrium dendrobaditis (Bd). We sought to determine whether microbial community traits are predictors of, or are affected by, Bd infection risk and intensity. Our results show that richness of putative Bd-inhibitory bacteria strongly predicts infection risk, while the proportion of putative Bd-inhibitory bacteria predicts future infection intensity. Variation in microbial community composition is high across time and individual, and bacterial prevalence is low. Our findings demonstrate how ecological community traits of host-associated microbiota may be used to predict infection risk by pathogenic microbes.

Identification of a rapidly-spreading triple mutant for high-level metabolic insecticide resistance in Anopheles gambiae provides a real-time molecular diagnostic for antimalarial intervention deployment.

Studies of insecticide resistance provide insights into the capacity of populations to show rapid evolutionary responses to contemporary selection. Malaria control remains heavily dependent on pyrethroid insecticides, primarily in long lasting insecticidal nets (LLINs). Resistance in the major malaria vectors has increased in concert with the expansion of LLIN distributions. Identifying genetic mechanisms underlying high-level resistance is crucial for the development and deployment of resistance-breaking tools. Using the Anopheles gambiae 1000 genomes (Ag1000g) data we identified a very recent selective sweep in mosquitoes from Uganda which localized to a cluster of cytochrome P450 genes. Further interrogation revealed a haplotype involving a trio of mutations, a nonsynonymous point mutation in Cyp6p4 (I236M), an upstream insertion of a partial Zanzibar-like transposable element (TE) and a duplication of the Cyp6aa1 gene. The mutations appear to have originated recently in An. gambiae from the Kenya-Uganda border, with stepwise replacement of the double-mutant (Zanzibar-like TE and Cyp6p4-236 M) with the triple-mutant haplotype (including Cyp6aa1 duplication), which has spread into the Democratic Republic of Congo and Tanzania. The triple-mutant haplotype is strongly associated with increased expression of genes able to metabolize pyrethroids and is strongly predictive of resistance to pyrethroids most notably deltamethrin. Importantly, there was increased mortality in mosquitoes carrying the triple-mutation when exposed to nets cotreated with the synergist piperonyl butoxide (PBO). Frequencies of the triple-mutant haplotype remain spatially variable within countries, suggesting an effective marker system to guide deployment decisions for limited supplies of PBO-pyrethroid cotreated LLINs across African countries.

Horizontal transmission of disseminated neoplasia in the widespread clam Macoma balthica from the Southern Baltic Sea.

Disseminated neoplasia (DN) is one of the most challenging and unrecognised diseases occurring in aquatic fauna. It has been diagnosed in four bivalve species from the Gulf of Gdansk (Southern Baltic Sea) with the highest frequency in Macoma balthica (formerly Limecola balthica), reaching up to 94% in some populations. The aetiology of DN in the Baltic Sea has not yet been identified, with earlier studies trying to link its occurrence with environmental pollution. Taking into account recent research providing evidence that DN is horizontally transmitted as clonal cells between individuals in some bivalve species, we aimed to test whether DN is a bivalve transmissible neoplasia (BTN) in the population of M. balthica from the Gulf of Gdansk highly affected with cancer. We examined mitochondrial cytochrome c oxidase I (mtCOI) and elongation factor 1α (EF1α) sequences of genomes obtained from haemolymph and tissues of neoplastic and healthy individuals. Sequence analysis resulted in detection of an independent transmissible cancer lineage occurring in four neoplastic clams that is not present in healthy animals. This study describes the first case of BTN in the clam M. balthica (MbaBTN), providing further insights for studies on this disease.

Amplification of pathogenic Leptospira infection with greater abundance and co-occurrence of rodent hosts across a counter-urbanizing landscape.

Land use change can elevate disease risk by creating conditions beneficial to species that carry zoonotic pathogens. Observations of concordant global trends in increased pathogen prevalence or disease incidence and landscape change have generated concerns that urbanization could increase transmission risk of some pathogens. Yet host-pathogen relationships underlying transmission risk have not been well characterized within cities, even where contact between humans and species capable of transmitting pathogens of concern occurs. We addressed this deficit by testing the hypothesis that areas in cities experiencing greater population loss and infrastructure decline (i.e., counter-urbanization) can support a greater diversity of host species and a larger and more diverse pool of pathogens. We did so by characterizing pathogenic Leptospira infection relative to rodent host richness and abundance across a mosaic of abandonment in post-Katrina New Orleans (Louisiana, USA). We found that Leptospira infection loads were highest in areas that harboured increased rodent species richness (which ranged from one to four rodent species detected). Areas with greater host co-occurrence also harboured a greater abundance of hosts, including the host species most likely to carry high infection loads, indicating that Leptospira infection can be amplified by increases in overall and relative host abundance. Evidence of shared infection among rodent host species indicates that cross-species transmission of Leptospira probably increases infection at sites with greater host richness. Additionally, evidence that rodent co-occurrence and abundance and Leptospira infection load parallel abandonment suggests that counter-urbanization can elevate zoonotic disease risk within cities, particularly in underserved communities that are burdened with disproportionate concentrations of derelict properties.

Unravelling virus community ecology in bats through the integration of metagenomics and community ecology.

The spillover of viruses from wildlife into agricultural animals or humans has profound socioeconomic and public health impact. Vampire bats, found throughout South America, feed directly on humans and other animals and are an important reservoir for zoonotic viruses, including rabies virus. This has resulted in considerable effort in understanding both the ecology of bat-borne viruses and the composition and associated correlates of the structure of entire virus communities in wildlife, particularly in the context of disease control interventions. In a From the Cover article in this issue of Molecular Ecology, Bergner et al. (2019) set out to reveal virus community dynamics in vampire bats by interrogating factors that affect the structure, diversity and richness of these communities. Due to the linkage of metagenomic sequence data with community ecology, this study represents an important advance in the field of virus ecology.

Sustained immune activation is associated with susceptibility to the amphibian chytrid fungus.

The disease chytridiomycosis caused by the fungus Bd has devastated amphibian populations worldwide. Functional genomic contributions to host susceptibility remain enigmatic and vary between species and populations. We conducted experimental Bd infections in Rana yavapaiensis, a species with intraspecific variation in chytridiomycosis susceptibility, to assess the skin and spleen transcriptomic response to infection over time. We predicted that increased immune gene expression would be associated with a positive disease outcome, but we instead found that surviving frogs had significantly reduced immune gene expression compared to susceptible frogs and to uninfected controls. MHC class IIß gene expression was also significantly higher in susceptible frogs compared to surviving frogs. Furthermore, susceptible frogs expressed a significantly larger number of distinct class IIß alleles, demonstrating a negative correlation between class IIß expression, functional diversity, and survival. Expression of the MHC class IIß locus previously associated with Bd disease outcomes was a significant predictor of Bd infection intensity at early infection stages but not at late infection stages, suggesting initial MHC-linked immune processes are important for ultimate disease outcomes. We infer through disease association and phylogenetic analysis that certain MHC variants are linked to the immune expression that was negatively associated with survival, and we hypothesize that frogs that did not express these alleles could better survive infections. Our study finds that MHC expression at early and late infection stages predicts Bd infection intensity, and suggests that generating a sustained immune response against Bd may be counterproductive for surviving chytridiomycosis in this partially susceptible species.

Two's company, three's a crowd: Exploring how host-parasite-microbiota interactions may influence disease susceptibility and conservation of wildlife.

A large body of research has demonstrated that host-associated microbiota-the archaeal, bacterial, fungal and viral communities residing on and inside organisms-are critical to host health (Cho & Blaser, 2012). Although the vast majority of these studies focus on humans or model organisms in laboratory settings (Pascoe, Hauffe, Marchesi, & Perkins, 2017), they nevertheless provide important conceptual evidence that the disruption of host-associated microbial communities (termed "dysbiosis") among wild animals may reduce host fitness and survival under natural environmental conditions. Among the myriad of environmental factors capable of inducing dysbiosis among wild animals (Trevelline, Fontaine, Hartup, & Kohl, 2019), parasitic infections represent a potentially potent, yet poorly understood, factor influencing microbial community dynamics and animal health. The study by DeCandia et al. in this issue of Molecular Ecology is a rare example of a host-parasite-microbiota interaction that impacts the health, survival and conservation of a threatened wild animal in its natural habitat. Using culture-independent techniques, DeCandia et al. found that the presence of an ectoparasitic mite (Otodectes cynotis) in the ear canal of the Santa Catalina Island fox (Urocyon littoralis catalinae) was associated with significantly reduced ear canal microbial diversity, with the opportunistic pathogen Staphylococcus pseudintermedius dominating the community. These findings suggest that parasite-induced inflammation may contribute to the formation of ceruminous gland tumours in this subspecies of Channel Island fox. As a rare example of a host-parasite-microbiota interaction that may mediate a lethal disease in a population of threatened animals, their study provides an excellent example of how aspects of disease ecology can be integrated into studies of host-associated microbiota to advance conservation science and practice.

Resource quality determines the evolution of resistance and its genetic basis.

Parasites impose strong selection on their hosts, but the level of any evolved resistance may be constrained by the availability of resources. However, studies identifying the genomic basis of such resource-mediated selection are rare, particularly in nonmodel organisms. Here, we investigated the role of nutrition in the evolution of resistance to a DNA virus (PiGV), and any associated trade-offs in a lepidopteran pest species (Plodia interpunctella). Through selection experiments and whole-genome resequencing, we identify genetic markers of resistance that vary between the nutritional environments during selection. We do not find consistent evolution of resistance in the presence of virus but rather see substantial variation among replicate populations. Resistance in a low-nutrition environment is negatively correlated with growth rate, consistent with an established trade-off between immunity and development, but this relationship is highly context dependent. Whole-genome resequencing of the host shows that resistance mechanisms are likely to be highly polygenic and although the underlying genetic architecture may differ between high and low-nutrition environments, similar mechanisms are commonly used. As a whole, our results emphasize the importance of the resource environment on influencing the evolution of resistance.

Comparative landscape genetics reveals differential effects of environment on host and pathogen genetic structure in Tasmanian devils (Sarcophilus harrisii) and their transmissible tumour.

Genetic structure in host species is often used to predict disease spread. However, host and pathogen genetic variation may be incongruent. Understanding landscape factors that have either concordant or divergent influence on host and pathogen genetic structure is crucial for wildlife disease management. Devil facial tumour disease (DFTD) was first observed in 1996 and has spread throughout almost the entire Tasmanian devil geographic range, causing dramatic population declines. Whereas DFTD is predominantly spread via biting among adults, devils typically disperse as juveniles, which experience low DFTD prevalence. Thus, we predicted little association between devil and tumour population structure and that environmental factors influencing gene flow differ between devils and tumours. We employed a comparative landscape genetics framework to test the influence of environmental factors on patterns of isolation by resistance (IBR) and isolation by environment (IBE) in devils and DFTD. Although we found evidence for broad-scale costructuring between devils and tumours, we found no relationship between host and tumour individual genetic distances. Further, the factors driving the spatial distribution of genetic variation differed for each. Devils exhibited a strong IBR pattern driven by major roads, with no evidence of IBE. By contrast, tumours showed little evidence for IBR and a weak IBE pattern with respect to elevation in one of two tumour clusters we identify herein. Our results warrant caution when inferring pathogen spread using host population genetic structure and suggest that reliance on environmental barriers to host connectivity may be ineffective for managing the spread of wildlife diseases. Our findings demonstrate the utility of comparative landscape genetics for identifying differential factors driving host dispersal and pathogen transmission.

In silico target network analysis of de novo-discovered, tick saliva-specific microRNAs reveals important combinatorial effects in their interference with vertebrate host physiology.

The hard tick Ixodes ricinus is an important disease vector whose salivary secretions mediate blood-feeding success on vertebrate hosts, including humans. Here we describe the expression profiles and downstream analysis of de novo-discovered microRNAs (miRNAs) expressed in I. ricinus salivary glands and saliva. Eleven tick-derived libraries were sequenced to produce 67,375,557 Illumina reads. De novo prediction yielded 67 bona fide miRNAs out of which 35 are currently not present in miRBase. We report for the first time the presence of microRNAs in tick saliva, obtaining furthermore molecular indicators that those might be of exosomal origin. Ten out of these microRNAs are at least 100 times more represented in saliva. For the four most expressed microRNAs from this subset, we analyzed their combinatorial effects upon their host transcriptome using a novel in silico target network approach. We show that only the inclusion of combinatorial effects reveals the functions in important pathways related to inflammation and pain sensing. A control set of highly abundant microRNAs in both saliva and salivary glands indicates no significant pathways and a far lower number of shared target genes. Therefore, the analysis of miRNAs from pure tick saliva strongly supports the hypothesis that tick saliva miRNAs can modulate vertebrate host homeostasis and represents the first direct evidence of tick miRNA-mediated regulation of vertebrate host gene expression at the tick-host interface. As such, the herein described miRNAs may support future drug discovery and development projects that will also experimentally question their predicted molecular targets in the vertebrate host.

A novel approach to wildlife transcriptomics provides evidence of disease-mediated differential expression and changes to the microbiome of amphibian populations.

Ranaviruses are responsible for a lethal, emerging infectious disease in amphibians and threaten their populations throughout the world. Despite this, little is known about how amphibian populations respond to ranaviral infection. In the United Kingdom, ranaviruses impact the common frog (Rana temporaria). Extensive public engagement in the study of ranaviruses in the UK has led to the formation of a unique system of field sites containing frog populations of known ranaviral disease history. Within this unique natural field system, we used RNA sequencing (RNA-Seq) to compare the gene expression profiles of R. temporaria populations with a history of ranaviral disease and those without. We have applied a RNA read-filtering protocol that incorporates Bloom filters, previously used in clinical settings, to limit the potential for contamination that comes with the use of RNA-Seq in nonlaboratory systems. We have identified a suite of 407 transcripts that are differentially expressed between populations of different ranaviral disease history. This suite contains genes with functions related to immunity, development, protein transport and olfactory reception among others. A large proportion of potential noncoding RNA transcripts present in our differentially expressed set provide first evidence of a possible role for long noncoding RNA (lncRNA) in amphibian response to viruses. Our read-filtering approach also removed significantly more bacterial reads from libraries generated from positive disease history populations. Subsequent analysis revealed these bacterial read sets to represent distinct communities of bacterial species, which is suggestive of an interaction between ranavirus and the host microbiome in the wild.

Poor peripheral blood stem cell mobilization affects long-term outcomes in multiple myeloma patients undergoing autologous stem cell transplantation.

BACKGROUND: Peripheral blood stem cell (PBSC) mobilization is routinely undertaken prior to autologous stem cell transplantation (ASCT) in patients with multiple myeloma (MM). A number of studies have identified risk factors for poor PBSC mobilization, however, little data exists to correlate mobilization with disease-specific outcomes in this patient population. Prospective work in MM has demonstrated similar outcomes in a homogenous patient population. METHODS: In this single institution analysis, we retrospectively studied the impact of poor PBSC mobilization on progression free survival (PFS) and OS in MM patients undergoing PBSC mobilization. Poor mobilizers are defined as patients that collected < 4 × 106 CD34+ cells/kg over maximum of 5 apheresis days, or those that required ≥2 mobilization cycles to achieve this target. RESULTS: We confirm that poor PBSC mobilization is significantly associated with a shortened PFS (P = .0012) and OS (P = .0005) compared with good mobilizers. Our univariate analysis also shows that independent risk factors for poor mobilization include male gender, higher ideal body weight, and a greater median number of lines of chemotherapy prior to PBSC mobilization. However, by multivariate analysis, only number of prior lines of chemotherapy remains significantly predictive of poor mobilization (Odds ratio 1.857, P = .0095). The use of high-dose G-CSF (> 10 mcg/kg/day) and/or plerixafor can significantly improve mobilization and ASCT chances in this population. DISCUSSION: These data indicate that poor mobilization can be predictable and is associated with more aggressive disease biology and worse outcomes, warranting intensive post-ASCT management.

Urban landscapes can change virus gene flow and evolution in a fragmentation-sensitive carnivore.

Urban expansion has widespread impacts on wildlife species globally, including the transmission and emergence of infectious diseases. However, there is almost no information about how urban landscapes shape transmission dynamics in wildlife. Using an innovative phylodynamic approach combining host and pathogen molecular data with landscape characteristics and host traits, we untangle the complex factors that drive transmission networks of feline immunodeficiency virus (FIV) in bobcats (Lynx rufus). We found that the urban landscape played a significant role in shaping FIV transmission. Even though bobcats were often trapped within the urban matrix, FIV transmission events were more likely to occur in areas with more natural habitat elements. Urban fragmentation also resulted in lower rates of pathogen evolution, possibly owing to a narrower range of host genotypes in the fragmented area. Combined, our findings show that urban landscapes can have impacts on a pathogen and its evolution in a carnivore living in one of the most fragmented and urban systems in North America. The analytical approach used here can be broadly applied to other host-pathogen systems, including humans.

Dissecting the dynamics of trans-generational immune priming.

The transfer of immunity from mother to offspring is a central way to endow the offspring with increased protection against pathogens. This phenomenon is not only found within the vertebrate domain: in some circumstances, invertebrate mothers can also give their offspring an immune kick-start, which is termed trans-generational immune priming (TGIP). TGIP has been uncovered for a number of invertebrate species, but it is not ubiquitously evident. The reasons for which are not known. In this issue of Molecular Ecology, Tate, Andolfatto, Demuth, and Graham () probe the molecular underpinnings of TGIP in concert with the temporal dynamics of the response in the red flour beetle, Tribolium castaneum, infected with the bacterium Bacillus thuringiensis (Figure ). They provide previously lacking evidence for the repeatability of TGIP, meaning that when averaged across several experiments, the offspring of mothers infected with heat-killed bacteria had better survival when they themselves were infected with live bacteria than offspring from mothers that had not encountered the bacterium. In a detailed temporal examination of the offspring's acute infection phase (zero to 24 hr after infection), Tate et al. () follow T. castaneum's gene regulation responses to infection while simultaneously documenting bacterial load. Such an approach gives considerable insight into the physiological processes that occur in primed offspring, and a first glance at a potential role for tolerance and effects on host metabolism that might even resemble trained immunity, which is a form of innate immune memory in vertebrates.

Population genetic structure and adaptation of malaria parasites on the edge of endemic distribution.

To determine whether the major human malaria parasite Plasmodium falciparum exhibits fragmented population structure or local adaptation at the northern limit of its African distribution where the dry Sahel zone meets the Sahara, samples were collected from diverse locations within Mauritania over a range of ~1000 km. Microsatellite genotypes were obtained for 203 clinical infection samples from eight locations, and Illumina paired-end sequences were obtained to yield high coverage genomewide single nucleotide polymorphism (SNP) data for 65 clinical infection samples from four locations. Most infections contained single parasite genotypes, reflecting low rates of transmission and superinfection locally, in contrast to the situation seen in population samples from countries further south. A minority of infections shared related or identical genotypes locally, indicating some repeated transmission of parasite clones without recombination. This caused some multilocus linkage disequilibrium and local divergence, but aside from the effect of repeated genotypes there was minimal differentiation between locations. Several chromosomal regions had elevated integrated haplotype scores (|iHS|) indicating recent selection, including those containing drug resistance genes. A genomewide FST scan comparison with previous sequence data from an area in West Africa with higher infection endemicity indicates that regional gene flow prevents genetic isolation, but revealed allele frequency differentiation at three drug resistance loci and an erythrocyte invasion ligand gene. Contrast of extended haplotype signatures revealed none to be unique to Mauritania. Discrete foci of infection on the edge of the Sahara are genetically highly connected to the wider continental parasite population, and local elimination would be difficult to achieve without very substantial reduction in malaria throughout the region.

Comparative study of host response to chytridiomycosis in a susceptible and a resistant toad species.

In the past century, recently emerged infectious diseases have become major drivers of species decline and extinction. The fungal disease chytridiomycosis has devastated many amphibian populations and exacerbated the amphibian conservation crisis. Biologists are beginning to understand what host traits contribute to disease susceptibility, but more work is needed to determine why some species succumb to chytridiomycosis while others do not. We conducted an integrative laboratory experiment to examine how two toad species respond to infection with the pathogen Batrachochytrium dendrobatidis in a controlled environment. We selected two toad species thought to differ in susceptibility - Bufo marinus (an invasive and putatively resistant species) and Bufo boreas (an endangered and putatively susceptible species). We measured infection intensity, body weight, histological changes and genomewide gene expression using a custom assay developed from transcriptome sequencing. Our results confirmed that the two species differ in susceptibility with the more susceptible species, B. boreas, showing higher infection intensities, loss in body weight, more dramatic histological changes and larger perturbations in gene expression. We found key differences in skin expression responses in multiple pathways including upregulation of skin integrity-related genes in the resistant B. marinus. Together, our results show intrinsic differences in host response between related species, which are likely to be important in explaining variation in response to a deadly emerging pathogen in wild populations. Our study also underscores the importance of understanding differences among host species to better predict disease outcomes and reveal generalities about host response to emerging infectious diseases of wildlife.

Response to Tibayrenc and Ayala: Reproductive clonality in protozoan pathogens--truth or artefact?

Tibayrenc and Ayala raised several interesting objections to an opinion piece we recently published in Molecular Ecology (Ramirez & Llewellyn 2014). Our piece examined the value of an alternative perspective to their theory of predominant clonal evolution (PCE) on the prevalence and importance of genetic exchange in parasitic protozoa. In particular, our aim was to establish whether population genetic signatures of clonality in parasites were representative of true biological/evolutionary processes or artefacts of inadequate tools and inappropriate or inadequate sampling. We address Tibayrenc and Ayala's criticisms and make a detailed response. In doing so, we deny the consensus that Tibayrenc and Ayala claim around their views and dismiss much of the language which Tibayrenc and Ayala have introduced to this debate as either arbitrary or inaccurate. We strongly reject accusations that we misunderstood and misquoted the work of others. We do not think the PCE provides a useful framework for understanding existing parasite population structures. Furthermore, on the eve of the population genomic era, we strongly urge Tibayrenc and Ayala to wait for the forthcoming wealth of high-resolution data before considering whether it is appropriate to refine or re-iterate their PCE hypothesis.

Heterozygote advantage at MHC DRB may influence response to infectious disease epizootics.

The effect of MHC polymorphism on individual fitness variation in the wild remains equivocal; however, much evidence suggests that heterozygote advantage is a major determinant. To understand the contribution of MHC polymorphism to individual disease resistance or susceptibility in natural populations, we investigated two MHC class II B loci, DQB and DRB, in the New Zealand sea lion (NZSL, Phocarctos hookeri). The NZSL is a threatened species which is unusually susceptible to death by bacterial infection at an early age; it has suffered three bacterial induced epizootics resulting in high mortality levels of young pups since 1997. The MHC DQB and DRB haplotypes of dead NZSL pups with known cause of death (bacteria, enteritis or trauma) were sequenced and reconstructed, compared to pups that survived beyond 2 months of age, and distinct MHC DRB allele frequency and genotype differences were identified. Two findings were striking: (i) one DRB allele was present only in dead pups, and (ii) one heterozygous DRB genotype, common in live pups, was absent from dead pups. These results are consistent with some functional relationship with these variants and suggest heterozygote advantage is operating at DRB. We found no association between heterozygosity and fitness at 17 microsatellite loci, indicating that general heterozygosity is not responsible for the effect on fitness detected here. This result may be a consequence of recurrent selection by multiple pathogen assault over recent years and highlights the importance of heterozygote advantage at MHC as a potential mechanism for fitness differences in wild populations.