Exploiting a Y chromosome-linked Cas9 for sex selection and gene drive

CRISPR-based genetic engineering tools aimed to bias sex ratios, or drive effector genes into animal populations, often integrate the transgenes into autosomal chromosomes. However, in species with heterogametic sex chromsomes (e.g. XY, ZW), sex linkage of endonucleases could be beneficial to drive the expression in a sex-specific manner to produce genetic sexing systems, sex ratio distorters, or even sex-specific gene drives, for example. To explore this possibility, here we develop a transgenic line of Drosophila melanogaster expressing Cas9 from the Y chromosome. We functionally characterize the utility of this strain for both sex selection and gene drive finding it to be quite effective. To explore its utility for population control, we built mathematical models illustrating its dynamics as compared to other state-of-the-art systems designed for both population modification and suppression. Taken together, our results contribute to the development of current CRISPR genetic control tools and demonstrate the utility of using sex-linked Cas9 strains for genetic control of animals.

The β2Tubulin, Rad50-ATPase and enolase cis-regulatory regions mediate male germline expression in Tribolium castaneum

Genetics-based pest management processes, including the sterile insect technique, are an effective method for the control of some pest insects. However, current SIT methods are not directly transferable to many important pest insect species due to the lack of genetic sexing strains. Genome editing is revolutionizing the way we conduct genetics in insects, including in Tribolium castaneum, an important genetic model and agricultural pest. We identified orthologues of β2Tubulin, Rad50-ATPase and enolase in T. castaneum. Using RT-PCR, we confirmed that these genes are predominantly expressed in the testis. PiggyBac-based transformation of T. castaneum cis-regulatory regions derived from Tc-β2t, Tc-rad50 or Tc-eno resulted in EGFP expression specifically in the T. castaneum testis. Additionally, we determined that each of these regulatory regions regulates EGFP expression in different cell types of the male gonad. Cis-regulatory regions from Tc-β2t produced EGFP expression throughout spermatogenesis and also in mature sperms; Tc-rad50 resulted in expression only in the haploid spermatid, while Tc-eno expressed EGFP in late spermatogenesis. In summary, the regulatory cis-regions characterized in this study are not only suited to study male gonadal function but could be used for development of transgenic sexing strains that produce one sex in pest control strategies.

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.

A Novel Genetic Sexing Strain of Anastrepha Ludens for Cost-Effective Sterile Insect Technique Applications: Improved Genetic Stability and Rearing Efficiency

Anastrepha ludens (Loew) is one of the most destructive insect pests damaging several fruits of economic importance. The sterile insect technique (SIT) is used under an area-wide integrated pest management approach, to suppress these pest populations. Mass rearing facilities were initially established to produce sterile males of bi-sexual strains in support of SIT. The first genetic sexing strain (GSS) for A. ludens, Tapachula-7, based on pupal color dimorphism, was a key development since the release of males-only significantly increases the SIT efficiency. In this study, we document the development of a novel pupal color-based GSS. Twelve radiation-induced translocation lines were assessed as potential GSS in terms of recombination rates and rearing efficiency at a small scale. The best one, GUA10, was cytogenetically characterized: it was shown to carry a single translocation between the Y chromosome and chromosome 2, which is known to carry the black pupae marker. This GSS was further evaluated at medium and large scales regarding its genetic stability, productivity and quality versus Tapachula-7. GUA10 presented better genetic stability, fecundity, fertility, production efficiency, flying ability, and male mating, clear indicators that GUA10 GSS can significantly improve the efficacy and cost-effectiveness of SIT applications against this pest species.

Marker-assisted mapping enables effective forward genetic analysis in the arboviral vector Aedes aegypti, a species with vast recombination deserts

Aedes aegypti is a major vector of arboviruses that cause dengue, chikungunya, yellow fever and Zika. Although recent success in reverse genetics has facilitated rapid progress in basic and applied research, integration of forward genetics with modern technologies remains challenging in this important species, as up-to-47% of its chromosome is refractory to genetic mapping due to extremely low rate of recombination. Here we report the development of a marker-assisted-mapping (MAM) strategy to readily screen for and genotype only the rare but informative recombinants, drastically increasing both the resolution and signal-to-noise ratio. Using MAM, we mapped a transgene that was inserted in a >100 Mb recombination desert and a sex-linked spontaneous red-eye (re) mutation just outside the region. We subsequently determined, by CRISPR/Cas9-mediated knockout, that cardinal is the causal gene of re, which is the first forward genetic identification of a causal gene in Ae. aegypti. This study provides the molecular foundation for using gene-editing to develop versatile and stable genetic sexing methods by improving upon the current re-based genetic sexing strains. MAM does not require densely populated markers and can be readily applied throughout the genome to facilitate the mapping of genes responsible for insecticide- and viral-resistance. By enabling effective forward genetic analysis, MAM bridges a significant gap in establishing Ae. aegypti as a model system for research in vector biology. As large regions of suppressed recombination are also common in other plant and animal species including those of economic significance, MAM will have broad applications beyond vector biology.

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.

Pupation Substrate Type and Volume Affect Pupation, Quality Parameters and Production Costs of a Reproductive Colony of Ceratitis capitata (Diptera: Tephritidae) VIENNA 8 Genetic Sexing Strain

Adequate pupation substrates and substrate volume are critical factors in the mass-rearing of insects for Sterile Insect Technique (SIT) applications. To identify an ideal pupation substrate for a reproductive colony of Ceratitis capitata (Wiedemann) VIENNA 8 genetic sexing strain, we first examined pupation in cellulose from recycled paper (cellulose I), sawdust, fine wheat bran, vermiculite and coconut fiber using a volume of 2.5–12.5 mL of substrate for each 5 mL volume of fly larvae. We found a positive relationship between substrate volume and pupation, with cellulose I generating the highest proportions of pupation and coconut fiber the lowest. Higher proportions of female flies (white pupae) pupated in sawdust. The proportion of female fliers increased as substrate volume rose in sawdust and coconut fiber, whereas it decreased in vermiculite and cellulose. In a second experiment, we tested three types of cellulose differing in physicochemical characteristics (celluloses I, II and III), sawdust, and fine wheat bran using a substrate:larvae ratio of 1:1. The three types of cellulose produced the highest pupation levels. The highest proportions of female fliers were observed in sawdust, and cellulose types III and II. Cellulose III and sawdust at relatively low volumes were more cost-effective to produce one million pupae than other substrates, including fine wheat bran used in a mass-rearing facility in Mexico.

Determining the Sterilization Doses under Hypoxia for the Novel Black Pupae Genetic Sexing Strain of Anastrepha fraterculus (Diptera, Tephritidae)

A common strategy used to maintain sterile fly quality without sacrificing sterility is to irradiate the insects under an oxygen-reduced atmosphere. So far, sterilizing doses for the South American fruit fly Anastrepha fraterculus have only been determined under normoxia. Our study reports for the first time the dose-sterility response under hypoxia for two different A. fraterculus strains. The pupae were derived from a bisexual strain (a Brazilian-1 population) and a recently developed genetic sexing strain (GSS-89). Two hours prior to irradiation, pupae were transferred to sealed glass bottles and irradiated when oxygen concentration was below 3%. Four types of crosses with nonirradiated flies of the bisexual strain were set to assess sterility for each radiation dose. For males from both strains, Weibull dose–response curves between radiation doses and the proportion of egg hatch, egg-to-pupa recovery, and recovery of adults were determined. The GSS males revealed high sterility/mortality levels compared to males from the bisexual strain at doses < 40 Gy, but a dose of 74 Gy reduced egg hatch by 99% regardless of the male strain and was considered the sterilizing dose. The fertility of irradiated females was severely affected even at low doses under hypoxia.

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.