RESEARCH OBJECTIVES FOR 1996/97

3 BIOLOGICAL CONTROL OF POSSUMS

The introduced Australian brushtail possum (Trichosurus vulpecula) is a very serious pest in New Zealand. It causes extensive damage to indigenous forests and acts as the major vector of bovine Tb to cattle and deer.

Forest ecosystems are being drastically altered by the selective attention of possums which can eliminate favoured browse-species from whole tracts of forest.

The potential reduction in returns from agricultural production, should restrictions on access for meat and dairy products be imposed by our trading partners due to the presence of bovine Tb, could be up to $500 million annually.

Currently about $32 million is spent annually on poisoning and trapping possums. However, this level of expenditure is not sustainable and these methods of control are becoming less socially and politically acceptable.

Recently research into possum biocontrol as a long-term cost-effective solution to the possum problem was initiated. The expectation is, however, that control will be, for the foreseeable future, dependent on a number of technologies used in an integrated manner.

The research into biological control of possums, funded through Vote: Agriculture, owes its origin to a National Science Strategy Committee meeting held in October 1992 at which clear research priorities for biological control were established. Subsequently, MAF/DoC sought Government funding for a programme to address the priorities and Government agreed to fund the programme for three years, 1 July 1993 through 30 June 1996, at $2 million p.a. via Vote: Agriculture.

A further three years of funding totalling $6.25 million, voted in 1996, will support the programme through until July 1999.

Most of the research programmes follow one of two directions for possum biocontrol. The first involves targeting physiological pathways, such as the possum reproductive system, while the second is looking for potential pathogens.

Most of the physiological research is focusing on the reproductive system. This includes studies on gametes, fertilization, embryonic development, endocrine control of reproduction and on lactation. Essentially, the search is on for possum-specific physiological pathways which could be targeted for disruption and at this stage much of the research is fundamental.

Studies investigating possum parasites and diseases are being conducted in both New Zealand and Australia. These studies seek to identify parasites and diseases which could have an adverse effect on possums. There is no expectation that a single disease causing agent will solve the problem but it is believed that a number of different organisms could, collectively, adversely affect possums and afford some level of control - especially when added to other environmental stresses.

The surveys are also seeking candidate vector organisms which could, possibly, be genetically manipulated in order to enable them to spread disruptive genes throughout the possum population. These genes would be expressed as biochemicals which would interfere with possum reproduction or health.

Research directions are set, and progress monitored, by an informal group of five which takes into account the priorities established by the Control of Possums and Bovine Tb National Science Strategy Committee (NSSC). Two members of the group, Drs Paul Livingstone (AHB) and Peter Kettle (MAF) are members of the NSSC. The three other members are Drs Ken McNatty, Richard Sadleir and Hugh Davies who are contracted by MAF to provide a technical overview of the research into possum physiology, Tb vaccine development and possum diseases.

MAF funded research into control of Tb and possums accounts for 21% of the national investment in this area ($14.5m for 1996/97).

3.1Physiology & Reproduction

3.1.1

Objective Title:Control of growth in possums

Research Leader:Associate Professor R Gemmell

Institution:University of Queensland

Aim:To understand the role of the maternal and fetal thyroid glands in the growth and development of the brushtail possum.

Researchers consider the thyroid hormone, thyroxine may be important in the development of the young during early in the lactation period. The structure of organs including lung, kidney and liver will be examined to obtain developmental patterns and to make correlations with the plasma concentration of thyroxine and the number of receptors for the thyroid hormones.

3.1.2

Objective Title:Control of thermoregulation in possums

Research Leader:Associate Professor R Gemmell

Institution:University of Queensland

Aim:To understand the role of the maternal and fetal thyroid glands in the development of thermoregulation in the brushtail possum.

The young possum within the pouch displays a circadian rhythm with the highest temperatures during the day and the lowest in the early evening. Using methimazole to block the production of thyroid hormones researchers have found that treatment with methimazole does not delay the young possum obtaining the adult circadian rhythm for body temperature but affects the ability of the young to thermoregulate. Researchers will continue to examine to look at the effect of methimazole from 100-150 days post partum.

3.1.3

Objective Title:Disruption of growth and thermoregulation in possums

Research Leader:Associate Professor R Gemmell

Institution:University of Queensland

Aim:To examine the effect of methimazole on growth and thermoregulation of the young brushtail possum.

Methimazole inhibits the growth of the possum and its ability to thermoregulate. Researchers have found that the body weights of possums from day 100 post-partum to day 198 given methimazole were lower than the control possums after day 155 post-partum. Researchers will now examine the effect of blocking thyroxine from the mother to the young early in lactation.

3.1.4

Objective Title:Purification of prolactin and growth hormone (from possum pituitary glands)

Research Leader:Dr J Curlewis

Institution:University of Queensland

Researchers have successfully purified possum growth hormone (GH) and will continue with further purifications to provide raw materials for radioimmunoassay development. The researchers have not been successful with the purification of prolactin, probably because the prolactin content in possum pituitary glands is extremely low. This approach will be abandoned for prolactin and will proceed with recombinant production of the hormone.

3.1.5

Objective Title:Clone possum prolactin and growth hormone cDNAs

Research Leader:Dr J Curlewis

Institution:University of Queensland

Because of the difficulties encountered with purification of prolactin from possum pituitary glands, the researchers have decided to adopt a molecular cloning strategy with the eventual aim of producing the hormone by recombinant synthesis. In addition, this approach will provide the researchers with molecular probes that will be useful, for example in studies of the endocrine control of lactation or growth. Because the techniques will be the same for prolactin and growth hormone, it is appropriate to proceed with both hormones at the same time.

3.1.6

Objective Title:Produce possum prolactin by recombinant synthesis

Research Leader:Dr J Curlewis

Institution:University of Queensland

Once a full length possum prolactin cDNA clone has been obtained, the gene will be inserted into a bacterial expression vector with the aim of producing large quantities of prolactin. While the researchers would also like to produce recombinant possum growth hormone, it is unlikely that sufficient time will be available.

3.1.7

Objective Title:Develop prolactin and growth hormone radioimmunoassays

Research Leader:Dr J Curlewis

Institution:University of Queensland

The purified hormones produced under 3.1.5 & 3.1.6, growth hormone and prolactin, will be used to immunise animals. Once high titre antibodies are obtained, hormone assays will be developed and then tested to prove they can measure plasma hormone concentrations.

3.1.8

Objective Title:Effect of lactation and season on prolactin and growth hormone gene expression in possums

Research Leader:Dr J Curlewis

Institution:University of Queensland

Prolactin and growth hormone gene expression will be determined by Northern analysis using cDNA probes for possum prolactin and growth hormone labelled with 32P. Autoradiograms will be quantified by computer densiometry following which the membranes will be re-probed for beta-actin or similar genes so that hormone mRNA levels can be corrected.

3.1.9

Objective Title:Disruption of ovarian function

Research Leader:A/Professor C Sernia

Institution:University of Queensland

In eutherian mammals oxytocin (OT) and vasopressin (AVP) are involved in the regulation of ovarian function. For example, in sheep OT is luteolytic. OT and AVP receptors are present in the possum ovary and both AVP and both AVP and mesotocin (MT) are produced by the corpus luteum. Therefore it seems probable that these two peptides are important in possum ovarian function.

It has been found that the corpus luteum has only AVP receptors and that MT receptors are found in the ovarian stroma. Researchers will continue to examine ovaries at different stages of the oestrous cycle and measuring AVP/MT content and receptor localization. Researchers also plan to look at the effect of the intravenous infusion of the oxytocin antagonist OTA on ovarian function.

The elucidation of uterine receptor changes in gestation/parturition was a major aim in 1995/96 and will continue to be during 1996/97. In 1996/97 researchers will start infusions with OTA, the OT receptor antagonist, to examine its effectiveness in disrupting parturition.

3.1.10

Objective Title:Prevention of parturition

Research Leader:A/Professor C Sernia

Institution:University of Queensland

There are high concentrations of oxytocin receptors in the uterus and medialvagina of the possum (S5). Since oxytocin is important for normal parturition in eutherian mammals, these receptors in the possum could be targets antagonists which would disrupt parturition. The marsupial uterus is also responsive to vasopressin; these could be an additional target site. Although it is known that receptors are present in the possum uterus/vaginae, we do not know how they change in gestation/parturition and oestrous cycle.

3.1.11

Objective Title:Effect of oxytocin antagonist on male testicular function

Research Leader:A/Professor C Sernia

Institution:University of Queensland

In 1995/96 researchers performed preliminary experiments in which male possums were infused intraperitonealy with saline or OTA. Initial data show decreases in testicular size and weight and increases in adrenal and kidney weights. The result are sufficiently encouraging to warrant a complete study in 1996/97.

3.1.12

Objective Title:Cloning of possum specific genes

Research Leader: Dr K McNatty

Institution:AgResearch

Aim:To provide a basic understanding of possum gonadal development, focusing on the migration of primodial germ cells (PGC) into gonad.

Researchers will:

  • the possum PGC specific marker gene c-kit from possum ovarian cDNA;
  • the c-kit gene by DNA sequencing and analysis; and
  • the expression of stem cell factor c-kit and oct 3/4 genes during gonadal development and PGC migration in the possum.

3.1.13

Objective Title:Isolation of Possum Gonadotrophin - Releasing Hormone cDNA Clones

Research Leader: Dr K McNatty

Institution:AgResearch

Aim: To provide a basic understanding of the endocrine factors controlling possum reproduction.

Researchers will:

  • possum genomic DNA;
  • possum hypothalamic RNA and synthesize 1st strand cDNA;
  • both possum genomic DNA and possum hypothalamic 1st strand cDNA templates continue ongoing efforts to amplify the possum GnRH gene using nested PCR;
  • and sequence any amplification products likely to encode the possum GnRH gene;
  • PCR primers for the amplification of the possum FSH ß gene;
  • possum pituitary RNA and synthesize 1st strand cDNA;
  • possum pituitary 1st strand cDNA as template amplify the possum FSH ß gene; and
  • and sequence any amplification products likely to encode the possum FSH ß gene.

3.1.14

Objective Title:The female possum reproductive tract

Research Leader:Dr B McLeod

Institution:AgResearch

Aim:Characterisation of the biochemical and functional changes in the female reproductive tract associated with oestrus, mating and pregnancy in the possum.

Researchers will:

  • using light and electron microscopy, changes in mucous secretory cells of the vaginal cul-de-sac associated with oestrus and mating in the female possum;
  • stereology to characterise quantitative changes in the size, volume and ultrastructure of the epithelial cells, and associated with mucous secretion during oestrus and mating;
  • biochemical analyses to quantify changes in glycosaminoglycan concentrations of mucous and of secretory tissue of the vaginal cul-de-sac;
  • in vitro cell cultures of reproductive tract epithelium cells from possums;
  • in viro cell cycle analysis of possum tissues with in vitro cell cycle times of reproductive tract epithelial cells; and
  • an in vivo technique for identifying glycosaminoglycan synthesis in mucoid secretions.

3.1.15

Objective Title:Synchronised oestrus and controlled mating of possums

Research Leader:Dr B McLeod

Institution:AgResearch

Aim:Evaluation of methods of synchronising oestrus in female possums,including its predictability and the degree of synchrony achieved, by accurate determination of the time of ovulation with repeated laparoscopy.

Researchers will:

  • the time of ovulation and return-to-oestrus following pouch young removal at different times of the breeding season;
  • the effect of steroid hormone treatment on synchrony of ovulation;
  • the effect of manipulating plasma FSH concentrations on antral follicle development;
  • the efficacy of exogenous hormones to artificially induce synchronised ovulation of preovulatory follicles at known stages of development; and
  • social facilitation effects on oestrus and ovulation synchrony, by the introduction of vasectomised or entire male possums to previously isolated females.

3.1.16

Objective Title:Field trial of reproductive inhibition in possums

Research Leader:Mr D Ramsey

Institution:Landcare

Aim:Assess whether reproductive inhibition can effectively reduce possum population abundance in the wild by:

  • the effects of sterilisations in year 1 on possum population parameters;
  • year 2 sterilisations and removal of juvenile immigrants;
  • the initial prevalence of Leptospira balcanica at two sites where either 0% or 80% of females have been sterilised; and
  • the effects of sterilisation on home ranges, denning behaviour and interactions of radio-tagged possums at sites where either 0% or 80% of females have been sterilised.

3.1.17

Objective Title:Lactation inhibition in possums

Research Leader:Dr T Fletcher

Institution:Landcare

Aim:Identify the role of the corpus luteum and pituitary gland in preparation of the possum mammary gland for lactogenesis and suckling of the new pouch young to evaluate potential methods of inhibiting lactation, by:

  • trial of the effects of bromocriptine given during pregnancy on the initiation of lactation;
  • prolactin uptake by the mammary gland in pregnancy and early lactation;
  • the effects of corpus luteum removal during pregnancy on establishment of lactation in possums; and
  • the fatty acid composition of possum milk in early and late lactation.

3.1.18

Objective Title:Artificial breeding techniques for possums

Research Leaders:Dr T Fletcher, Mr A Grazier & Dr F Molinia

Institution:University of Newcastle and CRC for Conservation & Management of Marsupials

Work will focus on the continued improvement of the methods to hormonally manipulate female possums and to broaden the use of laparoscopic insemination by:

  • whether priming with higher levels of PMSG or with FSH will improve ovarian stimulation and subsequent superovulation following LH treatment;
  • estrogen content of follicular fluid from possums undergoing normal and stimulated reproductive cycles; and
  • and contrasting fertility intra vaginal and intrauterine insemination of epididymal, electro ejaculated or frozen-thawed spermatozoa.

3.1.19

Objective Title:Sperm transport in vivo in possums

Research Leaders:Dr F Molinia with Ms M Nickel, PhD Student

Institution:University of Newcastle and CRC for Conservation & Management of Marsupials

Intrauterine and intra vaginal laparoscopic AI will be used to improve an understanding of general sperm transport and capacitation by:

  • the numbers of sperm, including the proportion capacitated, that are recovered from the vagina, uterus and oviduct at various time intervals before, during and after ovulation; and
  • a range of specific markers for different regions of the sperm cell, that may detect changes associated with sperm capacitation in vivo.

3.1.20

Objective Title:In vitro capacitation of possum sperm

Research Leaders:Dr M Lin with Ms M Nickel PhD Student

Institution:University of Newcastle and CRC for Conservation & Management of Marsupials

In vitro capacitation will be attempted by:

  • if, or under what culture conditions electro ejaculated or epididymal possum spermatozoa will adopt thumbtack morphology after incubation in oviduct or uterus organ culture; and
  • the biochemistry of this in vitro culture if successful.

3.1.21

Objective Title:Passive immunity to pouch bacteria in possums

Research Leader: Professor D Cooper

Institution:Macquarie University

Researchers will determine whether antibodies to pouch bacteria occur in the serum, milk and other saliva of the female. Investigations will be conducted to establish whether there is transfer of these antibodies to the pouch young.

3.1.22

Objective Title:Maternal immune attack upon neonate in possums

Research Leader:Professor D Cooper

Institution:Macquarie University

Researchers aim to clone possum androgen receptor (AR). The ideal antigenic component of AR would be a region which is highly immunogenic and possum (marsupial) specific. Immunisation with an appropriate AR peptide coupled to a larger molecule will take place in 1996/97 in an attempt to sterilise male offspring. Consideration is also being given to immunising against androgens themselves.

3.1.23

Objective Title:Structure and function of the reproductive tract in the female possum

PhD fellowship:Janet Crawford

Institution:University of Otago/AgResearch

This PhD study is focused on the structure and function of the female reproductive tract associated with oestrus, mating and pregnancy in the possum. The PhD fellow will:

  • using light and electron microscopy, the cellular location of mucous secretory activity of the vaginal cul-de-sac, uterus and oviduct in the female possum;
  • stereology to quantitatively define changes in the size of secretory epithelial cells (volume) and organelles associated with secretion (volume and membrane surface area) during oestrus, mating and pregnancy, and correlate these with hormonal changes over the reproductive cycle; and
  • and characterise mucoid secretions.

3.1.24

Title:Casein genes of the possum

PhD fellowship:Melanie Ginger

Institution:University of Otago

In this study the PhD fellow will investigate the control of lactation in the possum by:

  • the caseins of possum milk;
  • any changes in the expression of caseins in possum milk during lactation;
  • molecular probes for the caseins of possum milk; and
  • critical regulatory features of the genes of the possum caseins.

3.1.25

Objective Title:Possum egg coat antibodies: coat free embryo survival

Research Leader:Dr L Selwood

Institution:La Trobe University

In some marsupials, egg coats are essential for the long term embryonic survival in vitro. Researchers have been studying egg coats in possums for several years to determine whether for the possums disrupting the egg coats could be a means of biocontrol. In this study egg coats from possums will be collected and antibodies will be raised to them. Researchers will complete the electron microscope analysis of development of coat-free embryos in vitro.

3.1.26

Objective Title:OCT3/4 marker for normal development in possums

Research Leader:Dr L Selwood

Institution:La Trobe University

This year researcher will complete the preparation of the OCT3/4 probes for identification of embryonic versus trophoblast lineages. Researchers will also complete the identification of the relationship between secretion of extracellular matrix (EMC) and cell-cell and cell-zona adhesion in normal separation of the future embryo and extra-embryonic membranes and trophoblastic lineages during blastocyte formation. These processes are unique to marsupials and monotremes and essential to normal embryo formation.

3.1.27

Objective Title:Isolation of genes involved in possum testis differentiation

Programme Leader: Professor J M Graves/Dr Diana Hill

Institution:La Trobe University, School of Genetics and Human Variation

The researchers will identify and clone the possum homologues of genes involved in testis differentiation, as candidate genes for a biological programme. The first step in determination of male development in all mammals is the differentiation of the indifferent gonad into a testis under the influence of the "testis determining factor", located on the Y chromosome. This factor has recently been identified as the Y-borne SRY gene in humans and mice. Other steps in testis determination and differentiation seem to be under the control of related genes SOX3 and SOX9, and are suppressed in some patients by an increased dose of the gene DAX1. Mutation or altered dosage of any of these genes in chromosomally XY mammals, or disruption of their action, should produce physically normal but sterile females.

3.1.28

Objective Title:Isolation of possum genes involved in spermatogenesis

Research Leader:Professor J M Graves/Dr Diana Hill

Institution:La Trobe University

The researchers will identify and clone the possum homologues of three Y-borne genes thought to be critical for mammalian spermatogenesis. Deletions of regions of the Y containing these genes in humans and mice produce normal but sterile males, showing specific blocks to meiosis, sperm maturation ormotility. Candidate genes cloned from mouse include ZFY, UBE1Y, and SMCY, and those cloned from the human Y are TSPY and YRRM1 (now renamed RBM1) and most recently DAZ. Of these, RBM1 appears to be the most promising, but DAZ is still a strong candidate. Thus the researchers have given the highest priority to cloning and characterizing RBM1 and DAZ. If the homologues of these genes function in possum spermatogenesis, disruption of either should produce normal but sterile males.

3.1.29

Objective Title:Characterization of possum genes involved in sex determination

Research Leader:Professor J M Graves

Institution:La Trobe University

Each of the cloned possum candidate testis determining genes SRY, SOX3, SOX9 and DAX1, will be characterized to the extent that its homology with known human and mouse genes is confirmed, and its uniqueness, structure, sequence, expression pattern and location in the possum genome is established. The function, and therefore the potential of each of the candidate genes for use in the possum biocontrol programme can then be judged.

3.1.30

Objective Title:Characterization of possum genes involved in spermatogenesis

Research Leader:Professor J M Graves

Institution:La Trobe University

Each of the cloned possum candidate spermatogenesis genes will be characterized to the extent that its homology with known human and mousegenes is confirmed, and its copy number, structure, sequence, location and expression pattern in possum is established. The conservation and Y-location of candidate spermatogenesis genes may provide the best guarantee of important conserved male-specific function because genes on the Y chromosome are subject to extreme variation and loss, the detection of a conserved sequence on the Y would provide strong evidence for an important male-specific function in man, mouse and possum. The function, and therefore the potential of each of the candidate genes for use in the possum biocontrol programme can then be judged.

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