Study Of Midgut Trypsin Activities And Gene Expression In Oriental Fruit Moths During Larval Development

Grapholitha molesta is a cosmopolitan fruit pest,which mainly harm peach shoots and the fruits of peach,pear,apple,papaya and plum trees.The typical habit of seasonal transferto the host plants,is the main reason of a G.molesta outbreak.Proteases in the midgut play an important role in insects' growth and developmental processes.This research selected G.molesta as the material of study.First,the optimal pH of the intestinal protease of G.molesta larvae was determined by biochemical technology.Basedon that,G.molesta were fed with different host plants,and their growth and development conditionswere observed.In the meantime,the protease activities were determined bybiochemical technology,preliminarily making clear the role of trypsin activity in the insects' growth,development and host adaptability.Next,trypsin genes were clonedby molecular biological techniques,obtaining correlation information of gene sequences and determining the trypsin gene expression level in the midgut of G.molestafed with different host plants by real-time PCR.Knowing the molecular mechanism of the trypsin gene in the digestive process of insects may provide new ideas for using protease to target and control pest insects.The results are as follows:(1)By using biochemical technology,we obtained the optimum pH of midgut total protease of G.molesta larvae in the three buffers,Tris-HCl,KH2PO4/NaOH and glycine/NaOH,were 10.5,11.0 and 11.0,respectively;those of high-alkaline trypsin were 10.5,11.0 and 11.0,respectively;and those of low-alkaline trypsin were 8.5,9.0 and 9.0,respectively.(2)G.Molesta larvae were collected from 4 host plants in the fields(peach tops,Qinguan apples,pear and papaya).Artificially reared G.molesta by were fed with 6 host plants(Qinguan apples,Gala apples,Chinese flowering crabapples,peaches,nectarines and plums).Their total protease,high-alkaline trypsin,low-alkaline trypsin activities showed significant differencesin different instars.The protease activities of G.molesta collected from 4 host plants in the field were the highest in the 4th instar,then the 3rd instar,followed by the 5th instar.All proteases activities of G.molesta fed with peaches were the highest,followed by papayas.For artificially reared G.molesta were fed with 6 host plants,the total protease activities were significantly different in 4th instar,high-alkaline trypsin activities showed significant differences,and the activity characteristics were 4th instar >3rd instar >5th instar.However,low-alkaline trypsin showed no differences.The total protease and high-alkaline trypsin activities of artificially reared G.molesta fed with Qinguan apples were higher than for those fed with Gala apples.The total protease and high-alkaline trypsin activities of G.molesta collected from Qinguan apple trees in the fields were lower than for those artificially reared G.molesta fed with Qinguan apples.(3)The growth,development and reproduction status of artificially reared G.molesta fed with 6 host plants(Qinguan apples,Gala apples,Chinese flowering crabapples,peaches,nectarines and plums)and G.molesta collected from Qinguan apple trees in the fields fed with Qinguan apples were observed,significant differences were found due to the effect of host plants showed.The measurements of larval duration(9.79 d),adult duration(11.16 d),larval-adult total duration(29.09 d),larval survival rate(38.66%),emergence rate(68.86%),number of eggs per female(50.0)and pupa weight(8.96 mg)of artificially reared G.molesta fed with peaches were low,but the rate of increase(rm)was the highest.The pupation rate(98.13%)and number of eggs perfemale(67.8)of artificially reared G.molesta fed withQinguan apples were higher than that fed with Gala apples.The number of eggs per female(110.6)of artificially reared G.molesta fed with plums was the highest,The number of eggs per female of G.molesta fed with apples was higher than that for peaches.Plum is the most suitable host for G.molesta.(4)To observethe effects of the different host plants(artificial diet,peach shoots,apples,pears,papayas,Chinese flowering crabapples,peaches,nectarines,plums)on the expression level of trypsin genes,two trypsin genes: GMtrypsin-1 and GMtrypsin-2,were cloned in this study and were verified as G.molesta larvae midgut trypsin genes;they were 816 bp and 923 bplong respectively,encoding 257 and 259 amino acid residues,respectively.The predicted mature trypsin had molecular masses of 27.98 kDa and 27.93 kDa respectively.Sequence analysis results showed that two genes translated protein that trypsin has the characteristic of structure,such as signal peptide,catalytic triplet amino acids His-Asp-Ser and highly conservative actie sites RIVGG and GDSGGP in both ends of the sequence.The kinship between GMtrypsin-1 and soybean budworm Leguminivora glycinivorella was close relatively,but GMtrypsin-2 wasn't relatively closely related to other Lepidoptera species.(5)The expression levels of G.molesta trypsin GMtrypsin-1 and GMtrypsin-2genes had significant differences in different instars,and the gene expression level characteristics were 4th instar>3rd instar >5th instar.The GMtrypsin-1expression level of G.molesta collected from Qinguan apple trees in the field was higher than that for artificially reared G.molesta fed with Qinguan apples,however the GMtrypsin-1expression level was lower than the latter.Two gene expression levels of artificially reared G.molesta fed with Qinguan apples were both lower than those for Gala apples,and two gene expression levels of artificially reared G.molesta fed with peach tops,Qinguan apples and nectarines were high.(6)Comprehensive analysis shows that trypsin activities can affect the parameters of G.molesta,such as calendar period,emergence rate,pupa weight,promote the growth and development of G.molesta.The characteristic of G.molesta the trypsin activities and trypsin gene expression levels all were 4th instar >3rd instar >5th instar.The GMtrypsin-2 gene expression level of artificially reared G.molestafed with plums was the highest,and trypsin activities were high,and the growth was better.There is a positive correlation among the three aspects,but with some special phenomena indicating that trypsin activity and gene expression levels are different with different host plants and different species of the same host,and that these differences are associated with the growth and development of G.molesta.