Manipulation of the symbiont microbiota of insect pests for the development of sustainable control strategies in the agri-food chain

Manipulation of symbiotic associations in insect pests represents a promising approach within the framework of Integrated Pest Management (IPM). Indeed, by targeting the bacterial symbionts that play essential roles in the biology and survival of their host, it is possible to develop targeted pest control strategies reducing environmental impact and risks on non-target organisms. In this context, the present doctoral thesis explores the relationship of four insect pests with specific microorganisms, as a target for the development of sustainable control strategies. The four insect pests are relevant for primary production and for food storage: Halyomorpha halys damages a wide range of crops, including fruit and vegetables, Bactrocera oleae is a major pest of olive trees, and Lobesia botrana targets grapevines, while Periplaneta americana is ubiquitous in many urban and industrial environments and it is responsible for food contamination and hygiene issues in urban and industrial settings. Those pests contribute to important economic losses across agricultural sectors and food storage, as they reduce crop yields, compromise food quality, and increase management costs, resulting in a significant challenge for sustainable food systems. Three of the above-mentioned insects, namely H. halys, B. oleae, and P. americana are associated with microbial endosymbionts, which are vertically transmitted to the offspring and which occupy specific locations in the respective host. On the contrary, no stable associations for L. botrana have been confirmed. Taxonomically speaking, the known primary symbionts belong to different species and genera of family Enterobacteriaceae, in H. halys and B. oleae, and Flavobacteriaceae in P. americana. The in vitro uncultivability represents a significant challenge, as it limits both the ability to investigate their biological role for the insect host, and the possibility to screen antimicrobial compounds that could effectively interfere with bacterial viability. Given the specificities of each association between insect pests and related microbial symbionts, the four case studies were analyzed with different experimental strategies. The first study insect investigated was Halyomorpha halys (Heteroptera: Pentatomidae), the host of Candidatus Pantoea carbekii. The microbial presence on externally deposed eggs was the basis to develop an innovative pipeline of analyses to screen the effect of low-environmental-risk Plant Protection Products (PPPs). The integration of fast microbiological analyses directly on the egg masses (live/dead staining with fluorescence microscopy) and observations on egg hatching and nymphal viability provided a complete overview of effects of PPPs. As a proof-of-concept, fungicides and plant biostimulants were tested, with two application modes (spraying and dipping) and results showed that compounds with different use in agriculture could modulate the endosymbiont viability impairing also the insect development. The same compounds were also tested on the parasitoid Trissolcus japonicus, a biocontrol agent for H. halys. The results demonstrated that they did not affect parasitoid emergence nor their ability to parasitize treated eggs, highlighting the compatibility with symbiotic control. The second case study involved Bactrocera oleae (Diptera: Tephritidae) and its endosymbiont Candidatus Erwinia dacicola, located mainly in the esophageal bulb in adult. Given the similarity of the endosymbiont to the free-living species E. aphidicola, a novel selective growth medium was developed. Computational approaches, including phylogenomics, comparative genomics, and metabolic modeling, combined with the use of E. aphidicola as a proxy species, enabled the formulation of a completely new growth medium. Culturing trials were performed along two seasons and when insects where available. Growth was observed only in primary cultures, which allowed the identification of key nutritional factors necessary for cultivation. However, further optimization is needed to enable successful subculturing. As a complementary approach, E. aphidicola was employed as a proxy species to investigate the antimicrobial resistance pattern possibly shared with Ca. E. dacicola which could extend ineffective compounds to be used for symbiotic control. The Minimum Inhibitory Concentrations (MICs) were determined for the free-living E. aphidicola and genome-based analyses were performed to determine core genes shared by the two species. Results showed that E. aphidicola, exhibited the most concordant phenotypic resistance/susceptibility profile and is the most similar to the endosymbiont at the genetic level of resistance profiles suggesting that it could be considered a reliable tool to hypothesize antimicrobial effectiveness against Ca. E. dacicola. A comprehensive literature review was performed on L. botrana (Lepidoptera: Tortricidae). Unlike other pests, it does not appear to host obligate symbionts, and a previously suspected mutualism. All the available information on the microbiota associated with species of Lepidoptera in general and Tortricidae in particular, and possible differences during the developmental stages. Based on current knowledge, microbial agents could be applied in a form of biological control, in a framework of IPM, even though a “standard” symbiotic control is not possible due to the lack of stable symbionts. The last case study regarded P. americana (Blattodea: Blattidae), a relevant food-related pest, which hosts an obligate endosymbiont of the genus Blattabacterium. Given the availability of 97 genome sequences (more specifically, metagenome-assembled-genomes or MAG) of uncultured Blattabacterium spp., an analysis of metadata revealed that the broad range of cockroaches host species for different lineages of this genus. Further, a genome-based analysis showed that Blattabacterium associated with P. americana represent a distinct genomovar, genomically distinguishable from B. cuenoti, the species usually indicated as the symbiont. Finally, a survey of the current knowledge on the symbiosis was performed to evaluate the potential for developing symbiotic control strategies for this resilient insect in pest management for stored food products. The integration of microbiological, computational and entomological techniques allowed to pursue applied purpose like the proposal of a novel pipeline of analyses for compound testing and leveraged on the use of genome data to address culturability, investigate susceptibility/resistance to antimicrobial compounds as well as determine the correct identification of endosymbionts. Overall, the findings of this doctoral project contributed both to the scientific understanding of microbial endosymbiont characteristics and to the development of novel approaches in the light of sustainable pest management, including also the impact on non-target organisms, in line with the principles of IPM systems.