| The insects have strong ability to adapt to the environment and it is mostly attributed to their powerful ability of chemosensory.The insects depend on their chemosensory to accomplete some important physiological activities and behaviors such as mating,finding the host plants and defense.In moths,females of many species of moths(Insecta:Lepidoptera)secrete species-specific sex pheromones(specific components and proportions)from sex pheromone glands to attract conspecific males.Chilo suppressalis(Walker)belongs to Lepidoptera and Pyralidae,is a notorious rice pest in East Asian countries,causing great economic losses to rice crops.Deep understanding the molecular mechanism of the rice stem borer' sex pheromone communication system will be the advising to the pest control by using the sex pheromone in the field,and thus provide potential target genes for the development of efficient behavior control technology.By now,the related researches which belong to sex pheromone biosynthesis of C.suppressalis and its chemosensory mechanism of the ovipositor behavior were rare.In this study,the transcriptome sequencing of sex pheromone glands-ovipositor of C.suppressalis were done.Then by transcriptome analyses and PCR technology,a total of 32 genes related to sex pheromone biosynthesis and 30 putative chemosensory genes were identified.Among the 62 genes,56 were new.The tissue expression patterns of all these 62 genes were determined by RT-PCR or qPCR.In addition,in order to preliminary study with the chemosensory mechanism of the ovipositor and adult legs of C.suppressalis,the sensilla on the ovipositor and adult legs were observed with the scanning electron microscope.The main results are as follows:1.Transcriptome sequencing and bioinformatics analyses of pheromone glands-ovipositor of C.suppressalis.By transciptome sequencing with using Illumina HiSeqTM2000 platform and assemble the reads with using Trinity,a total of 37,619 Unigenes were got.Among the Unigenes,18,03 7were matched by the Blastx homology search to corresponding genes of other species,with 57.97%to Danaus plexippus sequences,6.49%to Bombyx mori,4.09%to Papilio xuthus and 2.57%to Tribolium castaneum.The Gene Ontology(GO)annotation was used to classify the 37,619 Unigenes into different functional groups using BLAST2GO.Based on the sequence homology,8,506 Unigenes(22.61%)could be annotated,and each Unigene was classified into one or more functional groups of the three biological processes.In total,31,874 annotation hits(Unigene-functional group)come to the biological process,18,952 to the cellular component and 9,639 to the molecular function.To further classified,more than half could align to "cellular process"(62.53%),"metabolic process"(52.92%),"binding"(51.98%),"cell"(50.48%),and "cell part"(50.48%).The results of the transcriptome sequencing provide important foundation for understanding the genes' function and related metabolic pathway of C.suppressalis.2.Identification,sequence analyses and expression pattern of putative sex pheromone biosynthesis genes of C.suppressalis.The sex pheromones of female C.suppressalis were identified as a binary mixture of Z11-16:Ald,Z13-18:Ald and Z9-16:Ald at the ratio of 48:6:5 had the maximum attraction.However,genes involved in the pheromone biosynthesis of C.Suppressalis have not been explored.By analyzing the transcriptomic data of sex pheromone gland-ovipositor,we identified 32 candidate genes related to sex pheromone biosynthesis,including 1 FAS,6 Dess,10 FARs,2 ACOs,1 ACC,4 FATPs,3 ACBPs and 5 ELOs.Gene expression patterns and phylogenetic analyses revealed that 2 Desaturases(CsupDes4 and CsupDes5)and 1 fatty acid reductases(CsupFAR2)were predominantly expressed in pheromone glands,and clustered with genes that already functionally determined to be involved in pheromone biosynthesis in other moths,suggesting that these 3 genes play important roles in C.suppressalis sex pheromone biosynthesis.3.Identification,sequence analyses and expression pattern of putative chemosensory genes of C.suppressalis.Ovipositor in moths has long been proposed to play important chemoreception roles in oviposition site selection.However,the chemoreception related proteins expressed in moth ovipositors and their functions are mostly unknown.Based on the transcriptomic data of pheromone glands-ovipositor,a total of 30 putative chemosensory genes were identified,including 8 OBPs,10 CSPs,2 ORs,1 SNMP,8 CXEs and 1 AOX.Among the 30 chemosensory genes which were identified in the present study,none was OV-PG specified and mostly were antennae-biased except for 2 CSPs(CsupCSP2 and CsupCSP10)were ovipositor-biased.In addition,there was no PBP detected in pheromone glands-ovipositor of C.suppressalis.4.Observation on the sensilla of ovipositor and adult legs of C.suppressalis with scanning electron microscopeMorphology of ovipositor and adult leg's sensilla of C.suppressalis were observed with scanning electron microscope.It is revealed that the ovipositor of C.suppressalis was a pseudo-ovipositor,and the tip of the ovipositor was relatively flat where with many long sensilla cheatica.Near the spawning hole,we found a possible sensilla trichodea which was relatively thick and long.2 types of sensilla were located on the tibiae of adult legs:sensilla cheatica and sensilla squamiformia.The sensilla cheatica were classified to 2 subtypes:A-sensilla cheatica(about 50?m)and B-cheatica(about 20-30?m).A-sensilla was the main type of sensilla on the fore-leg of female and was also distribute widely on the metapede of male,while B-cheatica was only found on male legs with very widely distribute.In addition,1 sensilla squamiformia was found on mid-leg and metapede of female respectively.Compared the quality of sensilla of female and male,the number of metapede's sensilla of male is much larger than female.In contrast,the number of fore-legs' sensilla of female is larger than male.The results provide morphological basis for the chemosensory of the ovipositor and legs of the female moths when they were in oviposition. |
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