| The oriental fruit fly,Bactrocera dorsalis(Hendel),one of the most invasive and polyphagous pests,causes substantial loss of more than 250 host fruits and vegetables crops,worldwide.The high invasive potential of B.dorsalis is characterized by a variety of factors such as wide host range,powerful reproduction rate,dispersal capacity,and rapid adjustability to environmental stresses.B.dorsalis has invaded the southern and northern cool areas in China,indicating that adaptation to cold environment may be a crucial factor for its geographic expansion.Gut symbiotic bacteria promotes host fitness and performance under environmental stress conditions in B.dorsalis and thereby impacts pest control strategies.However,the contribution of gut microbes to host fitness during long-term low-temperature stress is still unclear.This study was designed to identify the functions of key gut symbiotic bacteria,which helps the host B.dorsalis to promote its resistance to low-temperature stress of 10?.By combining transcriptomic and metabolomic approaches,we also explore that gut symbiotic bacteria stimulates the host metabolic pathway to enhance its resistance to low-temperature stress via influencing the mitochondrial functionality.1.Gut microbiota enhances the host resistance to low-temperature stress in adult Bactrocera dorsalisThe first part of this study examined the role of gut bacteria in host long-term low-temperature stress resistance in adult B.dorsalis.The results showed that after the gut bacteria of flies were removed via antibiotic treatment,the median survival time was significantly decreased to approximately 68%of that in conventional flies following exposure to a temperature stress of 10?.Furthermore,we found that Klebsiella michiganensis BD177 is a key symbiotic bacterium,whose recolonization in antibiotic treated(ABX)flies significantly extended the median survival time to 160%of that in the ABX control,and restored their lifespan to the level of conventional flies at 10?.Culture-dependent approach and qPCR analysis revealed the K.michiganensis BD177 is able to sustain in the gut,and benefit host health throughout life postexposure to 10?.After the recolonization of live K.michiganensis BD177 to ABX flies,the total amino acid and lipid levels were also significantly increased by 170%and 300%in the hemolymph compared with those in their ABX counterparts,respectively.The results of this study suggest that improvement in the host lifespan and physiological mechanisms at 10?,might be linked with the gut microbial community,especially K.michiganensis BD1772.Impact of gut microbiota on the metabolomic and transcriptomic responses of the host to low-temperature stress in Bactrocera dorsalisThe second part of this study was designed to assess the effect of gut bacteria on host molecular mechansims;therefore,we employed transcriptomic and metabolomic techniques to identify the genes and metabolites responsible for the host adaption to low-temperature stress of 10?.The results of metabolomic analysis revealed that a total of 1548 metabolites were detected in conventional,K.michiganensis BD177,and ABX fly hemolymph,including amino acids,multiple lipids and their derivatives,and carbohydrates.Furthermore,analysis of differentially expressed metabolites indicated that 201 metabolites in conventional vs.ABX,and 202 metabolites in K.michiganensis-reinfected vs.ABX groups were significantly enriched,and 133 metabolites were equally shared by both comparisons.Several amino acids,e.g.,histidine,proline,arginine,ornithine and putrescine were significantly upregulated in K.michiganensis-reinfected or conventional flies compared to that of ABX flies,suggesting that thses metabolites are absorbed by the host under low-temperature stress of 10?.However,glycerol-phospholipids,e.g.,LysoPE and carnitine derivatives,e.g.,L-Acetylcarnitine and L-Palmitoylcarnitine,and sugars,including Fructose,Egonol glucoside were significantly downregulated in conventional and K.michiganensis-reinfected flies compared with ABX flies,indicating that they are consumed by the symbiotic gut bacteria.Among the enriched pathways,arginine and proline metabolism,and monoterpenoid biosynthesis pathways were the top significantly enriched pathways in both groups.Whereas,the results of transcriptomic analysis showed that a total of 18,576 differentially expressed genes(DEGs)in conventional vs.ABX,and 10,760 DEGs between K.michiganensis-reinfected vs.ABX groups were identified.Among these significantly enriched genes,8306 DEGs were equally shared by both comparisons.Several DEGs associated with low-temperature stress resistance,encoding heat shock proteins(HSPs),zinc finger proteins(ZFPs),serine/threonine protein kinase(STKs),and arginine and proline metabolism(APMs)were significantly enriched in the current study.Gene enrichment analysis showed that most common DEGs enriched to metabolic pathways,including ribosome,longevity regulating pathway,protein processing in endoplasmic reticulum,arginine and proline metabolism,D-Arginine and D-ornithine metabolism pathways in both comparisons.3.Gut microbiota promotes host resistance to low-temperature stress by stimulating its arginine and proline metabolism pathway in adult Bactrocera dorsalisIn the third part,we pooled significantly enriched overlapping metabolic pathways,and found a common pathway 'arginine and proline metabolism',shared across both'omics' techniques.Notably,the relative levels of proline and arginine metabolites were significantly downregulated by 34-and 10-fold,respectively,in ABX flies compared with those in the hemolymph of conventional flies after exposure to a temperature stress of 10? whereas recolonization of ABX flies by K.michiganensis BD177 significantly upregulated the levels of proline and arginine by 13-and 10-fold,respectively,compared with those found in the hemolymph of ABX flies.qPCR analysis also confirmed that K.michiganensis-recolonized flies significantly stimulated the expression of transcripts from the arginine and proline metabolism pathway compared with the ABX controls,and RNAi mediated silencing of two key genes,pyrroline 5-carboxylate reductase(Pro-C)and argininosuccinate synthase(ASS),significantly reduced the survival time of conventional flies,postexposure to low-temperature stress.We show that microinjection of L-arginine and L-proline into ABX flies significantly increased their survival time following exposure to temperature stress of 10?.Transmission electron microscopy(TEM)analysis further revealed that low-temperature stress caused severe destruction in cristae structures and thus resulted in abnormal circular shapes of mitochondria in ABX flies gut,while the recolonization of live K.michiganensis BD177 helped the ABX flies to maintain mitochondrial functionality to a normal status,which is important for the arginine and proline induction.Moreover,K.michiganensis-recolonized flies significantly increased the ATP concentration in the gut tissues by 377%compared with the ABX flies,suggesting the crucial role of gut symbiont to maintain the normal metabolic mechanism of mitochondria under low-temperature stress.4.Characteristics and expression analysis of arginine and proline metabolism genes in Bactrocera dorsalisIn the fourth part of this study,we performed a series of experiments to identify and characterize the crucial regulatory roles of arginine and proline metabolism genes(APMs),which were stimulated by gut microbiota,in B.dorsalis development and in response to extreme temperature stresses.Following genes;namely pyrroline 5-carboxylate reductase(Pro-C),ornithine decarboxylase(ODC),ornithine aminotransferase(OAT),arginase(Arg)and arginine kinase(AK)were selected on the basis of their cryoprotectant roles in host development and survival under stressful conditions.The open reading frames of five B.dorsalis APMs were 815,1178,1283,1067 and 1172 bp in length,respectively,encoding proteins of 271,392,427,355 and 520 amino acids,respectively.Real-time quantitative PCR(RT-qPCR)analyses showed that ODC,OAT,Arg and AK transcripts were more abundant in adults,whereas the transcript levels of Pro-C was highest in 1-day-old larvae.Transcripts of three APMs,including ODC,OAT and Arg were highly expressed in the fat body by 36-,41-and 133-fold than that detected in the other tissues,while Pro-C and AK were abundantly expressed in the gut and testis,respectively.After cold shock treatment of 0? for 3 h,the mRNA expression levels of Pro-C,OAT and Arg were significantly upregulated by 2.30-,1.92-and 4.22-fold,respectively,whereas ODC expression was significantly incrased by 1.52-fold only after 10? shock treatment,in comparison with those under the normal treatment condition(27?),suggesting their important roles in B.dorsalis upon exposure to extreme temperature stressIn conclusion,our results suggest that gut microbiota plays a vital role in promoting the host resistance to low-temperature stress in B.dorsalis.The experimental observations led us to conclude that gut symbiont,K.michiganensis BD177,helps the host flies to sustain a dynamic equilibrium in mitochondria and promotes its metabolic functions that elevate arginine and proline levels sufficient enough to enhance B.dorsalis resistance to low-temperature stress.These results will provide a new strategy of using gut symbionts as biocontrol agents.Furthermore,identification and characterization of APMs would provide important informations to develop a novel control strategy of B.dorsalis,and to explore further functional studies in other insect species. |
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