Identification And Tissue Expression Profiles Of Chemosensory Genes In Blattella Germanica(Blattaria:Blattidae)

Olfaction is critical to many insect behaviors,such as avoiding predators,adjusting population density and locating various resources such as food,hosts,oviposition sites,and mating partners.For insect,antenna is the most important olfactory organ,which is covered with lots of olfactory sensilla.The periphery process of insect olfaction is commonly thought to include following main steps.Firstly,external hydrophobic odorants enter into the sensillum through the pores;secondly,odorants are bound and transported by odorant binding proteins(OBPs)or chemosensory proteins(CSPs)across the sensillum lymph that fills the cavity around the dendrites of olfactory sensory neuron(OSN);thirdly,the odorants or odorant/OBP(CSP)complexes activate the membrane bound olfactory receptor proteins(ORs),which causes cascade reaction and finally triggers the transductions of chemical signals into electric signals.Identification of those genes that are involved in the olfaction is of primary importance for exploring gene functions and the mechanisms of insect olfaction.The German cockroach,Blattella germanica(Blattaria:Blattidae)is an important sanitary insect pest.The identification of the chemosensory genes and further exploring the mechanisms underlying the olfaction of this insect will be significant,not only for elucidation of the chemosensory gene evolution,but also for development of new behavior-based control strategies for this pest.However,except for a paper by Zhou et al.,that described identification of portion of chemosensory genes,there is no other study reported in B.germanica.In our present work,based on an antennal transcriptomic analysis,we identified 82 putative chemosensory genes,71 of which were novel in B.germanica.In addition,we conducted a comprehensive investigation on the tissue expression profile of these genes by Reverse Transcription-PCR(RT-PCR)and Quantitative Real Time-PCR(qPCR),and found some genes that were antenna-specific or antenna-biased in expression.The results provided important foundation for further functional study and for elucidation of the molecular mechanisms of insect olfaction.Particularly,we found that more than half the chemosensory genes were expressed in the cerci,implying its important olfactory functions in B.germanica.The main results are as follows:1.Antennal transcriptomic sequencing and bioinformatics analyses of antennal of B.germanica.We carried out a transcirptomic sequencing project on a cDNA library constructed from the antennae of B.germanica using Illumina HiSeqTM2000 platform.The analysis of the sequencing data resulted in about 4.81GB reads and 47,925 Unigenes.Among these Unigenes,19,099 were matched by the Blastx homology search to corresponding genes of other species,with 14.08%to Tribolium castaneum,12.16%to Pediculus humanus corporis,6.19%to Megachile rotundata,5.24%to Nasonia vitripennis and 4.60%to Acyrthosiphon pisum.The Gene Ontology(GO)annotation was used to classify the 47,925 Unigenes into different functional groups using BLAST2GO.As a result,9,491 Unigenes could be annotated based on sequence homology,resulting in 37,299 alignments in biological process category,21,080 in cellular component category and 11,959 in molecular function category.To further classify,cellular process(62.05%)and metabolic process(49.76%)were the top 2 sub-categories with highest percentages of Unigenes in biological process category;binding(50.52%)and catalytic activity(50.15%)were the top 2 sub-categories in molecular function category;and cell(46.98%)and cell part(46.98%)were the top 2 in cellular component category.The results of the transcriptome sequencing provide important foundation for studying the molecular mechanism of chemosensory and other physiological processes of B.germanica.2.Identification and sequence analyses of putative chemosensory genes of B.germanica.Based on the antennal transcriptomic data of B.germanica,a total of 82 chemosensory genes were identified by using Blastx analysis,including 48 OBPs,9 CSPs,6 SNMPs,5 ORs,5 IRs and 9 GRs,and of these 71 genes(42 OBPs,5 CSPs,6 SNMPs,4 ORs,5 IRs and 9 GRs)were newly reported in the present study.Notably,Plus-C OBPs account for an exceptionally high proportion(39.58%)of the total 48 OBPs in this primitive insect.This proportion is much higher than 24%(12 of 51)in Drosophila melanogaster,11%(5 of 44)in Bombyx mori and 2%(1 of 49)in Tribolium castaneum,which is not agreeable with the hypothesis that the advanced species have higher Plus-C OBP proportion than the primitive species.3.Expression pattern of putative chemosensory genes of B.germanica.The tissue distribution profile could provide functional clues of a gene.To predict the chemosensory functions of the genes,a detailed global tissue expression profiling was investigated by RT-PCR.It showed that:1)of the 48 OBPs,only OBP1 was antenna specific,and OBP6,15 and 28 were highly antenna biased in expression.Eleven OBPs(OBP4,5,7,25,26,34,35,36,39,40 and 42)were only expressed in antennae,heads(with antennae removed)or cerci.In contrast,OBP3,16,20,27,32,38,41,43,45 and 46 were detected in all tested tissues.2)Among the 9 CSPs,C.SP9 displayed an antenna relatively biased expression.CSP1and CSP3 showed expression in all tested tissues except for head and thorax.The other six(CSP2,4-8)were expressed in all tested tissues.3)SNMP1 was evenly expressed in all tested tissues,while SNMP1 and SNMP1 showed a biased expression in antennae.4)Of the 5 OR genes,OR](Orco)was antenna specific and OR2 were expressed at much higher levels in antennae than in other tissues.In contrast,the other OR genes showed wide expression.5)Of the 5 IR genes,IR1,3 and 5 were highly antenna biased,while IR4 was commonly detected in tested tissues.Furthermore,we found that more than half the chemosensory genes were expressed in the cerci,implying the important chemosensory functions of the organ in B.germanica.The more accurate qPCR measurements for randomly selected 8 genes resulted in generally similar results as the RT-PCR measurement,while it revealed a higher antennal and female biased expression.Taken together,our study provided important bases for further functional study and for elucidation of the molecular mechanisms of insect chemosensation.