Science of Cane Toad Invasion and Control. Proceedings of the Invasive Animals CRC/ CSIRO/Qld NRM&W Cane Toad Workshop 5-6 June 2006, Brisbane Edited by Kerryn Molloy & Wendy HendersonInvasive Animals Cooperative Research Centre Disclaimer: The views and opinions expressed in this report reflect those of the authors and do not necessarily reflect those of the Australian Government or the Invasive Animals Cooperative Research Centre. The material presented in this report is based on sources that are believed to be reliable. Whilst every care has been taken in the preparation of the report, the authors give no warranty that the said sources are correct and accept no responsibility for any resultant errors contained herein, any damages or loss, whatsoever, caused or suffered by any individual or corporation. 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Proceedings of the IA CRC/CSIRO/QLD NRM&W Cane Toad Workshop June 2006 Brisbane ISBN 0-9775707-2-X August 2006 Invasive Animals CRC Animals Invasive Cane Toad Workshop Control of cane toads by sterile male release and inherited sterility Michael Mahony and John Clulow. School of Environmental and Life Sciences, The Newcastle University, University Drive, Callaghan 2308 NSW. Phone 02 49216014 [email protected] Abstract We consider the possibility of biological control of the invasive cane toad by application of a sterile male release program. Our approach was initiated by two primary observations, 1) sterile male release programs (commonly called the sterile insect technique) have been successful in controlling a number of insect pests in area wide programs, and 2) sterility occurs naturally in frogs under certain circumstances indicating that there are methods to produce sterile cane toads. Sterile male release as a form of biological control is a species specific and environmentally non-polluting method of population control that relies on the mass rearing, sterilization and release of a large number of individuals with fitness equivalent to wild type animals. Released sterile males compete for and mate with wild females, reducing their reproductive output and, ultimately, if enough sterile males are released for a sufficient length of time, eradication of the population is achieved. Sterile adult frogs have been detected in nature and indicate that there are means by which sterility can be induced in the cane toad. Specific cases involve the occurrence of chromosomal variants in the number of haploid genome complements. The majority of animals are diploid (2n) having two of each chromosome per nuclei. In a small number of cases species have evolved by duplication of entire genomes (polyploidization) which persist because the level of duplication is even (usually 4n) and therefore meiosis is balanced. Where even numbered polyploid taxa (4n) interact with their diploid progenitors (2n) they form triploid hybrids (4n x 2n = 3n) which are sterile due to uneven chromosome numbers. In other situations rare sterile triploids occur within diploid species. Triploids grow and develop similar to diploids and display normal fitness, however the occurrence of uneven numbered chromosome complements means that meiosis is disrupted and the animal sterile. We have investigated the development of a genetically modified stock of cane toads that would be ideal for a male-only sterile release program. To that end we have developed a method for making sterile male via triploid and tetraploidy. We outline the methods that would be necessary to produce only sterile males for a control program. Background Population control by sterile male release fulfils the most basic requirements of any form of biological control – species specificity. This is because mating in most species is species specific to the males and females of that species. This means that if there is a mechanism by which the majority of males can be rendered sterile and the mating system is non-promiscuous (i.e., not polyandrous) then most matings will fail to produce offspring. An allied approach to the sterile male technique is the inherited sterile offspring technique, also referred to as delayed sterility, F1 sterility, and partial sterility. This approach relies on a mechanism that produces fertile males whose offspring, rather than the parent, are infertile such that they would provide Cane Toad Workshop a means of driving down the size of a population. These approaches operating over one and two generations rely on similar genetic constructs to the male sterility technique but are likely to have different demographic outcomes if applied in nature, i.e., delayed but also possibly amplified effects on population growth rates. The daughterless male technique is an example of this technique. Specificity is the great strength of the sterile male technique. Unlike an introduced disease (either natural or genetically engineered) or poison that must be tested against a wide variety of native animals to ensure safety and specificity, the sterile male approach can be implemented with little risk. In the case of application to cane toads there is no requirement to test whether the technique affects native frogs or other native organisms. This in turn eliminates a considerable amount of time and cost in the deployment of the biological control agent. There is the added advantage of biosecurity, because with the sterile male approach there is no pathogen that can escape or be transported from the locality or country of release. The release of sterile males to control pest populations in area-wide programs has been most effectively applied to insects (e.g. screwworm fly). The general approach is to swamp a population with sterile males so that the eggs of females are not fertilized. The approach relies on mass rearing and release of sterile males. The released males compete for mates with the wild males; any wild female mating with a released sterile male has no progeny and if a sufficient proportion of females produce no offspring the population declines. If sufficient sterile males can be released for a long enough period, the target population will be controlled or eradicated. The sterile male method works most effectively in organisms that are not highly mobile, where reproduction is restricted to single pair copulation (non-promiscuous), where reproductive output is high, and the life cycle relatively short (Alphey and Andreasen 2005). The method has not been applied to vertebrate pests because they often do not meet these criteria. However, the cane toad meets several of these criteria, and in these features is more akin to the insect models than other vertebrates. The cane toad has a high reproductive output (up to 40,000 eggs per mating), reproduction for the female, as far as is known, is restricted to one single partner per mating (sperm competition is not likely), and perhaps only one mating per season, and adults are relatively sedentary around established breeding sites. A male may achieve several matings in a season. The sterile male and sterile offspring techniques, if effective, control highly fecund species, such as the cane toad, by reducing reproductive potential. In 1994 we suggested investigation of this mode of biological control for the cane toad (CSIRO Cane Toad Taskforce). Our objectives were to: 1) investigate genetic means to produce sterile male cane toads that have libidos equal to or greater than normal males, and; 2) produce male toads that confer sterility on their offspring. Two primary observations led us to propose this approach. The first was the successful application of the sterile insect technique (SIT) in the control of insect pests. The second was the observation of sterile frogs in nature. In extremely rare events sterile frogs result from hybridisation between bisexual tetraploid and diploid species (Mahony and Robinson, 1980,Mahony 1986), and occasionally occur within diploid species (Green et al. 1984). Their occurrence indicated that sterility could be produced in male frogs and by extension that the technique of sterile male release was possible. We will briefly review the SIT technique and the observation of male sterility in frogs and then bring these themes together to develop the proposal that sterile male release provides a safe method for the biological control of cane toads. Invasive Animals Cooperative Research Centre Biological control and the sterile insect technique Successful, field applied SIT programs have been conducted against the New World screwworm fly (Cochliomyia hominivorax)(Knipling 1985, Lindquist 1993, Wyss 2000), the Mediterranean fruit fly (Ceratitis capitata)(Hendrichs et al. 1995), and the tsetse fly (Glossina spp.)(Masangi et al. 2000). SIT has also been applied to ten species of disease vector mosquitoes in the period between 1960 and 1990 (see summary table in Benedict and Robinson 2003) and is being used, has been used, or is being developed for as many as 18 agricultural pests, mostly dipterans but also ticks and weevils (see papers in Graham et al. 1985). In most of these cases releases were aimed at answering specific research questions and did not anticipate immediate population suppression, and consequently many were of limited scale of release and area of application.