| Aphidius gifuensis is an endoparasite natural enemy insect that shows permanent effect against aphids that infest vegetables,fruit trees and crops.Diapause is a survival strategy adopted by insects during the long-term evolution to withstand harsh conditions.In the field of biological control,diapause has been known to effectively extend the shelf life of natural enemy insect products,facilitate the storage and transportation,and improve the performance of these products.A.gifuensis can accumulate glycogen and convert it into trehalose during the preparation period for diapause.Trehalose is not only an important energy source during diapause of insects,but also an important stress-resistant substance,so studies on the accumulation of sugar are of essential importance to reveal the molecular mechanisms of diapause.Studies have shown that the metabolism of carbohydrates is mainly regulated by the trehalose signaling pathway and the downstream glycogen metabolism pathway,among which trehalose6-phosphate synthase(TPS),glycogen phosphorylase(GP)and glycogen synthase(GYS)are the key regulatory enzymes involved in glycogen synthesis and conversion of glycogen to trehalose.At present,the functions and interactions of these three enzymes in diapause of A.gifuensis have not been clarified.Based on transcriptome sequencing results of A.gifuensis,primers were designed to obtain the sequences of AgTPS,AgGP,and AgGYS from the late 4~thh instar larvae of the insect by PCR amplification,RACE and sequencing,and bioinformatics analysis was performed.Relative expression of the 3 diapause-related genes at different developmental stages under diapause-inducing condition,and was compared to those during non-diapause-inducing condition,to clarify the expression pattern of these 3 genes in preparation phase,maintenance phase,and termination phase of diapause.At last,alternative host plants were screened for A.gifuensis as conventional host plant tobacco requires long incubation period,large space,and high humidity that cause extra cost.The main findings are as follows:1.Full-length sequence of the AgTPS,AgGP and AgGYS were obtained.The AgTPS gene is 2971 bp long,including a 2385 bp open reading frame(ORF)that encodes 794 amino acids;the AgGP gene is2590 bp long,including a 2535 bp ORF that encodes 844 amino acids;and the AgGYS gene is 2529 bp long,including a 2073 bp ORF that encodes 690 Amino acids.The proteins encoded by the 3 genes are highly conserved,and are more than 90%identical to homologous proteins of other insects.2.The stability of 5 commonly used internal reference genes(Gapdh,PPI,β-tub,RPL13 and RPII)in different developmental stages and diapause status of A.sinensis was evaluated.No amplification curves or dissolution curves of PPI,β-tub and RPII genes were observed in the fluorescence PCR analysis.Analysis of the remaining 2 internal reference genes with BestKeeper and Normfinder softwares suggested that Gapdh was more stable and can be used as an internal reference gene in this study.3.The expression patterns of AgTPS,AgGP and AgGYS genes showed significant differences under diapause-inducing and non-diapause-inducing conditions.Expression levels of all 3 genes were significantly lower under diapause-inducing condition in 3rd instar larva;but AgGP and AgGYS expression significantly increased in 4~thh instar larva,presumably to prepare for the diapause during which A.gifuensis accumulates large amount of glycogen and synthesizes cold-resistant substances;AgTPS expression significantly increased at the end of 4~thh instar larvae,AgGP expression remained stable,and AgGYS expression decreased,presumably to facilitate trehalose accumulation.During the maintenance phase,expression of all genes was significantly reduced which was in line with decreased metabolic activity;then in the termination phase,expression of AgTPS and AgGP remained low,while AgGYS expression significantly increased,suggesting that glycogen play an important role in post-diapause development of the insect.4.The development duration and fecundity of Myzus persicae on four legumes:Pisum sativum,Vicia faba,Glycine max and Vigna radiata were tested,and then a comparison was conducted amongst the important biological characteristics,such as emergence rate,body type,adult lifespan,and parasitic capacity,of the Aphidius gifuensis reared on leguminous plants and tobaccos,as well as the progeny obtained by letting the wasp rared on broad bean return parasitize aphids on tobaccos.The results showed that the development durations of aphids on soybean and mung bean were shorter,and the total litter size of a single aphid was also significantly higher than that of Garden pea and broad bean.There was a significant difference in the propagation speed.Vigna radiata was not a suitable host plant for the propagation of aphid and wasp.The emergence rate of mummy on Garden pea and soybean was significantly lower than that of other plants.There was no significant difference in the sex ratio of the progeny aphid on different plants.The adult lifespan of the progeny wasp on leguminous plants were significantly shorter than that of the tobacco group,not to mention that they were also found to develop smaller bodies.The parasitic capacity of Aphidius gifuensis on different host plants can be ranked as follows:Nicotiana tabacum>Vicia faba>Pisum sativum>Glycine max.Each indicator of the progeny obtained by the return parasitization was not lower than those of the tobacco group.In conclusion,considering such factors as rearing cycle,time and space utilization rate,and economic cost,Vicia faba can be regarded as the most suitable host plant for the mass rearing of Aphidius gifuensis. |
