Identification And Function Analysis Of Chemosensory Proteins And Gustatory Receptor Genes From Dendroctonus Armandi(Coleoptera:Scolytidae)

Chinese white pine beetle?Dendroctonus armandi Tsai and Li?is an important coniferous forest pest that harms large areas of Pinus armandi forests,causing huge economic losses and serious ecological damage.D.armandi resides in the phloem of Pinus armandi across all life stages except for a brief dispersal period to mate and find new hosts,which is highly hidden and difficult to be controled.It is urgent to develop an effective and environment-friendly way to control this pest.D.armandi uses aggregation pheromones to coordinate mass attacks on host trees,while odorants from host and non-host trees modulate pheromone response.D.armandi responds to volatiles emitted from both host and non-host plants,as well as insect pheromones.Sensitive chemosensory system of D.armandi plays an important role in foraging,mating,searching for spawning grounds,avoiding toxins and escaping natural enemies.However,the olfactory recognition mechanism of how host plant volatiles and pheromones were perceived by the D.armandi is still ambiguous.The identification and functional analysis of chemosensory protein?CSP?and gustatory receptor?GR?genes of D.armandi can help us to understand the molecular mechanism of the chemoreception of D.armandi.More importantly,the research can provide a theoretical basis for the prevention and control of D.armandi by interfering with their chemoreception.In this paper,in order to identify the mechanisms of CSPs and GRs genes of D.armandi for identifying volatiles and pheromones of host plants,phylogenetic of CSPs and GRs genes,the differences in expression of CSPs and GRs genes at different developmental stages and tissues of D.armandi,and the differences in expression of CSPs and GRs genes of D.armandi under feeding or starvation condition,as well as the electrophysiological responses to volatiles after CSP RNAi of D.armandi were studied.The main results were as follows:1.Nine full-length sequences of CSPs from D.armandi were obtained by RT-PCR and RACE techniques.The amino acid sequence of the D.armandi CSP genes are highly homologous to other insects of Dendroctonus and they may have similar expression profiles and functions.However,amino acid sequences of 9 CSPs genes exhibited considerable variation in identity,indicating that they may have multiple functions.2.The CSPs of D.armandii have different expression patterns at different developmental stages and tissues.The expression levels of DarmCSP4,DarmCSP5 and DarmCSP6 in mature larvae and pupae were significantly higher than other developmental stages,and DarmCSP2 and DarmCSP9 had a sudden upregulation before eclosion.These results suggest that these five genes may be involved in the metamorphosis development of D.armandi.The relative expression levels of DarmCSP1,DarmCSP3,DarmCSP7 and DarmCSP8 in the adult stage were higher than those in other periods,suggesting that they may be related to the specific chemosensory behaviors of D.armandi,such as searching for new host trees and mating.DarmCSP1,DarmCSP2,DarmCSP3,and DarmCSP7 were highly expressed in antennae,with DarmCSP3 almost exclusively found there,suggesting that these DarmCSPs may be involved in recognizing pheromones and plant volatiles.DarmCSP2,DarmCSP4,DarmCSP5,DarmCSP8,and DarmCSP9 genes were enriched in mouthparts.These DarmCSPs may play a role in gustation,recognizing non-volatile food sources or detecting close-range odors.In sum,tissue and developmental expression profiles indicate that DarmCSPs serve numerous functions in metamorphosis,olfaction,and gustation.3.The CSP genes of D.armandi has a variety of expression patterns in the feeding and starvation treatment of emerged adults.The relative expression levels of DarmCSP genes showed the trend of down-regulation?DarmCSP1 and DarmCSP2 in both sexes?or first down-regulation and post-upregulation?DarmCSP3 and DarmCSP6 in both sexes and DarmCSP4 in males?in different feeding stages.These results suggest that these DarmCSP genes may coordinate with each other and regulate the transport of odor molecules when D.armandi are feeding.Other genes?DarmCSP8 in both sexes and DarmCSP4 in females?showed upregulation trend during the feeding,suggesting that they may be associated with identification and transportion of non-volatile secondary metabolites of host plants.In starvation treatment,the relative expression levels of DarmCSP2,DarmCSP6,and DarmCSP9 genes of both sexes of D.armandi were upregulated,and the up-regulation time was different,indicating that they coordinated each other to participate in the search for food and host.4.The results of fluorescence competition assay showed that DarmCSP2 and DarmCSP9had strong affinity with the selected 10 host volatiles and 4 pheromones,and compare with DarmCSP9,DarmCSP2 had stronger affinity to all tested volatiles,indicating that these two proteins could bind to both the host volatiles and the pheromones of D.armandii.DarmCSP2and DarmCSP9 play an important role in the chemical sensation of D.armandii and may be the carrier of these volatiles.In the 3D model of DarmCSP2,I73 and W80 of DarmCSP2corresponded to active sites of SgerCSP4,which are the key binding sites of the pheromone,suggesting that I73 and W80 of DarmCSP2 may be involved in pheromone binding.5.The importance of DarmCSP2 in binding to major volatiles was further confirmed by RNAi experiment of D.armandi emerged adults.The relative expression levels of DarmCSP2was significantly reduced after RNAi,and antennae subjected to RNAi experienced significantly reduced EAG activity in response to six tested host volatiles[?+?-?-Pinene,?+?-?-Pinene,?-?-?-Pinene,?+?-Camphene,?+?-3-Carene,and Myrcene],but not in response to pheromones.It suggested that DarmCSP2 may play a key role in the olfaction of D.armandi and that it collaborates with multiple carrier proteins?including other CSPs and OBPs?to transport numerous compounds.6.A full-length sequence of the GRs gene of D.armandii was obtained by RT-PCR and RACE techniques.By aligning the amino acid sequences of other insects and phylogenetic analysis,it can be determined that the obtained GR belongs to CO₂ GR.And it is homologous to DmelGR63a of Drosophila and GR3 of other insects.Therefore,named as DarmGR3.The expression level of DarmGR3 was significantly higher in emerged adults and feeding adults than in other developmental stages,and the expression level of DarmGR3 was significantly higher in the antennae of emerged adults than in other tissues.These results indicated that DarmGR3 plays roles in finding new hosts,spouses,and spawning locations.After 72 h of starvation,the relative expression level of DarmGR3 was significantly up-regulated in both sexes emerged adults,suggesting that DarmGR3 may be involved in searching for host plants.