GUT-BACTERIA SYMBIOSIS IN INSECT PESTS

Insects are one of the most fascinating taxa on Earth: their diversity, diffusion, colonization of different niches are unparalleled in the animal kingdom. Besides, they have a remarkable impact on human life: they are parasites for people, animals and crops, vectors of diseases, pollinators, and even breeding animals (e.g. honeybees, silkworms). This extraordinary evolutionary success and diversification is partially due to the symbiotic relationships that insects have with a wide range of bacteria. These symbionts can be divided into primary, secondary symbionts and gut bacteria. Primary symbionts are found in very specialized cells (the bacteriocytes), strictly maternally transmitted and not cultivable. They are essential for their host, and vice-versa: they can actually be considered part of a single organism called \u201cholobiont\u201d. Secondary symbionts are not necessary for the host survival, although often beneficial, and they can inhabit various organs and tissues. In this category fall also reproductive parasites, as Wolbachia, which spreads in the population by maternal transmission, manipulating the reproduction of the host to favour the birth of infected daughters. Finally, gut bacteria are a more vague category, comprising organisms that live in the insect intestine because they are ingested with the diet, but also symbionts that establish a close relationship with the host, being essential for its survival and development. The roles of all these microorganisms are, to different extents, important for the insect physiology. Primary symbionts are generally essential to complement unbalanced diets and secondary ones contribute to the host fitness, while reproduction parasites deeply affect the reproduction mode of their hosts. Even commensals have been demonstrated to influence the development, mating choice and immune responses in Drosophila flies. For these reasons, the understanding of the biology of an insect can not do without the characterisation of its microbiota. In the second chapter of my PhD thesis, a review on the microbial ecology techniques applied to the study of insect microbial communities gives an overview on the methods that can be applied to this purpose. On one hand, molecular analyses based on the 16S gene sequencing, such as 16S rRNA barcoding (pyrotag) and Denaturing Gradient Gel Electrophoresis (DGGE) are the most powerful methods to get a complete picture of the microbial community composition and structure. Microscopic localisation of symbionts can be also achieved by Fluorescent In Situ Hybridisation. On the other hand, the isolation of bacteria allows to deeply characterize the cultivable fraction, verifying through direct in vitro tests the activities of the strains. Taking advantage of a strain collection isolated from the target insect, the symbiotic relationship can be investigated through in vivo experiments. The more common ones involve i) the labeling of the strains with fluorescent proteins and the recolonization of the insects, to evaluate their localisation and colonisation ability, ii) the assessment of the detrimental effects of symbionts deprivation on the hosts, and iii) the comparison of insects monoassociated with different strains to check the effects on host fitness. To further analyse the interaction between bacteria and their hosts from a genetic point of view, advanced techniques, such as Signature Tagged Mutagenesis or In Vivo Expression Technology, can be performed. Many of these techniques have been applied in the case studies here presented, in which the microbial communities associated to three insect pests have been characterised. In the third chapter is presented a study on the spotted-wing fly Drosophila suzukii. Unlike its relative D. melanogaster, which feeds on rotten fruit, this fly feeds and lays eggs on healthy fruits. The most damaged crops are members of the Drupaceae family (e.g. cherries) and berries (strawberries, raspberries, blueberries). The bacterial community associated to this pest have been characterised with a focus on acetic acid bacteria (AAB), important symbionts of many sugar-feeding insects. According to our findings, D. suzukii harbours a diverse community of AAB, detected both in the isolate collection and in culture-independent screenings (pyrotag, DGGE). They are primarily localised in the gut, attached to the peritrophic matrix, as showed by FISH micrographs. The ability of three AAB species (Gluconobacter oxydans, Acetobacter tropicalis and Acetobacter indonesiensis) to colonise the gut has been proved by recolonization experiments of the insect using GFP-marked strains. In the fourth chapter, the bacterial community of the wood-feeding beetle Rhynchophorus ferrugineus has been analysed. Commonly named Red Palm Weevil (RPW), this insect is an important pest for palm trees. The plants are damaged mainly by the larvae, which dig tunnels in the trunks until pupation. Bacteria associated to the red palm weevil have been studied primarily by molecular means (pyrotag). Our results outline that the bacteria hosted by R. ferrugineus are mainly acquired from the environment while feeding. Indeed, a sharp difference has been registered between field-caught and bred specimens. While field caught RPW harbour more bacterial taxa which are in common with their feeding plants, the animals fed on apple in the laboratory show a higher prevalence of lactic acid and acetic acid bacteria, which presumably grow on the rotten fruit. The latter result is further confirmed by the bacterial isolations performed on apple-fed specimens. Besides, the DNA sequence of a primary symbiont, Candidatus Nardonella, has been detected. This bacterium has been shown to inhabit a wide range of insects of the same family of the RPW, Curculionidae. The fifth chapter is about the gut bacterial community of Psacothea hilaris hilaris. Native of Japan and east China, this longicorn beetle (family: Cerambicidae) arrived in Italy as a consequence of the wood trade, and settled as a stable population in a small area in Como province. Its larvae dig tunnels in the trunks of the trees of the Moraceae family, while the adults feed on leaves. The most damaged by its feeding habits are mulberry and fig trees. This beetle hosts a variegate gut microbiota, that, as shown by DGGE, greatly changes according to the diet and to the gut tract examined. The cultivable fraction of this microbiota has been tested for several activities that proved the capability of the community as a whole to exploit the food sources in the insect gut (primarily, sugars from plant cell walls) and to assist their host in carbon and nitrogen absorption. Thus, even if acquired from the environment, these bacteria seem to be adapted to a symbiotic lifestyle. From the comparison among these three studies, some conclusions can be drawn. All three case studies outline the importance of the diet in shaping the insect microbial community. In detail, wild insects always show higher diversity and individual variability in their associated microbiota. Reared insects appear, on the contrary, dominated by the species that can rapidly grow on laboratory diets, such as Lactobacillales and Enterobacteriales. Secondly, these studies depict a more accurate image of the commensal bacteria, which are not merely acquired by chance through feeding, but are capable to actively colonize insect guts, and to efficiently exploit this niche to multiply and spread in the environment. Finally, the research data point out that the origin and the function of many of the organisms detected in insects are yet poorly understood. For this reason, these studies can be considered a basis to for future research, aimed to a more in-depth understanding of the roles of these bacteria and their interactions with the hosts

Microbial ecology-based methods to characterize the bacterial communities of non-model insects

Among the animals of the Kingdom Animalia, insects are unparalleled for their widespread diffusion, diversity and number of occupied ecological niches. In recent years they have raised researcher interest not only because of their importance as human and agricultural pests, disease vectors and as useful breeding species (e.g. honeybee and silkworm), but also because of their suitability as animal models. It is now fully recognized that microorganisms form symbiotic relationships with insects, influencing their survival, fitness, development, mating habits and the immune system and other aspects of the biology and ecology of the insect host. Thus, any research aimed at deepening the knowledge of any given insect species (perhaps species of applied interest or species emerging as novel pests or vectors) must consider the characterization of the associated microbiome. The present review critically examines the microbiology and molecular ecology techniques that can be applied to the taxonomical and functional analysis of the microbiome of non-model insects. Our goal is to provide an overview of current approaches and methods addressing the ecology and functions of microorganisms and microbiomes associated with insects. Our focus is on operational details, aiming to provide a concise guide to currently available advanced techniques, in an effort to extend insect microbiome research beyond simple descriptions of microbial communities

Contribution of gut symbionts to the host physiology of a wood-boring beetle

Psacothea hilaris hilaris is a xylophagous beetle belonging to the Cerambycidae family. Its larvae grow inside mulberry tree and fig trunks, reaching the length of up to three centimetres and damaging the plants to death. In Japan, this is a serious pest for sericulture, while its recent establishment in northern Italy could threaten the fig cultivation in the Mediterranean basin. In this study, we characterized the gut bacterial communities associated to wild and laboratory-reared P. h. hilaris larvae by molecular and culture-dependant methods. PCR-Denaturant Gradient Gel Electrophoresis (DGGE) was applied to assess the significant effect of the diet and the gut tract in modifying the taxonomical composition of the gut bacterial community. Field-caught larvae and larvae reared on diet in presence of antibiotics and preservative showed richer communities than larvae reared on artificial diet, not exposed to antibiotics and/or preservatives; in this last case the larvae were dominated by Enterococcus and Leuconostoc sequences. This probably was due to the overgrowth of these microbes on the diets where antibiotics and/or preservatives were removed. PCR-DGGE analysis showed a significant difference in the bacterial community composition between the midgut and the hindgut, probably correlated to the different physiological conditions. By using a collection of bacterial isolates from the guts of wild larvae we evaluated the possible contribution of the isolates to the host physiology (in terms of contribution to carbon or nitrogen uptake) through in vitro tests. Results suggested that symbiont isolates can contribute to the cellulose digestion or exploit the by-products of the degradation of cell wall compounds and help their host to absorb nitrogen converting waste molecules (uric acid and urea) or proteins to ammonia and smaller peptides, or even fixing atmospheric nitrogen. From our observations, the cultivable gut bacterial community of P. h. hilaris appears to include many different commensals, which are suitable to exploit the food sources in the gut and to give a contribution to the host metabolism

Errors in ribosomal sequence datasets generated using PCR-coupled 'panbacterial' pyrosequencing, and the establishment of an improved approach

Universal bacterial primers are often used in PCR-coupled sequencing approaches to investigate environmental and host-associated bacterial communities. Some of these primers can also amplify eukaryotic DNA. This is leading to the submission of datasets to public databases which are erroneously annotated as prokaryotic sequences. The present note sends a message about the risk of submitting incorrectly annotated sequence data and suggests a reliable approach for the sequencing of 16S rRNA genes and identification of bacteria within complex communities