| Blattella germanica(L.)is an acknowledged worldwide health pest with life history characteristics such as short generation cycle,strong reproductive capacity,protection of maternal egg-bearing pod,and has become the dominant species of indoor cockroach.Blattella germanica carries and spreads a variety of parasites and pathogenic microorganisms as a mechanical carrier,and its epidermis and secretions contain allergens,which can directly or indirectly lead to a variety of diseases in human.Chemical insecticides make the Blattella germanica population generally have different degrees of drug resistance,which increases the difficulty of comprehensive management of Blattella germanica.Behavioral resistance,as a non-physiological resistance mechanism,plays a vital role in biological adaptation to the environment.The olfactory system is the main structural basis for insects’ behavioral response to changes in the external environment,which has a potential close relationship with the formation and development of behavioral resistance of Blattella germanica.However,there is little research on the role and function of olfactory-related genes and proteins in the formation of behavioral resistance.Therefore,how to find olfactory-related targets for integrated control of Blattella germanica against its resistance status is very necessary.In view of the previous research result that the last instar male Blattella germanica showed the strong drug resistance,the Sensitive(S)strain and the beta-cypermethrin resistance(R)strain of the last instar male Blattella germanica were taken as the research subjects,and the morphological structure of antennae,the expression level of olfactory-related genes and behavioral performance were studied through morphology observation,molecular biology detection and behavioral verification.the main research contents include:(1)The ultrastructure of the type,number and distribution of the antennal sensilla of different strains of Blattella germanica was observed by scanning electron microscopy,and the relationship between the antennal sensilla and drug resistance was determined based on the changes of the antennal sensilla.(2)Carrying out transcriptome sequencing on the antennal tissues of the last instar male Blattella germanica,comparing differentially expressed genes of sensitive and resistant strains through biological information analysis,further clarifying the possible roles of genes encoding the differential proteins in the drug resistance of the Blattella germanica,and searching for olfactory related genes and proteins which are related to the behavior resistance.(3)Comprehensive analysis,screening and determination of the Snmp2 gene of sensory neuron membrane protein,and RNAi at the early stage to study the relationship between the change of relative gene expression and behavioral manifestations such as tropism and avoidance of toxicity.The main results are as follows:1.Comparison of antennal sensilla between two strains of the last instar Blattella germanicaSensitive strain and resistant character The antennae of the last instar male Blattella germanica are divided into stipe,peduncle and whip.Scanning electron microscopy showed that there were 8 species of sensilla in the antennae of the two strains of late-age Blattella germanica:sensilla chaetica Ⅰ,sensilla chaetica Ⅱ,trichoid sensilla,cambered sensilla,sensilla basiconica,elliptical sensilla,marginal sensilla and Bohm′s bristles.The number of trichoid sensilla was significantly reduced in the more sensitive strain(22.00±0.58)of the resistant strain(14.00±2.31)(P<0.05),and there was no difference between the two strains of other types of sensilla(P>0.05).2.Analysis of antennal transcriptome of two strains of the last instar Blattella germanicaRNA-seq technique was used to perform transcriptome sequencing on the antenna tissue of sensitive strain and resistant the last instar male Blattella germanica,and 57092552,54982970,54191036,55813716,55514566,51570906 Clean reads were obtained respectively.The percentage of Total mapped reads data is about 88%.The distribution of each group of reads in each position of the reference genome and the distribution of genes in each position have good repeatability.Based on comparative analysis of biological information,1252 differentially expressed genes were screened out,among which 420 genes were significantly up-regulated and832 genes were significantly down-regulated for S relative to R strain.The GO functional annotation showed that the number of genes with binding,catalytic activity and transporter activity was the highest among the molecular functions.KEGG enrichment analysis showed that the percentage of signal transduction genes was the highest in environmental information processing,and the above results were in line with the functional characteristics of insect antennae.In the up-regulation mode of top30 in the enrichment analysis of differential gene GO,the functional distribution mainly focuses on:(1)positive regulation of synaptic maturation,DNA replication initiation and DNA replication in biological processes;(2)extracellular region,cell surface and extracellular space in cell components;(3)Carbohydrate binding in molecular function,single-stranded DNA dependent ATP enzyme activity,extracellular matrix structural components.In the down-regulation mode of top30 in the enrichment analysis of differential gene GO,the functional distribution mainly focuses on:(1)chitin-based cuticle development,fatty acid biosynthesis and long-chain fatty acyl-Co A metabolism in biological processes;(2)protein-carbohydrate complex,extracellular matrix and extracellular region in cell components;(3)the activity of trans-hexa-isoprene transferase in the molecular function,the structural components of the cuticle,and the structural composition of the chitin-based larval cuticle.In the up-regulation pattern of top20 in the enrichment analysis of differential gene KEGG,the number of genes involved in neuroactive ligand-receptor interaction and lysosome is the largest,and DNA replication is the most significant.In the top20 downregulation pattern of differential gene KEGG enrichment analysis,the peroxidase body contains the most genes,and the biosynthesis of cutin,sulfite and wax has the greatest significance.Through further annotation and analysis of differentially expressed genes,27olfactory-related differentially expressed genes were identified,including 3 olfactory odor receptor genes(ORs),1 ionic receptor gene(IRS)and 1 sensory neuron membrane protein gene(Snmp2),among which Snmp2 may have potential functions in pheromones and general odorant reactive receptors;Fifteen differentially expressed genes participate in olfactory transduction pathway,among which the most significant difference is Pkg21D＿0 and Pkg21D＿1 of PKG protein family,both of which are protein kinases with down-regulated pattern.Referring to the transcriptome sequencing data and combining with the q RT-PCR verification results,the sensory neuron membrane protein gene Snmp2 was selected for subsequent experiments.3.Correlation analysis between Snmp2 gene and behavioral resistanceThe RNAi efficiency of the last instar male Blattella germanica was detected by q RT-PCR,and the relative expression level of Snmp2 gene was significantly down-regulated and stabilized on the 4th day.Using olfactometer to test the tropism,it was found that the tropism RAte of Blattella germanica in 2μg/μL si Snmp2 interference group(RA)to the toxic area decreased significantly(P<0.05)as compared with no significant change among r strain,s strain,DEPC water group(RD)and 2μg/μL si NC negative control group(RN).The disc device was used to test the toxicity avoidance.It was found that the activity time of Blattella germanica in the toxicity area of RA group decreased significantly with the increase of the days of interference(P<0.05).In addition,in the process of behavior test,RA group often accompanied by phenomena such as increased frequency of antennal combing,slower movement speed,decreased exploration ability,increased aggregation behavior,etc.The above results indicate that the expression level of Snmp2 gene will affect the behavioral adaptability of Blattella germanica,such as odor recognition,movement speed,aggregation,exploration ability.It is speculated that the odor recognition disorder may be caused by the weakening of olfactory response mechanism.To sum up,with the increase of the resistance of Blattella germanica,the number and distribution range of trichoid sensilla on the antennae decreased,and the number of differentially expressed genes involved in epidermis formation and nerve conduction showed a significant increase trend,indicating that the development of resistance affected the body development and olfactory regulation mechanism of Blattella germanica,and sensory neuron membrane protein Snmp2 gene would enhance the aggregation of Blattella germanica and weaken the olfactory response mechanism,action speed and exploration ability of Blattella germanica.however,the specific pathway and influence mechanism of Snmp2 gene in nerve conduction need further research and discussion. |
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