Studies On Diapause Mechanism Related With Physiology And Biochemistry Of Wheat Blossom Midge, Sitodiplosis Mosellana (Gehin)

The wheat blossom midge,Sitodiplosis mosellana(Gehin),is one of the intermittentlydisastrous insect pests injuring wheat.Two times of broad and disastrous damages wererecorded in China history in 1950's and 1980's respectively.In about ten years recently,thedisaster caused by the wheat blossom midge in a broad area was controlled effectiviely,butthe local damage was still often found in some wheat-growing regions.A trend extending itsdistribution area is obvious.Therefore,the potential threat of the wheat blossom midge to thewheat production did not disappear.It is said that diapause is one of key factors causingrampancy endangerment with intermittent and regional characteristics.However,manyproblems about the diapause mechanisms in wheat blossom midge remain uncertain.In thispaper,diapause mechanism of wheat blossom midge was conducted by means of methods andtechniques of biochemistry,proteomics,electron microscopy and radioimmunoassay on thebasis of previous studies.The present research will provide a foundation for further studies ondiapause regulation mechanism,forecasting,control of the wheat blossom midge.The mainresults were as follows.1.The comassie brilliant blue dying,SDS-PAGE and PAGE techniques were used toanalyze protein contents and compositions in pre-diapause,diapause and post-diapause larvaeof the wheat blossom midge.The protein content in different diapause stages wassignificantly different,which in pre-diapause and post-diapause larvae were higher than thosein diapause larvae.However,no obvious difference of protein content was observed between1st and 2nd year of diapause larvae,as well as between cocooned and non-cocooned larvae inthe same diapause periods.Based on the protein contents,the larval diapause in the wheatblossom midge larvae could be divided into two stages during diapause cycle:stage 1 is fromJune to December,protein average content 19.2269μg/individual in cocooned larvae and19.5734μg/individual in non-cocooned larvae.The stage 2 from late February to mid-May ofnext year,average 20.9578 and 20.7153μg/individual respectively,both higher than stage 1.According to the electrophoresis diagrams,expressions levels of two protein bands(18.3 and32.0 kD)from SDS-PAGE,and four protein bands from PAGE,as well as relativeexpressions amounts of protein bands 18.3 and 19.8 kD from SDS-PAGE obviously changed at various periods of pre-diapause,diapause and post-diapause,but their expressions weresimilar in different stages of diapause period.2.The proteomic approach was adopted to investigate proteins extracted from larvae ofwheat blossom midge at defferent developmental stages including pre-diapause,over-summering diapause,over-wintering diapause and post-diapause.On 2-DE gels stained bycoomassie brilliant blue,about 300 protein spots were detected in pre-diapause,and 275protein spots in each of other periods.There were 91,92 and 95 protein spots that showedmore than 2-fold changes in abundance in larvae at over-summering diapause,over-winteringdiapause and post-diapause in comparison with pre-diapause larvae.Eight protein spots,which were with most significant differences in larvae at different diapause periods,wereanalyzed by Matrix-assisted laser desorption ionization time of flight mass spectrometry(MALDI-TOF-MS).Among these eight proteins,three were up-regulated or unique duringpre-diapause,two were down-regulated or absent during pre-diapause,one was unique duringover-wintering diapause period,and the other two were up-regulated during over-summeringand over-wintering diapause stages.Seven of the eight protein spots were successfullyidentified from peptide mass fingerprints on the basis of NCBInr database.They wereproopiomelanocortin,NADH dehydrogenase subunit 1,F10F2.5,IKK interacting proteinisoform 2,GA10647-PA,purple CG16784-PB isoform B and B0228.6,respectively.Theseproteins play important roles in wheat blossom midge.3.The activities of protective enzymes peroxidase(POD),superoxide dismutase(SOD)and catalase(CAT)in pre-diapause,diapause and post-diapause larvae of wheat blossommidge were determined by using the protective enzyme testing kits.The results indicated thatactivities of the three protective enzymes showed a decreasing trend from pre-diapause toearly diapause.In one-year cycle,SOD and CAT activities of diapause larvae had the sameresponses to environment temperature,i.e.,increased at lower temperature but decreased athigher temperature,while POD activity was related to both environmental temperature anddevelopment situation of the larvae.A similar seasonal variation trend was observed in thethree protective enzymes of both no-cocooned and cocooned larvae in one-year cycle.Thethree protective enzyme activities of no-cocooned larvae were higher than those of cocoonedlarvae at the same diapause stage.No significant difference of the three protective enzymeactivities was found between 1st and 2nd year diapause larvae.The activities of the threeprotective enzymes increased gradually with the development of post-diapause larvae.4.The activities of metabolic enzymes Hexokinase(HK),Aldolase(ALD),Glycogenphosphorylase(Gpase)and Phosphofructokinase(PFK)in pre-diapause,diapause andpost-diapause larvae of wheat blossom midge were measured using testing kits.Flexible metabolic rearrangement was observed in diapause processing of S.mosellana.Gpase activitywas strengthened significantly,and activities of glycolysis enzymes(HK,ALK,PFK)wereweakened significantly after entering diapause,which ensured more carbon flowing intotrehalose synthesis.On the contrary,Gpase activity decreased,and activities of glycolysisenzymes increased after diapause termination,which ensured to form glycogen from trehaloseto supply energy for development.In one-year diapause cycle,activities of four enzymes wereall related with development situation of diapause larvae.Gpase and PFK were alsoinfluenced by environmental temperature.At the same diapause stage,activities of HK,ALKand PFK of no-cocooned larvae were higher than those of the cocooned larvae.On thecontrary,Gpase activities of no-cocooned larvae were lower than those of the cocooned larvae.No obvious difference of four metabolic enzyme activities was found between 1st and 2ndyear diapause larvae.5.The ecdysteroid titers in pre-diapause,diapause and post-diapause larvae of wheatblossom midge were determined by radioimmunoassay.No obvious difference of ecdysteroidtiters was found between 1 st and 2nd year of diapause larvae,as well as between no-cocoonedand cocooned larvae in the same diapause stages.In one-year cycle,the ecdysteroid titersshowed obviously a dynamic changes,which decreased significantly after diapausing,andthose of over-wintering larvae were highest(about 7.0pg/larva)during diapausing,whilethose of other season were low and had a minor difference.The ecdysteroid titers reducedsignificantly after diapause termination,and increased gradually with the development andreached peak level(15.22 pg/larva)about one week before pupation.6.By means of electron microscopy,ultrastructure of silk glands in S.mosellana larvaecollected from wheat ear and cocooned larvae from soil were observed.The results showedthat parallelly rough endoplasmic reticulum,golgi apparatus and mitochondrion were all verydeveloped in silk glands cell of larvae both from wheat ear and soil.Moreover,annularlyrough endoplasmic reticulum,which was very complex,was found in silk gland cells oflarvae from wheat ear.Glycogenosome,secretory granules around golgi apparatus,and silkmaterial of glandular cavity were richer in silk glands cell of larvae from wheat ear than thoseof cocooned larvae.