| Palembus dermestoides(Fairmaire)belongs to Coleoptera,Tenebrionidae,Diaperinae,Diaperini,and distributes widely at home and abroad.Originally,it was known as a kind of storage pest,but now it has turned into an important resource insect for its very high medicinal and forage value.In this paper,Palembus dermestoides was chosen to be the research project,RT-PCR technology was used to clone ecdysone receptor(EcR)gene sequence fragment from P.dermestoides,and then phylogenetic analysis and bioinformatics analysis was carried out.Quantitative real-time PCR technology was used to analyze EcR and hormone receptor 3(HR3)gene mRNA expression in different development stage of P.dermestoides.Different development stages of P.dermestoides were treated by injecting different concentration exogenous 20-hydroxyecdysone(20E),and the external morphological changes,mortality,eclosion rate,etc.were discussed after treatment.Transmission electron microscope(TEM)was uesd to investigate integument ultrastructure changes of P.dermestoides pupa treated by 20E.Midgut ultrastructure changes of P.dermestoides last instar larva after injecting 20E were also taken into research.After fully discussed and analyzed of experimental results in each chapter,we drew the main conclusions as follows:1.A 738bp P.dermestoides EcR gene(PdEcR)sequence fragment was obtained,with a Genbank accession no.JQ666843,which showed high similarity to EcR-A isoform encoding a 244 amino acid protein.Its longest open reading frame(ORF)contained 700bp(39bp-737bp)sequence,encoded 233 amino acid with a predicted molecular mass 25686.5Da and a molecular formula C1099H1738N314O370S13.Theoretical isoelectric point(PI)of PdEcR was 7.67,that proved PdEcR was an alkalinity protein.There was no signal peptides in PdEcR protein,which made PdEcR as non secretory type protein.On the whole,PdEcR performed hydrophilcity,but there were a few hydrophobic regions in functional domain.PdEcR protein contained no transmembrance domains,but had conservative function structure in DNA binding domain that nuclear receptor family peculiarly have.EcR genes from insects of the same order have close evolutionary relationship,and the differences between EcR isoforms of the same species are smaller than that in different species.2.EcR gene expressed in different development periods of female and male individuals of P.dermestoides was studied.In female individuals,the highest expression of EcR gene appeared in 2ndd female pupa,and the expression gradually declined from 3 rdd to 6thd female pupa.In 6thd female pupa,EcR gene expression was already lower than that in the last instar larva,and in the lstd female adult,EcR gene expression slightly went up,but started to decline in 3rdd female adult until slightly rising again in 6thd female adult.Last instar larva had higher EcR gene expression than in female adult individuals.In male individuals,both lstd male pupa and 2ndd male pupa had a very high EcR gene expression,and the expression of 1std male pupa was even or higher than that in 20dd male pupa.In the 3rdd male pupa,EcR gene expression sharply decreased,and slightly raised again in the 4thd male pupa.The EcR gene expression level in the 4thd male pupa Was a little bit higher than that in the last instar larva,but in the 5thd male pupa,EcR gene expression decreased again.EcR gene expression changed weakly in the 6thd male pupa and in adults,slightly lower than that in the last instar larva.Combined the results of EcR gene expression in female and male individuals,we could easily find out that,in pupa and adult stage,the female individual had only one EcR gene expression peak which is in the 2ndd female pupa,but male individual had 2 peaks which is showed at 1Std and 2ndd male pupa respectively.3.HR3 gene expressed in different development periods of female and male individuals of P.dermestoides was also studied.In female individuals,there were 3 expression peaks of HR3 gene,which were showed in the preliminary pupa,the 4thd pupa and the 6thd pupa respectively,and the highest HR3 gene expression peak was showed in the 4thd pupa.The highest HR3 gene experession level was 3 times higher than that in preliminary female pupa,2 times higher than that in 6thd female pupa,and 30 times higher than that in the last instar 'larva.The relative HR3 gene expression decreased progressively in female adults.In the 1std female adult,HR3 gene expressed 4 times higher than that in the last instar larva,but the expression in the 6thd female adult became lower than that in the last instar larva.In male individuals,there were 2 expression peaks of HR3 gene,which were in preliminary pupa and 4rhd pupa,and the peak in 4thd mare pupa was extremely high,which exceeded 5 times higher than that of highest expression peak of female.The relative HR3 expression slightly decreased in male adults,but their expression level was always higher than that in the last instar larva.From the trends of HR3 expression in female and male individuals,the peak expression in female individuals and male individuals were both in the 4thd pupa,and the peak expression level in male individuals was much higher than that in the female individuals.4.The toxic effects under the dosage of 20E used in this research on the last instar larvea of P.dermestoides were unconspicuous.Even at the 96th after treated by 10?g/?L 20E(the highest concentration be used in this experiment),the cumulative correction mortality was only about 30%.During the 6thh-96thh after treatment,the higher the concentration of 20E used,the sooner the 20E caused larvea dead.During the 12thh-36thh after treating by 10?g/?L 20E,dead bodies appeared due to cannibalism.The ratio of motality caused by cannibalism is more than 50%in the total mortality.However,there were no cannibalism phenomenon in the control goup and the other two dosage treatment groups.Therefore,it could be concluded that a certain concentration of 20E could enhance cannibalisms of P.dermestoides larva.The last instar larva treated by 10?g/?L 20E started to show molting behaviors earlier than the individuals in the control group,and usually caused those treated insects death due to the unfinished molting.1?pg/?L and 5?g/?L dosage of 20E usually caused small effection on mortality of pupa.However,20E of 10?g/?L caused about 60%pupa cumulative correction mortality in 96thh after treatment,and the toxic action was 6 times higher than that of 5?g/?L of 20E.Dead individuals were in copper brown colour,stiff,shrivelled condition.Early eclosion appeared in both 1?g/?L and 5?g/?L dosage treated group,but did not appear in 10?g/?L dosage treated group.Within 96th after treating by different concentration of 20E on female and male adults of P.dermestoide,except for 1 ?g/?L dosage treated group,male adults died earlier than female adults in 5?g/?and 10?g/?L dosage treated group,and male adults cumulative correction mortality was greater than that in female adults of the same time.The results revealed that,male adults were more sensitive than female adults treated by 5?g/?L and 10?g/?L dosage of 20E.5.Changes on P.dermestoides pupa integument structure during 6thh-96thh after treating by 10?g/?L dosage of 20E were observed.The results showed that,at the 6thh after treatment,integument changes were not obvious compared to that of the control group,cuticle synthetized thinly,the cytoplasm area of epidermis was rich in organelles,epidermal cells in vigorous secretory activity secreted toward cuticle and formed new cuticle layer.Microvilli apexes formed new endocuticle lamellar structure.Basement membrane was in dense integrity,and tightly connected with epidermal cells.After treating 12thh,epidermis departed from cuticle,and ecdysial space showed.Microvilli swelled into mushroom shape protuberance.Basement membrane connected tightly with epidermis.After treating 24thh,ecdysial space between cuticle and epidermis largened.Intercellular space appeared,abundant of organelles in epidermal cell cytoplasm,dense electron concentration mitochondrion,basement membrane was in good condition and connected tightly with epidermis.After treating 36thh,apical membranes of epidermal cell were destroyed,moulting fluids and materials degested by old endocuticle in ecdysial space scattered.There were abundant of organelles in epidermal cell cytoplasm,such as dense electron concentration mitochondrion,lots of electron lucent vesicles.Gaps between karyotheca and tenuigenin appeared,basement membrane structure became thinner,and new cuticle did not start to synthetize.After treating 48thh,new cuticle preliminarily formed,cavity appeared between old cuticle and new cuticle.Lots of ecdysial granules appeared in apolysis lucent structure,pore cannal extanded from epidermis to new formed cuticle,nutrition or synthesis materials delivered from epidermis to cuticle through pore cannals.Gaps between karyotheca and tenuigenin largened,mitochondrion electron concentration increased,the number of electron lucent vesicle increased.Large-sized blank area appeared in epidermis near basement membrane,and basement membrane became thiner.After treating 72ndh,basement membrane was destroyed,and wasn't be seen.New cuticle and epidermis connected loosely or apart.There were large-sized blank areas in epidermis,apolysis lucent structure did not depart from new cuticle.After treating 96th,clear area in cytoplasm of epidermal cell largened,the number of electron lucent vesicles increased,nucleus was seen,but organelles structures were difficult to recognize,and basement membrane structure was also destroyed and was not seen,epidermal cell area on the verge of degradation further worsened.6.Midgut ultrastructure of the last instar larvae of P.dermestoides treated by 10?g/?L dosage of 20E were observed within 96thh after treatment.The results showed that,microvilli of midgut epithelial cells from treated insects were seriously destroyed in preliminary intoxication stage,chromatin were uniformly-distributed,intercellular junctions were still tight,and abundant organelles structures in dense cytoplasm could be find.However,the more serious toxic-symptom treated insects got,the more obvious changes in midgut ultrastructure revealed.In the middle intoxication stage,intercellular spaces showed up,nucleus shrinked,nucleolus irregularly largened,chromatin condensed,clear areas appeared nearby nucleus in cytoplasm and between karyotheca and tenuigenin,electron concentration of mitochondrion increased.In nearly dead intoxication stage,intercellular spaces between cells largened,cytomembranes were destroyed,and bareness nucleus showed up,membrane organelles were destroyed seriously in cytoplasm,mitochondrion swelled with brokage range,and cells gradually disorganized,cytoarchitectures were damaged only with bareness nucleus in.Therefore,we can conclude that one of the toxic mechanisms of 20E is due to the destructive effects on microvilli,cytomembrane,membrane organelles(eg.endoplasmic reticulum)etc.membrane system.It was the first time that EcR gene sequence fragment was cloned from P.dermestoides of Coleoptera,and,mRNA expression of EcR and HR3 gene in different developing stages of P.dermestoides were analyzed by using quantitative real-time PCR technology.Macroscopic differences and microcosmic differences of P.dermestoides were observed after treating by 20E,and the results showed above.These results provide data references on specific function of ecdysterone and nuclear receptor in insects for further study,and on development and utilization of plant source pesticides of molting hormone. |
PDF Full Text Request