Molecular Mechanisms Of Wolbachia Influencing Sleep Behavior And Learning And Memory Capacity In Drosophila

Wolbachia are genus of bacteria found within the tissues of several groups of arthropods,they are mainly distributed in the reproductive and nervous system of insects.Previous studies focused on the effects of Wolbachia on reproductive system of the hosts.They found that Wolbachia could regulate the reproduction mode of the host through various mechanisms,such as cytoplasmic incompatibility(CI),parthenogenesis,feminination and male killing.They typically infect the reproductive tissues of insects where they can manipulate reproduction of their hosts to enhance the vertical transmission of Wolbachia from mother to offspring.The effects of Wolbachia on sleep behavior or learning and memory capacity(LMC)of insect hosts are not well documented,although they are also widely distributed in the host nervous system,including the brain.Our study on the effect of Wolbachia on host behavior,it would contribute to a better understanding of the biological processes of host/endosymbion interactions,particularly the effects of Wolbachia on neurobiology in invertebrate hosts.First,we found that wMel Wolbachia were widely distributed in the brains of Drosophila melanogaster(Dmel wMel)by transmission electron microscopy and fluorescence in situ hybridization.Then,we used the Drosophila activity monitor system(DAMS)system to detect the sleep time and circadian rhythm of Drosophila melanogaster,and to detect the sleep homeostasis and arousal threshold of Drosophila by mechanical vibration.The results indicated that Wolbachia infection caused an increase of total sleep time in Drosophila melanogaster,compared to the control flies(Dmel T),especially an increase in the number of nighttime sleep bouts or episodes,but not in sleep bout duration.Correspondingly,Wolbachia infection also reduced the arousal threshold of their fly hosts.They were easy to wake up due to external stimuli,and the sleep latency was significantly prolonged,in other words,the interval between wake and the next sleep was prolonged.However,neither circadian rhythm nor sleep rebound following deprivation was influenced by Wolbachia infection.As studies have shown that sleep is associated with the dopamine pathway,we adopted RT-PCR(qRT-PCR)to test the expression level of two essential genes,Pale and Ddc in dopamine biosynthesis pathway,they were significantly upregulated in Wolbachia-infected flies,compared to the control flies.Together,these results indicate that Wolbachia decreases the sleep quality of their insect hosts and mediates the expression of dopamine related genes.Some studies found that sleep behavior are usually closely related to learning and memory,since Wolbachia infection affected the sleep behavior of host flies by dopamine pathway,we wondered that whether Wolbachia could influence the learning and memory ability of its host Drosophila.We adopted a conditional stimulus learning model that combines two odors(3-octanol and 4-methylcyclohexanol)with sucrose(as a reward),using the T-maze to detect the learning and memory ability of Dmel wMel and Dsim wRi.Learning and memory capacity are assessed using the Performance Index(PI),which is calculated by subtracting the number of flies destined for unconditional odor from the number of flies that are conditional odor,and dividing by the total number of flies.The learning and memory capacity of Dmel wMel and control group were 0.254±0.01 and 0.189± 0.009(p<0.01),respectively.The learning and memory capacity of Dsim wRi and control group were 0.247 ± 0.005 and 0.177 ± 0.006(p<0.01),respectively.It indicated that Wolbachia infection could significantly improve the learning and memory ability of Drosophila hosts.To dissect the molecular mechanism of LMC changes when there is a Wolbachia infection of D.melanogaster.We first decided to test the expression of 13 well-characterized genes known to be related to LMC in the head of D.melanogaster.qRT-PCR showed that among these genes,five of them:crebB,DopR,rutabaga(rut),dunce and crebA were significantly up-regulated in the head of Dmel wMel flies compared to Dmel T flies.Of the five up-regulated genes,crebA was up-regulated most in the presence of Wolbachia.We then examined whether the improvement of LMC in Dmel wMel was due to increased expressiorn of crebA by Wolbachia infection.Hence,we knocked down crebA in Wolbachia-infected flies by using a Wolbachia-infected actGal4 driver line;We then overexpressed crebA in Wolbachia-free flies.Results indicate knockdown of crebA in Wolbachia-infected flies significantly reduces LMC while overexpression of crebA can indeed significantly improve LMC in Drosophila.It indicate that up-regulation of crebA expression may be one of the main reasons for the improvement of learning and memory in Drosophila while Wolbachia infection.It has been reported that Wolbachia infection might alter transcription of host microRNAs(miRNAs)to regulate expression of host genes to affect learning behaviour.The up-regulation of crebA in Wolbachia-infected Drosophila led us to hypothesize that Wolbachia may regulate the expression of host miRNAs targeting crebA to affect host fitness or learning.Using in silico homology searches(RNA22,RNAHybrid),a putative miRNA(dme-miR-92b)that may target crebA was identified.Target sequences with complete complementarity to the dme-miR-92b seed region were predicted in the 3'UTR of crebA at nucleotides 1557-1564.Expression of dme-miR-92b was confirmed using qRT-PCR,with the expression level significantly lower in Dmel wMel flies than in Dmel T flies(p<0.01).These results suggest that dme-miR-92b may act as an inhibitor of crebA expression in D.melanogaster.To further determine whether crebA mRNA is repressed by dme-miR-92b,we performed two independent experiments:(1)Wolbachia-free Drosophila S2 cells were transfected with a specific synthetic mimic or inhibitor(reverse complementary sequence)of dme-miR-92b.Control cells were transfected with an unrelated miRNA sequence(mimic NC)or miRNA negative control(inhibitor NC).After 48 h,we observed a significantly lower transcript level of crebA in cells transfected with the miRNA mimic than in cells transfected with control transfections.In contrast,crebA expression was significantly up-regulated after dme-miR-92b inhibitor was transfected.These results confirm that dme-miR-92b acts as an inhibitor of crebA expression.(2)To test whether crebA is a direct target of dme-miR-92b,fragments of the 3'UTR seed region of wild-type crebA and crebA containing a 4-bp mutation in the seed region were respectively cloned into the psiCHECK2 dual luciferase reporter plasmid.Luciferase reporters were co-transfected with either dme-miR-92b mimic or miRNA negative control(miRNA NC)into S2 cells.As shown in results,co-transfection of dme-miR-92b mimic with the crebA 3'UTR reporter resulted in an extremely significant decrease(more than 50%)in luciferase activity compared to other groups.No decrease in luciferase activity was observed when dme-miR-92b mimic was transfected together with the mutant reporter or null plasmid,indicating that the predicted site in crebA is a direct target of dme-miR-92b.Taken together,these results suggest that the dme-miR-92b might directly regulate crebA expression by targeting the 3'UTR of its transcript in D.melanogaster.To further investigate the molecular mechanism by which Wolbachia infection improves LMC,we synthesized the agomir and antagomir ofdme-miR-92b,and injected them into adult flies.The mRNA level of crebA in Dmel wMel was significantly decreased after injection of miR-92b agomir;correspondingly the LMC was reduced significantly.In contrast,the mRNA level of crebA in Dmel T was significantly increased after injection of miR-92b antagomir,and the LMC was also improved compared to the control group.These results indicate that Wolbachia infection improves LMC by increasing crebA expression through down-regulation of dme-miR-92b.These results further suggest that Wolbachia may improve LMC in Drosophila by altering host gene expression through an miRNA-target pathway.In summary,Wolbachia may afifect the sleep behavior of host flies through the dopamine pathway,increasing the sleep time of flies and reducing the sleep quality of flies.At the same time,Wolbachia may affect the learning and memory ability of the host fly by regulating the expression of crebA through dme-miR-92b.Our results contribute to a better understanding of the biological processes of host/endosymbion interactions,particularly the effects of Wolbachia on neurobiology in invertebrate hosts.It also helped us understand coevolution between Wolbachia and the host.Since many hosts(eg,arthropods,nematodes)are intermediate hosts for many vertebrate pathogens,this study was also of great significance in the biological control of pests.