Publications archive - Biodiversity
Key departmental publications, e.g. annual reports, budget papers and program guidelines are available in our online archive.
Much of the material listed on these archived web pages has been superseded, or served a particular purpose at a particular time. It may contain references to activities or policies that have no current application. Many archived documents may link to web pages that have moved or no longer exist, or may refer to other documents that are no longer available.
D. Hazell, R. Nott and M. F. Shannon
Department of the Environment and Heritage, August 2003
The methodology and effectiveness of the project research to:
A breeding colony of Bufo marinus has been successfully established at CSIRO Sustainable Ecosystems, Gungahlin. Breeding techniques were established with the help of Megan Brady, a recent graduate from the University of Queensland. However, the first attempt at establishing a colony was unsuccessful due to a parasitic infection. The colony was re-established and had supplied material for the generation of a cDNA library and RNA samples for micro array screening. Recently, a fungal infection has affected the colony but it is hoped that this can be eliminated and that the colony can continue to breed and supply appropriate materials for molecular biology work.
The fact that a there have been a number of difficulties in establishing an ongoing colony of cane toads raises several issues including the following:
Attempts to attenuate a ranavirus have been carried out in two ways:
This latter work required considerable background work, searching for 'non-essential genes' in a ranavirus using comparisons with other large DNA viruses, cloning these genes from the ranavirus and using recombination techniques to insert a foreign gene into the virus. To date a neomycin resistance gene has been inserted into a selected gene but experiments are underway to insert the neomycin resistance gene into 3 other areas of the virus genome. It was also necessary to find a viral promoter to 'drive' the expression of the reporter gene.
This has been successfully achieved. The insertion of foreign DNA into a ranavirus is a 'world first' for ranaviruses. Clearly, the ability to create a recombinant ranavirus has been demonstrated.
Both of the viruses generated above have been tested for attenuation in Litoria infrafrenata. This experiment involved bathing frogs in 105 TCID50/ml/frog. All the frogs infected with wild type virus died whereas none of the frogs infected with the 'attenuated' viruses died. This is a very promising result and needs to be repeated. It may be that the 'attenuated' viruses did not infect the animals as initial screens for virus using PCR were negative. This will need to be further tested. From this initial experiment it appears that the ranavirus may be able to be attenuated but more experiments will need to be carried out to confirm this.
This part of the project is progressing very well and if the initial result is repeated and the modified virus is in fact still infective then this can move to the next phase of research as outlined in plans for the next year and 3 years.
Micro array technology was used to identify genes involved in toad development. This is a very feasible approach and has already yielded some interesting and potentially useful results. Details of the technology are given in the Major Review Document for the project (November 2002). The technology has now been well established by the researchers.
To date, a number of genes specifically expressed in adult frogs and not in tadpoles have been identified. Some of these genes may be useful candidates for insertion into the viral vector to block metamorphosis. However, more needs to be done to screen for expression across different developmental stages to ensure that potential genes are expressed at limited stages of development. In addition, subtractive cDNA libraries need to be made in order to screen for genes that are less highly expressed. Overall, there has been excellent progress in this part of the project.
A previous publication has shown that injection of the adult beta-globin protein into Bullfrog tadpoles blocked development (Maniatis et al 1969, Science, 165, 67-69). The adult beta-globin gene was cloned from the cane toad, protein generated in vitro and injected into cane toad tadpoles as a test of the principal involved. The survival rate for those animals injected with beta-globin or mock-injected was identical. The reason for this was investigated and it was found that while the adult cane toad was no longer producing adult beta-globin, it maintained expression of the tadpole globin, thus allowing survival. Thus, while the globin gene may not be a good target for blocking metamorphosis in cane toads, this experiment shows that injecting an adult gene into a tadpole can alter gene expression. Whether this is through an immune-related mechanism or another mechanism is unknown.
The focus now needs to be on the identification of the most suitable gene(s) that can potentially inhibit metamorphosis.