This study investigates the impact of the three most widely used antibiotics in the beekeeping sector (oxytetracycline, tylosin, and sulfonamides) on the honeybee gut microbiota and on the spread of antibiotic resistance genes. The research represents an advance to the present literature, considering that the tylosin and sulfonamides effects on the gut microbiota have never been studied.

Nosema ceranae is a major pathogen in the beekeeping sector, responsible for nosemosis. This disease is hard to manage since its symptomatology is masked until a strong collapse of the colony population occurs. Conversely, no medicaments are available in the market to counteract nosemosis, and only a few feed additives, with claimed antifungal action, are available. New solutions are strongly required, especially based on natural methods alternative to veterinary drugs that might develop resistance or strongly pollute honey bees and the environment. This study aims at investigating the nosemosis antiparasitic potential of some plant extracts, microbial fermentation products, organic acids, food chain waste products, bacteriocins, and fungi. Honey bees were singularly infected with 5 × 10${}^4$ freshly prepared N. ceranae spores, reared in cages and fed ad libitum with sugar syrup solution containing the active ingredient. N. ceranae in the gut of honey bees was estimated using qPCR. The results showed that some of the ingredients administered, such as acetic acid at high concentration, p-coumaric acid, and Saccharomyces sp. strain KIA1, were effective in the control of nosemosis. On the other hand, wine acetic acid strongly increased the N. ceranae amount. This study investigates the possibility of using compounds such as organic acids or biological agents including those at the base of the circular economy, i.e., wine waste production, in order to improve honeybee health.

The role of a balanced gut microbiota to maintain health and prevent diseases is largely established in humans and livestock. Conversely, in honeybees, studies on gut microbiota perturbations by external factors have started only recently. Natural methods alternative to chemical products to preserve honeybee health have been proposed, but their effect on the gut microbiota has not been examined in detail. This study aims to investigate the effect of the administration of a bacterial mixture of bifidobacteria and Lactobacillaceae and a commercial product HiveAlive^TM on honeybee gut microbiota. The study was developed in 18 hives of about 2500 bees, with six replicates for each experimental condition for a total of three experimental groups. The absolute abundance of main microbial taxa was studied using qPCR and NGS. The results showed that the majority of the administered strains were detected in the gut. On the whole, great perturbations upon the administration of the bacterial mixture and the plant-based commercial product were not observed in the gut microbiota. Significant variations with respect to the untreated control were only observed for Snodgrassella sp. for the bacterial mixture, Bartonella sp. in HiveAlive^TM and Bombilactobacillus sp. for both. Therefore, the studied approaches are respectful of the honeybee microbiota composition, conceivably without compromising the bee nutritional, social and ecological functions.

Ground beetles of the genus Poecilus (Carabidae) play key ecological roles in pest control and soil health. However, their gut microbiome remains largely unexplored despite increasing interest in insect-associated microbiota and its environmental implications. This study used next-generation sequencing and qPCR to characterise the gut microbiome of Poecilus beetles collected from organic and conventional tomato fields. Core microbiota were identified through prevalence–abundance filtering, revealing dominant genera including Gilliamella, Weissella, Enterobacter, and Enterococcus, alongside several low-abundance but consistently present taxa. Notably, Carnobacterium was detected for the first time in an insect host, and Nosema ceranae was identified for the first time in Carabidae, expanding the known host range of this microsporidian pathogen. Functional predictions based on 16S rRNA data and comparative genomic analysis showed enrichment in pathways related to amino acid synthesis, protein degradation, and monosaccharide metabolism. Significant inter-individual variation in microbial diversity and predicted functionality was observed, with lowest diversity and metabolic potential in beetles from conventionally managed fields, potentially indicating dysbiosis and environmental stress. The detection of Nosema and Serratia pathogens in some individuals adds new insights into pathogen dynamics within carabid beetles. Our findings reveal that the gut microbiome of Poecilus may be shaped by environmental factors and agricultural practices, influencing host health and ecological performance. These insights support the use of Poecilus as a bioindicator for soil ecosystem health and highlight the potential of microbiome-based metrics in agroecological monitoring.

Pollen collected by pollinators can be used as a marker of the foraging behavior as well as indicate the botanical species present in each environment. Pollen intake is essential for pollinators’ health and survival. During the foraging activity, some pollinators, such as honeybees, manipulate the collected pollen mixing it with salivary secretions and nectar (corbicular pollen) changing the pollen chemical profile. Different tools have been developed for the identification of the botanical origin of pollen, based on microscopy, spectrometry, or molecular markers. However, up to date, corbicular pollen has never been investigated. In our work, corbicular pollen from 5 regions with different climate conditions was collected during spring. Pollens were identified with microscopy-based techniques, and then analyzed in MALDI-MS. Four different chemical extraction solutions and two physical disruption methods were tested to achieve a MALDI-MS effective protocol. The best performance was obtained using a sonication disruption method after extraction with acetic acid or trifluoroacetic acid. Therefore, we propose a new rapid and reliable methodology for the identification of the botanical origin of the corbicular pollens using MALDI-MS. This new approach opens to a wide range of environmental studies spanning from plant biodiversity to ecosystem trophic interactions. Graphical Abstract

Vespa orientalis, the oriental hornet, is an emerging predator of honey bees whose ecological impact and microbial ecology remain poorly understood. Here, we present the first detailed characterisation of its gut microbiota by integrating 16S rRNA gene sequencing, predicted microbial function, pathogen screening, and a three-year beekeeper survey across urban and rural sites in Malta. Hornets were sampled from four locations and classified by observed foraging behaviour, either predation on honey bees or scavenging on cat food. Survey data confirmed consistent V. orientalis sightings and seasonal colony losses, particularly during peak foraging months. Microbiome analysis revealed a conserved core community dominated by Spiroplasma, Arsenophonus, and Rosenbergiella, with overall diversity stable across sites and diets. However, specific taxa varied with foraging behaviour. For example, Arsenophonus was enriched in bee-predating hornets, while Enterobacter and Serratia were more common in scavenging individuals, suggesting environmental and dietary influences on microbiota composition. Predicted functional profiles remained broadly conserved, reflecting robust nutrient metabolism and potential detoxification capabilities, with some variations related to the diet behaviour. Pathogen screening detected Nosema ceranae and Crithidia bombi in a substantial proportion of hornets, including those not observed feeding on bees. Although our findings do not demonstrate pathogen transmission, they support the hypothesis that V. orientalis may act as a transient carrier, potentially contributing to pathogen persistence via environmental exposure. Together, these results reveal the dietary flexibility and microbial flexibility within the gut microbiome of V. orientalis, and highlight its potential involvement in pollinator pathogen dynamics.