Functional Studies On The Sugar Gustatory Receptors In Heortia Vitessoides And Its Relatives

Insects use a chemosensory system for sensing various chemicals in the environment.Taste perception plays important role in insect behaviors such as feeding,mating,and oviposition.Gustatory receptors(GRs)are the main chemosensory genes for taste in insects but they were only reported in a few model species.In the Crambidae family(Lepidoptera),Heortia vitessoides,as an oligophagous pest,only feeds on a few woody plants,and is the main pest of Aquilaria sinensis;while the main agricultural pest of maize,Ostrinia furnacalis,has a wide range of hosts.Gustatory receptors may be related to the host range and feeding-preference.However,GRs and the gustatory mechanism are still unknown.In this study,GRs were identified using high-throughput sequencing in H.vitessoides and O.furnacalis.Function of sugar GRs were analyzed by Xenopus oocyte expression system.Finally,these sugar GRs were silenced by RNAi technology to verify their function associated with feeding behaviors.The specific research results are as follows:1.Identification of GRs using high-throughput sequencing technology in H.vitessoides and O.furnacalis.RNAseq were used to identify the GRs expressed in the antennae and mouthparts of two pests.As the results,17 and 15 GRs were identified H.vitessoides and O.furnacalis,respectively.Phylogenetic analysis indicated that GRs in CO2 receptor clade were more conserved among two species,but displayed more differences in sugar receptor clade and bitter receptor clade.In the fructose receptor clade,the four GRs were identified in H.vitessoides,but only one was found in O.furnacalis;in the sugar receptor clade,five GRs were identified in H.vitessoides,while four GRs were found in O.furnacalis.However,there was no homologous gene of OfurGR4 was found in H.vitessoides,but there were three GRs clustered with OfurGR5 in a branch;in the bitter receptor clade,five GRs were identified in H.vitessoides,while nine GRs were found in O.furnacalis.Among them,seven OfurGRs were clustered with HvitGR17,while OfurGR16,HvitGR13,HvitGR15 and HvitGR16 seemed to be the species-specific GRs compared with each other.In addition,other chemosensory genes were identified in H.vitessoides,with a total number of 107 genes including the odorant receptors(ORs).2.Expression profiles of GRs among different tissues.Taking the O.furnacalis as an example,the expression profiles of GRs in 10 tissues were analyzed:the CO2 receptor OfurGR3,OfurGR5,OfurGR7,OfurGR9,OfurGRll and OfurGR16 were highly expressed in female labial palp.However,in the antennae,OfurGR6,OfurGR7,OfurGR8,OfurGR14 and OfurGR16 are highly expressed in males.In the proboscis,most GRs equally expressed in males and females except OfurGR16,which is highly expressed in the females.In the genitalia,OfurGR4,OfurGR11,OfurGR13,OfurGR15 and OfurGR16 are highly expressed in females.3.Functional analysis of the sugar receptor GR by Xenopus oocyte expression system.Full-length of the five sugar receptor GRs including OfurGR4,OfurGR5,OfurGR6,OfurGR7 and OfurGR8 were obtained by RACE.The results showed that the fructose receptor OfurGR8 cannot detect fructose and any other tested sugar compounds,while the other sugar receptor GRs could detect both sucrose and fructose.In the unary expression system,only OfurGR6 was identified in response to sucrose and fructose.Moreover,we found that OfurGR7 has a synergistic effect on OfurGR6 for detecting sucrose and fructose.However,OfurGR7 did not response to any sugars if only been expressed alone,indicating that OfurGR7 may function as a co-receptor.4.Function of OfurGR6 and OfurGR7 for feeding behavior in O.furnacalis.The results showed that after silencing OfurGR6 by RNAi,the selectivity of sucrose and fructose for the 5th instar larvae were significant reduced.Interestingly,the feeding intakes of O.furnacalis were significantly increased.Similar results were obtained while silencing OfurGR7.It showed that OfurGR6 and OfurGR7 are both involved in the recognition of sugars,and affect the satiety in O.furnacalis.After gene silencing,the larvae were hungry and continued to feed.In summary,GRs from two pests with different feeding habits were identified by transcriptome analysis.The sugar receptor GRs were functionally analyzed by Xenopus oocyte expression system and RNAi,two GRs were identified in response to fructose and sucrose.This study provides a theoretical basis for clarifying the feeding recognition mechanism in pests,and provides a basis for the further study of taste perception mechanisms among different pests that have different feeding habits.