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Ashok Karuppannasamy,Bhargava Chikmagalur Nagaraja,Prasad Babu Karakatti,Rohan Wishard,Manamohan Malligopal,Rai Anil,Sanjay Kumar Pradhan,Parvathy Madhusoodanan Sujatha,Kennedy John Samuel,Asokan Rama 한국응용곤충학회 2023 Journal of Asia-Pacific Entomology Vol.26 No.1
Overuse of synthetic chemicals over a long period of time has not only resulted in control failures but also enormous ecological damage. This necessitates developing eco-friendly, effective alternatives for sustainable pest management. In this regard, potential of CRISPR/Cas9 mediated genome editing to introduce site specific mu tations that mostly resulting in loss of function has been successfully demonstrated in wide varieties of organisms including insects. This has opened a new avenue to design and implement futuristic pest management strategies like precision guided sterile insect technique (pgSIT) for an area wide suppression. This requires validation of target genes employing Ribonucleoprotein (RNP) complex containing sgRNA and Cas9 protein before under taking transgenesis for achieving pgSIT. In the present study, we have for the first time, supporting CRISPR/Cas9 mediated editing of the eye colour gene, Tryptophan 2, 3-dioxygenase of the eggplant shoot and fruit borer, Leucinodes orbonalis using RNP complex. The mutant moths of both sexes exhibited reddish brown eyed phenotype. Having established an RNP mediated editing system in L. orbonalis, the key genes involved in sex determination and spermatogenesis will be validated for developing a pgSIT system for L. orbonalis.
Wishard Rohan,Karuppannasamy Ashok,Asokan Ramasamy,Chikmagalur Nagaraja Bhargava,Chalapathi Pradeep,Dhawane Yogi,Kumar S Sampath,Maligeppagol Manamohan,Rai Anil 한국응용곤충학회 2023 Journal of Asia-Pacific Entomology Vol.26 No.2
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.
Muthukumar Muthamilselvan,Ashok Karuppannasamy,Samuel Kennedy John 한국응용곤충학회 2023 Journal of Asia-Pacific Entomology Vol.26 No.4
The fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), is considered a highly destructive pest of maize. It has its origin in the tropical and subtropical regions of America and has recently invaded many agricultural regions of India. One of the struggles in managing the pest is pheromone divergence between strains and pheromone blend variation between geographically different populations. The calling pattern, copulation behaviour and mating compatibility of different populations of S. frugiperda were investi gated using behavioural methods. EAG and GC-EAD were used to analyze the chemical composition of the female gland extracts and GC–MS was used to identify the active compounds. In closed arena bioassays, the virgin males’ behavioural responses to the GC-EAD compounds were further assessed. Three active compounds, Z9-14:OAc, Z11-16:OAc and Z7-12:OAc, were identified as major constituents of sex pheromone extracted from rice strain FAW which were found to be feeding on maize. All three compounds triggered intense male antennation. This study provides a deeper insight into the sexual behaviour of fall armyworm which will help in the development of more efficient lure-based pest management strategies.