| Apriona germari(Hope)is one of the important borer in China’s agriculture and forestry,which causing serious economic losses to a variety of forest.It is important to explore the pollution-free control technology of A.germari for reducing the use of chemicals in traditional control techniques.Chemical communication is an important way for insect host selection,mate identification,mating and finding spawning sites,which is an important way to develop behavioral regulation strategies for A.germari by understanding its’ reproductive behavior characteristics and the olfactory mechanism of sexual communication in depth.In this study,behavioral and electrophysiological techniques were used to explore the characteristics of mating behavior and mate recognition organs of A.germari,and to identify and analyze the olfactory related protein genes and their tissue expressions in the external genitalia of A.germari through molecular biology and bioinformatics,and to screen the key olfactory genes in mating behavior.It lays the foundation for further revealing the reproductive behavior mechanism of A.germari.The results are as follows:(1)Daily behaviors observation of A.germari showed a regular pattern,Feeding and resting were the mainly behaviors all the day,crawling,flying,holding and mating behaviors were less frequently,and feeding behaviors were higher during the day than at night,crawling and flying behaviors were higher at night than during the day;In different periods,the female adults of A.germari resting mainly occurred at 3:30-6:30 in the morning,crawling and flying mainly occurred at 19:30-3:30 in the evening,feeding mainly occurred at 16:30-19:30 in the evening;the rest of the male adult mainly occured at 10:30-16:30 during the day,and the main occurrence time of crawling,flying and feeding was the same as that of the female;the holding and mating behaviors mainly occured at 6:30-16:30 during the day.(2)The observation results of the mating behavior of A.germari showed that the maxillary,labial palpus and the external genitalia played a major role in the mate identification of A.germari,especially the contact of the external genitalia was the key to the successful mating,which indicating there were sensory organs and sex pheromone components in the external genitalia.(3)The EAG reaction among the five kinds of volatiles of A.germari showed that,the females mainly reacted strongly to Z-9-tricostriene,heptadecane and nonacane,and the optimal reaction concentrations were all 0.01 mg/ml;while the males mainly reacted to 20 Nonaconane and Z-9-tricostriene react strongly,and the optimal reaction concentrations were 0.1 mg/ml and 0.001 mg/ml respectively.And heptadecane had a strong EAG response only to females.Females were much more sensitive to heptacosane,1-docosene,and Z-9-tricostriene than males,and less sensitive to 1-nonadecene.(4)Through scanning electron microscope observation,a total of 5 types and 10 species of sensilla were found on the external genitalia of A.germari.There were sensilla trichodea,sensilla chaetica,sensilla basiconica,sensilla gemmiformium,sensilla campaniformia and sensilla squamiformia.Among them,there are 2 subtypes of sensilla trichomes,and 4 subtypes of sensilla chaetica.And sensilla chaetica Ⅲ and sensilla campaniformia are unique to male adults,while sensilla chaetica Ⅳ,sensilla basiconica and sensilla gemmiformium were unique to female adults.(5)Analyzing the transcriptome data of the external genitalia of A.germari,a total of 69,406 unigenes genes with an average length of 1813 bp were obtained,and there were 41983,30963,30099,29678,9480,5341 unigenes genes in Nr,KEGG,Swiss-prot,GO,COG,and Pfam were successfully respectively annotated in the six databases.(6)Using bioinformatics software to analyze and process the transcriptome data of the external genitalia of A.germari,a total of 20 odorant binding protein genes were identified,including 6 sex pheromone binding protein genes,and 4 chemosensory protein genes,8 odorant receptor genes.(7)The RT-qPCR results of the sex pheromone binding protein gene of A.germari showed that,Ager PBP1 and Ager PBP6 were highly expressed in the antennae of male and female,Ager PBP2 was highly expressed in the antennae of male and the maxillary and labial palpus of male and female.Ager PBP5 was highly expressed in the antennae of female,while Ager PBP3 and Ager PBP4 was specifically expressed in the maxillary and external genitalia of male respectively.The results successfully screened out the key olfactory protein genes that may play a role in the mating behavior of A.germari,and laid an important foundation for later reseach on the gene function. |
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