High variability in the MHC class II DA beta chain of the brushtail possum (<Emphasis Type="Italic">Trichosurus vulpecula</Emphasis>)

The diversity of class II major histocompatibility complex (MHC) loci was investigated in the brushtail possum, an important marsupial pest species in New Zealand. Immunocontraception, a form of fertility control that generates an autoimmune response, is being developed as a population control method for the possum. Because the immune response is partly under genetic control, an understanding of immunogenetics in possum will be crucial to the development of immunocontraceptive vaccines. MHC molecules are critical in the vertebrate immune response. Class II MHC molecules bind and present exogenously derived peptides to T lymphocytes and may be important in the presentation of immunocontraceptives. We used polymerase chain reaction primers designed to amplify the peptide binding region of possum class II MHC genes to isolate sequences from 49 animals. We have previously described 19 novel alleles from the DAB locus in the possum (Holland et al., Immunogenetics 60:449–460, 2008). Here, we report on another 11 novel alleles isolated from possum DAB, making this the most diverse marsupial locus described so far. This high level of diversity indicates that DAB is an important MHC locus in the possum and will need to be taken into account in the design of immunocontraceptive vaccines.     

Identification of novel major histocompatibility complex class I sequences in a marsupial,the brushtail possum (<Emphasis Type="Italic">Trichosurus vulpecula</Emphasis>)

The major histocompatibility complex (MHC) is an essential part of the vertebrate immune response. MHC genes may be classified as classical, non-classical or non-functional pseudogenes. We have investigated the diversity of class I MHC genes in the brushtail possum, a marsupial native to Australia and an introduced pest in New Zealand. The MHC of marsupials is poorly characterised compared to eutherian mammal species. Comparisons between marsupials and eutherians may enhance understanding of the evolution and functions of this important genetic region. We found a high level of diversity in possum class I MHC genes. Twenty novel sequences were identified using polymerase chain reaction (PCR) primers designed from existing marsupial class I MHC genes. Eleven of these sequences shared a high level of homology with the only previously identified possum MHC class I gene TrvuUB and appear to be alleles at a single locus. Another seven sequences are also similar to TrvuUB but have frame-shift mutations or stop codons early in their sequence, suggesting they are non-functional alleles of a pseudogene locus. The remaining sequences are highly divergent from other possum sequences and clusters with American marsupials in phylogenetic analysis, indicating they may have changed little since the separation of Australian and American marsupials.     

Infection of naïve, free-living brushtail possums (Trichosurus vulpecula) with the nematode parasite Parastrongyloides trichosuri and its subsequent spread

Despite the importance of spatial processes in host-parasite interactions, parasite dispersal has been the subject of few experimental studies. Introduced marsupial common brushtail possums (Trichosurus vulpecula) are a major environmental and agricultural pest in New Zealand. Parastrongyloides trichosuri, an intestinal rhabdiasoid nematode parasite specific to possums, is being evaluated as a self-disseminating delivery system for engineered fertility control vaccines. This study addressed whether an artificial infection could be established in a na?ve, free-living possum population, by measuring the post-release dynamics of possum-parasite interactions at the release site, and by following the spread of the parasite into surrounding possum populations. Infection was established efficiently by applying infective larvae to the skin of possums on a single occasion. All experimentally infected possums recaptured 3 weeks after infection had parasite eggs in their faeces. Over the subsequent 2.5 years, infection spread steadily over an area of about 6000 ha. Infection persisted at the original release site for the 3.5 years of the study and at a nearby site infected by natural spread for more than 3 years. Seasonal changes in faecal egg counts were similar at the two sites. The rapid establishment of the parasite and its spread provide additional support for its ongoing development as a vaccine delivery system.     

Quantifying Short-Term Foraging Movements in a Marsupial Pest to Improve Targeted Lethal Control and Disease Surveillance

In New Zealand, the introduced marsupial brushtail possum (Trichosurus vulpecula) is a pest species subject to control measures, primarily to limit its ability to transmit bovine tuberculosis (TB) to livestock and for conservation protection. To better define parameters for targeted possum control and TB surveillance, we here applied a novel approach to analyzing GPS data obtained from 44 possums fitted with radio-tracking collars, producing estimates of the animals’ short-term nocturnal foraging patterns based on 1-, 3- or 5-nights’ contiguous data. Studies were conducted within two semi-arid montane regions of New Zealand’s South Island High Country: these regions support low-density possum populations (<2 possums/ha) in which the animals’ home ranges are on average larger than in high-density populations in forested habitat. Possum foraging range width (FRW) estimates increased with increasing monitoring periods, from 150-200m based on a single night’s movement data to 300-400m based on 5 nights’ data. The largest average FRW estimates were recorded in winter and spring, and the smallest in summer. The results suggest that traps or poison-bait stations (for lethal control) or monitoring devices (for TB surveillance), set for > 3 consecutive nights at 150m interval spacings, would likely place >95% of the possums in this type of habitat at risk of encountering these devices, year-round. Modelling control efficacy against operational expenditure, based on these estimations, identified the relative cost-effectiveness of various strategies that could be applied to a typical aerial poisoning operation, to reduce the ongoing TB vectorial risk that possums pose in the High Country regions. These habitat-specific findings are likely to be more relevant than the conventional pest control and monitoring methodologies developed for possums in their more typical forested habitat.     

Spatial variation in impacts of brushtail possums on two Loranthaceous mistletoe species

Browsing by introduced brushtail possums is linked to major declines in mistletoe abundance in New Zealand, yet in some areas mistletoes persist, apparently unaffected by the presence of possums. To determine the cause of this spatial variation in impact I investigated the abundance and condition (crown dieback and extent of possum browse) of two mistletoes (Alepis flavida, Peraxilla tetrapetala) and abundance and diet of possums in two mountain beech (Nothofagus solandri var. cliffortioides) forests in the central-eastern South Island of New Zealand. Mistletoe is common and there are long-established uncontrolled possum populations in both forests. Mistletoes were abundant (216?1359 per hectare) and important in possum diet (41?59% of total diet), but possum density was low (c. 2 per hectare) in both areas. Possum impacts were slight with low browse frequencies and intensities over much of the study sites. However, impacts were significantly greater at a forest margin, where possum abundance was highest, and at a high-altitude site where mistletoe density was lowest. Mistletoe crown dieback was inversely proportional to intensity of possum browsing. These results suggest that the persistence of abundant mistletoe populations at these sites is due to mistletoe productivity matching or exceeding consumption by possums in these forests of low possum-carrying capacity, rather than low possum preference for the local mistletoe populations.     

Identifying eradication units in an invasive mammalian pest species

Eradication of invasive species is an important component for species conservation and ecosystem restoration. Success of eradications is dependent on knowledge of population connectivity in order to determine reinvasion pathways, and hence populations requiring simultaneous eradication (eradication units). The common brushtail possum (Trichosurus vulpecula) was introduced into New Zealand from Australia and Tasmania, and now occupies a wide range of habitats across the majority of New Zealand. Possums are one of the most destructive invaders within New Zealand, with extensive control operations occurring throughout the country. Understanding the population connectivity of possums on mainland New Zealand (North and South Islands) will enhance the success of planned eradications. We examined the genetic population structure of invasive possums to identify gene flow, thus reinvasion pathways, between seven populations around Dunedin and on the Otago Peninsula where eradication of possums is occurring. Genetic variation at 12 microsatellite loci was comparable between all sampled populations and exhibited a significant isolation by distance pattern. Bayesian clustering methods supported the existence of two population clusters, indicating the presence of a reinvasion pathway onto the Otago Peninsula from urban areas at the Southern end of the Peninsula. To avoid recolonisation, all possums on the Otago Peninsula should be eradicated simultaneously, with the implementation and ongoing maintenance and monitoring of an urban buffer zone. We recommend pre-eradication genetic analyses be adopted by all pest managers to define appropriate eradication units, thereby maximising eradication success and avoiding costly failures.     

Native plant/herbivore interactions as determinants of the ecological and evolutionary effects of invasive mammalian herbivores: the case of the common brushtail possum

In their native range, mammalian herbivores exist in a suite of direct and indirect ecological and evolutionary relationships with plant populations and communities. Outside their native range these herbivores become embedded in a multitude of new ecological and evolutionary interactions with native plant species in the new range. Sound knowledge of the plant/herbivore interactions in the herbivores’ native range provides an ideal framework to better understand their effects in the introduced range. The example of the common brushtail possum (Trichosurus vulpecula) and its introduction to New Zealand from Australia provides an excellent case study. In Australia, the common brushtail possum is a widespread generalist herbivore and it is thought that this generalist lifestyle has equipped the species well for successful colonisation of New Zealand. In Australia the brushtail possum has co-existed with highly chemically defended foliage since the Oligocene and recent papers have supported the role of possums as agents of selection on eucalypt defences. While the chemical profile of New Zealand foliage is comparatively poorly understood, possums do show clear selectivity between and within populations and some of these interactions may be mediated by the animals ability to ‘cope’ with PSMs, coupled with maintaining its generalist diet. While possums have had less time to effect evolutionary change in New Zealand species, their impacts on plant fitness have been well documented. However, further knowledge on variation and heritability of foliage traits driving possum preferences is needed to elucidate the ecological and evolutionary plant/possum interactions in the invasive range.     

Trans-species polymorphism and evidence of selection on class II MHC loci in tuco-tucos (Rodentia: Ctenomyidae)

Balancing selection acting over the evolutionary history of a lineage can result in the retention of alleles among species for longer than expected under neutral evolution. The associated pattern of trans-species polymorphism, in which similar or even identical alleles are shared among species, is often used to infer that balancing selection has occurred. The genes of the major histocompatibility complex (MHC) are thought to be subject to balancing selection that maintains alleles associated with response to specific pathogens. To explore the role of balancing selection in shaping MHC diversity in ctenomyid rodents, we examined allelic variability at the class II DRB and DQA loci in 18 species in the genus Ctenomys. Previous studies of four of these species had revealed significant within-population evidence of positive selection on MHC loci. The current study expands upon these analyses to (1) evaluate among-species evidence of positive selection and (2) explore the potential for balancing selection on MHC genes. Interspecific nucleotide sequence variation revealed significant evidence of positive selection on the DRB and DQA loci. At the same time, comparisons of phylogenetic trees for these MHC loci with a putative species tree based on mitochondrial sequence data revealed multiple examples of trans-specific polymorphism, including sharing of identical DRB and DQA alleles among distantly related species of Ctenomys. These findings suggest that MHC genes in these animals have historically been subject to balancing selection and yield new insights into the complex suite of forces shaping MHC diversity in free-living vertebrates.     

Low MHC class II diversity in the Tasmanian devil (Sarcophilus harrisii)

The largest remaining carnivorous marsupial, the Tasmanian devil (Sarcophilus harrisii), is currently under threat of extinction due to a fatal contagious cancer-devil facial tumour disease. Low major histocompatibility complex (MHC) class I diversity is believed to have contributed to the transmission of the tumour allograft through devil populations. Here, we report low MHC class II variability in this species, with DA β chain genes (Saha-DAB1, 2 and 3) exhibiting very limited diversity and the sole α chain gene (Saha-DAA) monomorphic. Three, six and three alleles were found at Saha-DAB1, 2 and 3, respectively, with a predominant allele found at each locus. Heterozygosity at these three loci is low in the eastern population and modestly higher in northwestern individuals. The results are indicative of a selective sweep likely due to an infectious disease resulting in the fixation of selectively favoured alleles and depletion of genetic diversity at devil class II loci. Several attempts were made to isolate the other marsupial classical class II gene family, namely, DB, resulting in only one DBB pseudogene being found. These findings further support the view that this species has a compromised capacity to respond to pathogen evolution, emerging infectious diseases and environmental changes.     

Endogenous type D retrovirus in a marsupial, the common brushtail possum (Trichosurus vulpecula)

We have sequenced and characterized an endogenous type D retrovirus, which we have named TvERV(D), from the genome of an Australian marsupial, the common brushtail possum (Trichosurus vulpecula). Intact TvERV(D) gag, pro, pol, and env open reading frames were detected in the possum genome. TvERV(D) was classified as a type D retrovirus, most closely related to those of Old World monkeys, New World monkeys, and mice, based on phylogenetic analyses and genetic organization. Approximately 30 TvERV(D) proviruses are present in the genomes of possums, as detected by Southern hybridization. However, variability in fragment patterns between possums was observed and suggests recent (or ongoing) retrotranspositional activity.     

Two patterns of variation among MHC class I loci in Tuatara (Sphenodon punctatus)

The genes of the major histocompatibility complex (MHC) are a central component of the immune system in vertebrates and have become important markers of functional, fitness-related genetic variation. We have investigated the evolutionary processes that generate diversity at MHC class I genes in a large population of an archaic reptile species, the tuatara (Sphenodon punctatus), found on Stephens Island, Cook Strait, New Zealand. We identified at least 2 highly polymorphic (UA type) loci and one locus (UZ) exhibiting low polymorphism. The UZ locus is characterized by low nucleotide diversity and weak balancing selection and may be either a nonclassical class I gene or a pseudogene. In contrast, the UA-type alleles have high nucleotide diversity and show evidence of balancing selection at putative peptide-binding sites. Twenty-one different UA-type genotypes were identified among 26 individuals, suggesting that the Stephens Island population has high levels of MHC class I variation. UA-type allelic diversity is generated by a mixture of point mutation and gene conversion. As has been found in birds and fish, gene conversion obscures the genealogical relationships among alleles and prevents the assignment of alleles to loci. Our results suggest that the molecular mechanisms that underpin MHC evolution in nonmammals make locus-specific amplification impossible in some species.     

Comprehensive characterization of MHC class II haplotypes in Mauritian cynomolgus macaques

There are currently no nonhuman primate models with fully defined major histocompatibility complex (MHC) class II genetics. We recently showed that six common MHC haplotypes account for essentially all MHC diversity in cynomolgus macaques (Macaca fascicularis) from the island of Mauritius. In this study, we employ complementary DNA cloning and sequencing to comprehensively characterize full length MHC class II alleles expressed at the Mafa-DPA, -DPB, -DQA, -DQB, -DRA, and -DRB loci on the six common haplotypes. We describe 34 full-length MHC class II alleles, 12 of which are completely novel. Polymorphism was evident at all six loci including DPA, a locus thought to be monomorphic in rhesus macaques. Similar to other Old World monkeys, Mauritian cynomolgus macaques (MCM) share MHC class II allelic lineages with humans at the DQ and DR loci, but not at the DP loci. Additionally, we identified extensive sharing of MHC class II alleles between MCM and other nonhuman primates. The characterization of these full-length-expressed MHC class II alleles will enable researchers to generate MHC class II transferent cell lines, tetramers, and other molecular reagents that can be used to explore CD4+ T lymphocyte responses in MCM.     

Characterization of MHC class II B polymorphism in bottlenecked New Zealand saddlebacks reveals low levels of genetic diversity

The major histocompatibility complex (MHC) is integral to the vertebrate adaptive immune system. Characterizing diversity at functional MHC genes is invaluable for elucidating patterns of adaptive variation in wild populations, and is particularly interesting in species of conservation concern, which may suffer from reduced genetic diversity and compromised disease resilience. Here, we use next generation sequencing to investigate MHC class II B (MHCIIB) diversity in two sister taxa of New Zealand birds: South Island saddleback (SIS), Philesturnus carunculatus, and North Island saddleback (NIS), Philesturnus rufusater. These two species represent a passerine family outside the more extensively studied Passerida infraorder, and both have experienced historic bottlenecks. We examined exon 2 sequence data from populations that represent the majority of genetic diversity remaining in each species. A high level of locus co-amplification was detected, with from 1 to 4 and 3 to 12 putative alleles per individual for South and North Island birds, respectively. We found strong evidence for historic balancing selection in peptide-binding regions of putative alleles, and we identified a cluster combining non-classical loci and pseudogene sequences from both species, although no sequences were shared between the species. Fewer total alleles and fewer alleles per bird in SIS may be a consequence of their more severe bottleneck history; however, overall nucleotide diversity was similar between the species. Our characterization of MHCIIB diversity in two closely related species of New Zealand saddlebacks provides an important step in understanding the mechanisms shaping MHC diversity in wild, bottlenecked populations.     

Factors influencing brushtail possum (

Damage by introduced brushtail possums (Trichosurus vulpecula) to Pinus radiata trees was assessed in 41 compartments of a commercial forestry plantation on the Coromandel Peninsula, New Zealand. All the trees assessed were less than 3 years old. Possum damage in the compartments was low (median prevalence 3.3%) but highly variable (range 0?30%). Eight of 37 measured habitat factors differed significantly (P < 0.05) between the sites with damaged and undamaged trees. The best predictor of mean damage was stand age, but this explained only 21% of the variation in damage among compartments. Including both stand age and New Zealand bracken (Pteridium esculentum) cover improved the model significantly and explained 36% of variation in damage. Damage was apparently unrelated to compartment size, distance from the compartment boundary, and possum den-site availability. Surprisingly, the relationship between browse damage and a trap-catch index of possum abundance was weakly negative (r_S = −0.53, P = 0.05). The dense understorey associated with young pine stands tends to increase possum damage to associated P. radiata trees, but the possums in such stands may be less mobile at ground level and thus less easily trapped. Assessment of stand age and understorey characteristics, together with visual inspection for early signs of damage, is likely to be more cost-effective than possum surveys for identifying forest compartments at risk from possum browse.     

Brodifacoum residues in possums (

The presence of brodifacoum residues in possum (Trichosurus vulpecula) livers following routine possum control was investigated. Possums were poisoned in six nature reserves in the Wellington Region, New Zealand, using cereal baits containing 20 mg kg(-1) brodifacoum dispensed from bait stations. Thirty-five surviving possums, and five dead possums were sampled from the reserves following poisoning, and their livers analysed for the presence of brodifacoum. The majority (83%) of samples contained brodifacoum at concentrations ranging from 0.007 mg kg(-1) to 6.2 mg kg(-1). The presence of significant quantities of brodifacoum in possum carcasses following poisoning operations creates potential for secondary and tertiary poisoning of non-target species.     

Assessing Movements of Brushtail Possums (Trichosurus vulpecula) in Relation to Depopulated Buffer Zones for the Management of Wildlife Tuberculosis in New Zealand

In New Zealand, managing the threat of bovine tuberculosis (TB) to livestock includes population reduction of potentially infectious wildlife, primarily the brushtail possum (Trichosurus vulpecula). Population control is often targeted on forested buffer zones adjacent to farmland, in order to limit movements of possums across the buffer and reduce the risk of disease transmission to livestock. To assess the effectiveness of buffers in protecting livestock we analysed GPS telemetry data from possums located in untreated forest adjacent to buffers, and used these data to characterise patterns of movement that could lead to possums reaching farmland during the season when most dispersal occurs. Analyses of movement data showed that the direction of dispersal by sub-adult and adult possums and the extent of long exploratory movements were not biased toward forest buffers, even though these provided vacant habitat as suitable for possums as untreated forest. Instead, dispersal and exploratory movements were uncommon even for sub-adult possums and such events typically lasted <10 days. Dispersing possums settled predominantly in river valleys. A simulation model was developed for the 3-6-month dispersal season; it demonstrated a probability of <0.001 that an infected possum, originating from a low-density population with low disease prevalence in untreated forest, would move across 3 km of recently controlled forest buffer to reach farmland. Our results indicate short-term reduction in the risk of TB transmission from possums to livestock in New Zealand by the use of depopulated buffer zones, while acknowledging that the threat of disease spread from untreated forest is likely to increase over time as possum population density and, potentially, TB prevalence among those possums, increase in the buffer zone.     

中华白海豚3 个MHC 基因座位遗传变异的初步分析

主要组织相容性复合体(Major histocompatibility complex,MHC) 基因是由一组紧密连锁的基因组成,是哺乳动物免疫系统中最重要的组成部分。本文选择3 个MHC 基因座位的第二外元,即:MHC-I 类基因和II 类基因的DRA 和DQB 座位,初步调查濒危物种中华白海豚的遗传变异。共鉴定了2 个DRA、2 个DQB 和7 MHC-I等位基因。DRA 座位遗传变异非常低,而DQB 和MHC-I 座位具有相对较高水平的遗传变异。并且,在DQB 和MHC-I 基因座位的假定的抗原结合位点(Antigen binding sites,ABS),非同义替代明显大于同义替代,提示平衡选择(Balancing selection)维持这两个座位的多态性,而在DRA 座位上,并没有检测到平衡选择。系统发生分析表明中华白海豚的MHC 等位基因没有聚在一起,而是和其他的物种聚在一起,符合MHC 跨种进化(Transspecies evolution)的模式。     

Absence of Ross River virus amongst Common brushtail possums (<Emphasis Type="Italic">Trichosurus vulpecula</Emphasis>) from metropolitan Sydney,Australia

Ross River virus (RRV) is a mosquito-transmitted Alphavirus emerging in urban centres throughout Australia. The Common brushtail possum (Trichosurus vulpecula), a native marsupial that has successfully adapted to human settlement, has been implicated as a maintenance reservoir for RRV. In the present study, RRV exposure was assessed amongst 72 urban-adapted possums from Northern Sydney and ten possums from a woodland area, remote from urbanisation. Serological screening was performed using an enzyme-linked immunosorbent assay to detect RRV antibodies in possum sera. Findings indicated that both possum populations from urban and woodland habitats were negative for the presence of RRV antibodies. Lack of exposure to RRV highlights that the host status of possums is contingent upon factors other than their abundance and proximity to human settlement. In view of the potential for climate change to favour transmission of mosquito-borne disease in Australia, identification of wildlife populations entirely absent of RRV may prove useful for monitoring the predicted spread of the virus.     

Contrasting patterns of selection acting on MHC class I and class II DRB genes in the Alpine marmot (Marmota marmota)

The major histocompatibility complex (MHC) genes code for proteins that play a critical role in the immune system response. The MHC genes are among the most polymorphic genes in vertebrates, presumably due to balancing selection. The two MHC classes appear to differ in the rate of evolution, but the reasons for this variation are not well understood. Here, we investigate the level of polymorphism and the evolution of sequences that code for the peptide-binding regions of MHC class I and class II DRB genes in the Alpine marmot (Marmota marmota). We found evidence for four expressed MHC class I loci and two expressed MHC class II loci. MHC genes in marmots were characterized by low polymorphism, as one to eight alleles per putative locus were detected in 38 individuals from three French Alps populations. The generally limited degree of polymorphism, which was more pronounced in class I genes, is likely due to bottleneck the populations undergone. Additionally, gene duplication within each class might have compensated for the loss of polymorphism at particular loci. The two gene classes showed different patterns of evolution. The most polymorphic of the putative loci, Mama-DRB1, showed clear evidence of historical positive selection for amino acid replacements. However, no signal of positive selection was evident in the MHC class I genes. These contrasting patterns of sequence evolution may reflect differences in selection pressures acting on class I and class II genes.     

Independent evolution of functional MHC class II DRB genes in New World bat species

Genes of the major histocompatibility complex (MHC) play a pivotal role in the vertebrate immune system and are attractive markers for functional, fitness-related, genetic variation. Although bats (Chiroptera) represent the second largest mammalian order and are prone to various emerging infectious diseases, little is known about MHC evolution in bats. In the present study, we examined expressed MHC class II DRB sequences (exons 1 to 4) of New World bat species, Saccopteryx bilineata, Carollia perspicillata, Noctilio albiventris and Noctilio leporinus (only exon 2). We found a wide range of copy number variation of DRB loci with one locus detected in the genus Noctilio and up to ten functional loci observed in S. bilineata. Sequence variation between alleles of the same taxa was high with evidence for positive selection. We found statistical support for recombination or gene conversion events among sequences within the same but not between bat species. Phylogenetic relationships among DRB alleles provided strong evidence for independent evolution of the functional MHC class II DRB genes in the three investigated species, either by recent gene duplication, or homogenization of duplicated loci by frequent gene conversion events. Phylogenetic analysis of all available chiropteran DRB exon 2 sequences confirmed their monophyletic origin within families, but revealed a possible trans-species mode of evolution pattern in congeneric bat species, e.g. within the genera Noctilio and Myotis. This is the first study investigating phylogenetic relationships of MHC genes within bats and therefore contributes to a better understanding of MHC evolution in one of the most dominant mammalian order.