Sex and the single toad

What do the following items have in common: cricket bats, golf clubs and tomthumb firecrackers? If you are from Queensland and grew up in the 1960s, '70s and '80s, you would be thinking only one thing - summer nights of backyard cane toad culling.

Professor Peter Koopman, from the University of Queensland's Institute for Molecular Bioscience, is now developing a new way to get rid of this Aussie pest that is more permanent and far more likely to pass an ethics committee. He is making a "daughterless" strain of cane toads.

Recently named as a Queensland icon by the National Trust, Bufo marinus, or the humble cane toad, is certainly a success story as far as introduced pest species go. As most locals are aware, they were brought from Hawaii to the north Queensland canefields in the 1930s to control another pest, the sugar cane beetle.

The toads were meant to chow down on the beetles but promptly ignored them, mainly because they could not hop up to where the beetles actually lived at the top of the cane stalks. The toads then bred like that other well-known Aussie bush pest and headed south.

Their effectiveness as a pest just improved from there. Adaptable, happy to eat anything, especially dried pet food, toxic to predators and fiercely competitive for resources with native species such as frogs (most native frog tadpoles cannot live in the same water as the poisonous cane toad tadpoles), this amphibian invasion appears almost impossible to stop.

Cane toads have steadily moved both south and west over the past 70 years, reaching densities of 2000 toads per hectare in newly colonised areas. The current toad population Australia-wide is estimated at around 200 million individuals living in Queensland, the Northern Territory and parts of NSW.

Frogwatch co-ordinator Graeme Sawyer has warned that the species will appear in Western Australia within two to three years, depending on water sources. In fact, one of the few positives about the current drought crisis in eastern Australia is that it has probably slowed the inevitable arrival of Bufo marinus into the Murray-Darling river system to the south and Kakadu National Park to the west.

Sex to the rescue

So what does Professor Peter Koopman, internationally known researcher in the molecular genetics of sex determination and embryonic development, know about manipulating sex in toads to bring about their demise? Not much as it turns out ... yet. However, he has devised a plan is to genetically engineer toads in the laboratory to express a gene that causes female-to-male sex reversal, so the male offspring stay male and the female offspring reverse their sex.

The daughterless gene is passed on to the next generation in male-only progeny, and in this way the population eventually self-limits as the number of girl toads left to mate with dwindles. Cane toad numbers are essentially determined by the availability of females, each of which lays up to 35, 000 eggs at a time. Thus, limiting the females in a generation would significantly affect the total toad population.

Koopman began the laboratory phase of this ambitious and exciting new project in May 2006, although it is something he has thought about for some time. If anyone can succeed in this endeavour, this is the man. Koopman was part of the team that discovered the Y-chromosomal sex-determining gene SRY in the early 1990s and his group at the University of Queensland has been studying sex determination mechanisms for the last 15 or so years.

Most of these studies have been done in the mouse, which is a good model for human sexual development and disorders of sexual determination. By studying mice over a long period of time, Koopman knows an awful lot about the molecular pathways and genes governing sex determination and gonad development in mammals.

Over the years Koopman has explored ideas for using this knowledge in a more practical way, in the biotech or medical arena, "because most of my studies are basic research and are fairly esoteric, and that is where the cane toad project comes in", he says.

Female-free zone

The "daughterless" strategy is already underway in carp to control numbers in the Murray-Darling basin, initiated by one of Koopman's collaborators, Ron Thresher. Koopman was brought in early on in the carp project to advise on the genes that are important for sexual development to help select a good molecular target for the strategy.

"This was a powerful combination because I know the ins and outs of manipulating sex in transgenic animals, whereas Ron knows all about fish," Koopman says.

What it also did was remind Koopman that such a strategy, although not possible in mammals because sex-reversed mammals are sterile and therefore not able to propagate a daughterless gene through a population, was potentially applicable in other non-mammals.

Cane toads were of course the obvious choice - their public status as a true pest is legendary (even the stuff of movies) and it satisfied Koopman's long-held desire to solve a significant biological problem. So, with help from the newly established CRC for Pest Control, the daughterless toad project was kicked off in early 2006.

The first stage is the laboratory phase, to genetically engineer a cane toad carrying the daughterless gene. "We are really starting this from scratch," Koopman says.

The researchers know a lot about the genes that trigger sexual development in mammals and to some extent in other species like chicken and fish, but the cane toad is another kettle of ... well, amphibians.

The scientists first must establish how toads as a species decide if they are going to be male or female: whether it is chromosomal or gene-based, or perhaps involving environmental or social factors.

"The same cast of genes tend to be used for gonad development throughout evolution," Koopman says, assuming that the molecular pathways of sexual development are conserved between mammals and amphibian. Known genes will be cloned and sequenced the old-fashioned way (there is no genome sequence published for Bufo marinus), and then analysed for temporal and spatial expression patterns in male and female toads. Such information is necessary as a first step in selecting a molecular target for the daughterless strategy.

Koopman's team has already sequenced six genes of the toad that are known to be involved in mammalian sexual development, and are currently studying the expression of these genes. At the same time the lab is preparing to make transgenic animals, again learning as they go. Initially they will try the same techniques already established in the standard Xenopus amphibian model. They will first have to establish a breeding colony, working out all the optimal conditions for housing, feeding, and mating toads. "This is all very basic stuff so we have had to go right back to square one."

Having said that, Koopman is "very confident" of making a daughterless strain of cane toad in the lab within two to three years, based on what is known and the science involved. "The hard part really starts from there," he says.

The hard part

The second stage of the project will involve working quite closely with population modellers, ecologists, park rangers and so on to figure out whether this approach is actually going to work in the field and exactly how to execute it.

"This is a very clever, 'Smart State' strategy if you like and in some ways it is may be too clever in the sense that it is never going to work by itself," Koopman admits.

The team will have to produce a lot of transgenic toads - no one is even sure how many - then work out how, where, when and even how often to release them so that the transgenic animals have the best chance of competing with the blokey-er wild-type toads so the transgene is passed and starts affecting whole populations.

One of the things that Koopman and colleagues need to establish early on, preferably in the laboratory, is the fitness of the transgenic animals in terms of survival, whether they can mate competitively or even if they will be as attractive to females as the 'normal' toads.

Koopman stresses that the second, field stage of the strategy will involve a "multi-pronged attack on the cane toads" in conjunction with trapping, poisons or whatever works. Reducing the numbers of incumbent toads in a given area will give Koopman's team the best chance of attracting a mate and passing on the transgene.

"In the short term it is even going to look like we are making the problem worse and not better as we will be releasing toads into the environment, and the more we release the better chance we have that it is going to work, so it is a little bit counterintuitive."

Koopman emphasised that the daughterless strategy is non-toxic, species-specific and will not adversely affect other species such as native frogs or mammals that try to eat the toads. This is very important for the public acceptance of this strategy and also because of the devastation already caused by the cane toad.

"So, it is pretty ambitious and it is going to have to be done together with other strategies, and will involve a lot of work for a lot of people," he says. "Balanced against that though is the fact that if we don't try something it will continue to be an environmental disaster.

"The nice thing about this solution is that no cane toad actually has to die" ... except perhaps of loneliness and too much beer.