Exploring the influence of abiotic factors on the community structure and antimicrobial resistance spread in the insect gut microbiome

This PhD thesis research project is aimed at studying the influence of different abiotic factors on the gut microbial community of insects, with a focus on the structure of the gut microbiome and on the spread of antibiotic resistance genes. The research project will focus on the study of the influence of the diet and of different categories of pesticides on the gut microbial community structure and on antimicrobial resistance (AMR) spread in beneficial insects. Studio dell’influenza di fattori abiotici sulla struttura della comunità microbica intestinale e sulla diffusione della resistenza agli antimicrobici nel microbioma intestinale degli insetti Questo progetto di tesi di Dottorato è volto allo studio dell’influenza di diversi fattori abiotici sul microbioma intestinale degli insetti, considerando in particolare la struttura della comunità microbica intestinale e la diffusione di geni di resistenza agli antibiotici. Il progetto sarà focalizzato sullo studio dell’influenza della dieta e di diverse categorie di agrofarmaci sulla struttura della comunità microbica intestinale e sulla diffusione della resistenza agli antimicrobici in insetti benefici. 1. State-of-the-Art The gut microbiome of insects has a wide range of important roles, contributing to the development of the host, to its nutrition, and physiology (Engel and Moran, 2013). The gut microbial community of insects can be influenced by different factors, such as the developmental stage of the insect, the degree of sociality, the dietary regime and the environmental conditions (Engel and Moran, 2013). For example, Chandler et al. (2011) studied the effect of different diets on the composition of the gut microbiome of wild and laboratory-reared Drosophila melanogaster specimens and found that the host diet plays a pivotal role in shaping the gut bacterial community of the insect. Certain factors can lead to a condition of dysbiosis: in fact, it has been recently pointed out that the exposure to pesticides may cause perturbations in the insect gut microbiome, therefore compromising insect health (Motta and Moran, 2020). This aspect is especially relevant for pollinators, such as honey bees and bumble bees, which could be directly exposed to a wide variety of xenobiotics during foraging activities (Motta et al., 2018). Moreover, Qiu et al. (2022) found that that pesticides could have a role in enhancing antimicrobial resistance in microorganisms and that pesticides exposure promotes the spread of bacterial antimicrobial resistance, mainly through horizontal gene transfer (HGT). This mechanism could be relevant for the dissemination of antimicrobial resistance at different environmental levels (Qiu et al., 2022). Mobile genetic elements such as plasmids can be involved in HGT: it is important to consider that plasmid persistence is more probable in communities with higher levels of bacterial diversity (Brockhurst and Harrison, 2022). Hence, it would be interesting to study the response of the gut microbiome of insects in terms of bacterial diversity and correlate that to plasmid-mediated HGT. In general, up to now the influence of abiotic factors on HGT in the insect gut has been poorly studied. Thus, the aim of this PhD thesis research project will be to gain a better understanding of the impact of different abiotic factors on the insect gut microbiome and of the effect of these abiotic factors on HGT and antibiotic resistance genes (ARGs) dissemination within the insect gut microbiome. The model insect Drosophila melanogaster will be considered, for its ease of rearing and manipulation under laboratory conditions, together with beneficial insects such as the most important crop pollinator, Apis mellifera, and other less characterized insect pollinators. 2. PhD Thesis Objectives and Milestones Within the overall objective mentioned above this PhD thesis project can be subdivided into the following activities according to the Gantt diagram given in Table 1: A1) Study of the influence of abiotic factors on the structure of the gut microbial community of Drosophila melanogaster, the honeybee Apis mellifera and other insect pollinators: The gut microbial community of insects will be studied through quantitative PCR analyses on 16S rRNA gene, to evaluate if the abiotic factors cause a shift in the abundance of gut bacteria of the insects. A metataxonomic analysis on 16S rRNA gene will be also performed to observe any possible shift in the composition of bacterial community. A2) Resistome profile characterization: An initial survey for the presence of specific antibiotic resistance genes through quantitative PCR on field-collected pollinators will be conducted to obtain their resistome profile. Other molecular techniques (e.g. ResistoMap) will be also used to detect a wider range of antibiotic resistance genes. A3) HGT experiments: Conjugation experiments will be carried out to study the spread of antibiotic resistance genes: initially, the gut microbiome of Drosophila melanogaster specimens will be considered for in vitro and in vivo conjugation experiments. Then, conjugation experiments will be optimized for the gut bacterial communities of pollinators. A4) Writing and Editing of the PhD thesis, scientific papers and oral and poster communications. Table 1 Gantt diagram for the PhD thesis project in the next two years. Activity Months 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 A1) Study of the influence of abiotic factors on the insect gut microbiome 1) Evaluation of gut bacterial abundance 2) Evaluation of taxonomic composition of the gut microbiome A2) Resistome profile of pollinators 1) qPCR analyses on ARGs 2) Analysis of ResistoMap output A3) HGT experiments 1) Conjugation set-up with D. melanogaster 2) Conjugation experiments on pollinators A4) Thesis and Paper preparation 3. Selected References Brockhurst MA, Harrison E (2022) Ecological and evolutionary solutions to the plasmid paradox, Trends Microbiol 30(6): 534-543. Chandler JA, Morgan Lang J, Bhatnagar S, Eisen JA, Kopp A (2011) Bacterial Communities of Diverse Drosophila Species: Ecological Context of a Host-Microbe Model System, PLoS Genet 7 (9): 1-18. Engel P, Moran NA (2013) The gut microbiota of insects – diversity in structure and function, FEMS Microbiol Rev 37: 699-735. Motta EVS, Raymann K, Moran NA (2018) Glyphosate perturbs the gut microbiota of honey bees, PNAS 115 (41): 10305-10310. Motta EVS, Moran NA (2020) Impact of Glyphosate on the Honey Bee Gut Microbiota: Effects of Intensity, Duration, and Timing of Exposure, mSystems 5 (4): 1-16. Qiu D, Ke M, Zhang Q, Zhang F, Lu T, Sun L, Qian H (2022) Response of microbial antibiotic resistance to pesticides: An emerging health threat, Sci Total Environ 850: 1-8.