Development Of Genetic Sexing Strains Research Articles

Deep orange gene editing triggers temperature-sensitive lethal phenotypes in Ceratitis capitata

BackgroundThe Mediterranean fruit fly, Ceratitis capitata, is a significant agricultural pest managed through area-wide integrated pest management (AW-IPM) including a sterile insect technique (SIT) component. Male-only releases increase the efficiency and cost-effectiveness of SIT programs, which can be achieved through the development of genetic sexing strains (GSS). The most successful GSS developed to date is the C. capitata VIENNA 8 GSS, constructed using classical genetic approaches and an irradiation-induced translocation with two selectable markers: the white pupae (wp) and temperature-sensitive lethal (tsl) genes. However, currently used methods for selecting suitable markers and inducing translocations are stochastic and non-specific, resulting in a laborious and time-consuming process. Recent efforts have focused on identifying the gene(s) and the causal mutation(s) for suitable phenotypes, such as wp and tsl, which could be used as selectable markers for developing a generic approach for constructing GSS. The wp gene was recently identified, and efforts have been initiated to identify the tsl gene. This study investigates Ceratitis capitata deep orange (Ccdor) as a tsl candidate gene and its potential to induce tsl phenotypes.ResultsAn integrated approach based on cytogenetics, genomics, bioinformatics, and gene editing was used to characterize the Ccdor. Its location was confirmed on the right arm of chromosome 5 in the putative tsl genomic region. Knock-out of Ccdor using CRISPR/Cas9-NHEJ and targeting the fourth exon resulted in lethality at mid- and late-pupal stage, while the successful application of CRISPR HDR introducing a point mutation on the sixth exon resulted in the establishment of the desired strain and two additional strains (dor 12del and dor 51dup), all of them expressing tsl phenotypes and presenting no (or minimal) fitness cost when reared at 25 °C. One of the strains exhibited complete lethality when embryos were exposed at 36 °C.ConclusionsGene editing of the deep orange gene in Ceratitis capitata resulted in the establishment of temperature-sensitive lethal mutant strains. The induced mutations did not significantly affect the rearing efficiency of the strains. As deep orange is a highly conserved gene, these data suggest that it can be considered a target for the development of tsl mutations which could potentially be used to develop novel genetic sexing strains in insect pests and disease vectors.

Silencing transformer and transformer-2 in Zeugodacus cucurbitae causes defective sex determination with inviability of most pseudomales

transformer is a switch gene for sex determination in many insects, which cooperates with transformer-2 that is expressed in both sexes to regulate female differentiation, particularly in dipterans. Zeugodacus cucurbitae (Coquillett) is a very destructive pest worldwide, however, its sex determination pathway remains largely uncharacterized. Here, we show that the female sex ratio is sharply reduced with knockdown of either transformer or transformer-2 by RNA interference in early embryos of Z. cucurbitae. Most of the males grown from the embryos with transient transformer and transformer-2 suppression mated with wild-type females and produced mixed sex progeny, with one exception that produced only female progeny, and all of the few remaining males failed to mate with wild-type females and produced no progeny. The exceptional male and those males with mating failure were XX pseudomales as determined by the detection of Y chromosome-linked Maleness-on-the-Y, indicating that most XX pseudomales are not viable. The phenotypes of transformer and transformer-2 suggest that they play a key role in regulating sex determination and are required for female sexual development of Z. cucurbitae. Our results will be beneficial to the understanding of sex determination in Z. cucurbitae and can facilitate the development of genetic sexing strains for its biological control.

Maleness-on-the-Y (MoY) orthologue is a key regulator of male sex determination in Zeugodacus cucurbitae (Diptera: Tephritidae)

The initiation of sex differentiation in insects is regulated by primary sex determination signals. In the Medfly Ceratitis capitata and other Tephritids, Maleness-on-the-Y (MoY) is the master gene for male sex determination. However, the primary signal in Zeugodacus cucurbitae (Coquillett), a very destructive Tephritid pest across the world, remains ambiguous. In this study, we have isolated and characterized the Medfly MoY homolog in Z. cucurbitae, ZcMoY. ZcMOY protein shows high sequence conservation to its homologs in Bactrocera species. ZcMoY transcription begins and peaks at very early embryonic stages and then becomes undetectable except the testes and heads of day 1 male adults. Silencing ZcMoY in early embryos by RNAi causes abnormal external genitalia and interior reproductive organs, giving rise to intersexes and feminization of XY individuals. The expression pattern and knockdown phenotypes of ZcMoY indicate that ZcMoY plays a key role in regulating sex determination of Z. cucurbitae males. Our findings will help the understanding of sex determination in Z. cucurbitae and facilitate the development of genetic sexing strains in its biological control.

Transcriptome analysis of sex-biased gene expression in the spotted-wing Drosophila, Drosophila suzukii (Matsumura).

Sexual dimorphism occurs widely throughout insects and has profound influences on evolutionary path. Sex-biased genes are considered to account for most of phenotypic differences between sexes. In order to explore the sex-biased genes potentially associated with sexual dimorphism and sexual development in Drosophila suzukii, a major devastating and invasive crop pest, we conducted whole-organism transcriptome profiling and sex-biased gene expression analysis on adults of both sexes. We identified transcripts of genes involved in several sex-specific physiological and functional processes, including transcripts involved in sex determination, reproduction, olfaction, and innate immune signals. A total of 11,360 differentially expressed genes were identified in the comparison, and 1,957 differentially expressed genes were female-biased and 4,231 differentially expressed genes were male-biased. The pathway predominantly enriched for differentially expressed genes was related to spliceosome, which might reflect the differences in the alternative splicing mechanism between males and females. Twenty-two sex determination and 16 sex-related reproduction genes were identified, and expression pattern analysis revealed that the majority of genes were differentially expressed between sexes. Additionally, the differences in sex-specific olfactory and immune processes were analyzed and the sex-biased expression of these genes may play important roles in pheromone and odor detection, and immune response. As a valuable dataset, our sex-specific transcriptomic data can significantly contribute to the fundamental elucidation of the molecular mechanisms of sexual dimorphism in fruit flies, and may provide candidate genes potentially useful for the development of genetic sexing strains, an important tool for sterile insect technique applications against this economically important species.

Sex separation ofAedesspp. mosquitoes for sterile insect technique application: a review

AbstractSeparation of the sexes is necessary for the application of the sterile insect technique (SIT) in mosquitoes due to the hematophagous habits and disease vector activity of the females. In this review we analyze the history, current status, and future perspectives for the development of genetic sexing strains (GSS) ofAedesmosquitoes (Diptera: Culicidae). Various genetic control methods for mosquitoes are reviewed, as are their need for sex‐separation methods. We focus on areas of opportunity where GSS developed with classical genetic methods can be used. Regulatory restrictions and social acceptance of various control methods are analyzed. We conclude that the development of GSS by classical methods represents the most viable option for separation of the sexes and the application of large‐scale SIT programs within an area‐wide integrated vector management (AW‐IVM) approach.

The Insect Pest Control Laboratory of the Joint FAO/IAEA Programme: Ten Years (2010-2020) of Research and Development, Achievements and Challenges in Support of the Sterile Insect Technique.

Simple SummaryThe Insect Pest Control (IPC) Section and its associated laboratory (IPCL) is part of the Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture. Its mandate is to develop and implement the sterile insect technique (SIT) for selected key insect pests, and thereby to reduce the use of insecticides, to reduce animal and crop losses, to protect the environment, to facilitate international trade in agricultural commodities and to improve human health. With this aim, the IPCL has been implementing research in relation to the development of the SIT package for insect pests of crops, livestock and human health. This paper provides a review of the research carried out between 2010 and 2020 at the IPCL.The Joint FAO/IAEA Centre (formerly called Division) of Nuclear Techniques in Food and Agriculture was established in 1964 and its accompanying laboratories in 1961. One of its subprograms deals with insect pest control, and has the mandate to develop and implement the sterile insect technique (SIT) for selected key insect pests, with the goal of reducing the use of insecticides, reducing animal and crop losses, protecting the environment, facilitating international trade in agricultural commodities and improving human health. Since its inception, the Insect Pest Control Laboratory (IPCL) (formerly named Entomology Unit) has been implementing research in relation to the development of the SIT package for insect pests of crops, livestock and human health. This paper provides a review of research carried out between 2010 and 2020 at the IPCL. Research on plant pests has focused on the development of genetic sexing strains, characterizing and assessing the performance of these strains (e.g., Ceratitis capitata), elucidation of the taxonomic status of several members of the Bactrocera dorsalis and Anastrepha fraterculus complexes, the use of microbiota as probiotics, genomics, supplements to improve the performance of the reared insects, and the development of the SIT package for fruit fly species such as Bactrocera oleae and Drosophila suzukii. Research on livestock pests has focused on colony maintenance and establishment, tsetse symbionts and pathogens, sex separation, morphology, sterile male quality, radiation biology, mating behavior and transportation and release systems. Research with human disease vectors has focused on the development of genetic sexing strains (Anopheles arabiensis, Aedes aegypti and Aedes albopictus), the development of a more cost-effective larvae and adult rearing system, assessing various aspects of radiation biology, characterizing symbionts and pathogens, studying mating behavior and the development of quality control procedures, and handling and release methods. During the review period, 13 coordinated research projects (CRPs) were completed and six are still being implemented. At the end of each CRP, the results were published in a special issue of a peer-reviewed journal. The review concludes with an overview of future challenges, such as the need to adhere to a phased conditional approach for the implementation of operational SIT programs, the need to make the SIT more cost effective, to respond with demand driven research to solve the problems faced by the operational SIT programs and the use of the SIT to address a multitude of exotic species that are being introduced, due to globalization, and established in areas where they could not survive before, due to climate change.

Development and characterization of a pupal-colour based genetic sexing strain of Anastrepha fraterculus sp. 1 (Diptera: Tephritidae)

BackgroundArea-wide integrated pest management programs (AW-IPM) incorporating sterile insect technique (SIT) have been successful in suppressing populations of different fruit fly species during the last six decades. In addition, the development of genetic sexing strains (GSS) for different fruit fly species has allowed for sterile male-only releases and has significantly improved the efficacy and cost effectiveness of the SIT applications. The South American Fruit Fly Anastrepha fraterculus (Diptera: Tephritidae) is a major agricultural pest attacking several fruit commodities. This impedes international trade and has a significant negative impact on the local economies. Given the importance of sterile male-only releases, the development of a GSS for A. fraterculus would facilitate the implementation of an efficient and cost-effective SIT operational program against this insect pest species.ResultsFor potential use in a GSS, three new morphological markers (mutants) were isolated in a laboratory strain of A. fraterculus sp. 1, including the black pupae (bp) gene located on chromosome VI. The black pupa phenotype was used as a selectable marker to develop genetic sexing strains by linking the wild type allele (bp+) to the Y-chromosome -via irradiation to induce a reciprocal Y-autosome translocation. Four GSS were established and one of them, namely GSS-89, showed the best genetic stability and the highest fertility. This strain was selected for further characterization and cytogenetic analysis.ConclusionsWe herein report the development of the first genetic sexing strain of a major agricultural pest, A. fraterculus sp. 1, using as a selectable marker the black pupae genetic locus.

Irradiation induced inversions suppress recombination between the M locus and morphological markers in Aedes aegypti

BackgroundAedes aegypti is the primary vector of arthropod-borne viruses and one of the most widespread and invasive mosquito species. Due to the lack of efficient specific drugs or vaccination strategies, vector population control methods, such as the sterile insect technique, are receiving renewed interest. However, availability of a reliable genetic sexing strategy is crucial, since there is almost zero tolerance for accidentally released females. Development of genetic sexing strains through classical genetics is hindered by genetic recombination that is not suppressed in males as is the case in many Diptera. Isolation of naturally-occurring or irradiation-induced inversions can enhance the genetic stability of genetic sexing strains developed through genetically linking desirable phenotypes with the male determining region.ResultsFor the induction and isolation of inversions through irradiation, 200 male pupae of the ‘BRA’ wild type strain were irradiated at 30 Gy and 100 isomale lines were set up by crossing with homozygous ‘red-eye’ (re) mutant females. Recombination between re and the M locus and the white (w) gene (causing a recessive white eye phenotype when mutated) and the M locus was tested in 45 and 32 lines, respectively. One inversion (Inv35) reduced recombination between both re and the M locus, and wand the M locus, consistent with the presence of a rather extended inversion between the two morphological mutations, that includes the M locus. Another inversion (Inv5) reduced recombination only between w and the M locus. In search of naturally-occurring, recombination-suppressing inversions, homozygous females from the red eye and the white eye strains were crossed with seventeen and fourteen wild type strains collected worldwide, representing either recently colonized or long-established laboratory populations. Despite evidence of varying frequencies of recombination, no combination led to the elimination or substantial reduction of recombination.ConclusionInducing inversions through irradiation is a feasible strategy to isolate recombination suppressors either on the M or the m chromosome for Aedes aegypti. Such inversions can be incorporated in genetic sexing strains developed through classical genetics to enhance their genetic stability and support SIT or other approaches that aim to population suppression through male-delivered sterility.

An Efficient Duplex PCR Method for Sex Identification of the European Grapevine Moth Lobesia botrana (Lepidoptera: Tortricidae) at Any Developmental Stage.

Many genetic studies in insects require sex identification of individuals in all developmental stages. The most common sex chromosome system in lepidopterans is WZ/ZZ; the W chromosome is present only in females. Based on two W chromosome-specific short sequences (CpW2 and CpW5) described in Cydia pomonella (L.) (Lepidoptera: Tortricidae), we identified homologous female-specific sequences in Lobesia botrana Den. & Schiff, a polyphagous and very harmful species present in Chile since 2008. From this starting point, we extended the sequence information using the inverse PCR method, identifying the first W-specific sequences described up to now for the moth. Finally, we developed a duplex PCR method for rapid and sensitive determination of sex in L. botrana from larva to adult. The method showed a detection limit of 1 pg of genomic DNA; a blind panel of samples exhibited exact correspondence with the morphological identification. These results will be very useful for studies requiring sex-specific analyses at any developmental stage, contributing also to the understanding of gene expression in the insect, as well as to the eventual development of control protocols against the moth, such as the development of genetic sexing strains for the implementation of the sterile insect technique.

Advances and Challenges of Using the Sterile Insect Technique for the Management of Pest Lepidoptera.

Over the past 30 years, the sterile insect technique (SIT) has become a regular component of area-wide integrated pest management (AW-IPM) programs against several major agricultural pests and vectors of severe diseases. The SIT-based programs have been especially successful against dipteran pests. However, the SIT applicability for controlling lepidopteran pests has been challenging, mainly due to their high resistance to the ionizing radiation that is used to induce sterility. Nevertheless, the results of extensive research and currently operating SIT programs show that most problems with the implementation of SIT against pest Lepidoptera have been successfully resolved. Here, we summarize the cytogenetic peculiarities of Lepidoptera that should be considered in the development and application of SIT for a particular pest species. We also discuss the high resistance of Lepidoptera to ionizing radiation, and present the principle of derived technology based on inherited sterility (IS). Furthermore, we present successful SIT/IS applications against five major lepidopteran pests, and summarize the results of research on the quality control of reared and released insects, which is of great importance for their field performance. In the light of new research findings, we also discuss options for the development of genetic sexing strains, which is a challenge to further improve the applicability of SIT/IS against selected lepidopteran pests.

Development of genetic sexing strain of the oriental fruit fly, Bactrocera dorsalis (Hendel)

A genetic sexing strain (GSS) of the oriental fruit fly, Bactrocera dorsalis (Hendel) has been developed in order to release only sterile males to increase the efficiency of fruit fly control by using sterile insect technique. To compare the quality of GSS with the genetic marker strain from a mass-reared colony, it was found that eclosion, flight ability and fertility of GSS equal to 69.57, 53.14 and 47.48 % respectively, which were significantly lower than those of the genetic-marker strain. The adult eclosion, flight ability and fertility of the genetic-marker strain equal to 93.66, 87.00 and 60.48 % respectively. To study the effects of irradiation, the 2-days-before-emergence pupae of GSS were irradiated at doses of 70 and 90 Gy. It was found that sterility and competitiveness index of GSS irradiated at 70 and 90 Gy were 96.15, 97.12 % and 0.51, 0.39 respectively.

Medfly Gut Microbiota and Enhancement of the Sterile Insect Technique: Similarities and Differences of Klebsiella oxytoca and Enterobacter sp. AA26 Probiotics during the Larval and Adult Stages of the VIENNA 8D53+ Genetic Sexing Strain

The Mediterranean fruit fly, Ceratitis capitata, is a major agricultural pest worldwide. The development of genetic sexing strains (GSSs) for this species that allows male-only sterile insects releases has boosted the effectiveness of the environmental friendly pest control method known as the sterile insect technique. The last generation of these strains, the VIENNA 7 and VIENNA 8, are currently used in all mass rearing facilities worldwide and are considered as models for such pest control applications. The sterile insect technique depends on the rearing of sufficient numbers of adequate “biological quality” laboratory flies to be released in the field. Currently, there is an increasing amount of studies focusing on the characterization of the symbiotic communities and development of probiotic diets. In our study, two bacterial isolates, an Enterobacter sp. (strain AA26) and a Klebsiella oxytoca strain, were used as probiotics in larval and adult diet. These strains have been shown to be beneficial, affecting several aspects related to the rearing efficiency and biological quality of the medfly VIENNA 8D53+ GSS. Our results demonstrate the effect of K. oxytoca on the developmental duration of the immature stages and, to some extent, on flight ability. On the other hand, our study does not support the presence of any beneficial effect of (a) K. oxytoca on pupal and adult recovery and adults’ survival under stress conditions when provided as a larval diet supplement and (b) K. oxytoca and Enterobacter sp. AA26 on mating competitiveness when provided as adult diet supplements. Possible explanations for inconsistencies with previous studies and the need for universalizing protocols are discussed. Our findings, combined with previous studies can support the sterile insect technique, through the improvement of different aspects of mass rearing and biological properties of laboratory reared insect pests.

A review of more than 30 years of cytogenetic studies of Tephritidae in support of sterile insect technique and global trade

AbstractCytogenetics of Tephritidae has contributed for more than 30 years to the efficient control of pest members of this family and the species delimitation among them. The sterile insect technique (SIT) is a species‐specific and environmentally friendly method for the population control of insect pest species, always as a component of integrated pest management approaches. The construction of polytene chromosome maps facilitated the development, characterization, and improvement of genetic sexing strains, which have boosted the effectiveness of SIT and are considered as models for developing genetic sexing methodologies in other target species. In parallel, characterization of mitotic karyotypes and the availability of polytene chromosomes in Diptera have been and are being used as supplementary evidence in taxonomic studies. Lately, under the common understanding that speciation can be achieved via various pathways and under the umbrella of integrative taxonomy, cytogenetic studies have contributed to multidisciplinary taxonomic approaches. Such studies can shed light on the borders between closely related species and/or incipient speciation, and this is crucial both for the implementation of SIT and the revision/improvement of quarantine policies. In this review, we summarize the cytogenetic status of Tephritidae and discuss the contribution of cytogenetics to the development of genetic sexing strains in this family.

Cryopreservation of Embryos of the Mediterranean Fruit Fly Ceratitis capitata Vienna 8 Genetic Sexing Strain.

The Mediterranean fruit fly, Ceratitis capitata, is one of the most serious pests of fruit crops world-wide. During the last decades, area-wide pest management (AW-IPM) approaches with a sterile insect technique (SIT) component have been used to control populations of this pest in an effective and environment-friendly manner. The development of genetic sexing strains (GSS), such as the Vienna 8 strain, has been played a major role in increasing the efficacy and reducing the cost of SIT programs. However, mass rearing, extensive inbreeding, possible bottleneck phenomena and hitch-hiking effects might pose major risks for deterioration and loss of important genetic characteristics of domesticated insect. In the present study, we present a modified procedure to cryopreserve the embryos of the medfly Vienna 8 GSS based on vitrification and used this strain as insect model to assess the impact of the cryopreservation process on the genetic structure of the cryopreserved insects. Forty-eight hours old embryos, incubated at 24°C, were found to be the most suitable developmental stage for cryopreservation treatment for high production of acceptable hatch rate (38%). Our data suggest the absence of any negative impact of the cryopreservation process on egg hatch rate, pupation rates, adult emergence rates and stability of the temperature sensitive lethal (tsl) character on two established cryopreserved lines (flies emerged from cryopreserved embryos), named V8-118 and V8-228. Taken together, our study provides an optimized procedure to cryopreserve the medfly Vienna 8 GSS and documents the absence of any negative impact on the genetic structure and quality of the strain. Benefits and sceneries for utilization of this technology to support operational SIT projects are discussed in this paper.

Genome size estimation with quantitative real‐time PCR in two Tephritidae species: Ceratitis capitata and Bactrocera oleae

AbstractThe medfly Ceratitis capitata and the olive fruit fly Bactrocera oleae belong to the Tephritidae family of Diptera, a family whose members cause severe damages in agriculture worldwide. For such insect pests, the utmost concern is their population control. The sterile insect technique (SIT) has been used in the Tephritidae family with varying degrees of success. Its efficient use usually depends on the development of genetic sexing strains and the release of only male flies. However, such advances are based on modern genetic, molecular and genomic tools. The medfly is clearly the prototype of the family, since such tools have advanced considerably, which has resulted in effective SIT efforts around the world. A whole‐genome sequencing project of this insect is already underway. In contrast, similar tools in the olive fly lag behind, even though the insect is considered a promising candidate for a next SIT target. An accurate estimate of genome size provides a preliminary view of genome complexity and indicates possible difficulties in genome assembly in whole‐genome projects. Taking advantage of a quantitative real‐time PCR approach, we determined the genome size of these two species C. capitata and B. oleae as 591 Mb (CI range: 577–605 Mb) and 322 Mb (CI range: 310–334 Mb) respectively.

Ionising radiation and area-wide management of insect pests to promote sustainable agriculture. A review

Despite the liberal use of broad-spectrum insecticides to keep many insect pests of agricultural and veterinary importance at bay, food losses, both pre- and post-harvest, due to these insect pests contribute significantly to the high prevalence of undernourishment in the world. New, innovative pest control tactics and strategies are therefore needed that are both effective and not detrimental to the environment. As part of the arsenal of environmentally-friendly control tactics, the sterile insect technique (SIT) has proven to be a very effective tool against selected insect pests when used as part of an area-wide integrated pest management (AW-IPM) approach. Likewise, the use of natural enemies for augmentative or inundative biological control is now a major component of pest control in many parts of the world. Both control tactics are complementary and even synergistic under certain circumstances, but their combined use has so far not been applied on an operational scale. Ionising radiation can be readily employed to effectively and safely induce sexual sterility in insects. Although the sterile insect technique has often been associated with an eradication strategy, major advances in rearing efficiency, and improved handling and release methods, have made the use of sterile insects economically feasible for insect pest suppression, prevention or containment. Recently, more emphasis has been placed on the quality of the sterile insect once released in the field rather than mainly assessing quality in the rearing facility. This combined with other innovations such as the development of genetic sexing strains, better understanding the impact of radiation on radio-resistant species such as Lepidoptera and the development of the F1 sterility concept, advances in monitoring the induced sterility, etc. have significantly increased the efficiency of the sterile insect technique for several insect species. The action of sterile insects is inversely dependent on the density of the target population, and sterile insects have the intrinsic capacity to actively search for and mate with the last individuals of a pest population. These two characteristics make them ideal to deal with outbreaks of invasive insect pests. The use of sterile insects presents no threat to the environment, but aspects such as diet and waste disposal in large rearing facilities or bio-security in cases where the rearing facility is located in an area that is already free of the pest require the necessary attention. Ionising radiation can also be applied to greatly improve the efficiency of mass-rearing, handling and shipment of insect parasitoids and predators. Area-wide integrated pest management programmes that use sterile insects or natural enemies are complex and management-intensive, and require a management structure that is exclusively dedicated to the programme. Past and current examples have shown the enormous benefit-cost ratios that these programmes can generate and their importance for enhanced agriculture is increasing in significance.

Improvement of the sterile insect technique for codling moth Cydia pomonella (Linnaeus) (Lepidoptera Tortricidae) to facilitate expansion of field application

AbstractThe codling moth Cydia pomonella (L.) (Lepidoptera Tortricidae) is a key pest of pome fruit (apple, pear and quince) and walnut orchards in most temperate regions of the world. Efforts to control the codling moth in the past mostly relied on the use of broad spectrum insecticide sprays, which has resulted in the development of insecticide resistance, and the disruption of the control of secondary pests. In addition, the frequent reliance and use of these insecticides are a constant threat to the environment and human health. Consequently, there have been increased demands from the growers for the development of codling moth control tactics that are not only effective but also friendly to the environment. In that respect, the sterile insect technique (SIT) and its derivative, inherited sterility (IS), are, together with mating disruption and granulosis virus, among the options that offer great potential as cost‐effective additions to available control tactics for integration in area‐wide integrated pest‐management approaches. In support of the further development of the SIT/IS for codling moth control, the Joint FAO/IAEA Programme of Nuclear Techniques in Food and Agriculture implemented a 5‐year Coordinated Research Project (CRP) entitled ‘Improvement of codling moth SIT to facilitate expansion of field application’. Research focussed on sterile codling moth quality and management (e.g. mobility and life‐history traits in relation to rearing strategy, dispersal, flight ability, radiosensitivity and mating compatibility) and a better understanding of the basic genetics of codling moth to assist the development of genetic sexing strains (e.g. cytogenetics, the development of dominant conditional lethal mutations, molecular characterization of the sex chromosomes, sex identification in embryos and cytogenetic markers). The results of the CRP are presented in this special issue.

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.

Development of Genetic Sexing Strains in Lepidoptera: from Traditional to Transgenic Approaches

Development of Genetic Sexing Strains in Lepidoptera: from Traditional to Transgenic Approaches

MEDFLY AREAWIDE STERILE INSECT TECHNIQUE PROGRAMMES FOR PREVENTION, SUPPRESSION OR ERADICATION: THE IMPORTANCE OF MATING BEHAVIOR STUDIES

The Sterile Insect Technique (SIT) is amongst the most non-disruptive pest control methods. Unlike some other biologically-based methods it is species specific, does not release exotic agents into new environments and does not even introduce new genetic material into existing populations as the released organisms are not self-replicating. However, the SIT is only effective when integrated on an areawide basis, addressing the total population of the pest, irrespective of its distribution. There has been considerable progress in the development and integrated application of the SIT against the Mediterranean fruit fly (medfly), Ceratitis capitata, as reflected by operational programs for prevention, suppression and eradication of this pest. There is however, considerable scope for improving the efficiency of medfly SIT, an indispensable requirement for increased involvement of the private sector in any future application. One way to achieve this has been the development of genetic sexing strains, making it possible to release only sterile males. Another is improving sterile male performance through a better understanding of the sexual behavior of this insect. Unlike other insects for which the SIT has been successfully applied, medfly has a complex lek-based mating system in which the females exert the mate choice selecting among aggregated and displaying wild and sterile males. With the objective of developing a better understanding of medfly mating behavior, an FAO/IAEA Coordinated Research Project was carried out from 1994 to 1999. Some of the resulting work conducted during this period with the participation of research teams from ten countries is reported in this issue.