Analysis Of Olfactory Function And Transcriptional Regulation Of Evolutionarily Conserved OBPs In Monochamus Alternatus And Its Natural Enemy Dastarcus Helophoroides

Tritrophic interactions among host plants,herbivores,and their natural enemies are essential to terrestrial ecosystems.In the tripartite coevolution,complex chemical communication networks were formed.Plant volatiles have received considerable attention as cues for herbivorous insects to locate host plants and for natural enemies to find prey or hosts.Insects have evolved sensitive and specific olfactory perception system to perceive chemical information.Several kinds of proteins function in the olfactory recognition.Odorant binding proteins(OBPs)are taking an important part in the complicated process via binding with odoriferous chemicals specifically and transporting them through the hydrophilic sensillar lymph.As a link between the environment and individual behavioral responses,their evolution is strongly associated with ecological adaptions.It has been reported that evolutionarily conserved OBPs exist in herbivores and their natural enemies,however,the molecular mechanism of olfactory perception about how these OBPs regulate the behavioral choice of herbivores and natural enemies remains unclear.Monochamus alternatus Hope is a serious pest feeding on several pine and fir species and a major vector of the pine wood nematode Bursaphelenchus xylophilus.To control the spread of M.alternatus has become one of the important strategies to control pine wood nematode disease.Dastarcus helophoroides(Fairmaire)is a prominent natural enemy of M.alternatus,which can parasitize late-instar larvae,pupae,and pharate adults of M.alternates in stressed pines.Previous studies have shown that some volatiles can lead to behavioral activation for both M.alternatus and D.helophoroides.Through transcriptome analyses,29 OBPs and 23 OBPs respectively from M.alternatus and D.helophoroides have been identified.Evolutionarily conserved OBPs of M.alternatus and D.helophoroides have been found in the initial analyses.We hypothesize that for M.alternatus and D.helophoroides existing in the same habitat for a long time,evolutionarily conserved OBPs may share some similar functions and regulatory mechanism in the olfactory recognition for chemical signals.In this study,evolutionarily conserved OBPs of M.alternatus and D.helophoroides were identified systematically.The comparative analyses of olfactory functions and transcriptional regulaion of them will help to understand the function of evolutionarily conserved OBPs in the establishment of tritrophic relationships and the adaptive evolution mechanism of OBP genes.Main results were as follows:1.Identification of evolutionarily conserved OBPs respectively from M.alternatus and D.helophoroidesPhylogenetic analysis was performed using amino acid sequences of 223 OBPs from ten species(including M.alternatus and D.helophoroides)of eight superfamilies in Coleoptera.Combining with the results of selection pressure analyses,3 pairs of evolutionarily conserved OBPs were identified: Malt OBP24 and DhelOBP10,Malt OBP19 and DhelOBP22,Malt OBP16 and DhelOBP16.Malt OBP24 and DhelOBP10,Malt OBP19 and DhelOBP22 were identified as Plus-C OBPs,while Malt OBP16 and DhelOBP16 were identified as Classic OBPs.Comparative analyses based on the sequences indicated many similarities in sequence features and physicochemical properties,including the length of coding sequence,molecular weight,the length of signal peptides,and isoelectric points.The high similarity in motif structures among these evolutionarily conserved OBPs implied their potential to bind with similar ligands.The analyses of the rate of molecular evolution revealed that Malt OBP24 and DhelOBP10 have evolved with the slowest evolution rate,which implied that they may take part in maintaining some conserved functions.2.Olfactory function analyses of evolutionarily conserved OBPs respectively from M.alternatus and D.helophoroidesThe expression profiles of the 3 pairs of evolutionarily conserved OBPs in different issues and different development stages were analyzed using q PCR.The high-level expression in olfactory organs implied that they were involved in olfactory recognition.Specifically,highly expressed Malt OBP24 in the antennae of sexually matured M.alternatus adults revealed that it may play a role in the process of host conversion from fresh pine trees to stressed pine trees.The highly expressed DhelOBP10 in the antennae of D.helophoroides newly emerged adults implied its potential function of host location in wide range.Competitive fluorescence binding assays and fluorescence-quenching assays are combinedly utilized to detect ligands binding affinities with 14 common pine volatiles using purified proteins.It was found that(+)-fenchone,α-terpinolene,3-carene showed high binding affinities with Malt OBP24 and DhelOBP10 in fluorescence binding assays according to the relatively low dissociation constant(Ki < 20 μM)and resulted in static quenching to Malt OBP24 and DhelOBP10 in fluorescence-quenching assays.Y-tube olfactometer bioassays and RNAi experiment suggested that Malt OBP24 and DhelOBP10,play an essential role in the olfactory recognition of(+)-fenchone for M.alternatus and D.helophoroides respectively.These results revealed that evolutionarily conserved Malt OBP24 and DhelOBP10 can participate in establishing the pine-M.alternatus-D.helophoroides tritrophic interactions through recognizing(+)-fenchone from stressed pines.Malt OBP19 was detected mainly in antennae and mouthparts,especially in mature stage of M.alternatus adults.DhelOBP22 was mainly expressed in antennae of D.helophoroides adults,especially in mature stage.Competitive fluorescence binding assays indicated that Malt OBP19 can bind with camphene and myrcene,and DhelOBP22 can bind with camphene.Y-tube olfactometer bioassays and RNAi experiment showed that Malt OBP19 functioned in the recognition of camphene for M.alternatus adults in the stage of shifting to stressed hosts,and DhelOBP22 functioned the recognition of camphene for mated female D.helophoroides.As for Malt OBP16 specifically expressed in the antenna of M.alternatus adults and DhelOBP16 with high-level expression in the antenna of matures D.helophoroides,they did not bind with the tested 14 volatiles according to the fluorescence binding assays,which implied that they may perform other functions.3.Transcriptional regulatory mechanism of evolutionarily conserved OBPs respectively from M.alternatus and D.helophoroidesThe 5’ non-coding sequence of DhelOBP22 gene comprising 1238 bp was cloned using Tail-PCR and truncated to several fragments of different lengths.The dual-luciferase reporter assay in Drosophila S2 cells revealed the promoter activity of-686～-553 fragment.The fragment was tested for potential binding sites for transcription factors using JASPAR,and several conserved binding sites,which also existed in the promoter of Malt OBP19 gene,were found.Among them,the binding site of BarH1 was worthy of note,because it has been proved that BarH1 can act on the Malt OBP19 promoter in vitro in previous studies.To examine the effect of BarH1 on the 5’ non-coding region of DhelOBP22 gene,BarH1 was expressed in Drosophila S2 cells using the vector p AC5.1-Myc.Dual-luciferase reporter assay revealed that BarH1 can enhance the transcriptional activity of DhelOBP22 promoter in vitro.The result of EMSA showed that BarH1 can bind with the 5’ regulatory region of DhelOBP22 gene in vitro.The expression profiles of BarH1 in M.alternatus and D.helophoroides both indicated certain degree of similarities with that of Malt OBP19 and DhelOBP22 in vivo,especially in the different antennal segments of M.alternatus.When the BarH1 gene was silenced by RNAi,the gene expression levels of Malt OBP19 and DhelOBP22 significantly decreased in female and male adults of M.alternatus and D.helophoroides.The attractive behavior of the females of M.alternatus and mated females of D.helophoroides to camphene disappeared,but the males of M.alternatus displayed avoidance behavior.Subsequent transcriptome analysis indicated that additional olfactory genes show different changes between female and male adults,which implied more complex regulation mechanism.In summary,this study affirmed that evolutionarily conserved OBPs respectively from M.alternates and D.helophoroides shared similar olfactory functions in recognizing volatiles of host pines.They could function in the important behavioral choice of M.alternates and D.helophoroides by specifically binding with the same volatile.In addition,it was found that the expression of evolutionarily conserved Malt OBP19 and DhelOBP22 was regulated by the same transcription factor BarH1.These results provide an important foundation for revealing the function of evolutionarily conserved OBPs respectively from herbivores and their natural enemies.