In Vitro Hepatic Assessment of Cineole and Its Derivatives in Common Brushtail Possums (Trichosurus vulpecula) and Rodents

Folivore marsupials, such as brushtail possum (Trichosurus Vulpecula) and koala (Phascolarctos cinereus), can metabolise higher levels of dietary terpenes, such as cineole, that are toxic to eutherian mammals. While the highly efficient drug metabolising enzymes, cytochrome P450 3A (CYP3A) and phase II conjugating enzymes (UDP-glucuronosyltransferase, UGT), are involved in the metabolism of high levels of dietary terpenes, evidence for inhibitory actions on these enzymes by these terpenes is scant. Thus, this study investigated the effect of cineole and its derivatives on catalytic activities of hepatic CYP3A and UGT in mice, rats, and possums. Results showed that cineole (up to 50 µM) and its derivatives (up to 25 µM) did not significantly inhibit CYP3A and UGT activities in mice, rats, and possums (both in silico and in vitro). Interestingly, basal hepatic CYP3A catalytic activity in the possums was ~20% lower than that in rats and mice. In contrast, possums had ~2-fold higher UGT catalytic activity when compared to mice and rats. Thus, these basal enzymatic differences may be further exploited in future pest management strategies.

Testing the behaviour of Australian brushtail possums (Trichosurus vulpecula) to candidate semiochemicals from conspecifics’ urine

<p>The Australian brushtail possum (Trichosurus vulpecula) is a prolific introduced mammalian pest. Current pest control methods involve traps and poisons, but are proving to be ineffective long term solutions. As semiochemical lures are thought to be more attractive than food-based lures at low densities, researchers are attempting to utilize semiochemicals for kill traps. The objectives of this thesis were to create a testing protocol for use in a research colony of possums and use that protocol to test the attractiveness of semiochemicals found in possum urine that had been positively associated with possum attraction and interest in urine.. I hypothesised that there would be an observer effect present in the colony due to the nature of the colony husbandry protocols. There was no statistically significant observer effect present in the colony for eating, interaction, sitting, grooming or enclosure use (front or back). However, there was a trend (across the colony) that sitting and grooming increased in the presence of an observer. From these results, I designed an active presentation protocol for testing semiochemicals. I predicted that the most attractive compounds would belong to the acids and alcohol chemical groups. Overall there was no statistically significant difference between the semiochemicals. However, when taken individually there was clear evidence that five of the semiochemicals (belonging to the ketone, alcohol and phenol chemical groups) were more attractive than their control and the standard lure. It has been shown in this thesis that the attractiveness of semiochemicals can be tested in a small research colony. However, limits on colony size greatly reduce statistical power. For future studies, I would recommend increasing the sample size or instead developing field-based bioassays capable of being repeated across a greater number of sites, populations and therefore, individual possums.</p>

Testing the behaviour of Australian brushtail possums (Trichosurus vulpecula) to candidate semiochemicals from conspecifics’ urine

<p>The Australian brushtail possum (Trichosurus vulpecula) is a prolific introduced mammalian pest. Current pest control methods involve traps and poisons, but are proving to be ineffective long term solutions. As semiochemical lures are thought to be more attractive than food-based lures at low densities, researchers are attempting to utilize semiochemicals for kill traps. The objectives of this thesis were to create a testing protocol for use in a research colony of possums and use that protocol to test the attractiveness of semiochemicals found in possum urine that had been positively associated with possum attraction and interest in urine.. I hypothesised that there would be an observer effect present in the colony due to the nature of the colony husbandry protocols. There was no statistically significant observer effect present in the colony for eating, interaction, sitting, grooming or enclosure use (front or back). However, there was a trend (across the colony) that sitting and grooming increased in the presence of an observer. From these results, I designed an active presentation protocol for testing semiochemicals. I predicted that the most attractive compounds would belong to the acids and alcohol chemical groups. Overall there was no statistically significant difference between the semiochemicals. However, when taken individually there was clear evidence that five of the semiochemicals (belonging to the ketone, alcohol and phenol chemical groups) were more attractive than their control and the standard lure. It has been shown in this thesis that the attractiveness of semiochemicals can be tested in a small research colony. However, limits on colony size greatly reduce statistical power. For future studies, I would recommend increasing the sample size or instead developing field-based bioassays capable of being repeated across a greater number of sites, populations and therefore, individual possums.</p>

Towards the Creation of a Transgenic Possum Parasite

<p>The brushtail possum. Trichosurus vulpecula, is New Zealand's most serious vertebrate pest; possums destroy native flora and fauna and are vectors of bovine Tb. Conventional control is considered to be unsustainable and, in the long term, biological control is seen as the only solution to reducing possum numbers. The aim of this project is to contribute to the development of a self-disseminating vector that will spread a control molecule throughout the possum population reducing fecundity or increasing mortality. The possum-specific parasite Parastrongyloides trichosuri has considerable potential a-s such a vector. A protein from P. trichosuri specifically, was found to be antigenic in possums. The antibodies to this protein were purified from positive possum serum and used to detect the antigen on the surface of infective larvae but not in the excretory/secretory products of either larvae or adults. The protein was isolated from crude infective larvae and found to show homology to the heat-shock 70 family of proteins. Genomic DNA was extracted, an oligonucleotide probe made and a genomic library screened for the Hsp70 gene. Several positive clones were found and DNA isolated and sequenced from one such clone. Five kilo bases of unambiguous sequence was obtained in which was an open reading frame of 2 kb. Theoretical translation of this gave a protein of 64 amino acids with 80% homology to the Hsp70A protein of C. elegans. The region upstream of the ATG initiator codon was amplified and 1.3 kb of the putative promoter region was cloned into a vector containing the gfp:lacZ reporter genes. This construct was microinjected, first into C. elegans to demonstrate promoter function, and then into both tree-living and parasitic adults of P. trichosuri. Reporter gene expression was shown in the progeny of microinjected parasitic adults. RNA was made from infective P. trichosuri larvae, reverse transcribed and the coding sequence for the PtHsp70 protein cloned into an expression vector and expressed in E. coli, The recombinant protein pattern had a similar pattern of trypsin digestion products as the native protein, as shown by MALDI-TOF mass spectrometry, but it was immunologically distinct from the native protein. The culmination of this project was the generation of a transgenic P trichosuri, the first vertebrate endoparasitic nematode to be heritably transformed. This is a necessary step in the development of a self-disseminating vector to be used in the biocontrol of possums.</p>

Towards the Creation of a Transgenic Possum Parasite

<p>The brushtail possum. Trichosurus vulpecula, is New Zealand's most serious vertebrate pest; possums destroy native flora and fauna and are vectors of bovine Tb. Conventional control is considered to be unsustainable and, in the long term, biological control is seen as the only solution to reducing possum numbers. The aim of this project is to contribute to the development of a self-disseminating vector that will spread a control molecule throughout the possum population reducing fecundity or increasing mortality. The possum-specific parasite Parastrongyloides trichosuri has considerable potential a-s such a vector. A protein from P. trichosuri specifically, was found to be antigenic in possums. The antibodies to this protein were purified from positive possum serum and used to detect the antigen on the surface of infective larvae but not in the excretory/secretory products of either larvae or adults. The protein was isolated from crude infective larvae and found to show homology to the heat-shock 70 family of proteins. Genomic DNA was extracted, an oligonucleotide probe made and a genomic library screened for the Hsp70 gene. Several positive clones were found and DNA isolated and sequenced from one such clone. Five kilo bases of unambiguous sequence was obtained in which was an open reading frame of 2 kb. Theoretical translation of this gave a protein of 64 amino acids with 80% homology to the Hsp70A protein of C. elegans. The region upstream of the ATG initiator codon was amplified and 1.3 kb of the putative promoter region was cloned into a vector containing the gfp:lacZ reporter genes. This construct was microinjected, first into C. elegans to demonstrate promoter function, and then into both tree-living and parasitic adults of P. trichosuri. Reporter gene expression was shown in the progeny of microinjected parasitic adults. RNA was made from infective P. trichosuri larvae, reverse transcribed and the coding sequence for the PtHsp70 protein cloned into an expression vector and expressed in E. coli, The recombinant protein pattern had a similar pattern of trypsin digestion products as the native protein, as shown by MALDI-TOF mass spectrometry, but it was immunologically distinct from the native protein. The culmination of this project was the generation of a transgenic P trichosuri, the first vertebrate endoparasitic nematode to be heritably transformed. This is a necessary step in the development of a self-disseminating vector to be used in the biocontrol of possums.</p>

Ancient Viral Integrations in Marsupials: A Potential Antiviral Defence

Marsupial viruses are understudied compared to their eutherian mammal counterparts, although they may pose severe threats to vulnerable marsupial populations. Genomic viral integrations, termed endogenous viral elements (EVEs) could protect the host from infection. It is widely known past viral infections and EVEs play an active role in antiviral defence in invertebrates and plants. This study aimed to characterise actively transcribed EVEs in Australian marsupial species, because they may play an integral role in cellular defence against viruses. This study screened publicly available RNA sequencing datasets (n=35) and characterised 200 viral transcripts from thirteen Australian marsupial species. Of the 200 transcripts, 188 originated from either Bornaviridae, Filoviridae or Parvoviridae EVEs. The other 12 transcripts were from putative active infections from members of the Herpesviridae and Anelloviridae, and Hepadnaviridae. EVE transcripts (n=188) were mapped to marsupial genomes (where available, n=5/13) to identify the genomic insertion sites. Of the 188 transcripts, 117 mapped to 39 EVEs within the koala, bare-nosed wombat, tammar wallaby, brushtail possum and Tasmanian devil genomes. The remaining eight animals had no available genome (transcripts n=71). Every marsupial have Bornaviridae, Filoviridae and Parvoviridae EVEs, a trend widely observed in eutherian mammals. Whilst eutherian bornavirus EVEs are predominantly nucleoprotein-derived, marsupial bornavirus EVEs demonstrate a surprising replicase gene bias. We predicted these widely distributed EVEs were conserved within marsupials from ancient germline integrations, as many were over 65 million years old. One bornavirus replicase EVE, present in six marsupial genomes, was estimated to be 160 million years old, predating the American-Australian marsupial split. We considered transcription of these EVEs through small non-coding RNA as an ancient viral defence. Consistent with this, in koala small RNA sequence datasets we detected Bornaviridae replicase and Filoviridae nucleoprotein produced piRNA. These were enriched in testis tissue, suggesting they could protect marsupials from vertically transmitted viral integrations.

Has the introduction of two subspecies generated dispersal barriers among invasive possums in New Zealand?

The introduction of species into new environments provides the opportunity for the evolution of new forms through admixture and novel selection pressures. The common brushtail possum, Trichosurus vulpecula vulpecula from the Australian mainland and T.v.fuliginosus from Tasmania, were introduced multiple times to New Zealand from Australia to become one of New Zealand’s most significant pests. Although derived from two subspecies, possums in New Zealand are generally considered to be a single entity. In a previous analysis, we showed that possums in the Hawkes Bay region of New Zealand appeared to consist of at least two overlapping populations. Here, we extend that analysis using a genotype-by-sequencing approach to examine the origins and population structure of those possums and compare their genetic diversity to animals sampled from Australia. We identify two populations of each subspecies in Hawkes Bay and provide clear evidence of a contact zone between them in which a hybrid form is evident. Our analysis of private alleles shows higher rates of dispersal into the contact zone than away from it, suggesting that the contact zone functions as a sink (and hence as a barrier) between the two subspecies. Given the widespread and overlapping distribution of the two subspecies across both large islands in New Zealand, it is possible that many such contact zones exist. These results suggest an opportunity for a more targeted approach to controlling this pest by recognising sub-specific differences and identifying the contact zones that may form between them.

Are physiological and behavioural responses to stressors displayed concordantly by wild urban rodents?

Understanding wild animal responses to stressors underpins effective wildlife management. In order for responses to stressors to be correctly interpreted, it is critical that measurements are taken on wild animals using minimally invasive techniques. Studies investigating wild animal responses to stressors often measure either a single physiological or behavioural variable, but whether such responses are comparable and concordant remains uncertain. We investigated this question in a pilot study that measured responses of wild-caught urban brown and black rats (Rattus norvegicus, Rattus rattus) to fur-based olfactory cues from a predator, the domestic cat (Felis catus); a novel herbivore, the koala (Phascolarctos cinereus); and a familiar herbivore and competitor, the common brushtail possum (Trichosurus vulpecula). Physiological responses, measured by assaying faecal glucocorticoid metabolites, were compared to behavioural responses observed via video recordings. We found that physiological and behavioural responses to stressors were expressed concordantly. There was no sizeable physiological response observed, and the behavioural response when considered across the night was negligible. However, the behavioural response to the predator and competitor cues changed across the observation period, with activity increasing with increasing hours of exposure. Our results indicate that responses of wild rodents to cues are nuanced, with stress responses modulated by behaviour changes that vary over time according to the severity of the perceived threat as animals gather further information. If the physiological response alone had been assessed, this moderated response may not have been evident, and in terms of wildlife management, vital information would have been lost.