Evaluation of partial sterility in mating performance and reproduction of the West Indian sweetpotato weevil,Euscepes postfasciatus

The sterile insect technique (SIT) is based on population and behavioral ecology and is widely used to suppress or eradicate target pest insect populations. The effectiveness of SIT depends on the ability of the released sterile males to mate with and inseminate wild females. The use of gamma‐radiation to induce sterility is, however, associated with negative impacts not only on reproductive cells but also on somatic cells. Consequently, irradiation for sterilization diminishes mating performance over time. In this study, we evaluated the balance between the irradiation dose and both fertility and mating propensity in Euscepes postfasciatus (Fairmaire) (Coleoptera: Curculionidae) for 22 days following irradiation. The mating propensity of males irradiated with a 150‐Gy dose, as currently used to induce complete sterility of E. postfasciatus in the SIT program in Okinawa Prefecture, was equal to that of non‐irradiated weevils for up to 6 days, and the mating propensity of males irradiated with a dose of 125 Gy was equal to that of non‐irradiated weevils for twice this period (12 days). The fertilization ability of weevils irradiated with a dose of 125 Gy was reduced by 4.6% in males and 0.6% in females, compared to the potential fertilization ability. We also discuss the possibility of the application of partially sterilized insects in eradication programs.     

Engineered Repressible Lethality for Controlling the Pink Bollworm, a Lepidopteran Pest of Cotton

The sterile insect technique (SIT) is an environmentally friendly method of pest control in which insects are mass-produced, irradiated and released to mate with wild counterparts. SIT has been used to control major pest insects including the pink bollworm (Pectinophora gossypiella Saunders), a global pest of cotton. Transgenic technology has the potential to overcome disadvantages associated with the SIT, such as the damaging effects of radiation on released insects. A method called RIDL (Release of Insects carrying a Dominant Lethal) is designed to circumvent the need to irradiate insects before release. Premature death of insects’ progeny can be engineered to provide an equivalent to sterilisation. Moreover, this trait can be suppressed by the provision of a dietary antidote. In the pink bollworm, we generated transformed strains using different DNA constructs, which showed moderate-to-100% engineered mortality. In permissive conditions, this effect was largely suppressed. Survival data on cotton in field cages indicated that field conditions increase the lethal effect. One strain, called OX3402C, showed highly penetrant and highly repressible lethality, and was tested on host plants where its larvae caused minimal damage before death. These results highlight a potentially valuable insecticide-free tool against pink bollworm, and indicate its potential for development in other lepidopteran pests.     

A dominant lethal genetic system for autocidal control of the Mediterranean fruitfly

The Sterile Insect Technique (SIT) used to control insect pests relies on the release of large numbers of radiation-sterilized insects. Irradiation can have a negative impact on the subsequent performance of the released insects and therefore on the cost and effectiveness of a control program. This and other problems associated with current SIT programs could be overcome by the use of recombinant DNA methods and molecular genetics. Here we describe the construction of strains of the Mediterranean fruit fly (medfly) harboring a tetracycline-repressible transactivator (tTA) that causes lethality in early developmental stages of the heterozygous progeny but has little effect on the survival of the parental transgenic tTA insects. We show that these properties should prove advantageous for the implementation of insect pest control programs.     

Assessment of effect of partial sterility on mating performance in sweetpotato weevil (Coleoptera: Curculionidae)

The sterile insect technique (SIT) is widely used to suppress or eradicate target pest insect populations. Although the effectiveness of SIT depends on the ability of released sterile males to mate with and inseminate wild females, the use of gamma radiation to induce sterility negatively impacts reproductive cells as well as somatic cells. Consequently, sterilization by irradiation drastically diminishes mating performance over time. In the current study, we evaluated the effect of irradiation dose intensity on fertility, mating propensity, and mating competitiveness in sweetpotato weevil, Cylas formicarius elegantulus (Summers) (Coleoptera: Curculionidae), for 16 d after irradiation. Although the mating propensity of males irradiated with 200 Gy, the dose currently used to induce complete sterility of C. f. elegantulus in the SIT program in Okinawa Prefecture, was equal to that of nonirradiated weevils for the first 6 d, the mating propensity of males irradiated with doses between of 75 and 150 Gy was maintained for the first 12 d. The potential fertilization ability of weevils was highly depressed compared with the control weevils, even in those treated with 75 Gy. Mating performance was severely compromised in weevils that were irradiated with a dose of 100 Gy or more. These results demonstrate that partial sterilization can be highly advantageous in eradication programs for the sweetpotato weevil. We discuss the advantages of the application of partial irradiation in insect eradication programs.     

Enhancing the stability and ecological safety of mass‐reared transgenic strains for field release by redundant conditional lethality systems

The genetic manipulation of agriculturally important insects now allows the development of genetic sexing and male sterility systems for more highly efficient biologically‐based population control programs, most notably the Sterile Insect Technique (SIT), for both plant and animal insect pests. Tetracycline‐suppressible (Tet‐off) conditional lethal systems may function together so that transgenic strains will be viable and fertile on a tetracycline‐containing diet, but female‐lethal and male sterile in tetracycline‐free conditions. This would allow their most efficacious use in a unified system for sterile male‐only production for SIT. A critical consideration for the field release of such transgenic insect strains, however, is a determination of the frequency and genetic basis of lethality revertant survival. This will provide knowledge essential to evaluating the genetic stability of the lethality system, its environmental safety, and provide the basis for modifications ensuring optimal efficacy. For Tet‐off lethal survival determinations, development of large‐scale screening protocols should also allow the testing of these modifications, and test the ability of other conditional lethal systems to fully suppress propagation of rare Tet‐off survivors. If a dominant temperature sensitive (DTS) pupal lethality system proves efficient for secondary lethality in Drosophila, it may provide the safeguard needed to support the release of sexing/sterility strains, and potentially, the release of unisex lethality strains as a form of genetic male sterility. Should the DTS Prosβ2¹ mutation prove effective for redundant lethality, its high level of structural and functional conservation should allow host‐specific cognates to be created for a wide range of insect species.     

Prolongation of the effective copulation period by fractionated‐dose irradiation in the sweet potato weevil,Cylas formicarius

The sterile insect technique (SIT), based on the principles of population and behavioral ecology, is widely used to suppress or eradicate target pest insect populations. The effectiveness of SIT depends on the ability of released sterile males to mate with and inseminate wild females; however, the use of gamma radiation to induce sterility negatively affects both somatic cells as well as reproductive cells. Consequently, sterilization by irradiation drastically diminishes mating performance over time. It is well known that fractionated‐dose irradiation, in which a sterilizing dose is delivered via a series of smaller irradiations, reduces radiation damage. In the present study, we evaluated the effect of fractionated‐dose irradiation on fertility, longevity, and mating propensity in Cylas formicarius (Summers) (Coleoptera: Brentidae) for 16 days after irradiation. Fractionated‐dose irradiation with 200 Gy induced full sterility regardless of the number of radiation doses. Although the mating propensity of males sterilized by a single 200 Gy dose (the current standard of the Okinawa Prefecture SIT program) was equal to that of non‐irradiated weevils for the first 6 days, the mating propensity of males sterilized by a series of three doses was maintained for at least the first 12 days. These results demonstrated that fractionated‐dose irradiation can be highly advantageous in C. formicarius eradication programs.     

Prospects for using genetic transformation for improved SIT and new biocontrol methods

The genetic manipulation of non-drosophilid insect species is possible by the creation of recombinant DNA constructs that can be integrated into host genomes by several transposon-based vector systems. This technology will allow the development and testing of a variety of systems that can improve existing biological control methods, and the development of new highly efficient methods. For programs such as sterile insect technique (SIT), transgenic strains may include fluorescent protein marker genes for detection of released insects, and conditional gene expression systems that will result in male sterility and female lethality for genetic sexing. Conditional expression systems include the yeast GAL4 system and the bacterial Tet-off and Tet-on systems that can, respectively, negatively or positively regulate expression of genes for lethality or sterility depending on a dietary source of tetracycline. Importantly, strains for male sterility must also incorporate an effective system for genetic sexing, since typically, surviving females would remain fertile. Models for the use of these expression systems and associated genetic material come from studies in Drosophila and, while many of these systems should be transferable to other insects, continued research will be necessary in insects of interest to clone genes, optimize germ-line transformation, and perform vector stability studies and risk assessment for their release as transgenic strains.     

Development of genetic sexing strains in Lepidoptera: from traditional to transgenic approaches

The sterile insect technique (SIT) is currently being used for the control of many agricultural pests, including some lepidopteran species. The SIT relies on the rearing and release of large numbers of genetically sterile insects into a wild population. The holokinetic chromosomes of Lepidoptera respond differently to radiation than do species where there is a localized centromere. This difference has enabled a variation of the SIT to be developed for Lepidoptera where a substerilizing dose of radiation is given to the insects before their release with the result that a certain level of sterility is inherited by the F1 offspring. The development of genetic sexing strains for fruit flies, enabling the release of males only, has resulted in enormous economic benefits in the mass rearing and has increased the efficiency of the field operations severalfold. This article outlines Mendelian approaches that are currently available to separate large numbers of males and females efficiently for different lepidopteran species and describes their difficulties and constraints. Successful transgenesis in several lepidopteran species opens up new possibilities to develop genetic sexing strains. The proposal to develop genetic sexing strains described in this article takes advantage of the fact that in Lepidoptera, the female is the heterogametic sex, with most species having aWZ sex chromosome pair, whereas the males are ZZ. This means that if a conditional lethal gene can be inserted into the W chromosome, then all females should die after the application of the restrictive condition. The assumptions made to accommodate this model are discussed, and the advantages to be gained for control programs are elucidated.     

Fractionated irradiation improves the mating performance of the West Indian sweet potato weevil Euscepes postfasciatus

• 1 The sterile insect technique (SIT) is widely used to suppress or eradicate target pest insect populations.
• 2 The effectiveness of SIT depends on the ability of released sterile males to mate with and inseminate wild females. The use of gamma radiation to induce sterility, however, negatively affects both somatic cells as well as reproductive cells. Consequently, mating performance of sterilized individuals decreases drastically over time. The mating propensity of sterilized Euscepes postfasciatus (Fairmaire) males irradiated with a single dose of 150 Gy (the current standard of the Okinawa Prefecture SIT programme) is equal to that of non‐irradiated weevils for the first 6 days.
• 3 Fractionated irradiation, in which a sterilizing dose is delivered over time in a series of smaller irradiations, reduces the damage of irradiation in insects. In the present study, we evaluated the effect of fractionated irradiation on male fertilization ability, longevity and mating propensity of E. postfasciatus for a period of 16 days after irradiation.
• 4 Although fractionated irradiation totalling 150 Gy was found to induce full sterility regardless of the number of individual doses, the mating propensity of male weevils sterilized by fractionated irradiation was maintained for the first 12 days. These results demonstrate that fractionated irradiation can be highly advantageous in programmes aimed at eradication of E. postfasciatus.

     

Genetic elimination of field-cage populations of Mediterranean fruit flies

The Mediterranean fruit fly (medfly, Ceratitis capitata Wiedemann) is a pest of over 300 fruits, vegetables and nuts. The sterile insect technique (SIT) is a control measure used to reduce the reproductive potential of populations through the mass release of sterilized male insects that mate with wild females. However, SIT flies can display poor field performance, due to the effects of mass-rearing and of the irradiation process used for sterilization. The development of female-lethal RIDL (release of insects carrying a dominant lethal) strains for medfly can overcome many of the problems of SIT associated with irradiation. Here, we present life-history characterizations for two medfly RIDL strains, OX3864A and OX3647Q. Our results show (i) full functionality of RIDL, (ii) equivalency of RIDL and wild-type strains for life-history characteristics, and (iii) a high level of sexual competitiveness against both wild-type and wild-derived males. We also present the first proof-of-principle experiment on the use of RIDL to eliminate medfly populations. Weekly releases of OX3864A males into stable populations of wild-type medfly caused a successive decline in numbers, leading to eradication. The results show that genetic control can provide an effective alternative to SIT for the control of pest insects.     

Conceptual framework and rationale

The sterile insect technique (SIT) has been shown to be an effective and sustainable genetic approach to control populations of selected major pest insects, when part of area-wide integrated pest management (AW-IPM) programmes. The technique introduces genetic sterility in females of the target population in the field following their mating with released sterile males. This process results in population reduction or elimination via embryo lethality caused by dominant lethal mutations induced in sperm of the released males. In the past, several field trials have been carried out for mosquitoes with varying degrees of success. New technology and experience gained with other species of insect pests has encouraged a reassessment of the use of the sterility principle as part of integrated control of malaria vectors. Significant technical and logistic hurdles will need to be overcome to develop the technology and make it effective to suppress selected vector populations, and its application will probably be limited to specific ecological situations. Using sterile males to control mosquito vector populations can only be effective as part of an AW-IPM programme. The area-wide concept entails the targeting of the total mosquito population within a defined area. It requires, therefore, a thorough understanding of the target pest population biology especially as regards mating behaviour, population dynamics, dispersal and level of reproductive isolation. The key challenges for success are: 1) devising methods to monitor vector populations and measuring competitiveness of sterile males in the field, 2) designing mass rearing, sterilization and release strategies that maintain competitiveness of the sterile male mosquitoes, 3) developing methods to separate sexes in order to release only male mosquitoes and 4) adapting suppression measures and release rates to take into account the high reproductive rate of mosquitoes. Finally, success in area-wide implementation in the field can only be achieved if close attention is paid to political, socio-economic and environmental sensitivities and an efficient management organization is established taking into account the interests of all potential stakeholders of an AW-IPM programme.     

Re-engineering the sterile insect technique

The mass release of sterile insects (the Sterile Insect Technique, SIT) is a highly effective area-wide method of pest control with a low environmental impact. SIT relies on the sterilization by irradiation of large numbers of insects. This has unavoidable costs in terms of the fitness of the irradiated insects and the financial requirements of constructing and operating the radiation facility. In many cases it is considered important to release only males, but large-scale sex-separation is also problematic. I have proposed that both of these difficulties can be overcome by using engineered strains of insects carrying a dominant, repressible, lethal gene or genetic system. As a proof of principle, my group and others have constructed strains of Drosophila melanogaster with the required genetic properties.     

Molecular technologies to improve the effectiveness of the sterile insect technique

The application of the Sterile Insect Technique (SIT) in area-wide integrated pest management (AW-IPM) programmes continues to increase. However, programme efficiency can still be considerably enhanced when certain components of the technology are improved, such as the development of improved strains for mass rearing and release. These include strains that (1) produce only male insects for sterilization and release and (2) carry easily identifiable markers to identify released sterile insects in the field. Using both classical and modern biotechnology techniques, key insect pests are targeted, where SIT programmes are being implemented. The pests include mosquitoes, the Mexican fruit fly, the codling moth, the oriental fruit fly and the pink bollworm. This special issue summarizes the results of research efforts aimed at the development and evaluation of new strains to a level where a decision can be made as to their suitability for use in large scale SIT programmes. Major beneficiaries will be operational AW-IPM programmes that apply the SIT against major insect pests.     

A transgene-based,embryo-specific lethality system for insect pest management

Biological approaches to insect pest management offer alternatives to pesticidal control. In area-wide control programs that cover entire regions, the sterile insect technique (SIT) can be used to successfully suppress economically important pest species by the mass release of sterilized pest organisms. However, conventional sterilization by ionizing radiation reduces insect fitness, which can result in reduced competitiveness of the sterilized insects. Here we report a transgene-based, dominant embryonic lethality system that allows for generation of large quantities of competitive but sterile insects without the need of irradiation. The system involves the ectopic expression of a hyperactive pro-apoptotic gene that causes embryo-specific lethality when driven by the tetracycline-controlled transactivator (tTA) under the regulation of a cellularization gene enhancer-promoter. We have successfully tested this system in Drosophila melanogaster. The embryonic lethality can be suppressed maternally, which will allow it to be combined with transgenic female-specific lethality systems to raise only vigorous but sterile males.     

Radiation biology of mosquitoes

There is currently renewed interest in assessing the feasibility of the sterile insect technique (SIT) to control African malaria vectors in designated areas. The SIT relies on the sterilization of males before mass release, with sterilization currently being achieved through the use of ionizing radiation. This paper reviews previous work on radiation sterilization of Anopheles mosquitoes. In general, the pupal stage was irradiated due to ease of handling compared to the adult stage. The dose-response curve between the induced sterility and log (dose) was shown to be sigmoid, and there was a marked species difference in radiation sensitivity. Mating competitiveness studies have generally been performed under laboratory conditions. The competitiveness of males irradiated at high doses was relatively poor, but with increasing ratios of sterile males, egg hatch could be lowered effectively. Males irradiated as pupae had a lower competitiveness compared to males irradiated as adults, but the use of partially-sterilizing doses has not been studied extensively. Methods to reduce somatic damage during the irradiation process as well as the use of other agents or techniques to induce sterility are discussed. It is concluded that the optimal radiation dose chosen for insects that are to be released during an SIT programme should ensure a balance between induced sterility of males and their field competitiveness, with competitiveness being determined under (semi-) field conditions. Self-contained ⁶⁰Co research irradiators remain the most practical irradiators but these are likely to be replaced in the future by a new generation of high output X ray irradiators.     

Transgenic sexing system for Ceratitis capitata (Diptera: Tephritidae) based on female-specific embryonic lethality

Fruit fly pest species have been successfully controlled and managed via the Sterile Insect Technique (SIT), a control strategy that uses infertile matings of sterile males to wild females to reduce pest populations. Biological efficiency in the field is higher if only sterile males are released in SIT programs and production costs are also reduced. Sexing strains developed in the Mediterranean fruit fly Ceratitis capitata (medfly) through classical genetics are immensely beneficial to medfly SIT programs but exhibit reduced fertility and fitness. Moreover, transfer of such classical genetic systems to other tephritid species is difficult. Transgenic approaches can overcome this limitation of classical genetic sexing strains (GSSs), but had resulted so far in transgenic sexing strains (TSSs) with dominant lethality at late larval and pupal stages. Here we present a transgene-based female-specific lethality system for early embryonic sexing in medfly. The system utilizes the sex-specifically spliced transformer intron to restrict ectopic mRNA translation of the pro-apoptotic gene hid^Ala5 to females only. The expression of this lethal effector gene is driven by a tetracycline-repressible transactivator gene tTA that is under the control of promoters/enhancers of early-acting cellularization genes. Despite observed position effects on the sex-specific splicing, we could effectively establish this early-acting transgenic sexing system in the medfly C. capitata. After satisfactory performance in large scale tests, TSSs based on this system will offer cost-effective sexing once introduced into SIT programs. Moreover, this approach is straight forward to be developed also for other insect pest and vector species.     

Genetic sexing through the use of Y-linked transgenes

Sterile insect technique (SIT)-based pest control programs rely on the mass release of sterile insects to reduce the wild target population. In many cases, it is desirable to release only males. Sterile females may cause damage, e.g., disease transmission by mosquitoes or crop damage via oviposition by the Mediterranean fruit fly (Medfly). Also, sterile females may decrease the effectiveness of released males by distracting them from seeking out wild females. To eliminate females from the release population, a suitable sexual dimorphism is required. For several pest species, genetic sexing strains have been constructed in which such a dimorphism has been induced by genetics. Classical strains were based on the translocation to the Y chromosome of a selectable marker, which is therefore expressed only in males. Recently, several prototype strains have been constructed using sex-specific expression of markers or conditional lethal genes from autosomal insertions of transgenes. Here, we describe a novel genetic sexing strategy based on the use of Y-linked transgenes expressing fluorescent proteins. We demonstrate the feasibility of this strategy in a major pest species, Ceratitis capitata (Wiedemann), and discuss the advantages and disadvantages relative to other genetic sexing methods and potential applicability to other species.     

Fluorescent sperm marking to improve the fight against the pest insect Ceratitis capitata (Wiedemann; Diptera: Tephritidae)

The Sterile Insect Technique (SIT) involving area-wide release of mass-reared and sterilized pest insects has proven successful to reduce, control and eradicate economically important pest species, such as the Mediterranean fruit fly (medfly). For the efficient application, effective monitoring to assess the number and mating success of the released medflies is essential. Here, we report sperm-specific marking systems based on the spermatogenesis-specific Ceratitis capitata beta2-tubulin (Ccbeta2t) promoter. Fluorescent sperm can be isolated from testes or spermathecae. The marking does not cause general disadvantages in preliminary laboratory competitiveness assays. Therefore, transgenic sperm marking could serve as a major improvement for monitoring medfly SIT programs. The use of such harmless transgenic markers will serve as an ideal initial condition to transfer insect transgenesis technology from the laboratory to field applications. Moreover, effective and easily recognizable sperm marking will make novel studies possible on medfly reproductive biology which will help to further improve SIT programs.     

piggyBac transformation of the New World screwworm, Cochliomyia hominivorax, produces multiple distinct mutant strains

Sterile insect technique (SIT) programs are designed to eradicate pest species by releasing mass-reared, sterile insects into an infested area. The first major implementation of SIT was the New World Screwworm Eradication Program, which successfully eliminated the New World screwworm (NWS), Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae), from the Continental US, Mexico and much of Central America. Ionizing radiation is currently used for sterilization, but transgenic insect techniques could replace this method, providing a safer, more cost-effective alternative. Genetic transformation methods have been demonstrated in NWS, and verified by Southern blot hybridization, PCR and sequencing of element insertion junctions. A lethal insertional mutation and enhancer detection-like phenotypic expression variations are presented and discussed. In addition to supporting the eradication efforts, transformation methods offer potential means to identify genes and examine gene function in NWS.     

Developing transgenic Anopheles mosquitoes for the sterile insect technique

In the last 10 years the availability of the genome sequence of Anopheles gambiae and the development of a transgenic technology for several species of Anopheles mosquitoes have, in combination, helped in enabling us to gain several insights into the biology of these mosquitoes that is relevant to their capacity as vectors of the malaria parasite. While this information is anticipated to inform many novel vector control strategies, the technique most likely to benefit in the near future from the availability of a reliable transgenic technology is the sterile insect technique (SIT), which relies on releasing large numbers of sterile insects to compete for mates in the wild, leading to population suppression. Although SIT has been proven to work reliably for many insects, the construction of suitable strains, and induction of sterility, has until now been a laborious process, combining classical genetics with radiation-induced sterility. Using transgenesis to create strains of Anopheles suitable for SIT could potentially offer several advantages over current approaches, in that the basic design of transgenic constructs designed for other insects should be rapidly transferable to mosquitoes, and induction of sterility as a product of the transgenic modification could obviate the requirement for radiation and its associated deleterious effects. In this paper the progress of different transgenic approaches in constructing tools for SIT will be reviewed.