| The Propylaea japonica Thunberg can prey on a variety of aphids,eggs and young larvae of some insects such as lepidoptera,hemiptera and diptera,and it has the characteristics of strong adaptability,high temperature resistance and hunger resistance,etc.It is a kind of natural enemy insect with great application prospects and its large-scale production is the key for biological control.Artificial diet has many significant advantages,such as reducing the cost of large-scale rearing natural enemy insects and effective controlling of natural enemy insect growth uniformity,etc,and it is the important foundation of large-scale insect rearing,but the artificial diets of the P.japonica have many problems currently,e.g.slow growing,preoviposition period extension,no eggs or the quantity of eggs is little,etc,which greatly limit its application in biological control.Based on this,the larval and adult rearing schemes of P.japonica were respectively optimized on the basis of artificial diet,which has Mythimna separate Walker mostly.After that,the optimized artificial diet rearing scheme was evaluated by the sub-generational rearing,the verification of predation function and the mass centralized rearing of the P.japonica,and the differentially expressed genes of the nutrient and reproduction-related metabolic pathways in the P.japonica of different nutritional sources were investigated by transcriptome sequencing analysis.In addition,the vitellogenin(Vg)gene of P.japonica was cloned,and the expression of this gene and the Vitellin(Vt)content in the ovary of the P.japonica of different nutritional sources were measured.The results of this paper lay a foundation for the large-scale propagation of the P.japonica,and also provide a certain theoretical basis for the further optimization,improvement and exploration of its nutrition-reproduction relationship.The specific research results are as follows:1.Optimization of artificial diet for P.japonicaIn this study,the rearing effects of the artificial diets to P.japonica,which were based on M.separate,pig liver and Tenebrio molitor Linnaeus were firstly compared,and it was concluded that the rearing effect of the artificial diet based on M.separate was the best,with a survival rate of 66.5%.On this basis,the artificial diet in larval and adult stages were optimized respectively to further improve the survival rate and the fecundity of P.japonica.Finally,the rearing scheme of artificial diet for P.japonica was determined as follows: for larval stage,on the basis of the artificial diet based on M.separate,two frozen aphids were added everyday in the first and second instars;In the adult stage,10% trehalose solution and 5frozen aphids were added everyday on the basis of the artificial diet which has M.separate mostly.Under this rearing scheme,the survival rate of P.japonica was 87.3%,and the number of eggs laid by the single female was 221.7,which has no significant difference from that of the control group(fresh aphids).This method can save 80% of the natural prey,and the frozen aphids has many advantages,such as,they are not restricted by(and the frozen aphids can reduce the dependence on the climate and host plants,and they are convenient to be stored)the host plants and they can be stored,etc,so,they have a better application prospect in the future mass production of P.japonica.2.Effect evaluation of rearing P.japonica with artificial dietIn order to evaluate the application prospect of the optimized artificial diet in the large-scale propagation of P.japonica,this experiment continuously reared six generations of P.japonica with the help of this diet,and the conclusion can be reached that,the indexes such as survival rate,fecundity,instrinsic growth rate and the predation function had no degradation;During mass centralized rearing experiments,it was found that there was no significant difference in survival rate between the artificial diet rearing group and the fresh aphid control group with the same density,and the trend was the same,the survival rate was the highest when there are 30～50 heads in each cage,and the survival rate decreased with the increase of population density of P.japonica.3.Transcriptome analysis of P.japonica of different nutrient sourcesThrough transcriptome sequencing analysis of P.japonica of different nutrient sources(pjad: artificial diet + water;pjads: artificial diet +10% trehalose solution;pjsad: artificial diet+10% trehalose solution +5 frozen aphids/day;pjck: both larvae and adults are fresh aphids),conclusion can be reached that,there were 8,071,5,910 and 8,705 differentially expressed genes in the groups pjad,pjads,pjsad and control group(pjck group),respectively.Furthermore,the expression of differentially expressed genes related to nutrient metabolism,juvenile hormone synthesis and nutrient regulation pathway in P.japonica of different nutritional sources was analyzed,and the results showed that,in the pathway related to carbohydrate metabolism,the majority of differentially expressed genes of P.japonica of pjad group were up-regulated.In the pathway related to amino acid metabolism,the differentially expressed genes related to the intermediate of glucose metabolism which is as a synthetic material in the P.japonica of the pjad group were generally up-regulated;In the pathway related to amino acid metabolism,the differentially expressed genes related to the conversion of sugars into amino acids in P.japonica of the pjad group were generally up-regulated,but the differentially expressed genes related to the direct synthesis of amino acids were generally down-regulated,but the differentially expressed genes related to the direct synthesis of amino acids were generally down-regulated.In the pathways related to lipid metabolism,differentially expressed genes related to fatty acid metabolism and fatty acid degradation in P.japonica in the pjad group were significantly up-regulated.The gene expression level of Juvenile hormone acid methyl transferase(JHAMT)was down-regulated in the combination of groups pjad and pjads,and the pjsad group was close to the pjck group.But the enzymes involved in the decomposition of JH: the gene expression levels of juvenile-hormone esterase,juvenile hormone epoxide hydrolase and juvenile hormone diol kinase were up-regulated in the groups pjad and pjads.With the addition of trehalose(pjads group)and frozen aphids(pjsad group),the expression level of differentially expressed genes related to the above metabolic pathways in P.japonica gradually approached the level of control group(pjad group),but TOR(target of rapamycin),a key enzyme in the nutritional regulation pathway,was significantly down-regulated in the three groups of artificial diet.4.Cloning analysis of vitellogenin gene of P.japonicaBased on transcriptome data,the gene sequence of vitellogenin(Vg)of P.japonica was cloned and named pj Vg.Through analysis,the full length of pj Vg sequence open reading frame is 5349 bp,encoding 1782 amino acids,the molecular weight is 204.35 k Da,and theoretical isoelectric point is 8.31.Sequence alignment analysis showed that the pj Vg gene contained three typical structure domains of vitellogenin,and its sequence homology with the Vg of Harmonia axyridis Pallas was 65.23%,and that with the Vg of Tribolium castaneum Herbst was 53.39%.The expression levels of pj Vg in P.japonica of different nutrient sources were measured that,groups pjads,pjsad and pjck are 1.23,6.87 and 4.25 times of group pjad respectively.Vitellin(Vt)content in the ovaries of pjad,pjads,pjsad and pjck were 3.53μg/m L,4.90 μg/ml,8.78 μg/m L and 10.07 μg/m L,respectively,among which there was no significant difference between the groups pjsad and pjck. |
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