| The hispid leaf beetle,Octodonta nipae(Maulik),coconut leaf beetles,Brontispa longissima(Gestro)(Coleoptera:Chrysomelidae)and red palm weevil,Rynchophorus ferrugineus(Curculionidae)are recognized as major devastating pests of palm cultivation worldwide.Understanding the dynamics of the symbiotic microbiota is essential to unravel the complex inter:play between invasive pests and its bacterial symbionts.In the first part of this study,we used 454 pyrosequencing approach to identify the diversity and dynamics of bacterial symbionts,and the results yielded a total 2,075,281 raw reads across different life stages and reproductive organs of O.nipae.Clustering analysis at>97%similarity threshold generated at least 3,959 Operational Taxonomic Units(OTUs)that belonged to five different phyla.Proteobacteria,Actinobacteria,and Firmicutes have represented the bulk of taxa but underwent notable changes during metamorphosis at lower taxonomic level.Enterobacteriaceae and Dermabacteraceae were the most abundant families in immature stages(eggs,larvae,and pupae),while Anaplasmataceae family was dominated in adults(male and female)and reproductive organs(ovaries and testis).The genus Serratia and Lactococcus were most abundant in eggs,whereas Pantoea and Brachybacterium represented the bulk of larvae and pupae microbiota.Interestingly the genus Wolbachia found positive to all tested samples and was recorded extremely high(>64%)in the adult and reproductive organs.These outcomes foster our understanding of the intricate symbiotic associations between bacteria and O.nipae and will incorporate in devising novel pest control strategies against this palm pest.As higher rate of abundance,we highlighted here the Wolbachia associations in O.nipae.Endosymbiotic bacteria in the genus Wolbachia(Rickettsiaceae:Rickettsiales)are arguably one of the most abundant bacterial group associated with arthropods.Owing to its critical effects on host reproduction(male killing,parthenogenesis,cytoplasmic incompatibility and feminization),Wolbachia has garnered much attention as a prospective future tool for insect pest management.However,their association,infection dynamics,and functionality remain unknown in many invasive pests.Here,we diagnosis for the first time,the infection prevalence,and occurrence of Wolbachia in O.nipae.Experimental evidence by the exploration of wsp gene vindicate that O.nipae is naturally infected with bacterial symbiont of genus Wolbachia,showing a complete maternal inheritance with shared a common Wolbachia strain(wNip).Moreover,MLST(gatB,fbpA,coxA,ftsZ,and hcpA)analysis enabled the detections of new sequence type(ST-484),suggesting a particular genotypic association of O.nipae and Wolbachia.Subsequently,quantitative real-time PCR(qPCR)assay demonstrated variable infection density across different life stages(eggs,larvae,pupae and adult male and female),body parts(head,thorax,abdomen),and tissues(ovaries,testes,and guts).Phylogeny of Wolbachia infection associated with O.nipae rectified from all tested life stages were unique and fall within the same monophyletic supergroup-A of Wolbachia clades.With the continuity of prevalence trend,Wolbachia-mediated hosts such as coconut leaf beetles,Brontispa longissima(Gestro)(Coleoptera:Chrysomelidae)which has garnered attention as a serious pest of palms was also subjected to this interrogation.By adopting Wolbachia surface protein(wsp)and multilocus sequence type(MLST)genotypic systems,we determined the Wolbachia infection density within host developmental stages,body parts and tissues and the results revealed that all the tested samples of B.longissima were infected with the same Wolbachia strain(wLog)suggesting complete vertical transmission.Whereas,the MLST profile elucidated two new alleles(ftsZ-234 and coxA-266)that define new sequence type(ST-483)which indicates the particular genotypic association of B.longissima and Wolbachia.The quantitative real-time PCR(qPCR)analysis revealed higher infection density in eggs and adult stage followed by abdomen and reproductive tissues,respectively.However,no significant differences were observed in the infection density between sexes.Moreover,the wsp and concatenated MLST alignment analysis of this study with other known Wolbachia-mediated arthropods revealed similar clustering with distinct monophyletic supergroup B.The infection density of symbiont is among the valuable tool to understand their biological influence on hosts,and these latest discoveries would facilitate the future investigations to understand the host-symbiont complications and its prospective role as a microbiological agent to reduce pest populations.In the context of broad existence and unique phenotypic actions to alter reproductive systems of various insect,we aimed to determine the intriguing possibility of this endosymbiont from one of the deadliest palm pest,i.e.red palm weevil(RPW),Rynchophorus ferrugineus(Curculionidae)from different geographical locations using polymerase chain reactions(PCR)with four sets of Wolbachia-specific primers(SPs)along with one pair of universal bacterial primer(BP).Parallel analysis was also carried out with Wolbachia strain isolated from white-backed plant hopper,Sogatella furcifera(Homoptera:Delphacidae),as a positive control.Our analysis confirmed the absence of Wolbachia sp.across the various life stages of RPW reared in laboratory or captured from the field.Moreover,the phylogenetic analysis of all closely related Wolbachia-mediated weevils were compiled and retrieved from NCBI database indicates the extent of transfection of this bacterium into RPW for the future work on biological control of RPW.This study may facilitate to understand further evolutionary consequences of Wolbachia infection in weevils.Also,we comprehensively surveyed the Wolbachia-infection prevalence and mitochondrial DNA(mtDNA)polymorphism in B.longissima from five different geographical locations,including China's Mainland and Taiwan,Vietnam,Thailand,Malaysia and Indonesia.A total of 540 sequences were screened in this study through three different genes,i.e.,cytochrome oxidase subunit I(COI),Wolbachia outer surface protein(wsp)and multilocus sequencing type(MLST)genes.The COI genetic divergence ranges from 0.08%to 0.67%,and likewise,a significant genetic diversity(?=0.00082;P=0.049)was noted within and between all analyzed samples.In the meantime,ten different haplotypes(H)were characterized(haplotype diversity=0.4379)from 21 different locations,and among them,H6(46 individuals)have shown a maximum number of population clusters than others.Subsequently,Wolbachia-revalence results indicated that all tested specimens of B.longissima were found positive(100%),which suggested that B.longissima was naturally infected with Wolbachia.Wolbachia sequence results(wsp gene)revealed a high level of nucleotide diversity(?=0.00047)under Tajima's D test(P=0.049).Meanwhile,the same trend of nucleotide diversity(?=0.00041)was observed in Wolbachia concatenated MLST locus.Furthermore,phylogenetic analysis(wsp and concatenated MLST genes)revealed that all collected samples of B.longissima attributed to same Wolbachia B-supergroup.Our results strongly suggest that Wolbachia bacteria and mtDNA were highly concordant with each other and Wolbachia can affect the genetic structure and diversity within the B.longissima populations.The last part of our examination was focused to establish Wolbachia genotyping analysis along with host cytochrome oxidase subunit I(COI)gene for accurate identification between the individuals of the same family(Chrysomelidae)member,B.longissima and O.nipae.The B.longissima(Gestro)is biologically and morphologically identical to O.nipae(Maulik),and are known as the most harming nuisances of palm cultivation worldwide.Here,we have cloned and sequenced a gene coding Wolbachia surface protein(wsp)and COI gene regions amplified from both species by PCR.The nucleotide sequences were directly determined(?600bp for wsp and ?804bp for COI)and aligned using the multiple alignment algorithms in the ESPript3 package and the MEGA5 program.Comparative sequence analysis indicated that the representative of wsp and COI sequences from these two beetles were highly variable.To ensure this bacterial variation,multilocus sequence typing(MLST)of bacterial genes were conducted,and the results vindicated the same trend of variations.Furthermore,the phylogenetic analysis also indicates that B.longissima and O.nipae being the two different species harbors two distinct Wolbachia supergroups B and A,respectively.The present outcomes quickly discriminate between these two species.Considering its simplicity and cost-effectiveness,it can be used as a diagnostic tool for discriminating such invasive species particularly B.longissima and O.nipae which has overlapping morphologic characters. |
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