GM pink bollworms are a prelude to developing female-killing traits to control bollworm pests. Dr. Mae-Wan Ho and Prof. Joe Cummins explain the genetics and hazards of female killing systems.
The female-killing systems developed out of the sterile insect technique (SIT), which has been used in biological pest control since the 1950s. It involves mass rearing and release of insects made sterile with X-irradiation and other methods. Initially, sterile insects of both sexes were released, but sterile females were thought to be detrimental to pest control. One main reason is that a single male can mate with a large number of females, while each female will mate with only a few males. Hence, genetic sorting mechanisms (GSMs) were invented to kill the females. Until quite recently, all GSMs have involved radiation-induced X-Y translocation, ie moving of part of a normal X chromosome to the Y chromosome. The resultant Y chromosome then acts as a dominant selectable marker in a population in which all the X chromosomes carry a gene that is 'conditional lethal' in double dose. A conditional lethal is a gene that kills only under certain 'non-permissive' conditions, as for example, exposure to heat. Thus, when the population is heat shocked, the males (XY) survive, while the females (XX) die.
Recently, other conditional lethal systems have been considered for kill off females. Such systems could be introduced into any insect pest species with the help of genetic engineering. As a result, the GM insects could be released directly without pre-sterilisation. One method involves creating a strain that carries a conditional, sex-specific lethal gene, ie, a lethal gene that is expressed only in one sex under 'non-permissive' conditions. The design is such that the non-permissive condition is one that is normally found in nature, whereas the permissive condition (one that permits survival) depends on certain chemicals that could be added to the diet in the insect factory. Researchers have constructed such a system in Drosophila.
They make use of special transcription control elements (ie promoters) and transcription factors, proteins that bind to promoters to enhance transcription [1]. First, the transcription factor, tTa, a protein that interacts with tetracycline, is placed under the control of a promoter, Yp3, which is active in female larvae and adults, but not in males. Next, a reporter gene lacZ, coding for -galactosidase is placed under another promoter, the tetracycline responsive element, tRe. In the absence of tetracycline, tTa binds to tRe causing the reporter gene to become expressed. In the presence of tetracycline, however, the tetracycline binds to tTa, thereby preventing it from binding to tRe, and the reporter gene is not expressed. (Note: the convention is that genes are in italics, whereas the corresponding gene products are non-italics.)
Strains of flies homozygous for the constructs, Yp3-tTa and tRe-lacZ, respectively were crossed with each other. The resulting progeny were raised in the presence and absence of tetracycline in the culture medium. Adults were stained for -galactosidase activity. Females grown on normal diet without tetracycline stained strongly for the enzyme, whereas females raised on tetracycline and all males were negative.
To engineer the killing of females, a toxic gene product, Ras64B, was placed under the control of tRe; and a line with tRe-Ras64B was constructed. (Ras1 is a gene that codes for a protein that plays a key role in regulating transcription in the cell. Ras 64B is a defective mutant allele of Ras1. Ras homologues are oncogenes contributing to human cancer.) The tRe-Ras64B line was crossed with another line in which the tTa was placed under a nonspecific (constitutive) promoter. The progeny grown on tetracycline were viable and fertile. On normal medium, however, no progeny survived, ie, both males and females died. When the tRe-Ras64B line was crossed to the Yp3-tTa line, the male progeny but not the female survived in the absence of tetracycline.
Subsequently, the researchers constructed another line homozygous for both Yp3-tTa and tRe-Ras64B on the same chromosome, which was maintained on medium with tetracycline to inhibit the expression of Ras64B. When the flies were transferred to medium without tetracycline, no female progeny were recovered in a sample of more than 5000 males. The genetic system also worked with gene products that are specifically toxic to females.
The males were fertile when mated to other females. This is important for spreading the female-killing gene throughout the pest population. However, it would also spread the gene to related species. The potential also exists for horizontal transfer to unrelated species.
The proposal to engineer these genes into promiscuous transposon vectors will greatly multiply the risks of horizontal transfer to unrelated species, with potentially disastrous effects on biodiversity through the killing of females.