The Metabolic Role And Action Mechanism Of Gustatory Receptors In The Intestinal Organ Of Helicoverpa Armigera (Hübner)

Taste perception plays an essential role in multiple life activities include feeding,mating,and oviposition.Taste preception of insects is accomplished by gustatory receptors,which could be grouped into four categories: sugar receptors,fructose receptors,carbon dioxide receptors,and bitter receptors.Among these categories,sugar receptors and fructose receptors are sensitive to sugars,which is essential to feeding behavior.Typically,gustatory receptors are distributed in the taste organs such as mouthparts,antenna,legs,and anterior wing margins.However,in recent years,studies have found that some GRs are present in non-gustatory organs,which may have atypical sensory functions not limited to taste sense.The in-depth study on GRs in non-gustatory organs helps elucidate the mechanism of feeding,mating,and oviposition,which could contribute to our understanding of insect adaptive and nutritional regulation.Besides,the study will provide theoretical support and an experimental basis for the development of green chemical products in pest control.The cotton borer Helicoverpa armigera is one of the most destructive pests worldwide with high fecundity,strong adaptability to environments,and the ability of distant migration,which causes huge losses in agricultural production.Due to the increased pesticide resistance of H.armigera,the development of new pesticide targets is urgently needed.With the completion of whole-genome sequencing of H.armigera,we had an opportunity to make a genome-wide analysis of the gustatory receptor gene family.In this study,we choose H.armigera as the research object and the work we had done is present as follows: 1)we identified the gustatory receptor gene family from the whole-genome of H.armigera based on third-generation sequencing.2)we have cloned the full length of sugar receptors and the fructose receptor gene and examed their spatiotemporal expression in H.armigera.3)we knocked-out the Harm GR9 and screened out the homozygous.4)we tested the feeding behavior and reproductive behavior of the homozygous.5)we performed a comparative transcriptome analysis of foregut between wild type and homozygous using RNA-seq technology.The results of our study are detailed as follows:(1)We identified 235 gustatory receptor genes from the whole-genome of H.armigera.The phylogenetic analyses showed that the gustatory receptor gene family includes 3 carbon dioxide receptors,8 sugar receptors,2 fructose receptors and the remaining are all bitter receptors.The chromosome localization analysis showed that the gustatory receptors scattered on 16 out of 31 chromosomes.Sugar receptors and fructose receptors were located on the same chromosome respectively.Carbon dioxide receptors and bitter receptors distributed on different chromosomes and a significant expansion was observed in bitter receptors.The gene structure analysis revealed that carbon dioxide receptors,sugar receptors,and fructose receptors contain a higher number of introns,which corresponds to their functional conservation.(2)We cloned the GRs in response to sugar and fructose in H.armigera and obtained the full-length of five sugar receptors(Harm GR5、Harm GR6、Harm GR7、Harm GR11、Harm GR12)and one fructose receptor(Harm GR9).The expression analysis in various tissues indicated that the gustatory receptors were distributed widely among peripheral taste organs and non-gustatory organs.A sugar receptor Harm GR6 was highly expressed in the midgut and hindgut and Harm GR12 had a high expression level in the fat body and malpighian tubules of larvae.The fructose receptor Harm GR9 was highly-expressed in the foregut of larvae and abdomen of adults.The receptor is also expressed in the sexual gland of adults.(3)The fructose receptor Harm GR9 showed a high expression level in digestive organs over different developmental periods.To explore the function of Harm GR9 in feeding behavior and metabolism of H.armigera,we knocked-out the Harm GR9 using clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)system.Different editing types with base insertion or deletion were generated in generation one and the mutant rate was 75%.By further screening,we obtained a homozygous mutant line with the genotype of 2 bp deletion.The mutant caused a frameshift and lead to early termination of the protein translation.The mutant type and wild type showed no difference in their body length and body size.However,there is an obvious distinction in body color.The mutants were colored in light green while the wild type was colored in brown.Besides,the developmental period during larval stages was significantly shorter in the mutant line and the pupae of mutants were larger than those of the wild-type individuals.(4)We performed multiple behavioral tests to study the function of Harm GR9 in different tissues.The two choice leaf-disk preference test showed that the mutants lose their ability to perceive multiple kinds of sugar on the leaf.This suggests that Harm GR9 in the peripheral taste organs plays a role in feeding selectivity.Feeding assays showed the food consumption was significantly increased in both larvae and adults of mutants,which indicated that the Harm GR9 could inhibit feeding under the satiety condition.Reproductive behavioral tests showed that the Harm GR9 participates in the selection of oviposition sites and had no effect on the sexual gland volatiles.(5)In order to clarify the molecular mechanism of Harm GR9 affecting the growth and metabolism in larvae,we performed a comparative transcriptome analysis of foregut between wild type and homozygous.The result indicated that the loss of Harm GR9 mainly affects the carboxylic acid metabolic process,organic acid metabolic process,and oxoacid metabolic process.These processes could further affect a variety of biological processes include small molecule metabolic process,alpha-amino acid metabolic process,and cellular amino acid metabolic process.