Occurrence of Nosema ceranae, Ascosphaera apis and trypanosomatids in Vespa orientalis linneus 1771.

Vespa orientalis is spreading across the Italian and European territories leading to new interactions among species, which could lead to the transmission of pathogens between species. Detection of honey bee viruses in V. orientalis has already been revealed in both adults and larvae, while no information is available regarding parasitic occurrence. Sixty adult hornets collected across apiaries in the South of Italy were subjected to cytological, histopathological and biomolecular examination to evaluate the occurrence of Nosema ceranae, Ascosphaera apis, Lotmaria passim, Crithidia mellificae, and Crithidia bombi. Cytological examination revealed the presence of Nosema spores in 38.33% of individuals while histopathological analysis showed the presence of L. passim-like elements in the rectum of two examined specimens and the presence of fungal hyphae in the small intestine of another hornet. Biomolecular investigation revealed that N. ceranae was the most prevalent pathogen (50.0%), followed by A. apis (6.66%), L. passim (6.66%) and C. bombi (6.0%).

Using honey bee colonies to monitor phenotypic and genotypic resistance to colistin.

Colistin is a polymyxin antimicrobic mainly used to treat infection caused by multi-drug resistant Gram-negative bacteria. Mechanisms of colistin resistance are linked to the mobile colistin resistance (mcr) genes, which are transferable within mobile plasmids. Currently, there is limited research on the environmental dissemination of these genes. The behavioural and morphological characteristics of Apis mellifera L. make honey bees effective environmental bioindicators for assessing the prevalence of antimicrobial-resistant bacteria. This study aims to evaluate the colistin phenotypic and genotypic resistance in environmental Gram-negative bacteria isolated from foraging honey bees, across a network of 33 colonies distributed across the Emilia-Romagna region in Italy. Phenotypic resistances were determined through a microdilution assay using the minimum inhibitory concentration (MIC) with dilutions ranging from 0.5 µg/ml to 256 µg/ml. Strains with MIC values gather than 2 µg/ml were classified as resistant. Also, the identification of the nine mcr genes was carried out using two separate multiplex PCR assays. The study found that 68.5% of isolates were resistant and the genus with the higher resistance rates observed in Enterobacter spp. (84.5%). At least one mcr gene was found in 137 strains (53.3%). The most detected gene was mcr5 (35.3%), which was the most frequently detected gene in the seven provinces, while the least observed was mcr4 (4.8%), detected only in two provinces. These results suggested the feasibility of detecting specific colistin resistance genes in environmentally spread bacteria and understanding their distribution at the environmental level, despite their restricted clinical use. In a One-Health approach, this capability enables valuable environmental monitoring, considering the significant role of colistin in the context of public health.

Residues of agrochemicals in beebread as an indicator of landscape management.

The agricultural intensification represents a major threat to biodiversity, with negative effects on the ecosystem. In particular, habitat loss and degradation, along with pesticide use have been recognised as primary factors contributing to the actual global decline of pollinators. Here we investigated the quality of agroecosystems in the Emilia-Romagna region (Northern Italy) within the national monitoring project BeeNet. We analysed pesticide residues in 100 samples of beebread collected in 25 BeeNet stations in March and June 2021 and 2022. We evaluated diversity and concentration of these chemicals, their risk (TWC) to honey bees, and their correlation with land use. Overall, in 84 % of the samples we found 63 out of 373 different pesticide residues, >90 % of them belonging to fungicides and insecticides. The TWC exceeded the risk threshold in seven samples (TWCmix), mostly due to only one or two compounds. We also found 15 compounds not approved in the EU as plant protection products (PPPs), raising concerns about illegal use or contamination through beeswax recycling. Samples collected in 2021 and in June presented a significantly higher number of active ingredients and TWC than those collected in 2022 and in March. The TWC calculated on single compounds (TWCcom) exceeded the risk threshold in case of four insecticides, namely carbaryl, fipronil, imidacloprid and thiamethoxam (although each detected in only one sample). Finally, both TWC and number of active ingredients were moderately or highly positively correlated with the percentage of area covered by orchards. Considering that we found on average more than five different molecules per sample, and that we ignored potential synergistic effects, the results of this work highlight the alarming situation regarding pesticide treatments and toxicity risk for bees linked to the current agricultural practices, and the need for implementing sustainable and pollinator-friendly strategies.

Ecological and social factors influence interspecific pathogens occurrence among bees.

The interspecific transmission of pathogens can occur frequently in the environment. Among wild bees, the main spillover cases are caused by pathogens associated with Apis mellifera, whose colonies can act as reservoirs. Due to the limited availability of data in Italy, it is challenging to accurately assess the impact and implications of this phenomenon on the wild bee populations. In this study, a total of 3372 bees were sampled from 11 Italian regions within the BeeNet project, evaluating the prevalence and the abundance of the major honey bee pathogens (DWV, BQCV, ABPV, CBPV, KBV, Nosema ceranae, Ascosphaera apis, Crithidia mellificae, Lotmaria passim, Crithidia bombi). The 68.4% of samples were positive for at least one pathogen. DWV, BQCV, N. ceranae and CBPV showed the highest prevalence and abundance values, confirming them as the most prevalent pathogens spread in the environment. For these pathogens, Andrena, Bombus, Eucera and Seladonia showed the highest mean prevalence and abundance values. Generally, time trends showed a prevalence and abundance decrease from April to July. In order to predict the risk of infection among wild bees, statistical models were developed. A low influence of apiary density on pathogen occurrence was observed, while meteorological conditions and agricultural management showed a greater impact on pathogen persistence in the environment. Social and biological traits of wild bees also contributed to defining a higher risk of infection for bivoltine, communal, mining and oligolectic bees. Out of all the samples tested, 40.5% were co-infected with two or more pathogens. In some cases, individuals were simultaneously infected with up to five different pathogens. It is essential to increase knowledge about the transmission of pathogens among wild bees to understand dynamics, impact and effects on pollinator populations. Implementing concrete plans for the conservation of wild bee species is important to ensure the health of wild and human-managed bees within a One-Health perspective.

Predictive statistical models for monitoring antimicrobial resistance spread in the environment using Apis mellifera (L. 1758) colonies.

The rise of antimicrobial resistance (AMR) is one of the most relevant problems for human and animal health. According to One Health Approach, it is important to regulate the use of antimicrobials and monitor the spread of AMR in the environment as well. Apis mellifera (L. 1758) colonies were used as bioindicators thanks to their physical and behavioural characteristics. During their foraging flights, bees can intercept small particles, including atmospheric particulate matter, etc., and also microorganisms. To date, the antimicrobial surveillance network is limited to the sanitary level but lacks into environmental context. This study aimed to evaluate the use of A. mellifera colonies distributed throughout the Emilia-Romagna region (Italy) as indicators of environmental antimicrobial-resistant bacteria. This was performed by creating a statistical predictive model that establishes correlations between environmental characteristics and the likelihood of isolating specific bacterial genera and antimicrobial-resistant strains. A total of 608 strains were isolated and tested for susceptibility to 19 different antimicrobials. Aztreonam-resistant strains were significantly related to environments with sanitary structures, agricultural areas and wetlands, while urban areas present a higher probability of trimethoprim/sulfamethoxazole-resistant strains isolation. Concerning genera, environments with sanitary structures and wetlands are significantly related to the genera Proteus spp., while the Escherichia spp. strains can be probably isolated in industrial environments. The obtained models showed maximum values of Models Accuracy and robustness (R2) of 55 % and 24 %, respectively. The results indicate the efficacy of utilizing A. mellifera colonies as valuable bioindicators for estimating the prevalence of AMR in environmentally disseminated bacteria. This survey can be considered a good basis for the development of further studies focused on monitoring both sanitary and animal pathology, creating a specific network in the environments of interest.

Seasonal trends of the ABPV, KBV, and IAPV complex in Italian managed honey bee (Apis mellifera L.) colonies.

Acute bee paralysis virus (ABPV), Kashmir bee virus (KBV), and Israeli acute paralysis virus (IAPV) usually persist as covert infections in honey bee colonies. They can cause rapid bee mortality in cases of severe infection, often associated with high Varroa destructor infestation, by which they are transmitted. In various countries, these viruses have been associated with colony collapse. Despite their potential danger, these viruses are often disregarded, and little information is available on their occurrence in many countries, including Italy. In 2021, 370 apiaries representing all of the Italian regions were investigated in four different months (June, September, November, and March) for the presence of ABPV, KBV, and IAPV. IAPV was not found in any of the apiaries investigated, whereas 16.45% and 0.67% of the samples tested positive for ABPV and KBV, respectively. Most ABPV cases occurred in late summer-autumn in both northern and southern regions. We observed a scattered pattern of KBV-positive colonies that did not allow any seasonal or regional trends to be discerned. Differences observed among regions and months were potentially related to the dynamics of varroa infestation, viral genetic variations, and different climatic conditions resulting in variations in bee behaviour. This study improves our understanding of the circulation of bee viruses and will contribute to better disease prevention and preservation of bee health.

Effect of amino acid enriched diets on hemolymph amino acid composition in honey bees.

Amino acids (AAs) are an abundant class of nectar solutes, and they are involved in the nectar attractiveness to flower visitors. Among the various AAs, proline is the most abundant proteogenic AA, and γ-amino butyric acid (GABA) and ß-alanine are the two most abundant non-proteogenic AAs. These three AAs are known to affect insect physiology, being involved in flight metabolism and neurotransmission. The aim of this study was to investigate the effects of artificial diets enriched with either ß-alanine, GABA, or proline on consumption, survival, and hemolymph composition in honey bees belonging to two different ages and with different metabolism (i.e., newly emerged and foragers). Differences in feed intake among diets were not observed, while a diet enriched with ß-alanine improved the survival rate of newly emerged honey bees compared to the control group. Variations in the hemolymph AA concentrations occurred only in newly emerged honey bees, according to the diet and the time of hemolymph sampling. A greater susceptibility of young honey bees to enriched diets than older honey bees was observed. The variations in the concentrations of hemolymph AAs reflect either the accumulation of dietary AAs or the existence of metabolic pathways that may lead to the conversion of dietary AAs into different ones. This investigation could be an initial contribution to studying the complex dynamics that regulate hemolymph AA composition and its effect on honey bee physiology.

Nesting biology and nest structure of the exotic bee Megachile sculpturalis.

From the 1990s, the Southeast Asia native giant resin bee Megachile sculpturalis (Smith, 1853) was introduced first to North America, and then to many countries in Europe. Despite increasing studies on its invasive potential and geographical expansion, information on nesting behaviour of this species is still extremely scarce. To increase knowledge on the nesting biology of M. sculpturalis, we studied multiple aspects of nesting and pollen provisioning in three consecutive years in artificial nests in Bologna, Italy. We observed 166 bees visiting nests, and followed individual nesting behaviour and success of 41 adult females. We measured cavity diameter in 552 nests and characterised the structure in 100 of them. More than 95% of nest diameters ranged between 0.6 and 1.2 cm, overlapping with several sympatric species of cavity-nesting hymenopterans in the study area. Most nests had a first chamber from the entrance of variable length without brood, followed by an average of about two brood cells with a mean length of 2.85 ± 0.13 cm each. The pollen stored in brood cells was almost monofloral, belonging to the ornamental plant Styphnolobium japonicum (L.) Schott. We estimated that a single female should visit ≈180 flowers to collect enough pollen for a single brood cell. These results fill knowledge gaps on the nesting biology and nest structure of the exotic M. sculpturalis, and they are discussed in relation to possible competition with native bees for nesting sites and foraging resources.

Signals of adaptation to agricultural stress in the genomes of two European bumblebees.

Human-induced environmental impacts on wildlife are widespread, causing major biodiversity losses. One major threat is agricultural intensification, typically characterised by large areas of monoculture, mechanical tillage, and the use of agrochemicals. Intensification leads to the fragmentation and loss of natural habitats, native vegetation, and nesting and breeding sites. Understanding the adaptability of insects to these changing environmental conditions is critical to predicting their survival. Bumblebees, key pollinators of wild and cultivated plants, are used as model species to assess insect adaptation to anthropogenic stressors. We investigated the effects of agricultural pressures on two common European bumblebees, Bombus pascuorum and B. lapidarius. Restriction-site Associated DNA Sequencing was used to identify loci under selective pressure across agricultural-natural gradients over 97 locations in Europe. 191 unique loci in B. pascuorum and 260 in B. lapidarius were identified as under selective pressure, and associated with agricultural stressors. Further investigation suggested several candidate proteins including several neurodevelopment, muscle, and detoxification proteins, but these have yet to be validated. These results provide insights into agriculture as a stressor for bumblebees, and signal for conservation action in light of ongoing anthropogenic changes.

Occurrence of Honey Bee (Apis mellifera L.) Pathogens in Wild Pollinators in Northern Italy.

Diseases contribute to the decline of pollinator populations, which may be aggravated by the interspecific transmission of honey bee pests and pathogens. Flowers increase the risk of transmission, as they expose the pollinators to infections during the foraging activity. In this study, both the prevalence and abundance of 21 honey bee pathogens (11 viruses, 4 bacteria, 3 fungi, and 3 trypanosomatids) were assessed in the flower-visiting entomofauna sampled from March to September 2021 in seven sites in the two North-Italian regions, Emilia-Romagna and Piedmont. A total of 1,028 specimens were collected, identified, and analysed. Of the twenty-one pathogens that were searched for, only thirteen were detected. Altogether, the prevalence of the positive individuals reached 63.9%, with Nosema ceranae, deformed wing virus (DWV), and chronic bee paralysis virus (CBPV) as the most prevalent pathogens. In general, the pathogen abundance averaged 5.15 * 106 copies, with CBPV, N. ceranae, and black queen cell virus (BQCV) as the most abundant pathogens, with 8.63, 1.58, and 0.48 * 107 copies, respectively. All the detected viruses were found to be replicative. The sequence analysis indicated that the same genetic variant was circulating in a specific site or region, suggesting that interspecific transmission events among honey bees and wild pollinators are possible. Frequently, N. ceranae and DWV were found to co-infect the same individual. The circulation of honey bee pathogens in wild pollinators was never investigated before in Italy. Our study resulted in the unprecedented detection of 72 wild pollinator species as potential hosts of honey bee pathogens. Those results encourage the implementation of monitoring actions aiming to improve our understanding of the environmental implications of such interspecific transmission events, which is pivotal to embracing a One Health approach to pollinators' welfare.

On the road: Anthropogenic factors drive the invasion risk of a wild solitary bee species.

Complex biotic networks of invaders and their new environments pose immense challenges for researchers aiming to predict current and future occupancy of introduced species. This might be especially true for invasive bees, as they enter novel trophic interactions. Little attention has been paid to solitary, invasive wild bees, despite their increasing recognition as a potential global threat to biodiversity. Here, we present the first comprehensive species distribution modelling approach targeting the invasive bee Megachile sculpturalis, which is currently undergoing parallel range expansion in North America and Europe. While the species has largely colonised the most highly suitable areas of North America over the past decades, its invasion of Europe seems to be in its early stages. We showed that its current distribution is largely explained by anthropogenic factors, suggesting that its spread is facilitated by road and maritime traffic, largely beyond its intrinsic dispersal ability. Our results suggest that M. sculpturalis is likely to be negatively affected by future climate change in North America, while in Europe the potential suitable areas at-risk of invasion remain equally large. Based on our study, we emphasise the role of expert knowledge for evaluation of ecologically meaningful variables implemented and interpreted for species distribution modelling. We strongly recommend that the monitoring of this and other invasive pollinator species should be prioritised in areas identified as at-risk, alongside development of effective management strategies.

Donkey assisted interventions: A pilot survey on service providers in North-Eastern Italy.

CONTEXT: Animal Assisted Interventions (AAI), which are generally defined as 'pet therapy', focus on the improvement of human health and wellbeing through a multidisciplinary approach and the involvement of domestic animals. An Italian survey conducted in 2017 revealed that donkeys were the third most involved species in AAI. Nevertheless, data on the main features of the providers of Donkey Assisted Interventions (DAI), especially in Italy, and their organisational models are scarce. OBJECTIVE: This questionnaire-based pilot study aimed to collect information about the features of DAI providers in the Veneto Region to have a general framework about the field. STUDY METHOD: In this descriptive mixed-method pilot study, we interviewed DAI providers through a computer assisted telephone interview. Respondents were selected by integrating data from the Veneto Region Livestock Database and from the Italian official register of AAI professionals. The questionnaire was designed following the advice from Equator Network. MAIN OUTCOME MEASURES: Our outcome measures included the prevalent organisational system adopted by our respondents, main type of AAI programs, and users. Furthermore, we collected qualitative data about economic and income and respondents' personal opinions about DAI (strengths and weaknesses). MAIN RESULTS: Data analysis revealed a heterogeneous picture of the DAI field with a prevalence of non-profit organisations providing mainly Donkey Assisted Activities to a large range of user categories. Despite the poor economic framework and low income from DAI, DAI providers were strongly motivated and spent more time and efforts in delivering this service.

Honey bee (Apis mellifera L.) colonies as bioindicators of environmental SARS-CoV-2 occurrence.

SARS-CoV-2 is responsible for the COVID-19 pandemic. Airflows sustain the infection spread, and in densely urbanized areas airborne particulate matters (PMs) are deemed to aggravate the viral transmission. Apis mellifera colonies are used as bioindicators as they allow environmental sampling of different nature, PMs included. This experiment demonstrates for the first time the possible use of honey bee colonies in the SARS-CoV-2 monitoring. The trial was conducted in Bologna on 18 March 2021, when the third wave of the Italian pandemic was at its peak and environmental conditions allowed high PM concentrations in the air. Sterile swabs were lined up at the hive entrance to sample the dusty material on the body of returning foragers. All of them resulted positive for the target genes of viral SARS-CoV-2 RNA. Likewise, internal samples were taken, but they resulted in no amplification of the target sequences. This experiment does not support speculations about the role of honey bees or their products in SARS-CoV-2 transmission. However, it indicates a novel use of A. mellifera colonies in the environmental detection of airborne human pathogens, at least in a densely urbanized area, deserving better understanding and possible integration with data from automatic air samplers.

Hornets and Honey Bees: A Coevolutionary Arms Race between Ancient Adaptations and New Invasive Threats.

Hornets and honey bees have a long history of coevolution resulting in a plethora of captivating adaptations and counteradaptations between predator and prey. From simple physiological mechanisms to complex behavioral strategies, some Vespa hornets have specialized in hunting honey bees, while the latter have put in place effective defenses to counteract their attack. Both hornets and honey bees have evolved the ability to detect the odors and the pheromones emitted by the other to locate the prey or to spot foraging predators. Hornets often rely on their bigger size, heavily armored body and destructive attacks, while honey bees differentiated collective defense responses finely coordinated to deter or kill the hornet menace. However, when new species of hornets and honey bees come into contact, the absence of coevolution can have a heavy impact on the defenseless bees. The evolutionary arms race between hornets and honey bees provides not only compelling examples of adaptations and counteradaptations between predator and prey, but could also represent a starting point for the development of effective and sustainable strategies to protect honey bees and beekeeping activities and to control invasive alien species of hornets.

Beewatching: A Project for Monitoring Bees through Photos.

Bees play a key role in natural and agro-ecosystems and their diversity is worldwide threatened by anthropogenic causes. Despite this, there is little awareness of the existence of the numerous species of wild bees, and the common name "bee" is very often exclusively associated with Apis mellifera. Our aim was to create a citizen science project in Italy with the following objectives: (a) raising awareness of the importance and diversity of bees, (b) obtaining data on the biology, ecology and distribution of Italian species, and (c) launching the monitoring of alien bees. The first step of the project was to create a website platform with a section containing informative datasheets of the wild bee families and of the most common bee genera present in Italy, a form to send reports of observed bees and an interactive map with all citizen's reports. During the 2 years of the project 1086 reports were sent by 269 users, with 38 Apoidea genera reported on 190 plant genera; furthermore, 22 reports regarding the alien species Megachile sculpturalis arrived. The majority of bees (34 genera) were observed on spontaneous plants, including 115 genera native to Italy. Considering the increasing number of reports and data obtained in these first two years of the project, our objectives seem to be achieved. Future steps will be to outline the profile of beewatchers, to plan activities in a more targeted way, and also to start some sub-projects for conservation purposes.

Pathogens Spillover from Honey Bees to Other Arthropods.

Honey bees, and pollinators in general, play a major role in the health of ecosystems. There is a consensus about the steady decrease in pollinator populations, which raises global ecological concern. Several drivers are implicated in this threat. Among them, honey bee pathogens are transmitted to other arthropods populations, including wild and managed pollinators. The western honey bee, Apis mellifera, is quasi-globally spread. This successful species acted as and, in some cases, became a maintenance host for pathogens. This systematic review collects and summarizes spillover cases having in common Apis mellifera as the mainteinance host and some of its pathogens. The reports are grouped by final host species and condition, year, and geographic area of detection and the co-occurrence in the same host. A total of eighty-one articles in the time frame 1960-2021 were included. The reported spillover cases cover a wide range of hymenopteran host species, generally living in close contact with or sharing the same environmental resources as the honey bees. They also involve non-hymenopteran arthropods, like spiders and roaches, which are either likely or unlikely to live in close proximity to honey bees. Specific studies should consider host-dependent pathogen modifications and effects on involved host species. Both the plasticity of bee pathogens and the ecological consequences of spillover suggest a holistic approach to bee health and the implementation of a One Health approach.

Replicative Deformed Wing Virus Found in the Head of Adults from Symptomatic Commercial Bumblebee (Bombus terrestris) Colonies.

The deformed wing virus (DWV) is one of the most common honey bee pathogens. The virus may also be detected in other insect species, including Bombus terrestris adults from wild and managed colonies. In this study, individuals of all stages, castes, and sexes were sampled from three commercial colonies exhibiting the presence of deformed workers and analysed for the presence of DWV. Adults (deformed individuals, gynes, workers, males) had their head exscinded from the rest of the body and the two parts were analysed separately by RT-PCR. Juvenile stages (pupae, larvae, and eggs) were analysed undissected. All individuals tested positive for replicative DWV, but deformed adults showed a higher number of copies compared to asymptomatic individuals. Moreover, they showed viral infection in their heads. Sequence analysis indicated that the obtained DWV amplicons belonged to a strain isolated in the United Kingdom. Further studies are needed to characterize the specific DWV target organs in the bumblebees. The result of this study indicates the evidence of DWV infection in B. terrestris specimens that could cause wing deformities, suggesting a relationship between the deformities and the virus localization in the head. Further studies are needed to define if a specific organ could be a target in symptomatic bumblebees.

Next generation sequencing study on RNA viruses of Vespa velutina and Apis mellifera sharing the same foraging area.

The predator Asian hornet (Vespa velutina) represents one of the major threats to honeybee survival. Viral spillover from bee to wasp has been supposed in several studies, and this work aims to identify and study the virome of both insect species living simultaneously in the same foraging area. Transcriptomic analysis was performed on V. velutina and Apis mellifera samples, and replicative form of detected viruses was carried out by strand-specific RT-PCR. Overall, 6 and 9 different viral types were reported in V. velutina and A. mellifera, respectively, and five of these viruses were recorded in both hosts. Varroa destructor virus-1 and Cripavirus NB-1/2011/HUN (now classified as Triato-like virus) were the most represented viruses detected in both hosts, also in replicative form. In this investigation, Triato-like virus, as well as Aphis gossypii virus and Nora virus, was detected for the first time in honeybees. Concerning V. velutina, we report for the first time the recently detected honeybee La Jolla virus. A general high homology rate between genomes of shared viruses between V. velutina and A. mellifera suggests the efficient transmission of the virus from bee to wasp. In conclusion, our findings highlight the presence of several known and newly reported RNA viruses infecting A. mellifera and V. velutina. This confirms the environment role as an important source of infection and indicates the possibility of spillover from prey to predator.