Low Diversity of Major Histocompatibility Complex (MHC) Genes in Endangered Malayan Tapir (Tapirus indicus).

The Malayan tapir (Tapirus indicus) is listed as Endangered on the IUCN Red List due to multiple threats such as habitat loss and human disturbance that have led to its population decline. This decline increases the risk of inbreeding, which could result in the reduction of genome-wide genetic variation and negatively affect the gene responsible for immune response i.e., MHC gene. Class I and II MHC genes are responsible for encoding MHC molecules in the cells that recognise pathogenic peptides and present them to T-Cells on the cell surface for adaptive immune response. However, at present there is no study related to the MHC gene in Malayan tapir yet. This study characterises the MHC class I and II genes from seven individuals, investigates evidence of balancing selection and their relationships with homologous genes of other species. We identified at least one class I gene and four class II genes. Five sequences of alpha1 (α1) and four of alpha2 (α2) domains of class I alleles, two DRA, two DQA, three DRB and three DQB of class II alleles were isolated. α1 and α2 domains of class I and DRB domain of class II displayed evidence of selection with a higher rate of non-synonymous over synonymous substitutions. Within the DRB gene, 24 codons were found to be under selection where 10 are part of the codons forming the Antigen Binding Site. Genes sequences show species-specific monophyletic group formation except for class I and DRB genes with intersperse relationship in their phylogenetic trees which may indicate occurrence of trans-species polymorphism of allelic lineage. More studies using RNA samples are needed to identify the gene's level of expression.

Application of High-Throughput Sequencing (HTS) to Enhance the Well-Being of an Endangered Species (Malayan Tapir): Characterization of Gut Microbiome Using MG-RAST.

The Tapirus indicus, also known as Malayan tapir, has been listed as a rapidly declining animal species in the past decades, along with being declared and categorized as an endangered species by the International Union for Conservation of Nature (IUCN) 2016. This tapir species is geographically distributed across several countries in Southeast Asia such as Peninsular Malaysia, Indonesia (Sumatra), South Thailand, and Myanmar. Amongst these countries, the Peninsula Malaysia forest is recorded to contain the highest number of Malayan tapir population. Unfortunately, in the past decades, the population of Malayan tapirs has declined swiftly due to serious deforestation, habitat fragmentation, and heavy vehicle accidents during road crossings at forest routes. Concerned by this predicament, the Department of Wildlife and National Parks (DWNP) Peninsular Malaysia collaborated with a few local universities to conduct various studies aimed at increasing the population number of tapirs in Malaysia. Several studies were conducted with the aim of enhancing the well-being of tapirs in captivity. Veterinarians face problems when it comes to selecting healthy and suitable tapirs for breeding programs at conservation centers. Conventional molecular methods using high-throughput sequencing provides a solution in determining the health condition of Malayan tapirs using the Next-Generation Sequencing (NGS) technology. Unaware by most, gut microbiome plays an important role in determining the health condition of an organism by various aspects: (1) digestion control; (2) benefiting the immune system; and (3) playing a role as a "second brain." Commensal gut bacterial communities (microbiomes) are predicted to influence organism health and disease. Imbalance of unhealthy and healthy microbes in the gut may contribute to weight gain, high blood sugar, high cholesterol, and other disorders. In infancy, neonatal gut microbiomes are colonized with maternal and environmental flora, and mature toward a stable composition in two to three years. Interactions between the microorganism communities and the host allow for the establishment of microbiological roles. Identifying the core microbiome(s) are essential in the prediction of diseases and changes in environmental behavior of microorganisms. The dataset of 16S rRNA amplicon sequencing of Malayan tapir was deposited in the MG-RAST portal. Parameters such as quality control, taxonomic prediction (unknown and predicted), diversity (rarefaction), and diversity (alpha) were analyzed using sequencing approaches (Amplicon sequencing). Comparisons of parameters, according to the type of sequencing, showed significant differences, except for the prediction variable. In the Amplicon sequencing datasets, the parameters Rarefaction and Unknown had the highest correlation, while Alpha and Predicted had the lowest. Firmicutes, Bacteroidetes, Proteobacteria, Bacilli, and Bacteroidia were the most representative genera in Malayan tapir amplicon sequences, which indicated that most of the tapirs were healthy. However, continuous assessment to maintain the well-being of tapir for long term is still required. This chapter focuses on the introduction of 16S rRNA amplicon metagenomics in analyzing Malayan tapir gut microbiome dataset.

Social and Reproductive Behavior of Captive Malayan Tapirs' (Tapirus indicus): Interactions with Maternal Experience and Environmental Conditions.

Malayan tapirs are listed as endangered and are bred in captivity under governmental management. The success of captive breeding programs varies and the underlying causes are unclear. Here, we investigate how tapir reproduction is affected by previous breeding experience, enclosure type/size and visitor numbers so that appropriate steps can be taken to achieve self-sustaining captive populations. Data on social and reproductive behaviors were collected from six tapirs (three males, three females), from different breeding centers in Peninsular Malaysia for 18 weeks. Results revealed that social and reproductive behavior of both sexes was significantly influenced by social and environmental conditions. Larger enclosure size tended to increase social and reproductive behaviors, whereas high number of visitors reduced initial interaction between males and females. No specific breeding month was confirmed; however, reproductive behaviors were highest in April. Overall, this study contributes to a better understanding of the relationships between social and reproductive behaviors, and captive environments on Malayan tapirs. In future, frequency of sexual interactions should be monitored regularly to identify animals exhibiting below-average frequency and who might, therefore, be prone to reproductive difficulties.

Molecular sexing and preliminary assessment of population sex ratio of the endangered Malayan tapir (Tapirus indicus) in Peninsular Malaysia.

A molecular sexing method by polymerase chain reaction (PCR) amplification of a portion of the sex-determining region Y (SRY) and the zinc finger (ZF) gene, as well as six equine Y-chromosome-specific microsatellite markers, were tested in the Malayan tapir (Tapirus indicus). While the microsatellite markers did not yield any male-specific amplicons for sex-typing, the SRY/ZF marker system produced reliable molecular sexing results by accurately sex-typing 31 reference Malayan tapirs, using whole blood, dried blood spot (DBS), or tissue samples as materials for DNA extraction. The marker system was also tested on 16 faecal samples, and the results were in general consistent with the pre-determined sexes of the animals, despite some amplification failures. A preliminary estimation of wild Malayan tapir population sex ratio was estimated from the Wildlife Genomic Resource Bank (WGRB) database of the Malaysian Department of Wildlife and National Parks (PERHILITAN), zoos, and the Sungai Dusun Wildlife Conservation Centre (WCC), as well as from the results of molecular sexing 12 samples of unknown sex. The overall sex ratio favoured females, but the deviation from parity was statistically not significant when tested using the binomial test (p > 0.05), which may be due to reduced statistical power caused by small sample sizes.

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.

Heterozygosity-fitness correlations in a wild mammal population: accounting for parental and environmental effects.

HFCs (heterozygosity-fitness correlations) measure the direct relationship between an individual's genetic diversity and fitness. The effects of parental heterozygosity and the environment on HFCs are currently under-researched. We investigated these in a high-density U.K. population of European badgers (Meles meles), using a multimodel capture-mark-recapture framework and 35 microsatellite loci. We detected interannual variation in first-year, but not adult, survival probability. Adult females had higher annual survival probabilities than adult males. Cubs with more heterozygous fathers had higher first-year survival, but only in wetter summers; there was no relationship with individual or maternal heterozygosity. Moist soil conditions enhance badger food supply (earthworms), improving survival. In dryer years, higher indiscriminate mortality rates appear to mask differential heterozygosity-related survival effects. This paternal interaction was significant in the most supported model; however, the model-averaged estimate had a relative importance of 0.50 and overlapped zero slightly. First-year survival probabilities were not correlated with the inbreeding coefficient (f); however, small sample sizes limited the power to detect inbreeding depression. Correlations between individual heterozygosity and inbreeding were weak, in line with published meta-analyses showing that HFCs tend to be weak. We found support for general rather than local heterozygosity effects on first-year survival probability, and g2 indicated that our markers had power to detect inbreeding. We emphasize the importance of assessing how environmental stressors can influence the magnitude and direction of HFCs and of considering how parental genetic diversity can affect fitness-related traits, which could play an important role in the evolution of mate choice.

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.