| Bradysia impatiens Johansen is a kind of polyphagous pest.It was first recorded in foreign countries because of its harm to Impatiens balsamina,while it was first found in edible and medicinal mushroom cultivation greenhouses in China.The recent survey found that B.impatiens caused serious damage to protected vegetables（such as,Allium tuberosum,A.fistulosum,A.sativum,etc.）,as well as protected fruits and flowers.It occurs annually in the greenhouse environment with high temperature and high humidity,and has overlapping generations.The most commonly used control methods in agricultural production are still chemical pesticide irrigation or direct spraying.However,the long-term frequent use of pesticides has significantly increased the drug resistance of B.impatiens,resulting in serious environmental pollution,excessive pesticide residues in vegetables,fruits and other agricultural products,threatening people’s health.Therefore,there is a great demand for new green prevention and control technology in production.Allyl isothiocyanate（AITC）,a newly discovered botanical extract,has been widely used in fumigation control of a variety of storage pests because of its green,safety,low toxicity,low residue and easy degradation.In addition,it also has good activity on underground pests,weeds,nematodes and pathogenic bacteria.Carbon dioxide（CO2）is an important gas to regulate insect respiration.The increase of CO2 concentration（10%or higher）will make insects breathe frequently or their spiracular valves remain permanently open.The airtightness of ecological environment in greenhouse facilities was relatively fine,which could provide a favourable condition for indoor fumigation and soil fumigation of AITC to prevent and control B.impatiens.Meanwhile,people often take measures to enrich CO2 in greenhouse crops to enhance photosynthesis and improve yield and quality.Therefore,the appropriate increase of CO2 concentration is beneficial to protected crops.Based on finding that insect spiracular valves remain permanently open in CO2of 10%or higher,we speculate that the presence of CO2 with AITC may result in a higher uptake of fumigant via the spiracles and enhance the AITC toxicity to B.impatiens.In this present study,we detected the response mechanism of B.impatiens to AITC from the perspectives of toxicology,ecology,transcriptomics and metabolomics;examined the effect of AITC on the fumigation efficiency of B.impatiens under elevated CO2concentration.In addition,the response of B.impatiens to the elevated CO2 concentration was studied from the ecological and physiological aspects.The main results are as follows:1.Allyl isothiocyanate（AITC）has good fumigation activities on eggs,larvae,pupae,females and males of the B.impatiens,especially on adults.The LC50 value of AITC to 3rdlarvae was 10.400μL/L by fumigation and 13.632μL/L by stomach poison of leaf-dipping.In addition,after the 3rd instar larvae were treated with sublethal concentration of AITC(LC25and LC50),there were obviously adverse effects on growth,development and reproduction.For instance,the larval and pupal stages were prolonged,pupal weight was reduced,pupation rate and adult emergence rate were significantly reduced,and the fecundity was significantly inhibited.Moreover,the sublethal influence on fecundity was more significant by fumigation than that of leaf dipping.2.The results of transcriptome sequencing indicated that 480 differentially expressed genes were found in female and 14856 in male of Bradysia impatiens treated with LC25 of AITC.Through the analysis of KEGG database,we found that:（1）The up regulated genes of differential expression in the female of B.impatiens were significantly enriched in the insect hormone biosynthesis pathway,in which the hormone esterase（JHE）gene was induced to be upregulated;（2）The differentially expressed up-regulated genes were also significantly enriched in drug metabolism-cytochrome P450,the metabolism of exogenous substances by cytochrome P450 and glutathione metabolism,among which GST gene was all up-regulated;（3）Real time quantitative polymerase chain reaction（RT-q PCR）was used to verify the accuracy of transcriptome data.These results suggest that AITC induces the expression of genes related to various biological processes,such as hormone,oviposition and detoxification,in which JHE may be the key gene regulating AITC to inhibit oviposition of B.impatiens,and GST plays an active role in the detoxification of AITC.3.A large number of different metabolites were produced after AITC treatment,and 22metabolites with significantly increased expression were identified by hierarchical cluster analysis.KEGG was used to analyze the pathways of different metabolites,and 15 pathways were found to have significant effects.The identified key metabolites mainly include L-serine,L-methionine,L-leucine,L-asparagine,DL serine and taurine,which may play an important role in the pathogenesis or lethal mechanism of B.impatiens treated with AITC,which provides a new way for the prevention and control of this insect.4.With the increase of CO2 concentration,the fumigation effect of AITC on B.impatiens was improved,and the mortality rate was 100%with the extension of treatment time.The activities of three antioxidant enzymes（SOD,CAT,POD）were significantly induced in the high CO2 concentration.After AITC fumigation,the activities of SOD,GST and Car E were significantly induced.After the combination of AITC with high CO2 concentration,SOD,GST and Car E activity increased significantly.5.With the increase of CO2 concentration,the duration of pre-adult and pre-total oviposition were shortened first and then prolonged,they were 19.01 and 20.43 d,respectively,under normal CO2 concentration（NC=400 ppm）;14.48 and 15.59 d,respectively,under medium CO2 concentration（MC=600 ppm）;and 19.98 and 21.41 d,respectively,under high CO2 concentration（HC=800 ppm）.The longevity of female was decreased with the increase of CO2 concentration.The numbers of oviposition were 92.50,99.33 and 75.50 eggs from 400 ppm to 800 ppm.The value of intrinsic rate of increase,finite rate of increase and net reproduction rate of the population were all the highest under MC concentration,while the mean generation time and the population doubling time were the shortest under MC concentration.The values of life table population parameters are opposite to HC concentration.6.The increase of CO2 concentration affected the nutrient content of host plants,and then indirectly affected the physiological and biochemical indexes of B.impatiens.With the increase of CO2 concentration,the contents of soluble sugar and free amino acids remarksblely enhanced in Chinese chive leaves,while the soluble protein declined gradually,and there was no significant effect on free fatty acid.The contents of glycogen,soluble sugar and free amino acid increased significantly in the body of B.impatiens,soluble protein decreased significantly,while the contents of trehalose and total fat increased first and then decreased with elevated CO2.Besides that,the contents of physiological indexes in the body of B.impatiens were correlated with the contents of nutrients in Chinese chive leaves,and the correlation was differed under different CO2 concentration.The results provide theoretical basis and technical support for the green control of B.impatiens,scientific basis for the development and large-scale application of plant pesticide AITC,and reference for the suitability of B.impatiens to the elevated CO2 concentration. |
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