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Sterile insect technique

Sterile Insect Technique (SIT)

Definition and principles; The Sterile Insect Technique (SIT) (or sterile male technique) is an autocidal insect control method. It is a species-specific genetic “birth control” method that is inversely density-dependent, becoming more effective as the size of the target population decreases.

Dominant lethal mutations: Ionizing radiation or chemosterilants induce chromosomal aberrations and abnormalities in subsequent nuclear divisions. The exposed germ cells produce viable sperms or ova with dominant lethal mutations. Timing and dose of the treatment determines the relative effect on both germ and somatic cells. Timing and dose can be controlled in order to maximize the effect without affecting sperm motility, concomitantly minimizing the effect on survival, motility, mating ability and competitiveness of the treated insects. The desired outcome is an insect that searches and finds mates, copulates and transfers sperm successfully. As the sperm is defective, such fertilized eggs fail to develop.

The fundamentals of SIT: To successfully employ SIT, several prerequisites are necessary. The target population should be delimited and ecologically isolated. The most successful projects were carried out on maritime islands or in areas surrounded by deserts or high mountains. Females should preferably, although not necessarily, mate only once during their life time. The released sterile insects should be able to mix freely, and mate with the local population. The released insects should not themselves be pestiferous (e.g. female mosquitoes). The objective in SIT projects is to flood the target area with sterile male insects that compete with the native male insects for mates. Increasing the ratio of “sterile matings” (i.e. matings between sterile and native insects) will reduce the birth rate and reduce the population in subsequent generations. SIT is based upon the continuous presence of high numbers of sterile insects in the target area; hence it is a preventive and not a reactive control method. The efficacy of the sterile insects depends on their quality and ratio in the target population. A ratio of 10-100 sterile insects for each native insect is considered satisfactory. Behavioral, physiological and biological traits are components of the quality of the sterile insects, which is measured by their mating propensity and competitiveness, sperm transfer, dispersal, survival, and level of sterility.

Several models have been proposed to demonstrate and elucidate the effect of SIT on target populations. The Knipling model is intended to calculate the effect of recurrent releases of sterile individuals into a target population. The model assumes a stable native population in equilibrium with the environment (i.e. the replacement rate = 1), with a 1:1 sex ratio. The model also assumes complete isolation with no migration. Released males are assumed to be 100% sterile, equally competitive with native males and with equal probability of finding a female mate. The amount of the released insects remains constant in subsequent releases. Initial densities of the native population, and initial release ratios, may be varied. For example, the model shows that with an initial population of 2 million native insects and an initial release ratio of 2:1 (i.e. initial release of 4 million sterile insects), less than 1 female will remain in the fifth generation and the population would become extinct. The model does not include competitive values of released insects, nor does it take into account migration rates. Most current SIT projects rely on release ratios of 100:1 and the continuous maintenance of high levels of sterile insects in the environment. Eradication, when and if achieved, requires many more than 5 generations of the native population. The Knipling model was refined by other scientists in order to incorporate more realistic components. The Curacao model, developed by Sawyer and co-workers in 1987, is an extension of the Knipling model. It incorporates several variables that make it more realistic. The user can alter release ratios, competitive values of the released insects, estimates of the native population, spatial effects such as variable density of the native population, migration rates, etc. A simulation model was published in the Internet-based “Radcliffe’s IPM World Textbook” and can be downloaded from http://ipmworld.umn.edu/chapters/SirSimul.htm

Sterile insect releases and genetic sexing: SIT seems to be more effective when only sterile males are released for the following reasons:

  1. Males are more efficient carriers of the sterility trait, as they often copulate more than once, whereas females may be restricted in the number of their matings.

  2. When only males are released, assortative mating amongst the laboratory-reared insects can be avoided, increasing the exploitation of the sterile sperm.

  3. When no females are released, males disperse more rapidly in the native (wild) population.

  4. Female insects might damage crops even if they are sterile, as they could cause oviposition wounds.

  5. Rearing only males reduces the cost of mass-rearing, provided females can be eliminated early during the rearing process.

  6. “Genetic sexing” methods have been developed in order to produce all-male batches for release, and are most advanced with Ceratitis capitata (Wiedemann), the Mediterranean fruit fly (Medfly). Chromosomal translocations have been induced and selected so that selective mutations, which are located usually on one of the autosomes, became linked to the male Y-chromosome, and hence sex-linked. The earliest “genetic sexing” strains linked pupal color traits to the Y-chromosome. As a result, male pupae were heterozygous for the dominant, brown allele, whereas females were homozygous for the recessive white-pupa alleles. The male and female pupae could then be separated automatically by commercially available sorting machines. “Genetic sexing” was advanced by linking a conditional deleterious trait, e.g. the temperature sensitive lethal gene (tsl), to the sex chromosome. As a result, only female eggs were killed when all eggs were exposed to higher temperatures.

Current research is focused on genetic engineering, with the intention of transferring conditional genes from other organisms to the Medfly. “Genetic sexing” strains are currently replacing the conventional bisexual strain in most Medfly rearing facilities. Pupal color as well as tsl strains are used in Medfly SIT projects, the former being gradually being replaced by the more economical tsl strains. The major problem encountered with the “genetic sexing” strains is their stability, and research is focusing on enhancing their stability.

Highlights of SIT: SIT to control insect pests was first employed to eradicate the New World screwworm (Cochliomyia hominivorax (Cockerell)) from the southern USA. The first project was initiated by Knipling and Bushland in 1955, and is still active. It resulted in the complete eradication of the pest from the USA, Mexico, Guatemala and other Central American countries. This success prompted SIT research projects with various pest species. Major efforts were directed towards dipteran pests affecting humans and animals, including several species of mosquitoes, the house fly (Musca domestica Linnaeus) the stable fly (Stomoxys calcitrans (Linnaeus)), the hornfly Haematobia irritans (Linnaeus) and tsetse flies (Glossina spp.). The latter were the subject of a large scale, six-year-long project on the island of Zanzibar, where complete eradication was declared in 2000. SIT of herbivorous pests was mainly directed towards fruit fly species of the genera Ceratitis, Bactrocera, Anastrepha and Rhagoletis. Some lepidoteran pests such as the pink bollworm Pectinophora gossypiella, the corn earworm, Helicoverpa zea, the Gypsy moth (Lymantria dispar) and the codling moth, Cydia pomonella, were subjected to research and field testing of SIT. The method was also attempted on numerous other pest species, but has not so far graduated from laboratory and preliminary testing. Several large-scale projects are currently being conducted to control, eradicate or prevent the establishment of the Medfly. Most are conducted in South, Central and North America (Argentina, Chile, Guatemala, Mexico, USA), and another project is carried out on the island of Madeira (Portugal). The pink bollworm SIT program in California, initiated in 1968, consists of sterile adults that are released during the cotton growing season in the San Joaquin valley. The objective is neither eradication nor control, but creating a barrier against migrating populations from Arizona, southern California and Mexico.

Components of an SIT Project

Mass rearing: The mass-production of insects (bisexual strains or “Genetic sexing” strains) is essential in all SIT projects. No SIT project will thus be realized without developing an efficient mass-rearing method, including an appropriate facility which has to be planned and constructed. This requires the mutual efforts and knowledge of diverse disciplines, such as insect physiology, biology, behaviour and nutrition, industrial and mechanical engineering, logistics, production economy and production engineering, quality control etc. A mass-rearing facility is not simply an enlarged insect laboratory. It is a factory that has to provide a relatively non-expensive product (because it must compete with prevailing control methods), of good quality and should be available throughout the year in large enough quantities.

A mass-rearing facilities must have five basic components.

  1. Oviposition cages and egg collection systems; 2. Egg maturation containers; 3. Larval rearing trays; 4. Pupation containers; 5. Equipment for marking for identification, irradiation and packing. There are currently 36 mass-rearing facilities in 25 countries around the world. The capacities of these facilities range from over 1 billion insects per week (El Pino, Guatemala) to less than 1 million insects per week. The majority of the facilities rear one or more strains of the Medfly, including the tsl strain. Other fruit flies such as Anastrepha spp., the olive fly {Bactrocera oleae (Gmelin)} and other Bactrocera spp. and Ceratitis rosa (Karsch), are reared in 15 facilities. Species of the the tsetse fly are reared in 5 facilities, mainly in Africa. Two North American facilities rear the codling moth and the pink bollworm.

The directory of the mass-rearing facilities, including information on the insect strains reared, contacts and addresses (pdf files) can be downloaded from http://www.iaea.or.at/programmes/nafa/d4/public/sit- fac.pdf

Sterilization: The mass-reared insects are sterilized by exposure to ionizing radiation using Cobalt60 or Cesium137 sources. Both isotopes emit gamma rays that cause breakages in the chromosomes of the germ cells. Dosage, timing of treatment and the environment in which the sterilizing treatment is applied are aimed to achieve maximum sterility with minimum effect on mating competitiveness, survival and dispersal capacity of the treated insects. They may be irradiated in air, or in its absence, where the irradiated insects (pupa or likewise) are packed tightly in plastic bags, so that the oxygen supply is almost entirely depleted. Some facilities irradiate under nitrogen. Numerous studies showed that anoxia, or a total nitrogen environment, increased the resistance of the insect to irradiation, hence enabling a higher dose to be applied with lesser affect on mating competitiveness. A wide range of irradiation dosages are used, depending on the treated organism. In most Medfly-rearing facilities a minimum absorbed dose of 90 - 149 Gy is applied; the facility in Perth, Australia, uses a dose of 180 Gy in nitrogen. For other fruitflies dosages of 50 - 120Gy are used. Tsetse flies (Glossina spp) are sterilized with 120Gy. Moths, which have holokinetic chromosomes, are most resistant to ionizing radiation and require dosages of 200-300 Gy for satisfactory sterilization. Livestock pests, such as the new and old world screwworms (Chrysomyia bezziana and Cochliomyia hominivorax, respectively) require dosages of 40 and 80Gy, respectively. Irradiation is usually applied at a late pupal stage, 1-2 days prior to adult eclosion; the later the stage, the higher the sterilization level. However, when pupae have to be shipped for a considerable period, ample time should be allowed before adult eclosion occurs.

Most sterilized insects are marked prior to packing and irradiation so that they can be identified after release and distinguished from native individuals. This is essential for the monitoring of their dispersal and the decline in the native population as a result of releases. In SIT projects of flies (e.g. fruit flies or livestock pests), a small amount of fluorescent dust is mixed with the pupae. Particles of the fluorescent marker can easily be detected in the adult under a UV lamp. Currently research is focusing on a search for inherent body markers (e.g. color) and on the insertion of fluorescence-conferring genes from other organisms using genetic engineering techniques.

Release: The sterile insects are released as sexually mature adults. Eclosion and sexual maturation take place at release centers, which should be located close to the SIT target area. The release center consists of a receiving unit, eclosion and rearing rooms, preparation area for release and a quality control laboratory. It also includes auxiliary units such as adult food preparation, storage area, equipment cleaning and a repair shop. At the receiving unit the shipped insects are unpacked, weighed and distributed to the eclosion and maturation containers. Samples of these insects are taken for quality control tests. In SIT projects of fruit flies the sterilized pupae are placed into paper bags, which are then placed into aerated plastic containers called PARC boxes. The paper bags serve to increase the surface area within the container thereby reducing competition for space between the eclosed individuals. Adult food is provided to the eclosion containers which are placed in a rearing chamber with the appropriate conditions (temperature and humidity). The eclosion and maturation chambers occupy the largest space in the release center, as it takes several days for the insects to eclose and mature sexually.

The adult insects are released either from the ground or from the air. Ground releases are carried out either by cars or other means of transportation. The insects are packed into small boxes, which are opened along the trail. Aerial release is more economical, can cover larger areas, and overcomes inaccessibility in the target area. Most SIT projects currently use the “chilled adult release” system. At the release center the insects are placed into room-size refrigerators and chilled to 4°C until they fall down to the bottom of the PARC boxes. This is the “knockdown” phase. They are then collected into a chill-box which is carried to the release airplane and attached to a cooler that keeps the insects motionless during the release period. The flies drop from the bottom of the chill-box into an auger system, which moves them through a chute located on the underside of the airplane fuselage. The release rate (insects per unit area) can be controlled via the revolution speed of the auger system. Insect mortality in this system is negligible and dispersal is satisfactory.

Monitoring: The released insects are marked as described above, and a network of monitoring traps is deployed throughout the target area to follow the dispersal of the released insects, as well as to make adjustment to the release flights. It is essential that the sterile insects are distributed evenly, to insure their presence at all times in large enough numbers throughout the target area. Monitoring is also essential to assessing the progress and the rate of success of the project. SIT results are not seen immediately, and contrary to pesticide use, neither dead insects nor a visible reduction in the size of the population is observed. Theoretically, when release numbers remain constant during an SIT project, the population remains stable, but the proportion of sterile to native insects changes. Monitoring traps are therefore used to assess the relative abundance of the native insects from all insects captured.

Insects collected in the traps are usually brought to a specialized laboratory, where released and native insects can be identified, by the marker or by dissection of the reproductive organs. Sampling of plants or livestock that might be damaged by the target pests is an essential component of the monitoring system in SIT projects.

Quality control: Quality control is applied during the production process in the factory and on the end product. The production line has to run smoothly, utilize its resources efficiently, and minimizes environmental hazards, pollution and contamination (escape of non-sterile insects from the factory). The end product (sterile insects) must be of high quality, almost completely sterile and yet able to survive, disperse in the target area, able to locate mates and mate competitively within the target populations. Quality control measures and tests are applied throughout the rearing process in the factory, at the receiving release center, and following the release in the target area.

Quality control tests have been employed since the beginning of the use of the SIT, and are constantly being refined. Production economy was considered to be of major importance during the early phases of the SIT, as the costs of sterile insects had to be competitive with other control tools. Research was therefore directed towards automation and cost reduction of the rearing process. Weight and size of the produced insects were (and still are) the first measured quality traits, but it was soon realized that behavioral traits were no less important. Various tests concerning mating ability, mating propensity and competitiveness were added to the quality control tests. Tests of survival under varying conditions, with and without food, were added to the list.

With the increased spread of the use of SIT world-wide, uniformity of such tests, e.g. standardization of quality control factors became necessary. As a result quality control manuals (QC manuals) were developed, published and accepted in most SIT projects and are continuously updated. Common QC components are size, weight, rate of adult eclosion, percent of males (or females), flight ability of the eclosed adults, survival and mating competitiveness. The latter is probably the ultimate test, but also the most cumbersome to conduct. The sterile and native insects are mixed either in small laboratory cages or in large outdoor cages, mating pairs are observed, sperm transfer is checked and the comparative data are transformed into mating competitiveness, mating index etc. Mating frequency between sterile and native insects is also used to test the sterility level of the released insets. A complete QC manual for fruit flies can be downloaded from http://www.iaea.or.at/programmes/nafa/d4/public/qc41.pdf

SIT projects in Israel and the Middle East: The Medfly is a major pest of citrus, deciduous and tropical fruit crops as well as vegetables in the Middle East and particularly in Israel. The pest takes a heavy toll when not controlled, and prevents the export of fresh produce to countries that impose quarantine on that pest. The citrus industry in Israel has carried out a centralized campaign to control the pest on citrus since 1961, relying heavily on the aerial application of the organophosphate malathion in a bait mixture.

SIT was tested on a small scale in 1972 and 1989 in Israel. Pupae and chilled adults were released in 1972 over an area of 1,000 ha from the ground and from the air. In 1989 a “genetic sexing” strain was used and males were released from the ground on an area of 500 ha. In 1994 the International Atomic Energy Agency (IAEA) proposed to initiate a regional cooperative SIT project for the control or eradication of the Medfly in the Near East. This project, EASTMED, was planned in detail in 1995. A pilot project in the Israeli and Jordanian lower rift valley, the Israeli Arava and the Jordanian Araba Valleys, stretching from the Dead Sea to the Red Sea, was initiated in 1997, the target area being approx. 6000 Km⊃. On the Israeli side the area encompasses 21 settlements, including a regional center and the city of Eilat,. On the Jordanian side it includes five villages and the city of Aqaba. A release center was established in the Arava valley (Israel), and sterile flies were purchased from Madeira (Portugal) and from Guatemala. Aerial releases of chilled adults were carried out since December 1997, at a rate of about 6 million males per week. This pilot project was expanded in January 2001 to cover the whole southern part of Israel, up to the city of Beer-Sheva in the north, and to the Egyptian border in the west. This is mostly a desert area, with several towns and agricultural settlements that are located near the Egyptian border. The Medfly populations in the Arava Valleys dropped considerably, being almost eradicated towards the end of 2000. However, the objective of the project has not completely been attained because a quarantine system, intended to prevent the introduction of the pest from the north, has not yet been established. Also, the long-distance hauling from Guatemala (50 - 60 hours from the factory to the release center) affected the quality of the sterile flies, reducing their performance. The realization of a regional project that requires the weekly release of hundred of millions of flies thus depends on the construction of a large-scale rearing facility, in or close to the release region. The pilot project has shown that SIT is a feasible method, and can replace the conventional chemical methods to control that pest.

References

Klassen W, Knipling E.F. and McGuire Jr J.U. 1970. The potential for insect-population suppression by dominant conditional lethals. Annals of the Entomological Society of America 63: 238-255.

Knipling E.F. 1955. Possibilities of insect control or eradication through the use of sexually sterile males. Journal of Economic Entomology 48: 459-462.

Knipling, E.F. 1959. Sterile male method of population control. Science 130: 902-904.

Rossler, Y., Ravins, E. and Gomes, P.J. 2000. Sterile Insect Technique (SIT) in the Near East - a transboundry bridge for development & peace. Crop Protection 19: 733-738.

Website

https://www.google.co.il/search?q=sterile+male+technique+image&biw=1280&bih=687&tbm=isch&tbo=u&source=univ&sa=X&ved=0ahUKEwibodT2mvTJAhVF1RQKHfv8C1kQsAQIHg

Rossler, Y.