ICAR-National Bureau of Agricultural Insect Resources

Collaborating Centre Principal Investigator (CC-PI)

Dr. M. Mohan

Principal Scientist

Principal Investigator involved in the project

Dr. M. Mohan

Principal Scientist

Co-Principal Investigators (Co-PIs)

Dr. T. Venkatesan

Principal Scientist & Head

Dr. R. Gandhi Gracy

Principal Scientist

Dr. Sagar D

Senior Scientist

Dr. Ramya R S

Scientist

Target Traits in Insect Pests

Research Methods & Expected Outcomes

Gene sequences of selected target genes were obtained from public databases, and overlapping primers were designed to amplify full-length coding sequences (CDS). DNA and RNA were successfully isolated from various life stages—egg, all larval instars, pupa, and adult—across 81 insect samples, followed by cDNA synthesis. Standardization of all overlapping primers was completed, and the target genes were cloned using TA cloning. These cloned fragments were sequenced using Sanger sequencing, with three biological and three technical replicates to ensure accuracy. Guide RNAs (gRNAs) for each target were designed with high specificity to minimize off-target effects, and their validation through in vitro cleavage assays is currently underway. In parallel, standardized protocols were established for ovipositional substrates and egg collection methods for all four target insect species. For one species, ultrastructural studies using scanning electron microscopy were conducted to characterize the egg micropylar region, facilitating microinjection of gRNAs. Housekeeping genes for normalizing target gene expression and reference genes for quantitative PCR (qPCR)-based analysis under various experimental conditions were identified and validated. Significant milestones were achieved in functional genomics. Phenotypic marker genes were amplified, cloned, and corresponding sgRNAs were successfully designed, synthesized, and validated. Functional validation studies of key reproductive and developmental genes are ongoing using dsRNA-mediated knockdown approaches. Concurrently, gRNAs for these targets have been designed and synthesis is in progress to facilitate CRISPR-Cas-based editing. Additionally, efforts are underway to evaluate male sterilization strategies using gamma irradiation, dsRNA, and CRISPR technologies in insect pests as part of a precision-guided Sterile Insect Technique (pgSIT) framework.