Study On The Sulfakinin Inhibits Activity Of Digestive Enzymes And Positively Modulates Takeout Gene That Inhibiting Feeding Behavior Of Nilaparvata Lugens

Nilaparvata lugens(St(?)l)is an important pest that harms rice.It can directly damages rice by feeding,which has caused significant economic losses to rice production.However,there is little known about the feeding behavior of brown planthoppers.Insect neuropeptide plays important roles in regulating different physiological and behavioral process.They exert their effects by binding on their receptors,mostly of them are G-protein coupled receptor.Insect sulfakinin(SK)involves in regulating satiety,food intake and intestinal functions.In the previous studies of our lab,it has demonstrated that sulfakinin inhibits feeding of brown planthoppers,but the molecular mechanism is still unknown.Therefore,this paper taking brown planthopper as the research object to explore the physiological,biochemical and molecular mechanisms of SK regulating feeding of brown planthopper through pharmacology,RNAi,transcriptomic sequencing,qPCR,enzyme activity measurement and feeding behavior assay,so as to provide a basis for the research and development of new feeding regulators.The main research results are described as follows:1.Effects of sulfakinin on gene transcription of brown planthopperOur previous studies have showed that sulfakinin can inhibit feeding behavior of brown planthopper.In order to clarify the molecular mechanism of sulfakinin in inhibiting the feeding of brown planthoppers.In this study,we investigated the impact of activation or silencing of Nlsk signaling system on transcriptomic profiles of brown planthopper.Our results showed that after injection of sulfakinin into brown planthopper,which activates Nlsk signaling system.166 differentially expressed genes were found and 87 of them were upregulated,while 79 of them were down-regulated.Silencing of Nlsk gene induced 190 differentially expressed genes.111 of-them were up-regulated,while 79 of them were downregulated.And Silencing of Nlskr gene induced 196 differentially expressed genes.122 of them were up-regulated,while 74 of them were down-regulated.Gene ontology and KEGG data showed that the majority of the differentially expressed genes are involved in energy metabolism,olfaction,gustation and feeding-related genes.We infer that Nlsk generally manipulate feeding behavior of brown planthopper by modulating these differentially expressed genes.2.Sulfakinin inhibits digestive enzymes activity and digestive enzyme genes expression in N.lugensIn insects,the release of digestive enzymes is regulated by feeding.Digestive enzymes are produced and released in response to specific ratios of nutrients,so the quantity and quality of food ingested is an important factor in the secretion and activity of digestive enzymes.In general,the enzyme activity and secretion of fed insects are relatively higher than that of unfed insects.But also neuropeptides and peptide hormones are important regulators of enzyme activity.In several insects,the neuropeptide sulfakinin(SK)is known to be a regulator of feeding and digestion similar to cholecystokinin in mammals.However,the roles of diet and SK in regulation of activity of digestive enzymes in the important pest insect,the brown planthopper(Nilaparvata lugens)are unknown.In this study,we identified six genes encoding different digestive enzymes and cloned three of these.We found that enzymatic activity and transcriptional levels of digestive enzyme genes were upregulated by refeeding animals for 5 hours after 24 hours starvation.Furthermore,injection of N.lugens SK reduces digestive enzyme activity and leads to a downregulation of digestive enzyme gene transcripts.This study provides new insights into the action of diet and SK in regulation of digestive enzymes in(hemimetabolous)insects.Taken together with the roles of SK in inducing satiety,our data strongly suggest that SK signaling is important in regulation of food ingestion and processing.3.Sulfakinin positively regulated takeout gene that inhibited feeding in N.lugensTakeout plays important roles in the regulating of insect’s feeding behavior.We cloned the full length of takeout gene.And we found that injection of sulfakinin could induce takeout gene expression.However,silencing of Nlsk and Nlskr gene inhibits gene expression of takeout,which is consistence with our transcriptome data.These results indicated that Nlsk positively regulated takeout gene expression.However,silencing of takeout gene have no significant impacts on gene expression of Nlsk and Nlskr.which indicated that takeout could not modulate gene expression of Nlsk and Nlskr.Furthermore,we observed the increasing feeding behavior when we silenced the takeout gene.Hence,Nlsk could positively regulated takeout gene that inhibited feeding in the brown planthopper.Quantitative polymerase chain reaction results demonstrated that takeout expression is regulated by circadian clock and it was expressed at higher levels in head and antenna than other tissues.