Molecular diagnostic markers are necessary for establishing highthroughput screening systems to support insecticide-resistant population management. Here, we identified single amino acid substitution mutations related to carbamate resistance in Laodelphax striatellus Fallén type-1 acetylcholinesterase (Lsace1) using carbofuran-selected strains. The phenotypic resistance profiles of the final selection strain (SEL9) compared to the susceptible strain revealed a 14-fold higher resistance ratio based on topical application, 1.2-fold higher general esterase activity, and 4.3- fold higher acetylcholinesterase insensitivity based on the 50% inhibitory concentration (I50), suggesting that insensitivity of the target site could occur as a resistance factor. Comparison of the nucleotide sequences of Lsace1 of five strains (SUS, SEL0, SEL3, SEL6, and SEL9) revealed two amino acid substitutions (F330Y and F331H). To understand the roles of these mutations, we determined the allele frequency of both point mutations in the selected strains using quantitative sequencing methods. In addition, several quantitative genotypic traits (e.g., gene copy numbers and transcript levels of Lsace1, Lsace2, and LS.CarE1) were assessed. A correlation analysis of genotypic and phenotypic traits revealed strong correlations between resistance level and I50 with F331H allele frequency. Interestingly, the F331H mutation was negatively correlated with transcript levels of Lsace1, suggesting that selection pressure might result in a reduction of the target gene. Overall, the F331H mutation and reduced mRNA are important factors in the development of carbamate resistance. Furthermore, the point mutation can be used to monitor rapid carbofuran resistance in conjunction with molecular diagnostic methods such as quantitative sequencing.

Laodelphax striatellus is an important pest of rice due to not only sucking rice seedlings, but also transmitting serious plant viruses. Among various kinds of insecticide groups (carbamates, organophosphorus, neonicotinoids, etc.), carbofuran, a systemic carbamate insecticide, has been most extensively used to control rice pests including L. striatellus, resulting in widespread carbamate resistance in Korea and other Northeast Asia countries. To identify the genes associated with carbofuran resistance, we obtained a 14-fold higher resistant strain (SEL9) from the mixed-field population (SEL0) by consecutive selection. A transcriptome-based analysis was conducted and differentially expressed genes (DEG) were compared between the SEL9 and SEL0 strains. A total of 96,185,150 reads were analyzed, of which 62,860,430 reads were mapped. From these reads, 15,356 transcripts were annotated. A total of 327 up-regulated and 275 down-regulated genes were identified in the resistant SEL9 strain compared to SEL0 strain by DEG analysis. Gene ontology (GO) analysis was performed using DEGs showing statistical significance (P < 0.05). The GO analysis identified 1,320 genes in biological process group, 1,100 genes in cellular component group and 428 genes in molecular_function group.