Application Of Sterile Insect Technique Research Articles

Factors affecting Anastrepha fraterculus female receptivity modulation by accessory gland products

In the context of the sterile insect technique (SIT), mass-rearing and male irradiation are imperative. Post-teneral treatments such as the addition of protein in adult′s male diet and male hormonal treatment are used to improve sexual performance and to accelerate sexual maturation. In this work we investigated the effect of male accessory glands products (AGPs) on female receptivity of the South American fruit fly Anastrepha fraterculus (Wiedemann), and the effect of strain rearing history, male irradiation, male diet and hormonal treatment on AGPs. Injections of aqueous extracts of male accessory glands into the abdomen of females reduced their receptivity. The AGPs from laboratory males were more effective in inhibiting female receptivity, compared to AGPs from wild males, irrespective of females’ origin. The AGPs from fertile males were more effective than AGPs from sterile males. The AGPs from protein-fed males were more effective than AGPs from sugar-fed males. Finally, the AGPs of males treated with juvenile hormone were less effective in inhibiting female receptivity than AGPs of untreated males. We conclude that inhibition of sexual receptivity of A. fraterculus mated females is mediated by products in male accessory gland’s and the way that these products act vary widely according to the effect of extrinsic factors. We discuss the results in the perspective of the SIT application for A. fraterculus.

Tsetse Salivary Gland Hypertrophy Virus: Hope or Hindrance for Tsetse Control?

Many species of tsetse flies (Diptera: Glossinidae) are infected with a virus that causes salivary gland hypertrophy (SGH), and flies with SGH symptoms have a reduced fecundity and fertility. The prevalence of SGH in wild tsetse populations is usually very low (0.2%–5%), but higher prevalence rates (15.2%) have been observed occasionally. The successful eradication of a Glossina austeni population from Unguja Island (Zanzibar) using an area-wide integrated pest management approach with a sterile insect technique (SIT) component (1994–1997) encouraged several African countries, including Ethiopia, to incorporate the SIT in their national tsetse control programs. A large facility to produce tsetse flies for SIT application in Ethiopia was inaugurated in 2007. To support this project, a Glossina pallidipes colony originating from Ethiopia was successfully established in 1996, but later up to 85% of adult flies displayed symptoms of SGH. As a result, the colony declined and became extinct by 2002. The difficulties experienced with the rearing of G. pallidipes, epitomized by the collapse of the G. pallidipes colony originating from Ethiopia, prompted the urgent need to develop management strategies for the salivary gland hypertrophy virus (SGHV) for this species. As a first step to identify suitable management strategies, the virus isolated from G. pallidipes (GpSGHV) was recently sequenced and research was initiated on virus transmission and pathology. Different approaches to prevent virus replication and its horizontal transmission during blood feeding have been proposed. These include the use of antiviral drugs such as acyclovir and valacyclovir added to the blood for feeding or the use of antibodies against SGHV virion proteins. In addition, preliminary attempts to silence the expression of an essential viral protein using RNA interference will be discussed.

Egg Parasitoids AttackingCactoblastis cactorum(Lepidoptera: Pyralidae) in North Florida

Interest in the natural enemies of Cactoblastis cactorum (Berg) has increased since the moth was found in Florida in 1989. Previous surveys for natural enemies in Argentina identified egg parasitoids in the family Trichogrammatidae as potentially important control agents of C. cactorum. A study was conducted in north Florida to identify and to assess occurrence of egg parasitoids attacking this invasive moth in its new homeland. Surveys undertaken at 6 locations in north Florida from Jul 2008 to Dec 2009 revealed that eggsticks of C. cactorum were attacked by egg parasitoids from the Trichogramma genus: T. pretiosum Riley, T. fuentesi Torre, and an additional unidentified Trichogramma species belonging to the T. pretiosum group. In order to assess the importance of these egg parasitoids, the fate of individual C. cactorum eggsticks was determined during weekly visits to each site. This assessment showed that the combined level of parasitism of C. cactorum eggsticks was very low with less than 0.2% of host eggs attacked at any one of the 6 sites. While parasitoids attacked smaller eggsticks, there was no correlation between the numbers of eggs in an eggstick attacked with increasing number of eggs/eggstick. Comparing the mean number of eggs/eggstick, there was no difference between the 3 flight periods of C. cactorum, but there was a difference between the 6 sites. Based on these results, the use of Trichogramma wasps as an inundative biological control agent, complementary to the Sterile Insect Technique application, is discussed.

Methoprene treatment reduces the pre‐copulatory period in Anastrepha fraterculus (Diptera: Tephritidae) sterile males

AbstractAnastrepha fraterculus is a major fruit pest in South America. Ongoing studies support the implementation of the sterile insect technique (SIT) against this pest. Sexual readiness of sterile males is a key point for SIT application. The pre‐copulatory period of A. fraterculus males has not been reported before, but it is expected to last several days. An acceleration of sexual maturation was achieved in other Anastrepha species after topical applications of juvenile hormone analogues, like methoprene. Here, we studied the effect of methoprene on male sexual maturation, mating duration and sperm transfer in A. fraterculus as well as the impact of acetone (methoprene solvent) on survival. We also explored a method to deliver methoprene massively. Pheromone‐calling and mating ability were evaluated daily from adult emergence, and used as indicators of sexual maturity. Anastrepha fraterculus males showed a long pre‐copulatory period (7 days approximately), as other Anastrepha species. This process was accelerated after methoprene treatment (2.5 μg/μl), both in non‐irradiated and irradiated males which matured 2–3 days earlier. Mating duration for methoprene‐treated males was longer than for mature untreated males, however, no differences in sperm transfer were detected. Survival was not affected by acetone. Dipping pupae in methoprene allowed emerging males to mature as fast as those receiving topical application as adults. Dipping of pupae is a promising method to deliver massively methoprene and should be further investigated.

Dispersal and Survival of Aedes albopictus (Diptera: Culicidae) Males in Italian Urban Areas and Significance for Sterile Insect Technique Application

The dispersal and survival of laboratory-reared Aedes albopictus Skuse males were investigated during the summer of 2007 in three Northern Italy urban localities by mark-release-recapture techniques. Two marking methods were compared: one group of males was dusted with fluorescent pigments on the body (FP), and the other group was obtained from a strain whose natural infection of Wolbachia had been removed (WB0). FP- and WB0-marked males were released as adults and pupae, respectively, in one fixed station at each locality. Recaptures were performed by skilled technicians, within a radius of 350 m from the release site, on days 4, 5, and 7 after the release, and the males were collected while flying around the technician's body or in swarms. Recapture rates ranged from 0.63 to 4.72% for FP males and from 2.39 to 11.05% for WB0 males. The mean distance traveled for WB0 males was significantly higher than for FP males; no difference was observed between the dispersal distance measured for the males recaptured on human host versus males recaptured while swarming. No further increase of the dispersal occurred during the postrelease period investigated (from day 4 to day 7 after release). The mean survival rate at the release was 0.51 for FP-marked males and 0.81 for WB0 males. The data obtained are discussed for their significance in planning sterile insect technique programs against Ae. albopictus.

Total body nitrogen and total body carbon as indicators of body protein and body lipids in the melon fly Bactrocera cucurbitae: Effects of methoprene, a juvenile hormone analogue, and of diet supplementation with hydrolyzed yeast

The application of methoprene, and providing access to diet including hydrolyzed yeast, are treatments known to enhance mating success in the male melon fly Bactrocera cucurbitae Coquillett (Diptera: Tephritidae), supporting their use in mass rearing protocols for sterile males in the context of sterile insect technique (SIT) programmes. The objective of the present laboratory study was to investigate the effect of methoprene application and diet supplementation with hydrolyzed yeast (protein) on the turnover of body lipids and protein to confirm the feasibility of their application in melon fly SIT mass-rearing programmes. While females had access to a diet that included hydrolyzed yeast (protein), males were exposed to one of the following treatments: (1) topical application of methoprene and access to diet including protein (M+P+); (2) only diet including protein (M−P+); (3) only methoprene (M+P−) and (4) untreated, only sugar-fed, control males (M−P−). Total body carbon (TBC) and total body nitrogen (TBN) of flies were measured at regular intervals from emergence to 35 days of age for each of the different treatments. Nitrogen assimilation and turnover in the flies were measured using stable isotope (15N) dilution techniques. Hydrolyzed yeast incorporation into the diet significantly increased male body weight, TBC and TBN as compared to sugar-fed males. Females had significantly higher body weight, TBC and TBN as compared to all males. TBC and TBN showed age-dependent changes, increasing until the age of sexual maturity and decreasing afterwards in both sexes. Methoprene treatment did not significantly affect TBC or TBN. The progressive increase with age of TBC suggests that lipogenesis occurs in adult male B. cucurbitae, as is the case in other tephritids. Stable isotope dilution was shown to be an effective method for determining N uptake in B. cucurbitae. This technique was used to show that sugar-fed males rely solely on larval N reserves and that the N uptake rate in males with access to diet including hydrolyzed yeast was higher shortly after emergence and then stabilized. The implications of the results for SIT applications are discussed.

Analysis of the antiviral drugs acyclovir and valacyclovir-hydrochloride in tsetse flies ( Glossina pallidipes) using LC–MSMS

A new simple, sensitive and precise liquid chromatography–tandem mass spectrometry method has been developed and validated for the determination of valacyclovir-HCl and acyclovir in tsetse flies ( Glossina pallipides). Tsetse flies were extracted by ultrasonication with acidified methanol/acetonitrile, centrifuged and cleaned up by solid phase dispersion using MgSO 4 and MSPD C 18 material. Samples were analysed using a Waters Alliance 2695 series HPLC with a C 18 Gemini analytical column (150 mm × 4.6 mm × 5 μm) and a guard cartridge column connected to a Waters Quattro-Micro triple-quadrupole mass spectrometer. The isocratic mobile phase consisted of methanol:acetonitrile:water (60:30:10, v/v/v) plus formic acid (0.1%) at a flow rate of 0.25 ml/min. The precursor > product ion transition for valacyclovir ( m/ z 325.1 > 152) and acyclovir ( m/ z 226.1 > 151.9) were monitored in positive electrospray multiple reaction monitoring mode. The method was validated at fortification levels of 0.5, 1 and 2 μg/g. The range of calibration for both drugs was 0.45–4.5 μg/g. The overall accuracy of the method was 92% for valacyclovir and 95% for acyclovir with corresponding within-laboratory reproducibilities of 4.4 and 3.4%, respectively. Mean recoveries were above 80% for both drugs and repeatability ranged from 0.7 to 6.1%. For both drugs the limits of detection and quantification were 0.0625 and 0.2 μg/g, respectively. The method was applied in experiments on the mass rearing of tsetse flies for sterile insect technique (SIT) applications, in which the flies were fed with blood meals containing acyclovir or valcyclovir-HCl prior to analysis to assess effects on Glossina pallidipes Salivary Gland Hypertrophy syndrome.

Effects of the juvenile hormone analogue methoprene and dietary protein on male melon fly Bactrocera cucurbitae (Diptera: Tephritidae) mating success

The effect of access to dietary protein (P) and the topical application of a juvenile hormone analogue (methoprene (M)) on mating behaviour of male melon fly Bactrocera cucurbitae was assessed in the laboratory and in field cages. Age, dietary protein and methoprene application increased the mating success and influenced the mating behaviour. Treatment with methoprene (M+) to protein-deprived (P−) males had only a modest effect on the acceleration of sexual maturity, but application of methoprene (M+) to protein-fed (P+) males greatly accelerated sexual maturity. Protein diet (P+) increased mating success of males in comparison to protein-deprived (P−) males. Protein and methoprene have a synergistic effect on mating behaviour, since M + P+ treated males exhibit reduced mating latency and achieved higher mating in younger ages than methoprene and/or protein-deprived males. Copulation duration was correlated with nutritional status and M + P+ males copulated longer at the age of advanced sexual maturity than M − P+ males. Our results suggest that in this species with a lek mating system, females discriminate between the males based on their sexual signals, which were influenced by protein in the adult diet, methoprene application and age. The results are discussed in the light of mating competitiveness of precocious treated young males and their relevance to Sterile Insect Technique application against this pest species.

F1 sterile insect technique: A novel approach for risk assessment of Episimus unguiculus (Lepidoptera: Tortricidae), a candidate biological control agent of Schinus terebinthifolius in the continental USA

Federal regulations mandate that researchers in the field of classical weed biological control follow the precautionary principle when proposing the release of an organism that can affect our environment. However, laboratory risk assessment experiments often predict a much broader host range than that which occurs in the field. Because open-field tests are prohibited in the area of introduction, the application of the F1 sterile insect technique (F1SIT) could be used to conduct field testing in the proposed release area in a safe and temporary manner. In this study, we determined the minimum dose of radiation required to field test the tortricid Episimus unguiculus (Clarke), a candidate for biological control of Brazilian peppertree, Schinus terebinthifolius Raddi (Anacardiaceae) in Florida. Male and female virgin E. unguiculus adults were treated with increasing doses of gamma radiation and either inbred or outcrossed to non-treated E. unguiculus adults. Pairs of adults were placed in oviposition cages and allowed to mate and oviposit. Data from fecundity and fertility counts were recorded. The dose at which treated females were 100% sterile was 200 Gy. The dose at which F1 females and males were 100% sterile was 225 Gy. As the dose of radiation increased, there was an increase in sterility, a decrease in fecundity for both treated female crosses, and a higher ratio of F1 males to females. The F1 sterile insect technique (F1SIT) could be suitably applied to other areas of pest management, including risk assessment of potential lepidopteran biological control agents of invasive, exotic weeds.

Influence of methoprene and protein on survival, maturation and sexual performance of male Ceratitis capitata (Diptera: Tephritidae)

AbstractThe Mediterranean fruit fly (medfly), Ceratitis capitata (Wied.), like many other polyphagous tephritids (Diptera: Tephritidae), preforms a lek as mating system. The sterile insect technique (SIT) requires released males to survive in the field, and to compete with wild males in attracting, courting and mating with wild females. The effects of the juvenile hormone analogue, methoprene, and the incorporation of hydrolyzed yeast protein into the adult diet, on survival, sexual maturation and sexual performance of male C. capitata were evaluated for the four possible combinations of methoprene‐treated or untreated, and protein‐fed or deprived flies. The incorporation of protein had a positive impact on laboratory survival and accelerates the sexual maturation of mass‐reared males. In addition, in the laboratory, male sexual performance was significantly higher in treatments with methoprene. However, field cage tests with mature and protein‐fed wild males and females failed to show any effects of either factor on male sexual performance. The increased survival and the earlier sexual maturation of sterile males due to the addition of protein to the adult diet can positively influence the efficacy of SIT application against C. capitata.

A PROTOCOL FOR STORAGE AND LONG-DISTANCE SHIPMENT OF MEDITERRANEAN FRUIT FLY (DIPTERA: TEPHRITIDAE) EGGS. I. EFFECT OF TEMPERATURE, EMBRYO AGE, AND STORAGE TIME ON SURVIVAL AND QUALITY

The operational use of Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann), genetic sexing strains in Sterile Insect Technique applications can be maximized by developing methods for effective shipment of eggs. This would enable a central production facility to maintain the relevant mother stocks and large colonies to supply eggs to satellite centers that would mass produce only males for irradiation and release. In order to achieve this, the survival of medfly embryos of different ages was assessed after storage at 5, 10, 15, 20, and 25°C in water for different periods of time. Survival was affected by all 3 variables, i.e., embryo age, water temperature, and length of storage. Storage of embryos at any temperature for 120 h resulted in almost no survival. Controlling the age of the embryo at the time of the temperature treatment is crucial for the success of this procedure. Embryos collected between 0 to 12 h after oviposition and pre-incubated at 25°C for 12 h provide a suitable 72 h window for shipment when maintained between 10 to 15°C. Under these conditions, no significant reductions in survival during all the developmental stages were observed.

CACTOBLASTIS CACTORUM (Lepidoptera: pyralidae): Observations of Courtship and Mating Behaviors at Two Locations on the Gulf Coast of Florida

Cactoblastis cactorum (Berg) has become an invasive pest of Opuntia spp. along the coastal areas of southeastern United States from the panhandle of Florida to South Carolina. Spread of this insect into cactus dominated natural areas of the United States and Mexico and into agricultural opuntia fields of Mexico is raising concerns within international governments and conservation organizations. Interest is growing in using the Sterile Insect Technique (SIT) to manage C. cactorum populations. Information on courtship and mating behaviors of this insect is important in the development and application of SIT. We conducted mating table studies and determined that this moth exhibits simple rather than elaborate mating behaviors and that courtship and mating take place briefly during morning twilight. Typically, females initiate calling, males respond to females, and copulation are complete before sunrise. Successfully mated females attract males within a short period (mean of 5.2 min), while unsuccessful females continue calling for about 40 minutes. Mating pairs remain in copula for a mean of 31.8 min. Generally, mated females are busy ovipositing the first few nights after mating, not exhibiting additional mating behaviors. A release of marked males revealed that males stay near the release site and can be recovered and identified for subsequent population estimate studies. This study on courtship/mating behavior is helpful to the ongoing C. cactorum research to develop a successful SIT program, identify the female calling pheromone, improve monitoring traps, and develop a technique to estimate adult moth population abundance.

Invasive arthropods in agriculture: problems and solutions

Part 1: agriculture in the larger context of ecology, Miguel A. Altieri history and recent changes in regulatory entomology, Paula Henstridge et al role and relationship of official and scientific information concerning pest status, Christina Devorshak and Robert Griffin pathways of introduction, Howard Frank determination of the origin of invasive species, Bruce McPheron intentional introductions of exotic species, Donald Thomas cost of invasive species, David Pimentel predicting pest status, Susan Worner impact of invasive species on Hawaiian agriculture, Peter Follett risk analysis, Ron Sequeira. Part 2 Prevention: crime and punishment (enforcement), Jeff Grode and Alan Christian the importance and use of attractants, David C. Robacker and Peter J. Landolt pest limits analysis, Richard Baker research needs in ecology and behaviour to prevent fruit fly introductions, R.L. Mangan determination of host status, Guy J. Hallman quarantine treatments - research and application, Guy J. Hallman. Part 3 Eradication and management: eradication or management?, Judith Meyers considerations in implementing eradication programs, Carl Bausch recent developments in the sterile insect technique (SIT), Jorge Hendrichs application and evaluation of SIT in the field, Pat Gomes the role of pesticide baits in eradication, Daniel Moreno insect management and eradication technology - perspectives for the future, Bob Staten and Thomas Miller long term management of invasive species in agriculture, Carrol O. Calkins et al new restraints on biological control, Pauline Syrett towards a rational approach to biological control, Ernest Delfosse.

A novel method for rearing the progeny of wild mediterranean fruit flies using artificial fruit

A technique for rearing the progeny of wildCeratitis capitata flies was developed. The method is based on wild captured flies which are allowed to oviposit in artificial fruit containing larval rearing medium. Flies develop in the fruit from eggs to last larval instar, then exit the fruit and pupate within vermiculite. This method was shown to be feasible and efficient, and to increase considerably the number of wild flies for testing purposes. Possible contribution to the Sterile Insect Technique (SIT) and potential applications of the method are discussed.

Case studies of emergency management of screwworm.

Screwworm myiasis, caused by infestation of even minor wounds by the obligative parasitic larval stages of the New World screwworm (NWS) (Cochliomyia hominivorax) or Old World screwworm (OWS) (Chrysomya bezziana) flies, is a major cause of livestock morbidity and mortality in tropical and sub-tropical regions of the world. The two parasites occur in different hemispheres but are remarkably homologous. Animal health emergencies result from the invasion of new territories by the parasites or, in the case of NWS, reinfestation of areas from which the parasite had been eradicated after great effort and expense. The author reviews the biology of the parasites and the effects of screwworm, in addition to prevention of infestation upon the introduction of animals. Examples of three programmes or events are described. The first is the eradication of previously exotic NWS from an epizootic in Libya before the parasite spread to become enzootic in the Mediterranean Basin and eventually other areas of the Eastern Hemisphere. The second example reviews the serious consequences of the extension of the range of OWS into Iraq where conditions at the time were favourable for propagation and unfavourable for control. The third example describes the NWS programme strategy in North and Central America which, for forty years, has been to progressively achieve eradication and then protection of areas from north to south on that continent, employing the sterile insect technique (SIT). Outbreaks in areas where screwworm has already been eradicated divert costly programme resources and slow progress southwards, and are considered emergencies. Some problems encountered and the solutions found during the height of the eradication programme in Mexico are described. Although to date eradication of screwworms has only been accomplished with the application of SIT, this technique alone will not eradicate the pest. The author describes other elements which are required to control or eradicate screwworms. Programmes for this highly mobile parasite encompass large geographic areas and consequently require active and continuous international participation.

Increased effectiveness and applicability of the sterile insect technique through male‐only releases for control of Mediterranean fruit flies during fruiting seasons

AbstractOne of the main obstacles for a wider use of the Sterile Insect Technique (SIT) against the Mediterranean fruit fly (medfly) is the damage commercial fruit suffers due to sterile female stings. To overcome this obstacle, the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture has played a leading role in sponsoring and carrying out research to develop medfly genetic sexing strains that allow male‐only SIT releases.Recently, as a result of this continued FAO/IAEA effort, genetic sexing strains based on a temperature sensitive lethal (tsl) mutation have been developed at the IAEA Laboratories at Seibersdorf. Unlike previous pupal color sexing strains, these ‘second generation’ sexing strains allow female killing at an early (embryonal) stage. In addition, they are essentially stable under mass rearing conditions. This represents an important breakthrough because both of these attributes were considered indispensable for genetic sexing strains with any potential to replace conventional strains with both sexes in large scale sterile medfly production facilities. Besides the considerable savings in the costs of release and field monitoring, genetic sexing strains in field tests have shown severalfold increases in the effectiveness of the SIT as compared with the standard strains involving males and females. When releasing both males and females, sterile males are apparently not used effectively, because they use their limited sperm mostly to mate with sterile females and because they do not disperse widely in the presence of these females. When males only are released, however, they disperse much further in search of wild females and compete more intensely with wild males for wild females.As a result of the availability of usable male‐only strains, and the demonstration of their increased effectiveness, the applicability of the SIT against medfly has increased in two different ways. Highly developed commercial fruit growing regions, that previously had excluded application of SIT because of the fruit damage due to sterile female stings, are now reconsidering such free area/exclusion programs. More‐importantly, sterile male releases can now also be used for routine control purposes, rather than only for eradication programs, partially or fully replacing chemical bait‐sprays during the fruiting seasons.

Radiosensitivity of eggs of the rice moth, Corcyra cephalonica

The sensitivity of 1-day-old eggs of Corcyra cephalonica Staint., to ionizing radiation has been investigated in view of a possible future application of sterile-insect technique to the control of this species. Further, the irradiation effects on subsequent developmental stages were evaluated. A dose of 5 krad prevented hatching of eggs, while a dose of 4 krad was the highest dose that allowed an adequate percentage of the irradiated eggs (28.23) to complete development to the adult stage. While the mating percentage and mating frequency of resultant adults appeared to be unaffected, the percentage of multiple mating was variable and no clear pattern emerged. Egg production and fertility of P, adults was decreased significantly and all effects of the treatment were more severe for pairings of both sexes resulting from irradiated eggs than pairings of either sex with unirradiated partners. However, the results show that a dose of irradiation low enough to permit adequate numbers of adults to develop from irradiated eggs (4 krad) is insufficient to induce high levels of sterility. Therefore, irradiation of eggs for mass production of either sterile or partially sterile adults was found not to be feasible.

Gamma Irradiation of the Mediterranean Fruit Fly (Diptera: Tephritidae): Effects of Puparial Age under Induced Hypoxia on Female Sterility

Female Mediterranean fruit flies, Ceratitis capitata (Wiedemann), were subjected to sterilizing doses of 15 krad CO60 in a nitrogen atmosphere at the late puparial and newly emerged adult stages. Ovarian dissection and cross-mating experiments were conducted to determine whether egg development is enhanced during maturation of the puparial content and whether hypoxic atmospheres during irradiation might contribute to egg viability. When irradiated 2 days before eclosion, germinal ovarian tissue was severely damaged and no eggs occurred. However, a logarithmic increase in eggs was discovered in adult females from pupae irradiated 1 day before emergence, 3 h before emergence, and 3-h-old adults. Although no regeneration of germinal tissue was found, retention of eggs in the ovaries for varying lengths of time simulated “recovery of fertility.” Replicated cross matings with irradiated males of the same age distribution produced no offspring. However, matings of normal males A— females from pupae irradiated a few hours before emergence and newly emerged, produced viable offspring. The surviving F1, generation mated and produced a normal complement of eggs. This report describes discovery of a mechanism for remnant fertility which, if present in a pest species of such high reproductive potential, could adversely affect the success of sterile insect technique application.