Transgenic expression of Nix converts genetic females into males and allows automated sex sorting in Aedes albopictus

Aedes albopictus is a major vector of arboviruses. Better understanding of its sex determination is crucial for developing mosquito control tools, especially genetic sexing strains. In Aedes aegypti, Nix is the primary gene responsible for masculinization and Nix-expressing genetic females develop into fertile, albeit flightless, males. In Ae. albopictus, Nix has also been implicated in masculinization but its role remains to be further characterized. In this work, we established Ae. albopictus transgenic lines ectopically expressing Nix. Several were composed exclusively of genetic females, with transgenic individuals being phenotypic and functional males due to the expression of the Nix transgene. Their reproductive fitness was marginally impaired, while their flight performance was similar to controls. Overall, our results show that Nix is sufficient for full masculinization in Ae. albopictus. Moreover, the transgene construct contains a fluorescence marker allowing efficient automated sex sorting. Consequently, such strains constitute valuable sexing strains for genetic control.

Sterile Insect Technique (SIT) and Its Applications

Although most insect species have a beneficial role in the ecosystems, some of them represent major plant pests and disease vectors for livestock and humans. During the last six–seven decades, the sterile insect technique (SIT) has been used as part of area-wide integrated pest management strategies to suppress, contain, locally eradicate or prevent the (re)invasion of insect pest populations and disease vectors worldwide. This Special Issue on “Sterile insect technique (SIT) and its applications”, which consists of 27 manuscripts (7 reviews and 20 original research articles), provides an update on the research and development efforts in this area. The manuscripts report on all the different components of the SIT package including mass-rearing, development of genetic sexing strains, irradiation, quality control as well as field trials.

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

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.

Lessons from Drosophila: Engineering Genetic Sexing Strains with Temperature-Sensitive Lethality for Sterile Insect Technique Applications

A major obstacle of sterile insect technique (SIT) programs is the availability of robust sex-separation systems for conditional removal of females. Sterilized male-only releases improve SIT efficiency and cost-effectiveness for agricultural pests, whereas it is critical to remove female disease-vector pests prior to release as they maintain the capacity to transmit disease. Some of the most successful Genetic Sexing Strains (GSS) reared and released for SIT control were developed for Mediterranean fruit fly (Medfly), Ceratitis capitata, and carry a temperature sensitive lethal (tsl) mutation that eliminates female but not male embryos when heat treated. The Medfly tsl mutation was generated by random mutagenesis and the genetic mechanism causing this valuable heat sensitive phenotype remains unknown. Conditional temperature sensitive lethal mutations have also been developed using random mutagenesis in the insect model, Drosophila melanogaster, and were used for some of the founding genetic research published in the fields of neuro- and developmental biology. Here we review mutations in select D. melanogaster genes shibire, Notch, RNA polymerase II 215kDa, pale, transformer-2, Dsor1 and CK2α that cause temperature sensitive phenotypes. Precise introduction of orthologous point mutations in pest insect species with CRISPR/Cas9 genome editing technology holds potential to establish GSSs with embryonic lethality to improve and advance SIT pest control.

Genetic analysis of medfly populations in an area of sterile insect technique applications

The sterile insect technique (SIT) is widely used in integrated pest management programs for the control of the Mediterranean fruit fly (medfly), Ceratitis capitata. The genetic interactions between the released individuals from the genetic sexing strains (GSS), used for SIT applications worldwide, and wild individuals have not been studied. Under the hypothesis that a number of Vienna GSS individuals released to the field might not be completely sterile and may produce viable offspring, we have analyzed medfly Spanish field populations to evaluate the presence of Vienna strain genetic markers. To this goal, we have used contrasted nuclear and mitochondrial genetic markers, and two novel sets of nuclear polymorphisms with the potential to be markers to discriminate between Vienna and wild individuals. Nuclear Vienna markers located on the 5th chromosome of Vienna males have been found in 2.2% (19 from 875) of the Spanish wild medfly females captured at the area where SIT is applied. In addition, a female-inherited mitochondrial Vienna marker has been found in two from the 19 females showing nuclear Vienna markers. The detection of several of these markers in single individuals represents evidence of the introgression of Vienna strain into natural populations. However, alternative explanations as their presence at low frequency in wild populations in the studied areas cannot be fully discarded. The undesired release of non-fully sterile irradiated GSS individuals into the field and their interactions with wild flies, and the potential environmental implications should be taken into account in the application of the SIT.

Genetic sexing strains for the population suppression of the mosquito vector Aedes aegypti

Aedes aegypti is the primary vector of arthropod-borne viruses including dengue, chikungunya and Zika. Vector population control methods are reviving to impede disease transmission. An efficient sex separation for male-only releases is crucial for area-wide mosquito population suppression strategies. Here, we report on the construction of two genetic sexing strains using red- and white-eye colour mutations as selectable markers. Quality control analysis showed that the Red-eye genetic sexing strains (GSS) is better and more genetically stable than the White-eye GSS. The introduction of an irradiation-induced inversion (Inv35) increases genetic stability and reduces the probability of female contamination of the male release batches. Bi-weekly releases of irradiated males of both the Red-eye GSS and the Red-eye GSS/Inv35 fully suppressed target laboratory cage populations within six and nine weeks, respectively. An image analysis algorithm allowing sex determination based on eye colour identification at the pupal stage was developed. The next step is to automate the Red-eye-based genetic sexing and validate it in pilot trials prior to its integration in large-scale population suppression programmes. This article is part of the theme issue ‘Novel control strategies for mosquito-borne diseases’.

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

Background Aedes 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. Results For 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. Conclusion Inducing 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.

Precise single base substitution in the shibire gene by CRISPR/Cas9-mediated homology directed repair in Bactrocera tryoni

Background Pest eradication using the Sterile Insect Technique (SIT) involves high-density releases of sterilized males that mate with wild females and ultimately suppress the population. Sterilized females are not required for SIT and their removal or separation from males prior to release remains challenging. In order to develop genetic sexing strains (GSS), conditional traits such as temperature sensitive lethality are required. Results Here we introduce a known Drosophila melanogaster temperature sensitive embryonic lethal mutation into Bactrocera tryoni, a serious horticultural pest in Australia. A non-synonymous point mutation in the D. melanogaster gene shibire causes embryonic lethality at 29 °C and we successfully used CRISPR/Cas9 technology to recreate the orthologous shibire temperature sensitive-1 (shits1) mutation in B. tryoni. Genotypic analyses over three generations revealed that a high fitness cost was associated with the shits1 mutant allele and shits1 homozygotes were not viable at 21 °C, which is a more severe phenotype than that documented in D. melanogaster. Conclusions We have demonstrated the first successful use of CRISPR/Cas9 to introduce precise single base substitutions in an endogenous gene via homology-directed repair in an agricultural pest insect and this technology can be used to trial other conditional mutations for the ultimate aim of generating genetic sexing strains for SIT.

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

Background Area-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. Results For 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. Conclusions We 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.