Effects Of Intestinal Bacteria On Foraging Choice And Nutritional Ecology Of Bactrocera Dorsalis(Diptera:Tephritidae)

Gut bacteria associated with larval and adult stages of insects directly or indirectly affect the host use by provisioning missing nutrients,driving the sensory perception and modulating the feeding behavior of its host.While insect and gut microbiome share complex and mutually beneficial links,the role of bacteria in establishing and maintaining the host use in these patterns is poorly understood.Therefore,the purpose of this study was to assess the extent to which intestinal bacteria modulate the foraging behavior and the nutritional ecology of B.dorsalis.First,we created an imbalanced foraging environment(food drops that varied in their size and density)and adopted an approach based on direct observation of protein-starved flies,symbiotic,aposymbiotic or axenic feeding on dishes seeded with drops of full diet(containing all amino acids),non-essential amino acids diet or full diets supplemented with bacteria isolates at similar concentrations.Then,we evaluated how diet quality and gut bacteria interplay in shaping the foraging activity and nutritional adaptations of the fly.Finally,we allowed a single female fly to lay eggs on five different fruit types for 24h and monitored the bacterial population dynamics(within and between fruit types)and the larval development parameters(length,weight and development time)across five generations.Our study shows that gut bacteria drive the foraging behavior of B.dorsalis whenever the fly is confronted with constraining nutritional environments.Our results as summarized below were discussed in the context of previous studies on the effect of the gut microbiome on host behavior and nutrition,and suggest that these can aid in implementing an efficient bacteria-based control strategy of B.dorsalis.1.Identification of cultivable gut bacteria associated with larvae and adult B.dorsalisThe isolation and identification of gut bacteria is a crucial step toward studying their functions in host fitness and physiology.Here,we used the culture-dependent technique to isolate and identify cultivable bacteria inhabiting the gut of B.dorsalis(male,female and third instar larvae)that were used in the study of their functions.Forty-one different bacterial phylotypes were found(11 from the third instar larvae,18 and 12 from female and male populations,respectively)and divided into six bacterial families(Enterobacteriaceae,Enterococcaceae,Staphylococcaceae,Streptococcaceae,Micrococcaceae and Bacillaceae).Bacterial species from these families were differentially represented in various samples,except Klebsiella oxytoca,Enterobacter cloacae,Pantoea dispersa,and Enterococcus faecalis that were detected at all developmental stages.Overall,Enterobacteriaceae was the most dominant family in females and third instar larvae accounting for 57.89%and 26.32%,respectively,while Enterococcaceae was dominant in males with 75%of the total bacterial taxa.Moreover,99%of bacteria belonging to the Enterobacteriaceae family are gram-negative,whereas 99%of those from the Enterococcaceae family are gram-positive.The recurrence of some bacterial species in all developmental stages could be an indication of their ecological relevance for B.dorsalis.These results suggest that B.dorsalis possesses a considerable variety of cultivable bacteria in its gut.These bacteria were selected to explore their specific functions on host physiology and fitness.2.Effects of symbiotic population crash on microbiome composition and fly longevity of Bactrocera dorsalisAntibiotics treatment(aposymbiotic)or eggs sterilization(axenic)is generally used to produce aposymbiotic and axenic flies.This section aims to shed light on the adaptations plasticity of gut microbiome following antibiotic/eggs sterilization treatments and the effects on host longevity.Metagenomic analyses were carried out on gut homogenates to evaluate the structure of the remaining bacterial communities.Antibiotic treatments and egg disinfection resulted in a significant reduction of the vast majority of gut bacterial populations,especially Proteobacteria,Firmicutes,and Bacteroidetes.On the other hand,it allowed the persistence of Actinobacteria,Cyanobacteria and Acidobacteria populations.In untreated control flies,longevity was extended irrespective of diet quality in comparison to treated flies.Conversely,when gut bacteria were largely reduced(aposymbiotic and axenic flies),survival was decreased in the non-essential amino acids diet treatment versus slightly improved in the presence of a protein diet.These findings indicate antibiotics or eggs sterilization are essential for the study of the functions of gut symbionts and suggest that intestinal bacteria can modify their structure and composition to enhance the survival and adaptations capacity of the host fly.This type of plasticity could be targeted for initiating the management program of this pest.3.Intestinal bacteria modulate the foraging behavior of the oriental fruit fly Bactrocera dorsalis(Diptera:Tephritidae)Gut-associated bacteria are known to influence its host in a variety of ways,including the food habits and ecological adaptations.In this experiment,we studied how the presence or absence of gut symbionts affects foraging behavior and nutrient ingestion by B.dorsalis.As such,we offered to protein-starved flies,symbiotic or aposymbiotic,a choice between diets containing all amino acids or only the nonessential ones.The different diets were presented in a foraging arena as drops that varied in their size and density,creating an imbalanced foraging environment.Suppressing the microbiome resulted in significant changes in the foraging behavior of both male and female flies.Aposymbiotic flies responded faster to the diets offered in experimental arenas,spent more time feeding,ingested more drops of food,and were constrained to feed on time-consuming patches(containing small drops of food)when these offered the full complement of amino acids.These results indicate that the presence of gut microbiome maintains the foraging stability and modulate the host behavior according to the availability and density of food source.4.Intestinal probiotics motivate the foraging decision and promote fecundity and survival of Bactrocera dorsalis(Diptera:Tephritidae)The study of specific functions of gut symbionts requires their elimination and individual re-colonization of their original habitat.Here,we produced symbiotic(from normal eggs)and axenic flies(from sterilized eggs)and evaluated the effects of selected gut symbionts on foraging decision and fitness of Bactrocera dorsalis(female fecundity,and survival).The diets were offered on dishes containing drops of full diet or a full diet supplemented with selected bacteria(probiotics)at similar concentrations.The results showed that symbiotic status and diet types are essential factors in driving the foraging behavior of the experimental flies(symbiotic and axenic),yet preferences for probiotic diets were significantly higher in axenic flies to which they responded faster compared to full diet.All the flies consumed many probiotic drops to meet their nutritional goals and this correlated with an up-regulation of serine and trypsin-like protease genes,suggesting the capacity of bacteria to incite the consumption of profitable food by B.dorsalis.Moreover,females fed diet supplemented with Pantoea dispersa and Enterobacter cloacae laid more eggs but had shorter lifespan while female fed Enterococcus faecalis and Klebsiella oxytoca enriched diets lived longer but had lower fecundity compared to positive controls.Conversely,flies fed sugar diet(negative control)were not able to produce any eggs but lived longer than those from the positive control.These results suggest that intestinal bacteria possess the potentials to be used as probiotics to control the foraging decision in a way which promotes the reproduction and survival of B.dorsalis.5.Host plant as a suitable bacteria growth substrate that promotes the development of the fruit flyThe ability of host plant(fruits)to act as a substrate or media for larval development depends on how good it is at offering suitable nutrients for bacterial growth and proliferation.In this study,we evaluated the host suitability for larval development in relation with the bacterial community composition and diversity.We allowed a single female fly to lay eggs on five different host fruits and monitored the larval population dynamics across five generations and bacterial community structure at first and fifth generations.The results showed that the larval length and dry weight did not vary significantly across experimental generations,but were greatly affected by fruit types and larval stages.The larval development time was extended considerably in apple and tomato but shortened in banana and mango.There was a significant shift in bacterial community structure and composition across fruits and generations.Principal Coordinates Analysis revealed clusters of bacterial community of the same fruit and the analysis of similarity indicated that at first generation,the gut microbiome reared on apple and banana were similar to the parental female(predominance of Proteobacteria),but shifted significantly at fifth generation(dominance of Firmicutes),and the bacterial community diversity was higher compared to the first generation.Banana offered better suitability for larval development and bacterial growth compared to apple,suggesting its ability to supply nutrients for the larvae and bacteria.In contrast,reduced larval development was recorded in apple,suggesting that apple could be poor in necessary elements for bacterial growth.Our results indicate that the gut microbiome can shift its population in response to nutritional adaptations of the fly larvae.These data highlight the role of gut bacteria on host use and food habit of B.dorsalis and can help in initiating appropriate control methods against this pest.Conclusion:Our study demonstrates how gut bacteria drive the foraging behavior by allowing B.dorsalis to forage optimally while acquiring essential nutrients.It also shows that gut-associated bacterial possess a strong capacity to modulate nutritional ecology and increase the suitability of host fruits to act as substrate for larval growth and survival.This study adds a novel step to the nexus connecting the insect gut,its microbiome,chemoreception to individual patterns of foraging,and constitutes new vulnerabilities that could be targeted to control this pest.