Creating new evolutionary pathways through bioinvasion: the population genetics of brushtail possums in New Zealand.

Rapid increases in global trade and human movement have created novel mixtures of organisms bringing with them the potential to rapidly accelerate the evolution of new forms. The common brushtail possum (Trichosurus vulpecula), introduced into New Zealand from Australia in the 19th century, is one such species having been sourced from multiple populations in its native range. Here, we combine microsatellite DNA- and GIS-based spatial data to show that T. vulpecula originating from at least two different Australian locations exhibit a population structure that is commensurate with their introduction history and which cannot be explained by landscape features alone. Most importantly, we identify a hybrid zone between the two subspecies which appears to function as a barrier to dispersal. When combined with previous genetic, morphological and captive studies, our data suggest that assortative mating between the two subspecies may operate at a behavioural or species recognition level rather than through fertilization, genetic incompatibility or developmental inhibition. Nevertheless, hybridization between the two subspecies of possum clearly occurs, creating the opportunity for novel genetic combinations that would not occur in their natural ranges and which is especially likely given that multiple contact zones occur in New Zealand. This discovery has implications for wildlife management in New Zealand because multiple contact zones are likely to influence the dispersal patterns of possums and because differential susceptibility to baiting with sodium fluoroacetate between possums of different origins may promote novel genetic forms.

Chemical characterization of milk oligosaccharides of the common brushtail possum (Trichosurus vulpecula).

Structural characterizations of marsupial milk oligosaccharides have been performed in only three species: the tammar wallaby, the red kangaroo and the koala. To clarify the homology and heterogeneity of milk oligosaccharides among marsupials, 21 oligosaccharides of the milk carbohydrate fraction of the common brushtail possum were characterized in this study. Neutral and acidic oligosaccharides were separated from the carbohydrate fraction of mid-lactation milk and characterized by (1)H-nuclear magnetic resonance spectroscopy and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The structures of the 7 neutral oligosaccharides were Gal(ß1-3)Gal(ß1-4)Glc (3'-galactosyllactose), Gal(ß1-3)Gal(ß1-3)Gal(ß1-4)Glc (3", 3'-digalactosyllactose), Gal(ß1-3)Gal(ß1-3)Gal(ß1-3)Gal(ß1-4)Glc, Gal(ß1-3)Gal(ß1-3)Gal(ß1-3)Gal(ß1-3)Gal(ß1-4)Glc, Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (lacto-N-novopentaose I), Gal(ß1-3)Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (galactosyl lacto-N-novopentaose I), Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-3)Gal(ß1-4)Glc (galactosyl lacto-N-novopentaose II). The structures of the 14 acidic oligosaccharides detected were Neu5Ac(α2-3)Gal(ß1-3)Gal(ß1-4)Glc (sialyl 3'-galactosyllactose), Gal(ß1-3)(O-3-sulfate)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (lacto-N-novopentaose I sulfate a) Gal(ß1-3)[Gal(ß1-4)(O-3-sulfate)GlcNAc(ß1-6)]Gal(ß1-4)Glc (lacto-N-novopentaose I sulfate b), Neu5Ac(α2-3)Gal(ß1-3)Gal(ß1-3)Gal(ß1-4)Glc, Neu5Ac(α2-3)Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (sialyl lacto-N-novopentaose a), Gal(ß1-3)(-3-O-sulfate)Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc, Gal(ß1-3)Gal(ß1-3)[Gal(ß1-4)(-3-O-sulfate)GlcNAc(ß1-6)]Gal(ß1-4)Glc, Gal(ß1-3)[Neu5Ac(α2-6)Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (sialyl lacto-N-novopentaose b), Neu5Ac(α2-3)Gal(ß1-3)Gal(ß1-3)Gal(ß1-3)Gal(ß1-4)Glc, Gal(ß1-3)(-3-O-sulphate)Gal(ß1-3)Gal(ß1-3)Gal(ß1-3)Gal(ß1-4)Glc, Neu5Ac(α2-3)Gal(ß1-3)Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc, Gal(ß1-3)(-3-O-sulphate)Gal(ß1-3)Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc, Gal(ß1-3)Gal(ß1-3)Gal(ß1-3)[Gal(ß1-4)(-3-O-sulphate)GlcNAc(ß1-6)]Gal(ß1-4)Glc and Gal(ß1-3)Gal(ß1-3)[Neu5Ac(α2-6)Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (galactosyl sialyl lacto-N-novopentaose b). No fucosyl oligosaccharides were detected. Galactosyl lacto-N-novopentaose II, lacto-N-novopentaose I sulfate a, lacto-N-novopentaose I sulfate b and galactosyl sialyl lacto-N-novopentaose b are novel oligosaccharides. The results are compared with those of previous studies on marsupial milk oligosaccharides.

High variability in the MHC class II DA beta chain of the brushtail possum (Trichosurus vulpecula).

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.

Vkorc1 sequencing suggests anticoagulant resistance in rats in New Zealand.

BACKGROUND: Anticoagulant toxins are used globally to control rats. Resistance of Rattus species to these toxins now occurs in at least 18 countries in Europe, America and Asia. Resistance is often associated with single nucleotide polymorphisms (SNPs) in the Vkorc1 gene. This study gives a first overview of the distribution and frequency of Vkorc1 SNPs in rats in New Zealand. New Zealand is unusual in having no native rodents but three species of introduced Rattus - norvegicus Berk., rattus L. and exulans Peale. RESULTS: Sequence variants occurred in at least one species of rat at all 30 of the sites sampled. Three new SNPs were identified, one in kiore and two in ship rats. No SNPs previously associated with resistance were found in Norway rats or kiore, but seven ship rats were heterozygous and one homozygous for the A74T variant. Its resultant Tyr25Phe mutation has previously been associated with resistance to both first- and second-generation anticoagulants in ship rats in Spain. CONCLUSIONS: This is the first evidence of potential resistance to anticoagulant toxins in rats in New Zealand. Further testing using blood clotting response times in dosed rats is needed to confirm resistance potentially conferred by the Tyr25Phe mutation. Assessment is also needed of the potential of the other non-synonymous variants (Ala14Val, Ala26Val) recorded in this study to confer resistance to anticoagulant toxins. © 2016 Society of Chemical Industry.

Enhancing the effectiveness of biological control programs of invasive species through a more comprehensive pest management approach.

Invasive species are one of the greatest economic and ecological threats to agriculture and natural areas in the US and the world. Among the available management tools, biological control provides one of the most economical and long-term effective strategies for managing widespread and damaging invasive species populations of nearly all taxa. However, integrating biological control programs in a more complete integrated pest management approach that utilizes increased information and communication, post-release monitoring, adaptive management practices, long-term stewardship strategies, and new and innovative ecological and genetic technologies can greatly improve the effectiveness of biological control. In addition, expanding partnerships among relevant national, regional, and local agencies, as well as academic scientists and land managers, offers far greater opportunities for long-term success in the suppression of established invasive species. In this paper we direct our recommendations to federal agencies that oversee, fund, conduct research, and develop classical biological control programs for invasive species. By incorporating these recommendations into adaptive management strategies, private and public land managers will have far greater opportunities for long-term success in suppression of established invasive species. © 2016 Society of Chemical Industry.

Bird-repellent effects on bait efficacy for control of invasive mammal pests.

BACKGROUND: Repellents to reduce crop damage from birds and mammals have been investigated extensively, but their efficacy in reducing risk to non-target birds in aerial poisoning operations for control of mammal pests is less known. We assessed the impact on bait acceptability, palatability and kill efficacy for captive wild rats (Rattus rattus L.) and possums (Trichosurus vulpecula Kerr) of adding bird repellents (anthraquinone and d-pulegone) to baits used for their control in food choice trials. RESULTS: For possums, anthraquinone at 0.25% reduced acceptability and palatability but not the efficacy of poison baits, whereas d-pulegone at 0.17% had no significant effects. Rats showed little response to d-pulegone, but developed a marked aversion to prefeed baits containing anthraquinone at both 0.1 and 0.25%, such that almost no exposed rats ate poison baits and mortality was reduced significantly. The aversion induced by anthraquinone was generalised to the bait, as anthraquinone-exposed rats did not eat bait with only d-pulegone. CONCLUSION: Anthraquinone is not suitable for inclusion in bait for rat control at the concentrations tested, and also presents some risk to efficacy for possum control. D-pulegone would be suitable for inclusion in bait for possums and rats, but problems related to its volatility in bait manufacture and storage would need to be overcome. Further studies should focus on an alternative secondary repellent, or on establishing the maximum anthraquinone concentration that does not reduce efficacy for rats and testing whether or not that concentration is sufficient to repel native birds from baits reliably.

Quantifying the direct transfer costs of common brushtail possum dispersal using least-cost modelling: a combined cost-surface and accumulated-cost dispersal kernel approach.

Dispersal costs need to be quantified from empirical data and incorporated into dispersal models to improve our understanding of the dispersal process. We are interested in quantifying how landscape features affect the immediately incurred direct costs associated with the transfer of an organism from one location to another. We propose that least-cost modelling is one method that can be used to quantify direct transfer costs. By representing the landscape as a cost-surface, which describes the costs associated with traversing different landscape features, least-cost modelling is often applied to measure connectivity between locations in accumulated-cost units that are a combination of both the distance travelled and the costs traversed. However, we take an additional step by defining an accumulated-cost dispersal kernel, which describes the probability of dispersal in accumulated-cost units. This novel combination of cost-surface and accumulated-cost dispersal kernel enables the transfer stage of dispersal to incorporate the effects of landscape features by modifying the direction of dispersal based on the cost-surface and the distance of dispersal based on the accumulated-cost dispersal kernel. We apply this approach to the common brushtail possum (Trichosurus vulpecula) within the North Island of New Zealand, demonstrating how commonly collected empirical dispersal data can be used to calibrate a cost-surface and associated accumulated-cost dispersal kernel. Our results indicate that considerable improvements could be made to the modelling of the transfer stage of possum dispersal by using a cost-surface and associated accumulated-cost dispersal kernel instead of a more traditional straight-line distance based dispersal kernel. We envisage a variety of ways in which the information from this novel combination of a cost-surface and accumulated-cost dispersal kernel could be gainfully incorporated into existing dispersal models. This would enable more realistic modelling of the direct transfer costs associated with the dispersal process, without requiring existing dispersal models to be abandoned.

Vaccinia virus as a vaccine delivery system for marsupial wildlife.

Vaccines based on recombinant poxviruses have proved successful in controlling diseases such as rabies and plague in wild eutherian mammals. They have also been trialled experimentally as delivery agents for fertility-control vaccines in rodents and foxes. In some countries, marsupial mammals represent a wildlife disease reservoir or a threat to conservation values but, as yet there has been no bespoke study of efficacy or immunogenicity of a poxvirus-based vaccine delivery system in a marsupial. Here, we report a study of the potential for vaccination using vaccinia virus in the Australian brushtail possum Trichosurus vulpecula, an introduced pest species in New Zealand. Parent-strain vaccinia virus (Lister) infected 8/8 possums following delivery of virus to the oral cavity and outer nares surfaces (oronasal immunisation), and persisted in the mucosal epithelium around the palatine tonsils for up to 2 weeks post-exposure. A recombinant vaccinia virus construct (VV399, which expresses the Eg95 antigen of the hydatid disease parasite Echinococcus granulosus) was shown to infect 10/15 possums after a single-dose oronasal delivery and to also persist. Both parent vaccinia virus and the VV399 construct virus induced peripheral blood lymphocyte reactivity against viral antigens in possums, first apparent at 4 weeks post-exposure and still detectable at 4 months post-exposure. Serum antibody reactivity to Eg95 was recorded in 7/8 possums which received a single dose of the VV399 construct and 7/7 animals which received triple-dose delivery, with titre end-points in the latter case exceeding 1/4000 dilution. This study demonstrates that vaccinia virus will readily infect possums via a delivery means used to deploy wildlife vaccines, and in doing is capable of generating immune reactivity against viral and heterologous antigens. This highlights the future potential of recombinant vaccinia virus as a vaccine delivery system in marsupial wildlife.

Identification and evaluation of an infertility-associated ZP3 epitope from the marsupial brushtail possum (Trichosurus vulpecula).

Immunologically based fertility control vaccines against zona pellucida (ZP) proteins are being developed in New Zealand for biocontrol of the brushtail possum (Trichosurus vulpecula), an introduced Australian marsupial pest. We have shown that immunization of female possums with recombinant possum ZP3 protein (rZP3) reduced fertility by 79%. To enhance the specificity of possum immunocontraceptive vaccines, B-cell epitopes on possum ZP3 protein were mapped using sera of female possums immunized with possum rZP3 and subjected to a fertility trial. The amino acid sequence of the full-length possum ZP3 protein was used to synthesize a complete set of 83 (12-mer) biotinylated peptides each with an overlap of five amino acids with the neighboring peptides. The peptides were used in a modified enzyme-linked immunosorbent assay (ELISA) to identify continuous epitopes recognized by antibodies in the sera of possums immunized with possum rZP3. Sixteen epitopes were identified on the possum ZP3 protein. Comparison of the ELISA binding patterns of these peptides to antibodies in the individual sera with the fertility status of rZP3-immunized possums identified only one epitope (amino acids 156-172) to be associated with infertility. However, female possums immunized with this epitope showed no significant reduction in fertility. The possible reasons for the failure of this potential infertility epitope are discussed.

Humoral immune responses in brushtail possums (Trichosurus vulpecula) induced by bacterial ghosts expressing possum zona pellucida 3 protein.

The introduced common brushtail possum (Trichosurus vulpecula) is a major pest in New Zealand and immunocontraceptive vaccines are being developed for biocontrol of possum populations, with bacterial ghosts (BGs) being evaluated as a means of oral delivery. Recombinant BGs expressing possum zona pellucida 3 protein (ZP3) as an L' membrane-anchored protein (ZP3-L') or as an S-layer SbsA-fusion protein (MBP-SbsA-ZP3) were produced by the expression of the cloned bacteriophage phiX174 lysis gene E in E. coli NM522. The humoral immune responses of possums immunised with BGs expressing possum ZP3 were investigated following oral, intranasal/conjunctival, parenteral, and intraduodenal administration to evaluate the BG-ZP3 system for possum fertility control. Antibodies to possum ZP3 were detected in the serum, oviduct secretions, and follicular fluid of immunised animals. Intranasal/conjunctival immunisation elicited reliable antibody immune response in serum and at a key effector site, the ovarian follicular fluid. Intraduodenal administration of possum ZP3 BG vaccine as a priming immunisation elicited significant systemic immune responses, but oral immunisation did not, indicating that protection of BG vaccines from degradation by gastric acidity would enhance the effectiveness of orally delivered vaccines. The detection of antibodies at elevated levels at target sites in the reproductive tract following mucosal delivery demonstrates, for the first time, the potential of BGs as an effective system for vaccine delivery to wild animals, and intranasal/conjunctival immunisation as a promising means for delivery of immunocontraceptive vaccines to wild animals.

MHC haplotypes and response to immunocontraceptive vaccines in the brushtail possum.

The possum is a major invasive pest in New Zealand. One option for its control is the use of immunocontraceptive vaccines. Initial trials of vaccines have shown individual variation in response. The use of vaccines on wild populations could result in the evolution of a resistant population through selection for possums that remain fertile because of low or no response. Understanding the basis of this variation is therefore important. The major histocompatibility complex (MHC) is an important influence on the nature of immune responses. This study has investigated the relationship between MHC alleles and individual immune responses to immunocontraceptive vaccines comprising zona pellucida peptides. We identified MHC alleles and putative haplotypes, and compared these between individuals with measured responses to immunocontraceptive vaccines. Two haplotypes were found to associate significantly with differences in vaccine response. Possums that carried haplotype 6 showed reduced responsiveness to one vaccine, while possums that carried haplotype 9 showed increased responsiveness to a separate vaccine. The identification of MHC haplotypes associated with different responses to immunocontraceptive vaccines offers the opportunity to understand what factors trigger non-response and the persistence of fertility in some individuals, and may allow vaccines to be optimised to minimise non-responsiveness.

Novel alleles in classical major histocompatibility complex class II loci of the brushtail possum (Trichosurus vulpecula).

We have investigated the diversity of class II major histocompatibility complex (MHC) loci in the brushtail possum (Trichosurus vulpecula), an important marsupial pest species in New Zealand. Immunocontraceptive vaccines, a method of fertility control that employs the immune system to attack reproductive cells or proteins, are currently being researched as a means of population control for the possum. Variation has been observed in the immune response of individual possums to immunocontraceptives. If this variability is under genetic control, it could compromise vaccine efficacy through preferential selection of animals that fail to mount a significant immune response and remain fertile. The MHC is an important immune region for antigen presentation and as such may influence the response to immunocontraceptives. We used known marsupial MHC sequences to design polymerase chain reaction primers to screen for possum MHC loci. Alpha and beta chains from two class II families, DA and DB, were found in possums throughout New Zealand. Forty new class II MHC alleles were identified in the possum, and the levels of variability in the MHC of this marsupial appear to be comparable to those of eutherian species. Preliminary population surveys showed evidence of clustering/variability in the distribution of MHC alleles in geographically separate locations. The extensive variation demonstrated in possums reinforces the need for further research to assess the risk that such MHC variation poses for long-term immunocontraceptive vaccine efficacy.

Identification of novel major histocompatibility complex class I sequences in a marsupial, the brushtail possum (Trichosurus vulpecula).

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.

Bacterial ghosts as a delivery system for zona pellucida-2 fertility control vaccines for brushtail possums (Trichosurus vulpecula).

The introduced brushtail possum is a serious pest in New Zealand and there is much interest in the development of an immunocontraceptive vaccine for population control. Immunisation of female possums against recombinant possum zona pellucida protein-2 (ZP2) is known to reduce embryo production by 72-75% but successful development of fertility control will depend on a delivery system that is effective for field use. Bacterial ghost vaccine technology is a promising system to formulate a non-living vaccine for bait or aerosol delivery. The N-terminal (amino acid residues 41-316, ZP2N) and C-terminal (amino acid residues 308-636, ZP2C) regions of possum ZP2 were fused to maltose-binding protein and expressed in the periplasmic space of Escherichia coli NM522 bacterial ghosts. Female possums (n=20 per treatment group) were immunised with 20mg of either plain ghosts, ZP2N ghosts, or ZP2C ghosts in phosphate-buffered saline applied to the nostrils and eyes (nasal/conjunctival mucosa) at weeks 0, 2 and 4. Effects of immunisation on fertility were assessed following superovulation and artificial insemination. Both constructs evoked humoral (antibody) and cell-mediated immune responses in possums and significantly fewer eggs were fertilised in females immunised against ZP2C ghosts. Results in this study indicate that bacterial ghosts containing possum ZP antigens can reduce possum fertility when delivered by mucosal immunisation and offer a promising delivery system for fertility control of wild possum populations.

Immunogenicity and contraceptive potential of three infertility-relevant zona pellucida 2 epitopes in the marsupial brushtail possum (Trichosurus vulpecula).

In a previous study, three infertility-relevant epitopes of possum ZP2 (Pep12 (amino acids 111-125), Pep31 (amino acids 301-315), and Pep44 (amino acids 431-445)) were identified using sera from possums (Trichosurus vulpecula) immunized with recombinant possum zona pellucida 2 (ZP2) constructs, and a synthetic peptide library of possum ZP2 protein. In this study, the three peptides were conjugated to keyhole limpet hemocyanin and 300 mug of each conjugated peptide were administered subcutaneously to female possums (n = 20 per peptide) in complete Freund's adjuvant. Immunogen doses were repeated 3 and 6 weeks later using incomplete Freund's adjuvant. Control animals were immunized with either phosphate-buffered saline only (n = 10) or 300 mug keyhole limpet hemocyanin (n = 10), administered with the same adjuvants. Serum antibodies from animals immunized against these three epitopes bound to the corresponding possum ZP2 peptides, recombinant possum ZP2 protein constructs, and native zona. Possum fertility was assessed following superovulation and artificial insemination. Peptides Pep12 and Pep31 had no significant effects on fertility parameters (P > 0.05). However, animals immunized with Pep44 had lower egg fertilization rates (immunized 19.5% versus control 60.5%, P < 0.05) and produced significantly fewer embryos than control animals (immunized 0.5 embryos versus control 2.4 embryos, P < 0.05). The number of Pep44-immunized females that produced embryos was reduced by 64%. Identification and characterization of possum infertility-relevant epitopes on possum ZP2 protein will assist development of safe, humane, and possum-specific immunocontraceptive vaccines for controlling the introduced possums in New Zealand.

Plant-based immunocontraceptive control of wildlife--"potentials, limitations, and possums".

Possums (Trichosurus vulpecula), originally introduced from Australia, are spread over 90% of New Zealand and cause major economic and environmental damage. Immunocontraception has been suggested as a humane means to control them. Marsupial-specific reproductive antigens expressed at high levels in edible transgenic plant tissue might provide a safe, effective, and cheap oral delivery bait for immunocontraceptive control. As proof of concept, female possums vaccinated with immunocontraceptive antigens showed reduced fertility, and possums fed with potato-expressed heat labile toxin-B (LT-B) had mucosal and systemic immune responses to the antigen. This demonstrated that immunocontraception was effective in possums and that oral delivery in edible plant material might be possible. Nuclear transformation with reporter genes showed that transgenic carrot roots accumulate high levels of foreign protein in edible tissues, indicating their potential as a delivery vector. However, prior to attempts at large scale production, more effective immunocontraceptive antigen-adjuvant formulations are probably required before plant-based immunocontraception can become a major tool for immunocontraceptive control of overabundant vertebrate pests.