Mutations in NlInR1 affect normal growth and lifespan in the brown planthopper Nilaparvata lugens.

The brown planthopper (BPH) Nilaparvata lugens contains two insulin receptor homologues, designated NlInR1 and NlInR2. NlInR1 is strikingly homologous to the typical InR in insects and vertebrates, containing a ligand-activated intracellular tyrosine kinase catalytic domain. Herein, we report an optimized CRISPR/Cas9 system to induce mutations in the NlInR1 locus in BPH, consisting of a Cas9 plasmid that is specifically expressed in the germline via the Nlvasa promoter and versatile sgRNA expression plasmids under the control of the U6 promoter. We systematically evaluated the efficiency of injection mix compositions and demonstrated an appropriate combination of Cas9/sgRNA to target essential genes. Furthermore, we showed that homozygous mutants for the NlInR1 gene are early embryonic lethal, whereas heterozygous mutants grow more slowly, exhibit a severe reduction in body weight and wing size and live longer than the wild type. Interestingly, the severity of the mutant phenotype was different when targeting distinct important domains of the NlInR1 locus. The severity of the mutant phenotype is similar to that of insulin/insulin-like growth factor (IGF) signaling pathway deficiencies in vertebrates, suggesting a conserved function of NlInR1 in the regulation of development and longevity. Global expression profiling suggests that NlInR1 regulates many cellular processes in BPH, including insulin resistance, phototransduction, metabolism, endocytosis, longevity, biosynthesis and protein processing. Our results also pave the way for understanding the precise molecular mechanism of insulin signaling in wing polyphenism in insects.