Diet Analysis And Insect-Controlling Efficiency Of Wolf Spider Based On Molecular Biotechnology

This paper was supported by National Natural Science Fundation of China?No.31372159?.The perennial dominant species of Chinese farmland spiders--Pardosa pseudoannulata?Araneae:Lycosidae?and its prey was taken as the research object in this study.Based on the macro and micro aspects,the feeding behavior under different ambient conditions,digestion rate,recipe and predation efficiency of Pardosa pseudoannulata was explored.In this project,single factor analysis and L₉?3³?orthogonal test were used to analyze the macroscopic factors affecting the feeding behavior of Pardosa pseudoannulata,which included illumination intensity,ambient colour and ambient temperature.Moreover,frontier molecular biology methods were used to analyze the microscopic aspects,which included the predation and digestion efficiency of Pardosa pseudoannulata and prey lists in different habitats,such as end-point PCR,real-time PCR,DNA barcodes,NGS and digital PCR technology.An experimental method for Pardosa pseudoannulata's precise predation has been established,which contained five dominant pest species in the farmland ecosystem:Nilaparvata lugen,Laodelphax striatellus,Cnaphalocrocis medinalis,Sitobion avenae and Nephotettix ipunctatus.The research breaked through the puzzle that the predatory behavior of natural enemies such as Pardosa pseudoannulata was not easy to observe.The main findings are summarized below:?1?Macroscopically,the effects of different ambient factors on prey efficiency of Pardosa pseudoannulata were analyzed.The results of single factor experiments shown that the predator behavior of spiders was most active when the ambient conditions were 10Lx,red,and 27°C,respectively.In order to simulate the natural habitat of farmland,orthogonal experiment L₉?3³?was used to evaluate the comprehensive effects of various ambient factors on spider predation efficiency.The experimental results shown that the biggest range R was the ambient temperature.The predator behavior of spiders was most active under the optimized combination of 15 Lx-green-27°C.These results demonstrated that the predatory behavior of the Pardosa pseudoannulata is compatible with the ambient factors,which was the result of the continuous development of the species in the long-term evolution process.?2?Microscopically,the end point PCR method was used to investigate the digestive rate of Pardosa pseudoannulata after predation by Drosophila melanogaster.The results showed that after eating for 16 h,there were few Drosophila melanogaster left in the spider,and no residual DNA could be detected after 24 h.Subsequently,the end-point PCR method combined with real-time PCR method was used to detect the digestion efficiency of Pardosa pseudoannulata preying on Nilaparvata lugens under different digestion time and digestion temperature.The results showed that the DNA residue of Nilaparvata lugens in Pardosa pseudoannulata cephalothorax was relatively stable within 1 h² h after predation.The decay rate of the Nilaparvata lugens body fluid was accelerated at 2 h and tended to be gentle after 4 h.When predation after 2 h,the body fluid of Nilaparvata lugens began to enter the abdomen and digested rapidly.The digestion rate was the fastest at 4 h and became flat after 8 h.The digestion temperature had little effect on the Nilaparvata lugens DNA decay rate in Pardosa pseudoannulata cephalothorax.However,the DNA content of Nilaparvata lugens in the abdomen decreased gradually with increasing temperature,and the digestion rate was fastest at 27°C.Combined with the results of end-point PCR and real-time PCR,the prey can be better digested under the environmental conditions of 28°C±2°C.?3?NGS method was used to analyze the diet of Pardosa pseudoannulata in four habitats?a wetland,a tea plantation,an alpine meadow and a paddy field?.The results suggested that the prey taxa of Pardosa pseudoannulata was very wide and predominatly preyed on Coleoptera and Diptera insects.In all of the four habitats of this study,7 orders and 24 families of insects were detected.The living environment and season had a great impact on Pardosa pseudoannulata diet diversity,the rules of which could be concluded as follows:low altitude habitats>high altitude habitats;diversiform biocenoses habitats>simplex ones;fields with no pesticide use>fields with pesticide use;wetland habitats>dry land habitats;crop growth period habitats>alternate husbandry period habitats.?4?The digital PCR technique was used to quantitatively analyze the diet of Pardosa pseudoannulata.Based on the random sampled pests from the paddy fields of Hunan Academy of Agricultural Sciences,and COI gene was used as the target gene,universal primers were designed to identify 9 types of pests by DNA barcoding.It was found that the main dominant species of pests in rice fields in Hunan were Nilaparvata lugen,Laodelphax striatellus,Cnaphalocrocis medinalis,Sitobion avenae and Nephotettix ipunctatus.Under artificial intervention,Pardosa pseudoannulata were over-fed with the five pests mentioned above,and the maximum number of prey that the spider could prey within 24 h was obtained.The number of positive microdroplets were tested using digital PCR,followed by estimating the predation amounts of Pardosa pseudoannulata collected under natural conditions.Experimental data showed that the five Pardosa pseudoannulata had predated 13.2 Nilaparvata lugen,10.7 Laodelphax striatellus,0.8 Cnaphalocrocis medinalis,5.1 Sitobion avenae and 1.0 Nephotettix ipunctatus under natural conditions.Considering that the nucleic acid of preys would be degraded in the body,and the fierce nature of Pardosa pseudoannulata,which the preys would only be killed but not be eaten,it could be presumed that the actual number of insects controlled by Pardosa pseudoannulata would be greater than the experimental value.The characteristics and innovations of this research were as follow:The frontier molecular biology methods such as DNA barcodes,NGS and digital PCR technology were used successfully for the first time to detect the recipe and food intake of Pardosa pseudoannulata.An experimental method was established that the predation of Pardosa pseudoannulata for five major pests in paddy field under natural conditions could be precisely calculated.Moreover,an evaluation method and mathematical model for the pests-control efficiency of wolf spider was established under natural conditions.The research solved the problem that the traditional method could not fully study the spider recipes and precise predation.The results revealed the important role of Pardosa pseudoannulata in biological control,which was conducive to understanding the evolutionary relationship between spiders and their prey insects in the process of evolution,enriching the connotation of arachnology.This study provided practical information such as new topics and methods for developing IPM strategies in farmland ecosystems and provided new theoretical basis and scientific information for using spiders to control pests.This research was of great significance for reducing the amount of pesticides and protecting the ecological environment and human health.