Beneficial Bacteria and Plant Extracts Promote Honey Bee Health and Reduce Nosema ceranae Infection.

The research aims to give new insights on the effect of administering selected bacterial strains, isolated from honey bee gut, and/or a commercial plant extract blend (HiveAlive®) on Nosema ceranae. Analyses were first performed under laboratory conditions such as different infective doses of N. ceranae, the effect of single strains and their mixture and the influence of pollen administration. Daily survival and feed consumption rate were recorded and pathogen development was analysed using qPCR and microscope counts. Biomarkers of immunity and physiological status were also evaluated for the different treatments tested using one bacterial strain, a mixture of all the bacteria and/or a plant extract blend as treatments. The results showed an increase of abaecin transcript levels in the midgut of the honey bees treated with the bacterial mixture and an increased expression of the protein vitellogenin in the haemolymph of honey bees treated with two separate bacterial strains (Bifidobacterium coryneforme and Apilactobacillus kunkeei). A significant effectiveness in reducing N. ceranae was shown by the bacterial mixture and the plant extract blend regardless of the composition of the diet. This bioactivity was seasonally linked. Quantitative PCR and microscope counts showed the reduction of N. ceranae under different experimental conditions. The antiparasitic efficacy of the treatments at field conditions was studied using a semi-field approach which was adapted from research on insecticides for the first time, to analyse antiparasitic activity against N. ceranae. The approach proved to be reliable and effective in validating data obtained in the laboratory. Both the mixture of beneficial bacteria and its association with Hive Alive® are effective in controlling the natural infection of N. ceranae in honey bee colonies.

Pot experimental trial for assessing the role of different composts on decontamination and reclamation of a polluted soil from an illegal dump site in Southern Italy using Brassica juncea and Sorghum bicolor.

A pot experiment was carried out to evaluate the remediation potential of Brassica juncea and Sorghum bicolor in the decontamination of soil polluted with heavy metals such as copper, lead, tin, and zinc along with polychlorinated biphenyls, polycyclic aromatic hydrocarbons, and heavy hydrocarbons. Two composts obtained from different composting processes were tested as biostimulating agents. At the end of the trial, the effect of plant/compost combinations on soil microbial composition, contaminant removal, biochemical indicators, and plant biomass production was determined. The results highlighted that compost addition improved plant biomass despite slowing down plants' removal of organic and inorganic contaminants. In addition, compost partially enhanced the soil biochemical indicators and modified the relative abundance of the rhizosphere microorganisms. Sorghum showed better mitigation performance than Brassica due to its higher growth. The soil fertility level, the choice of plant species, and microbial richness were found fundamental to perform soil remediation. In contrast, compost was relevant for a higher crop biomass yield.

Combined effect of a neonicotinoid insecticide and a fungicide on honeybee gut epithelium and microbiota, adult survival, colony strength and foraging preferences.

Fungicides, insecticides and herbicides are widely used in agriculture to counteract pathogens and pests. Several of these molecules are toxic to non-target organisms such as pollinators and their lethal dose can be lowered if applied as a mixture. They can cause large and unpredictable problems, spanning from behavioural changes to alterations in the gut. The present work aimed at understanding the synergistic effects on honeybees of a combined in-hive exposure to sub-lethal doses of the insecticide thiacloprid and the fungicide penconazole. A multidisciplinary approach was used: honeybee mortality upon exposure was initially tested in cage, and the colonies development monitored. Morphological and ultrastructural analyses via light and transmission electron microscopy were carried out on the gut of larvae and forager honeybees. Moreover, the main pollen foraging sources and the fungal gut microbiota were studied using Next Generation Sequencing; the gut core bacterial taxa were quantified via qPCR. The mortality test showed a negative effect on honeybee survival when exposed to agrochemicals and their mixture in cage but not confirmed at colony level. Microscopy analyses on the gut epithelium indicated no appreciable morphological changes in larvae, newly emerged and forager honeybees exposed in field to the agrochemicals. Nevertheless, the gut microbial profile showed a reduction of Bombilactobacillus and an increase of Lactobacillus and total fungi upon mixture application. Finally, we highlighted for the first time a significant honeybee diet change after pesticide exposure: penconazole, alone or in mixture, significantly altered the pollen foraging preference, with honeybees preferring Hedera pollen. Overall, our in-hive results showed no severe effects upon administration of sublethal doses of thiacloprid and penconazole but indicate a change in honeybees foraging preference. A possible explanation can be that the different nutritional profile of the pollen may offer better recovery chances to honeybees.

The gut microbiome of solitary bees is mainly affected by pathogen assemblage and partially by land use.

Pollinators, including solitary bees, are drastically declining worldwide. Among the factors contributing to this decline, bee pathogens and different land uses are of relevance. The link between the gut microbiome composition and host health has been recently studied for social pollinators (e.g. honeybees), whereas the information related to solitary bees is sparse. This work aimed at the characterization of the gut microbiome of the solitary bees Xylocopa augusti, Eucera fervens and Lasioglossum and attempted to correlate the gut microbial composition with the presence and load of different pathogens and land uses. Solitary bees were sampled in different sites (i.e. a farm, a natural reserve, and an urban plant nursery) showing different land uses. DNA was extracted from the gut, 16S rRNA gene amplified and sequenced. Eight pathogens, known for spillover from managed bees to wild ones, were quantified with qPCR. The results showed that the core microbiome profile of the three solitary bees significantly varied in the different species. Pseudomonas was found as the major core taxa in all solitary bees analyzed, whereas Lactobacillus, Spiroplasma and Sodalis were the second most abundant taxa in X. augusti, E. fervens and Lasioglossum, respectively. The main pathogens detected with qPCR were Nosema ceranae, Nosema bombi and Crithidia bombi, although differently abundant in the different bee species and sampling sites. Most microbial taxa did not show any correlation with the land use, apart from Snodgrassella and Nocardioides, showing higher abundances on less anthropized sites. Conversely, the pathogens species and load strongly affected the gut microbial composition, with Bifidobacterium, Apibacter, Serratia, Snodgrassella and Sodalis abundance that positively or negatively correlated with the detected pathogens load. Therefore, pathogens presence and load appear to be the main factor shaping the gut microbiome of solitary bees in Argentina.

Environment or genetic isolation? An atypical intestinal microbiota in the Maltese honey bee Apis mellifera spp. ruttneri.

Introduction: Apis mellifera evolved mainly in African, Asian, and European continents over thousands of years, leading to the selection of a considerable number of honey bees subspecies that have adapted to various environments such as hot semi-desert zones and cold temperate zones. With the evolution of honey bee subspecies, it is possible that environmental conditions, food sources, and microbial communities typical of the colonized areas have shaped the honey bee gut microbiota. Methods: In this study the microbiota of two distinct lineages (mitochondrial haplotypes) of bees Apis mellifera ruttneri (lineage A) and Apis mellifera ligustica and carnica (both lineage C) were compared. Honey bee guts were collected in a dry period in the respective breeding areas (the island of Malta and the regions of Emilia-Romagna and South Tyrol in Italy). Microbial DNA from the honey bee gut was extracted and amplified for the V3-V4 regions of the 16S rRNA gene for bacteria and for ITS2 for fungi. Results: The analyses carried out show that the Maltese lineage A honey bees have a distinctive microbiota when compared to Italian lineage C honey bees, with the most abundant genera being Bartonellaceae and Lactobacillaceae, respectively. Lactobacillaceae in Maltese Lineage A honey bees consist mainly of Apilactobacillus instead of Lactobacillus and Bombilactobacillus in the lineage C. Lineage A honey bee gut microbiota also harbors higher proportions of Arsenophonus, Bombella, Commensalibacter, and Pseudomonas when compared to lineage C. Discussion: The environment seems to be the main driver in the acquisition of these marked differences in the gut microbiota. However, the influence of other factors such as host genetics, seasonality or geography may still play a significant role in the microbiome shaping, in synergy with the environmental aspects.

Alterations in the Microbiota of Caged Honeybees in the Presence of Nosema ceranae Infection and Related Changes in Functionality.

Several studies have outlined that changes in the honeybee gut microbial composition may impair important metabolic functions supporting the honeybees' life. Gut dysbiosis may be caused by diseases like Nosema ceranae or by other anthropic, environmental or experimental stressors. The present work contributes to increasing knowledge on the dynamics of the gut microbiome acquisition in caged honeybees, an experimental condition frequently adopted by researchers, with or without infection with N. ceranae, and fed with a bacterial mixture to control N. ceranae development. Changes of the gut microbiota were elucidated comparing microbial profile of caged and open-field reared honeybees. The absolute abundance of the major gut microbial taxa was studied with both NGS and qPCR approaches, whereas changes in the functionality were based on RAST annotations and manually curated. In general, all caged honeybees showed important changes in the gut microbiota, with [Formula: see text]-proteobacteria (Frischella, Gilliamella and Snodgrassella) lacking in all caged experimental groups. Caged honeybees infected with N. ceranae showed also a strong colonization of environmental taxa like Citrobacter, Cosenzaea and Morganella, as well as possibly pathogenic bacteria such as Serratia. The colonization of Serratia did not occur in presence of the bacterial mixture. The functionality prediction revealed that environmental bacteria or the supplemented bacterial mixture increased the metabolic potential of the honeybee gut microbiome compared to field and caged controls.

The ground beetle Pseudoophonus rufipes gut microbiome is influenced by the farm management system.

Intensive conventional farm management, characterized by high agrochemicals input, could alter the composition of microbial communities with potential negative effects on both functional traits and the ecosystem services provided. In this study, we investigated the gut microbial composition of a high ecological relevance carabid Pseudoophonus rufipes, sampled in two fields subjected to conventional and organic management practices. Carabids' gut microbiota was analyzed via qPCR and NGS. Profound differences between the microbial composition of organic and conventional samples were detected: the abundance of Tenericutes and Proteobacteria was significant higher in organic and conventional samples, respectively. Spiroplasmataceae and Bifidobacteriaceae families were significantly more abundant in samples from organic management, while Enterococcaceae, Morganellaceae and Yersiniaceae were more abundant in samples from conventional management. The diverse gut microbial composition of insects between the two management systems is related to the pressure of environmental stressors and it may representing an important bioindication of ecological functions and services provided by a carabid species.

Screening of Dietary Ingredients against the Honey Bee Parasite Nosema ceranae.

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 × 104 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.

Honeybee Exposure to Veterinary Drugs: How Is the Gut Microbiota Affected?

Several studies have outlined that a balanced gut microbiota offers metabolic and protective functions supporting honeybee health and performance. The present work contributes to increasing knowledge on the impact on the honeybee gut microbiota of the three most common veterinary drugs (oxytetracycline, sulfonamides, and tylosin). The study was designed with a semi-field approach in micro-hives containing about 500 honeybees. Micro-hives were located in an incubator during the day and moved outdoors in the late afternoon, considering the restrictions on the use of antibiotics in the open field but allowing a certain freedom to honeybees; 6 replicates were considered for each treatment. The absolute abundance of the major gut microbial taxa in newly eclosed individuals was studied with qPCR and next-generation sequencing. Antimicrobial resistance genes for the target antibiotics were also monitored using a qPCR approach. The results showed that the total amount of gut bacteria was not altered by antibiotic treatment, but qualitative variations were observed. Tylosin treatment determined a significant decrease of α- and ß-diversity indices and a strong depletion of the rectum population (lactobacilli and bifidobacteria) while favoring the ileum microorganisms (Gilliamella, Snodgrassella, and Frischella spp.). Major changes were also observed in honeybees treated with sulfonamides, with a decrease in Bartonella and Frischella core taxa and an increase of Bombilactobacillus spp. and Snodgrassella spp. The present study also shows an important effect of tetracycline that is focused on specific taxa with minor impact on alfa and beta diversity. Monitoring of antibiotic resistance genes confirmed that honeybees represent a great reservoir of tetracycline resistance genes. Tetracycline and sulfonamides resistance genes tended to increase in the gut microbiota population upon antibiotic administration. IMPORTANCE 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. Another original aspect lies in the experimental approach used, as the study looks at the impact of veterinary drugs and feed supplements 24 days after the beginning of the administration, in order to explore perturbations in newly eclosed honeybees, instead of the same treated honeybee generation. Moreover, the study was not performed with cage tests but in micro-hives, thus achieving conditions closer to real hives. The study reaches the conclusion that the most common veterinary drugs determine changes in some core microbiota members and that incidence of resistance genes for tetracycline and sulfonamides increases following antibiotic treatment.

Honeybees Exposure to Natural Feed Additives: How Is the Gut Microbiota Affected?

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 HiveAliveTM 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 HiveAliveTM 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.

Bifidobacterium xylocopae sp. nov. and Bifidobacterium aemilianum sp. nov., from the carpenter bee (Xylocopa violacea) digestive tract.

Social bees harbor a community of gut mutualistic bacteria, among which bifidobacteria occupy an important niche. Recently, four novel species have been isolated from guts of different bumblebees, thus allowing to suppose that a core bifidobacterial population may be present in wild solitary bees. To date there is sparse information about bifidobacteria in solitary bees such as Xylocopa and Osmia spp., this study is therefore focused on the isolation and characterization of bifidobacterial strains from solitary bees, in particular carpenter bee (Xylocopa violacea), builder bee (Osmia cornuta), and red mason bee (Osmia rufa). Among the isolates from Osmia spp. no new species have been detected whereas among Xylocopa isolates four strains (XV2, XV4, XV10, XV16) belonging to putative new species were found. Isolated strains are Gram-positive, lactate- and acetate-producing and possess the fructose-6-phosphate phosphoketolase enzyme. Full genome sequencing and genome annotation were performed for XV2 and XV10. Phylogenetic relationships were determined using partial and complete 16S rRNA sequences and hsp60 restriction analysis that confirmed the belonging of the new strains to Bifidobacterium genus and the relatedness of the strains XV2 and XV10 with XV16 and XV4, respectively. Phenotypic tests were performed for the proposed type strains, reference strains and their closest neighbor in the phylogenetic tree. The results support the proposal of two novel species Bifidobacterium xylocopae sp. nov. whose type strain is XV2 (=DSM 104955T=LMG 30142T), reference strain XV16 and Bifidobacterium aemilianum sp. nov. whose type strain is XV10 (=DSM 104956T=LMG 30143T), reference strain XV4.

Beneficial microorganisms for honey bees: problems and progresses.

Nowadays, honey bees are stressed by a number of biotic and abiotic factors which may compromise to some extent the pollination service and the hive productivity. The EU ban of antibiotics as therapeutic agents against bee pathogens has stimulated the search for natural alternatives. The increasing knowledge on the composition and functions of the bee gut microbiota and the link between a balanced gut microbiota and health status have encouraged the research on the use of gut microorganisms to improve bee health. Somehow, we are assisting to the transfer of the "probiotic concept" into the bee science. In this review, we examine the role of the honey bee gut microbiota in bee health and critically describe the available applications of beneficial microorganisms as pest control agents and health support. Most of the strains, mainly belonging to the genera Lactobacillus, Bifidobacterium and Bacillus, are isolated from honey bee crop or gut, but some applications involve environmental strains or formulation for animal and human consumption. Overall, the obtained results show the favourable effect of applied microbial strains on bee health and productivity, in particular if strains of bee origin are used. However, it is actually not yet possible to conclude whether this strategy will ever work. In particular, many aspects regarding the overall setup of the experiments, the dose, the timing and the duration of the treatment need to be optimized, also considering the microbiological safety of the hive products (i.e. pollen and honey). In addition, a deep investigation about the effect on host immunity and physiology is envisaged. Lastly, the final users of the formulations, i.e. beekeepers, should be taken into account for the achievement of high-quality, cost-effective and easy-to-use products.