| The leafhopper Cicadella viridis(Hemiptera,Cicadellidae)is a widespread pest,which vectors plant viruses.They can damage more than 160 host plants,especially the young fruit trees and forest.This thesis detected the species of endosymbionts,and evaluated the copy number of endosymbionts in different Cicadella viridis stages and anatomical organs by Absolute Quantification PCR(TaqMan).As well as,the paper studied the effect of different antibiotics on the removal of endosymbionts of Cicadella viridis,and explored effects of different antibiotics on the total content of amino acids,protein,protective enzyme,detoxifying enzymes and transaminase.The main purpose was to provide the theoretical basis and technical support for the discrepancy of endosymbionts of different geographical Cicadella viridis populations,the function of endosymbionts genes,and the correlation diversity between endosymbionts of Cicadella viridis and their mitochondrial genes.The main results are as follows: 1.Detection of endosymbionts and the sequences analysis of 16 S rRNA gene in the green leafhopper(Cicadella viridis)To understand the endosymbionts species in Cicadella viridis,PCR was performed using universal primers(27F-1513R)and specific primers for the bacterial 16 S rRNA gene from Cicadella viridis,followed by agarose gel electrophoresis,cloning,sequencing,sequence alignment,and analysis.The phylogenetic trees were generated by the Bayesian and maximum-likelihood methods.The results showed that:(1)Sulcia muelleri was classified as Flavobacteriaceae,Flavobacteriales,Flavobacteria,Bacteroidetes.The 16 S rRNA gene sequences of Sulcia muelleri got 13 new haplotypes and 14 variable sites;the identity rate and genetic distance of which were 99.40%-99.80% and 0.001-0.004,respectively.The phylogenetic tree revealed that Sulcia muelleri was associated with Sulcia muelleri detected from the leafhoppers of Cicadellinae.Sulcia muelleri had a low degree of variation,and all haplotypes belonged to Sulcia muelleri species.(2)Sodalis sp.belonged to Enterobacteriaceae,Enterobacteriales,Gammaproteobacteria,Proteobacteria.The 16 S rRNA gene sequences of Sodalis sp.obtained 5 new haplotypes and 20 variable sites in this study,and the identity rate and genetic distance were 96.10%-99.70% and 0.002-0.040,respectively.The phylogenetic tree showed that Sodalis sp.was most closely related to Sodalis glossinidius detected from the Tsetse flies.Sodalis sp.had a high degree of variation,and all haplotypes belonged one genus.(3)Rickettsia sp.belonged to Rickettsiaceae,Rickettsiales,Alphaproteobacteria,Proteobacteria.The 16 S r RNA gene sequences of Rickettsia sp.had 15 haplotypes and 35 variable sites,and the identity rate and genetic distance were 98.40%-99.90% and 0.001-0.015,respectively.The phylogenetic tree showed that all haplotypes belonged to Rickettsial bellii group.The analysis showed that Sulcia muelleri is Primary endosymbiont,Sodalis sp.and Rickettsia sp.are secondary endosymbionts,and especially,Rickettsia sp.was the first reported.2.Distribution of endosymbionts in the green leafhopper(Cicadella viridis)This study designed specific primers for Sulcia muelleri、Sodalis sp.and Rickettsia sp.,investigated the infection frequency of endosymbionts in different Cicadella viridis stages,and detected the copy number of endosymbionts in different Cicadella viridis stages and anatomical organs by using Absolute Quantification PCR(TaqMan).The results showed that:(1)The infection frequency of Sulcia muelleri、Sodalis sp.and Rickettsia sp.in all stages of Cicadella viridis(eggs,nymphs,females and males)were 100%.The copy number of endosymbionts gradually increased from eggs to nymph 2,while the copy number decreased from nymph 2 to nymph 3,and then the trend increased again from nymph 3 to nymph 5.The highest copy number of Sulcia muelleri and Rickettsia sp.was nymph 5,followed by female,and the lowest one was egg.The highest copy number of Sodalis sp.was female,followed by nymph 5,and egg contained the lowest copy number.(2)In different organs or tissues the highest copy number of Sulcia muelleri and Sodalis sp were both in Bacteriome,while the lowest one was in male midgut and salivary glands of female and male;the highest copy number of Rickettsia sp.was in the ovary.The results indicated that the different developmental stages and organs or tissues of cicadella viridis infected with 3 species of endosymbionts and their contents were different.3.Effect of three antibiotics on the removal of endosymbionts of Cicadella viridisCicadella viridis harbors primary and secondary endosymbionts which are used to provide nutrients and influence the development and reproduction of Cicadella viridis.This study designed specific primers for Sulcia muelleri,Sodalis sp.and Rickettsia sp.endosymbionts to investigate the infection frequency of endosymbionts in Cicadella viridis.The green leafhopper was treated with three antibiotics(Rifampicin,Oxytetracycline Hydrochloride,and Penicillin G potassium),and Absolute Quantification PCR(TaqMan)was used to detecte the copy number of endosymbionts in Cicadella viridis during different times.The results implied that the infection frequency of Sulcia muelleri,Sodalis sp.and Rickettsia sp.were both 100%.Rifampicin,Oxytetracycline Hydrochloride,and Penicillin G potassium could eliminate endosymbionts to some extent but failed to remove them entirely.Compared with the other two antibiotics,Oxytetracycline Hydrochloride eliminated Sulcia muelleri better,while there were some differences in the elimination effects of three antibiotics on Sodalis sp.The elimination effects of rifampicin were well on Sodalis sp.in the 5 d and 10 d,while Oxytetracycline Hydrochloride achieved the better effect to Sodalis sp.in 15 d,20 d,33-35 d and 35-37 d.For Rickettsia sp.,the elimination effect of rifampicin was better in the 5 d,while Oxytetracycline Hydrochloride achieved the better effect in the adult stage.Rifampicin,Oxytetracycline Hydrochloride and Penicillin G potassium could eliminate endosymbionts to some extent,but failed to remove entirely 4.Effect of antibiotic treatment on amino acid content and total protein content in Cicadella viridisCicadella viridis nymph 1 were treated by Oxytetracycline Hydrochloride to test Amino acid content and total protein content.The results showed that:(1)In the treatment total amino acid and non-essential amino acid content were lower than the control,while essential amino acid content was higher than the control after nymph 1 was treated by Oxytetracycline Hydrochloride.Seven essential amino acid content in treated male and female were higher than the control apart from lysine in female.For non-essential amino acid,cysteine,proline and tyrosine contents in treatment were lower than the control,and the other non-essential amino acid content were higher than the control.(2)In treatment total protein content was lower than the control during 5 days,but it was not obvious.Total protein content in female changed significantly,while the males did not.Amino acid content and total protein content in Cicadella viridis were changed after treated by antibiotic.5.Effects of antibiotic treatment on protective enzyme,detoxifying enzymes,and transaminase in Cicadella viridisProtective enzyme(POD,T-SOD and CTA),detoxifying enzymes(GST,CarE and AChE)and transaminase(GS,ALT and AST)were checked in the different periods after Cicadella viridis nymph 1 was treated by Oxytetracycline Hydrochloride.The results showed that protective enzyme,detoxifying enzymes and transaminase activities in Cicadella viridis increased during the early period,but these activities all decreased in the later period.(1)Protective enzyme: POD,T-SOD and CTA activities increased significantly on the fifth day.T-SOD and CTA activities decreased significantly in the fifteen days,but the variation of POD did not change significantly.Three protective enzymes activities reduced significantly or very significantly in the adult stage.(2)Detoxifying enzymes: GST,CarE and AChE activities increased significantly on the fifth day.In the fifteen days CarE and AChE activities in the nymph stage decreased significantly.In the adult stage three detoxifying enzymes activities declined significantly or very significantly.(3)Transaminase: GS,ALT and AST activities increased significantly on the fifth day.While in the fifteen day their activities decreased significantly.GS and AST activities in female declined significantly instead of ALT.While in male GS,ALT and AST activities decreased significantly or very significantly.The protective enzyme,detoxifying enzymes and transaminase activities in Cicadella viridis were changed after antibiotic treatment. |
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