Sessional Committee on Environment and Sustainable Development Written Submissions Received Volume 2 Issues associated with the progressive entry into the Northern Territory of Cane Toads October 2003
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Written Submissions Dr Mahony Volume 2 Cane Toad Inquiry Report 173 This effectively produces a diploid egg, with the incorporation of the sperm nucleus the zygote will be triploid. Shock treatment usually involves sudden temperature or pressure change (see Nishioka & Ueda, 1983, and reference therein). We have successfully used cold temperature shock to produce triploid cane toads. This method is not optimal and the use of pressure treatment as used on a large scale in the aquaculture industry may be most effective. Mature triploids have been obtained in numerous urodeles and anurans (see Kashiwagi, 1993, for a review). For example in three species of Hyla and four species of Rana, triploids were obtained by exposing eggs to low temperatures of 0 - 2oC for two hours, 20 minutes after insemination (Nishioka, 1972; Nishioka & Ueda, 1983; Kawamura, 1951a,b; Kawamura, Nishioka & Okumoto, 1983; Kashiwagi, 1993). Standard practice in the production of triploid salmon and trout is to use of hydrostatic pressure for a period of two hours, thirty minutes after artificial fertilisation (Purdom, 1983), but heat shock has also been successfully applied (Johnstone, 1985; purdom, Thompson & Lou, 1985). Using cold shock on artificially inseminated eggs of Rana rugosa, Kashiwagi (1993) produced 82% triploid offspring. The majority of these were raised to sexual maturity. No significant differences were observed between the triploids and control diploids in development and growth rate. All the triploids were male or hermaphrodites, which transformed into males, indicating that in this species the male is the heterogametic sex. IVF using sperm from eleven of these triploid males with eggs (2272) from normal diploid females resulted in 6% forming tadpoles, of which only one reached metamorphosis, i.e., they are effectively sterile. Chromosome counts revealed that the majority of the tadpoles were aneuploid. Female heterogamety has been reported in two species of the genus Bufo (B. bufo and B. japonicus)(Ponse, 1942; Muto, 1952). Muto (1952) found that the majority of triploids raised from cold-treated or heat-treated eggs were females. It is highly probably that in these species triploid females are ZZW or ZWW, and males ZZZ. If this is also the case in B. marinus it will be necessary to produce a stock of sex-reversed males (genetically male ZZ, but phenotypically female). This can be achieved by surgical removal of the testes in the sexually mature male toad. The Bidders organ which is located in the anterior part of the testes is the incompletely involuted cortex of the embryonic gonad. It has been compared to the rudimentary ovary. Furthermore, the Mullerian duct has been conserved. When the testis is removed, the Bidder's organ develops into a functional ovary and the Mullerian duct enlarges. Injection with female hormones would be expected to enhance the success of such animals. Step four Monitoring growth and development of the triploids. This step has not been conducted in our laboratory. Growth would need to be compared with the developmental stages of control diploids. Chromosome counts could be made on small sections of epithelium taken from the tail. The extracts are soaked in a hypotonic, colchicine solution for three hours and then squashed is acetic acid orcein stain. Measurements of erythrocytes and specific staining of the nucleolus organiser region in the nuclei can be used to determine the ploidy of individuals (Mahony & Robinson, 1981). Histology would follow standard procedures.