Roles Of Autophagy In Regulating Whitefly-symbiont Metabolic Interaction

Nutritional symbionts are restricted to specialized host cells called bacteriocytes in various insect orders.These symbionts can provide essential nutrients to the host.However,the cellular mechanisms underlying the regulation of these insect-symbiont metabolic associations remain largely unclear.The whitefly,Bemisia tabaci MEAM1,hosts Portiera and Hamiltonella bacteria in the same bacteriocyte.In this study,chemical treatment and LST8 silencing were used to induce autophagy in whitefly bacteriocytes.This induction of autophagy decreased symbiont titers,and essential amino acid and B vitamin contents.In contrast,the repression of autophagy in bacteriocytes via Atg8 silencing increased symbiont titers,and amino acid and B vitamin contents.Furthermore,dietary supplementation with non-EAAs or B vitamins alleviated autophagy in whitefly bacteriocytes,elevated TOR expression and increased symbiont titers.TOR silencing restored symbiont titers in whiteflies after dietary supplementation with B vitamins.These data suggest that Portiera and Hamiltonella evade autophagy of the whitefly bacteriocytes by activating the TOR pathway via providing essential nutrients.Taken together,we demonstrated that autophagy plays a critical role in regulating the metabolic interactions between the whitefly and two intracellular symbionts.Therefore,this study reveals that autophagy is an important cellular basis for bacteriocyte evolution and symbiosis persistence in whiteflies.These findings also provide new avenue for whitefly control via regulating autophagy in the future.The whitefly symbiosis unravels the interactions between cellular and metabolic functions of bacteriocytes.It may be ubiquitous that bacteriocyte autophagy mediates the metabolic interactions between various insects and their intracellular symbionts.The results are as follows:1.Effect of symbionts titer on content of essential amino acids and B vitamins in the bacteriocyte of whiteflyAfter rifampicin treatment,the titer of both Portiera and Hamiltonella decreased notably in B.tabaci.Except for isoleucine(Ile)and tryptophan(Trp),the content of eight essential amino acids(EAAs)and pantothenate in B.tabaci decreased significantly in whiteflies lacking Portiera and Hamiltonella.After treatment with three antibiotics(ampicillin,gentamicin and cefotaxime),the titer of Hamiltonella of B.tabaci decreased significantly,while the titer of Portiera remained unchanged.The content of seven EAAs in B.tabaci did not change significantly,the content of arginine(Arg),threonine(Thr)and methionine(Met)increased significantly,while the titers of biotin and folate decreased significantly in Hamiltonella-cured whiteflies.The results showed that the titer of Portiera significantly affected the contents of EAAs and pantothenate,while the titer of Hamiltonella significantly affected the contents of B vitamins such as biotin and folate.2.Relationship between symbiont titer of bacteriocyte in whitefly and autophagy levelBy searching and comparing the genomic databases of B.tabaci and Drosophila melanogaster,we found that there were 17 genes involved in autophagy pathway and 2 genes involved in TOR pathway in the genome of B.tabaci.The identity of amino acid sequences of Atg8,LST8 and TOR in B.tabaci and Acyrthosiphon pisum,Nilaparvata lugens and Trialeurodes vaporariorum was 91.46% 、 79.34% and80.89%,respectively.There is a conserved domain in Atg8,TOR and LST8 of B.tabaci;Phylogenetic analysis showed that the autophagy gene Atg8 and TOR pathway genes TOR and LST8 of B.tabaci clustered with the homologous gene of Hemiptera insect pea aphid A.pisum,brown planthopper N.lugens and greenhouse whitefly T.vaporariorum.The titers of Portiera and Hamiltonella in the female of B.tabaci reached the peak at the day 7 after emergence.From the 7th to 14 th days,the titer of Portiera remained stable,while the titer of Hamiltonella decreased significantly.From the 14 th to 28 th days,the titer of Portiera decreased gradually,while the titer of Hamiltonella remained at a low level.The expression of autophagy gene Atg8 in adult females at different ages was negatively correlated with the changes of titers of Portiera and Hamiltonella.Immunohistochemistry experiments revealed bacteriocytes had accumulation of autophagy marker Atg8,lysosomal marker RAB7 as well as intense and punctate lysotracker staining in whitefly bacteriocytes lacking Portiera and Hamiltonella,indicating that the decrease of symbionts titer of B.tabaci is closely related to the induction of autophagy.3.Autophagy induction reduces bacteriocyte symbiont titer,essential amino acid and B vitamin contents and whitefly fitnessAutophagy was induced by feeding B.tabaci with rapamycin and silencing the TOR pathway gene LST8 of B.tabaci.The results showed that expression of twelve genes involved in the whole autophagy pathway was significantly up-regulated in bacteriocytes of whiteflies after rapamycin treatment and LST8 silencing.The expression of twelve genes involved in the autophagy pathwayremained unchanged in heads and thoraxes of whiteflies after LST8 RNAi.Immunohistochemistry experiments revealed bacteriocytes had accumulation of autophagy marker Atg8,lysosomal marker RAB7,as well as intense and punctate lysotracker staining.after rapamycin treatment and LST8 gene silencing.Our transmission electronic microscopy confirmed induction of autophagy in the bacteriocytes of whiteflies treated with rapamycin and RNAi whiteflies.There were more autophagy structures including lysosome,autophagosome,autolysosome and lamellar bodies in the bacteriocytes of treated whiteflies,which showed that both treatment methods induced autophagy.The titers of Portiera and Hamiltonella of B.tabaci decreased significantly after autophagy induction.The contents of 10 EAAs,pantothenate and biotin decreased significantly after rapamycin treatment.After silencing LST8,arginine(Arg),valine(Val),isoleucine(Ile)and phenylalanine(Phe)contents were lower,but not significantly.Silencing the whitefly LST8 gene significantly reduced the contents of six of the ten EAAs,and of pantothenate and biotin.Moreover,after autophagy induced by the two treatments,the oviposition of female B.tabaci decreased significantly,the mortality increased and the life span shortened.The above results showed that autophagy induction could reduce the titer of symbionts and the contents of essential amino acids,pantothenic acid and biotin of B.tabaci,and reduce B.tabaci fitness.4.Autophagy inhibition increaces bacteriocyte symbiont titer,essential amino acid and B vitamin contents and enhances whitefly fitnessAutophagy was inhibited by ds Atg8 microinjection.The expression of three genes(Atg7,Atg8 and Atg9)involved in the autophagy pathway was significantly down-regulated and other nine genes remained unchanged in heads and thoraxes of whiteflies after Atg8 RNAi.The autophagy marker Atg8 was inhibited in whitefly bacteriocytes after microinjection with ds RNA.There was decline of lysosomal marker RAB7 and lysotracker staining was weak and diffuse in bacteriocytes after Atg8 silencing.TEM showed that there were relatively few autophagosomes,lysosomes,autophagy lysosomes and lamellar bodies,indicating that silencing Atg8 inhibited autophagy.The titers of Portiera and Hamiltonella of in the female adult whiteflies increased significantly after autophagy inhibition.UPLC analyses showed that the content of the amino acid,arginine(Arg),was elevated in RNAi-treated whiteflies,but not significantly.Silencing the whitefly Atg8 gene significantly increased the contents of nine of ten EAAs,and of pantothenate and biotin.After silencing Atg8 inhibited autophagy,the fecundity and longevity of whitefly females increased significantly.These results showed that autophagy inhibition could improve the titer of symbiotic bacteria,the content of essential amino acids,pantothenic acid and biotin and B.tabaci fitness.5.Dietary nutrition supplementation alleviates bacteriocyte autophagy and enhances symbiont titer and whitefly fitnessAfter supplementation of glutamine(Gln),asparagine(Asn)and proline(Pro)complexes and B vitamins,The autophagy marker Atg8 was repressed in the bacteriocytes of whiteflies.Additionally,there was decline of lysosomal marker RAB7 and reduction of RAB7 gene expression in bacteriocytes and the lysotracker staining became weak and diffuse in the bacteriocytes of whiteflies.Concomitantly,the titers of Portiera and Hamiltonella and the expression of TOR gene were significantly up-regulated.At the same time,TEM showed that there were relatively few autophagy related ultrastructure in bacteriocyte.After supplementing artificial diet with Gln + Asn + Pro,the contents of the other 8 amino acids increased significantly,while the contents of lysine(Lys)and tryptophan(Trp)remained unchanged.After supplementing an artificial diet with a B vitamin mix,the contents of Histidine(His),Methionine(Met),Valine(Val),Isoleucine(Ile)and Leucine(Leu)increased significantly,while the contents of arginine(Arg),Threonine(Thr),Lysine(Lys),Phenylalanine(Phe)and Tryptophan(Trp)remained unchanged.Microinjection with ds TOR restored the titers of Portiera and Hamiltonella in female adult whiteflies that fed on dietary B vitamin mix.After supplementation artificial diet with Gln + Asn + Pro,the number of egg per whitefly female increased significantly and the mortality decreased;After supplementation artificial diet with a mix B vitamins,the oviposition of whitefly female increased but not significantly,while the mortality decreased significantly.The above results show that supplementation dietary nutrition promote the synthesis of EAAs,activate TOR pathway and weaken autophagy,so as to make Portiera and Hamiltonella escape autophagy and improve B.tabaci fitness.In conclusion,this study,for the first time,demonstrated the important role of autophagy pathway and TOR pathway in the regulation of symbionts metabolism in bacteriocyte of B.tabaci.At the same time,this study revealed that in the long-term coevolution process,symbionts can activate TOR pathway by synthesizing essential amino acids and B vitamins to weaken autophagy,so as to maintain their titer in insect bacterocyte.This not only deepens our understanding of the interaction between insects and their intracellular symbionts,but also provides a new idea for the control of Hemiptera insects such as B.tabaci.