The oriental fruit fly (Bactrocera dorsalis) is an exceptionally hazardous pest that can cause up to 100% loss. Male annihilation using male attractant methyl eugenol (ME) is the most adopted control measure of this insect. The molecular process underlying this perception of insects remains unexplored. In this study, we focus on gene functionality of odorant-binding protein 13 (OBP13) in ME perception by knockout using CRISPR/Cas9 mediated embryonic editing through microinjection. The electrophysiological study of mutant flies through electroantennogram has shown highly depressed ME perception and further the genetic-level mutational confirmation stating that OBP13 is a major OBP involved in ME perception. The molecular docking of OBP13 with ME revealed the predicted key interactions involved, thus providing more scope in targeting gene editing and in engineering ME substitutes.

Pest management based on CRISPR/Cas9-mediated site-specific mutations is an effective and environmentally safer strategy to suppress the pest population. However, the potential of this approach is yet to be tested on many important agricultural pests such as Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), a fit candidate for area-wide pest management. Therefore, in the present study, 2 spermatogenesis-related genes viz. Testis-specific zinc finger protein (topi) and Testis-specific serine protein kinase 1 (Tssk1) of B. dorsalis were edited to impart male sterility and its impact on further progeny. In this regard, topi and Tssk1 mutant populations deposited significantly fewer eggs per day (6.12 +/- 0.36 and 3.60 +/- 0.24, respectively) as compared to the control (11.16 +/- 0.58 eggs per day). About the hatching rate, the above trend was observed, topi (44.51) and Tssk1 (30.04) as compared to the control (73.96). Furthermore, the total number of viable offspring for topi and Tssk1 populations decreased as a result of the cumulative progeny production ten days after the post-mating phase. It suggests that topi and Tssk1 from B. dorsalis could be potential targets for imparting male sterility in B. dorsalis. Graphical Abstract

The Fall armyworm, Spodoptera frugiperda , is a globally important invasive pest, primarily on corn, causing severe yield loss. Overuse of synthetic chemicals has caused significant ecological harm, and in many instances control has failed. Therefore, developing efficient, environmentally friendly substitutes for sustainable management of this pest is of high priority. CRISPR/Cas9-mediated gene editing causes site-specific mutations that typically result in loss-of-function of the target gene. In this regard, identifying key genes that govern the reproduction of S. frugiperda and finding ways to introduce mutations in the key genes is very important for successfully managing this pest. In this study, the pheromone biosynthesis activator neuropeptide ( PBAN ) gene of S. frugiperda was cloned and tested for its function via a loss-of-function approach using CRISPR/Cas9. Ribonucleoprotein (RNP) complex (single guide RNA (sgRNA) targeting the PBAN gene + Cas9 protein) was validated through in vitro restriction assay followed by embryonic microinjection into the G0 stage for in vivo editing of the target gene. Specific suppression of PBAN by CRISPR/Cas9 in females significantly affected mating. Mating studies between wild males and mutant females resulted in no fecundity. This was in contrast to when mutant males were crossed with wild females, which resulted in reduced fecundity. These results suggest that mating disruption is more robust where PBAN is edited in females. The behavioural bioassay using an olfactometer revealed that mutant females were less attractive to wild males compared to wild females. This study is the first of its kind, supporting CRISPR/Cas9 mediating editing of the PBAN gene disrupting mating in S. frugiperda . Understanding the potential use of these molecular techniques may help develop novel management strategies that target other key functional genes.

The Oriental fruit fly, Bactrocera dorsalis (Hendel), is a highly invasive pest of quarantine importance affecting the global fruit trade. In managing B. dorsalis, methods like cultural, biological, chemical, sterile insect technique (SIT), and semiochemical-mediated attract-and-kill are in use with varying success. The SIT approach is the method of choice for a chemical-free, long-term suppression of B. dorsalis, followed in many countries across the globe. The nonspecific mutations caused by irradiation affect the overall fitness of flies, thus requiring a more precise method for a heritable, fitness-not-compromising approach. In this regard, CRISPR/Cas9-mediated genome editing enables the creation of mutations at the precise genomic location/s through RNA-guided dsDNA cleavage. Of late, DNA-free editing employing ribonucleoprotein complex (RNP) is preferred to validate the target genes at G0 stage embryos in insects. It requires characterizing genomic edits from adults after completing their life cycle, which may entail a few days to months, depending on longevity. Additionally, edit characterization is required from each individual, as edits are unique. Therefore, all RNP-microinjected individuals must be maintained until the end of their life cycle, irrespective of editing. To overcome this impediment, we predetermine the genomic edits from the shed tissues, such as pupal cases, to maintain only edited individuals. In this study, we have shown the utility of pupal cases from five males and females of B. dorsalis to predetermine the genomic edits, which corroborated the edits from the respective adults.

Identification of novel approaches for managing the global pest, the Fall armyworm, Spodoptera frugiperda, is the need of the hour, as it defies many management strategies including synthetic chemicals, Bt transgenics, and so on. Recently CRISPR/Cas9-based genome editing opened up newer avenues to design novel pest management strategies such as precision-guided sterile insect technique (pgSIT). In this regard, genes governing sex determination, egg reproduction, and spermatogenesis could be the prime targets for genome editing. This requires validation of the target genes, preferably by a nontransgenic DNA-free editing, before the final application. One such important gene regulating sex determination in Drosophila is the Sex lethal (Sxl). However, the function of Sxl is not highly conserved in other insects and, in particular, we are beginning to comprehend its role in Lepidoptera with only one reference available in Spodoptera litura till date. In the present study, we have edited the sxl gene of S. frugiperda through the delivery of ribonucleoprotein complex (sgRNA + Cas9) at G0 stage embryo, targeting the conserved region of all the documented five splice variants. Results clearly showed that editing of sxl gene impacted the overall fecundity and hatching rate. Therefore, Sxl could be one of the target genes for developing pgSIT approach for the management of S. frugiperda.

The Fall armyworm, Spodoptera frugiperda is a significant global pest causing serious yield loss on several staple crops. In this regard, this pest defies several management approaches based on chemicals, Bt transgenics etc., requiring effective alternatives. Recently CRISPR/Cas9 mediated genome editing has opened up newer avenues to establish functions of various target genes before employing them for further application. The virgin female moths of S. frugiperda emit sex pheromones to draw conspecific males. Therefore, we have edited the key pheromone synthesis gene, fatty acyl-CoA Delta-9 desaturase (DES9) of the Indian population of S. frugiperda. In order to achieve a larger deletion of the DES9, we have designed two single guide RNA (sgRNA) in sense and antisense direction targeting the first exon instead of a single guide RNA. The sgRNA caused site-specific knockout with a larger deletion which impacted the mating. Crossing studies between wild male and mutant female resulted in no fecundity, while fecundity was normal when mutant male crossed with the wild female. This indicates that mating disruption is stronger in females where DES9 is mutated. The current work is the first of its kind to show that DES9 gene editing impacted the likelihood of mating in S. frugiperda.

The Oriental fruit fly, Bactrocera dorsalis is a serious, highly invasive pest of fruits affecting global trade. Areawide management of B. dorsalis by release of sterile males obtained through irradiation suffers from the disadvantage of somatic mutations. In this regard, insect pest management in the post genome editing era poised for producing precise mutations based on CRISPR/Cas for example precision guided sterile insect technique and gene drive. Development of these technologies depend on the genes in sex determination pathway and spermatogenesis in target species. In the sex determination pathway of B. dorsalis, transformer (tra & tra2) are involved in female specific splicing of double sex (dsx). CRISPR/Cas9 mediated loss-of-function of tra has been shown to influence sex determination while it is lacking in tra2. In the present study we have edited transformer2 gene through embryonic microinjection of CRISPR/Cas9 ribonucleprotein (Cas9 + sgRNA) targeting the sixth exon. Among the emergent G0 individuals, two showed an intersex phenotype, with a male-specific dorsal bristles in the third tergite on the left side of the abdomen and a degenerate ovipositor. The third individual manifested an unique phenotype, with a complete absence of the male specific dorsal bristles but a complete degenerate ovipositor. Sequencing results revealed both addition and deletions in all the three intersexes. Additionally, molecular karyotyping of these individuals, through Maleness on Y (MoY) PCR revealed a XX karyotype for all of them. Hence, we have shown that editing of the transformer2 locus in B. dorsalis interferes with the normal sex determination.