CRISPR/Cas9 mediated editing of pheromone biosynthesis activating neuropeptide (PBAN) gene disrupts mating in the Fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae).

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. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03798-3.

CRISPR/Cas9 mediated mutagenesis of the major sex pheromone gene, acyl-CoA delta-9 desaturase (DES9) in Fall armyworm Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae).

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.

First report on the utility of pupal case for early determination of CRISPR/Cas9 ribonucleoprotein mediated genomic edits in the oriental fruit fly, Bactrocera dorsalis (Hendel) (Tephritidae: Diptera).

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.