Also, during laboratory rearing, the males may be difficult to distinguish morphologically from the females. To solve this, RIDL (release of insects carrying a dominant lethal) technique is being developed. This involves insertion of dominant lethal genes that are repressible via certain chemical substances into insects. The usual lethal genetic system is designed to be female-specific. A pest population with this kind of system can be reared in the laboratory, where the growth medium used is supplied with the repressor of the lethal genetic system.
Upon reaching a desired population size, the repressor will be withdrawn from the medium, killing all females and leaving only males for SIT. These males are not sterile, but when they mate with wild females, any female offspring would inherit the lethal genetic system and die. Reduction of females would eventually lead to reproductive failure of the population. RIDL can make for easier separation of males from females during rearing. No sterilization is required; reducing expenses without reducing the reproductive ability of the released males. A system like this has been designed for Drosophila.
The system involves a lethal proapoptotic gene (hid) controlled by a tetracycline-activated transactivator (tTA).
The latter is then controlled by the female-specific enhancer from Drosophila yolk protein 1 (yp1).
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Experiments successfully show female specific lethality in flies inserted with the lethal genetic system. The technique is hypothesized to work with other insect pest species. INTRODUCTION Sterile insect technique (SIT) is a means of biological pest control. It involves mass rearing of pests in the laboratory. Males are then irradiated to sterilize them. These sterile males are then released into the environment.
They mate with wild females, producing no progeny. This reduces the population size of the pest; and with repeated releases, a pest population in a particular area can be eradicated. Males are the ones released as it is usually the female of the pest species that do damage (laying eggs on plants, acting as disease vectors).
SIT was developed by American entomologists Raymond Bushland and Edward Knipling during the 1950’s to combat screwworm infestation of North American livestock. During the 1930’s, the screwworm fly was decimating cattle herds along the American South.
The larvae of these flies enter open wounds in cattle and they start eating the flesh of the animal. Bushland and Knipling hypothesized that if they could disrupt the life cycle of screwworm by sterile male release, it would reduce the impact of pesticide use (or eliminate the need for chemical pesticides entirely).
Their research into this area was hampered by World War II, but was resumed in the early 1950’s. Shortly after resumption, their work succeeded in eradicating screwworm population in Sanibel Island, Florida. In 1954, they also succeeded in the island of Curacao, off the coast of Venezuela.
Up to the 1990’s, their technique worked in different American states, Mexico and North Africa. This initial SIT uses males sterilized by X-ray irradiation. Former US Secretary of Agriculture Orville Freeman has hailed SIT as “the greatest entomological achievement of the 20th century”, and for good reason. The application of SIT has resulted to near eradication in the pest species that have been targeted. Since no chemical pesticides are used, no harmful residues are left on the environment. The technique is species-specific; no other insects which may beneficial to an agricultural system are harmed.
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But SIT is not without its disadvantages. First, the technique is expensive. Data from the US Department of Agriculture show that SIT requires spending 73$/hectare. While cheaper than pesticide use (80$/hectare), it is still expensive. Underdeveloped countries which are in need of pest control may not afford SIT. Adding to the expense is the species-specificity of SIT. If an area has three pest species to be eradicated by SIT, three different SIT protocols must be developed. It might be cheaper to use a non-selective pesticide to wipe out all three species.
It is also very hard (in certain cases) to morphologically distinguish males from females in the laboratory. And lastly, sterilization via irradiation sometimes negatively affects the health of the reared males. When released into the environment, the sterile males cannot compete with wild males in getting mates. If this should happen, SIT would fail. To overcome these disadvantages, scientists are now looking to RIDL (release of insects carrying a dominant lethal).
This involves the molecular insertion of repressible dominant lethal genes into insects.
The insertion will kill the insect if the repressor (usually an antibiotic) is not present. The first step in RIDL is genetic engineering of the lethal genetic system. The system includes a lethal structural gene and a female-specific promoter inactivated by a repressor of choice. This gene construct will be inserted into the pest genome and mass laboratory rearing of insects (with selection) follows. Insects are grown in medium containing the repressor so they won’t die. Upon reaching a desired population size, the repressor is withdrawn from the diet of the insects.
All females would die, leaving only males for release. These males are not sterile but when they mate with wild females, any female offspring would inherit the lethal genetic system and die. RIDL is cheaper than conventional SIT. There is no need to physically separate males from females prior to release. No irradiation is needed, reducing expenses without affecting the health of the males. This ensures that released males have an equal chance of getting mates compared to wild females. A female-specific system has been developed for Drosophila (Heinrich and Scott, 2000).
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The genetic construct used contains the lethal hid (head involution defective) gene of Drosophila. The hid gene induces apoptosis (Grether etal. , 1995) in the cells where it is expressed. To control hid expression, they used a tetracycline-activated activator (tTA) which in turn is controlled by the female-specific and fat body-specific yolk protein 1 (yp1) enhancer. The inclusion of tTA causes hid to be expressed only when the antibiotic tetracycline is absent. The inclusion of yp1 ensures that only females are killed by hid. Two gene constructs were developed, tetO-hid and yp1-tTA.
Hid and yp1 were isolated from Drosophila genomic DNA and amplified with PCR. They were then inserted into readily available tetO and tTA transformation vectors. The developed constructs were then used to co-transform (via microinjection) Drosophila embryos. The transformed embryos were then grown and tested for two things: lethality only in the absence of tetracycline and female-specific lethality. RESULTS AND DISCUSSION Tetracycline-specific and female-specific lethality was confirmed by growing flies in both tetracycline mediums and normal mediums.
Complete viability was observed in tetracycline medium but in normal medium, fly populations were observed to be 99% males. This shows that the gene constructs severely reduce female viability. Specificity was further confirmed by including a reporter gene, lacz, in the constructs. Staining the lacz product (? -galactosidase) and subsequent microscopy show that only the females grown in normal medium express lacz. This shows that the promoters are working and that the constructs activate only in females in the absence of tetracycline.
