Neighbors sharing pathogens: the intricate relationship between Apis mellifera and ants (Hymenoptera: Formicidae) nesting in hives.

Ants are ubiquitous and eusocial insects that exhibit frequent physical contact among colony members, thereby increasing their susceptibility to diseases. Some species are often found in beehives and in their surroundings, where they exploit the food resources of honey bees. This intricate relationship may facilitate the interspecific transmission of honey bee pathogens to ants, although ants themselves may contribute to spillback phenomena. The objective of this study was to assess the presence and abundance of honey bee pathogens in ants sampled from Italian apiaries. A total of 37 colonies within 24 apiaries across 7 regions were monitored. In total, 6 pathogens were detected in adult ants and 3 in the brood. In particular, the study revealed a high prevalence of honey bee pathogens in ants, with DWV, BQCV, and CBPV being the most commonly encountered. The brood also tested positive for the same viruses. Notably, all analyzed viruses were found to be replicative in both adult ants and ant broods. Furthermore, co-infections were prevalent, suggesting complex pathogen interactions within ant populations. Statistical analysis indicated significant differences in pathogen prevalence and abundance among ant species and sample types. The findings highlight active infection in both the ants and the brood, suggesting a potential role of ants as reservoir hosts and vectors of honey bee pathogens emphasizing the need for further research to understand the implications of interspecific pathogen transmission on ant and bee health.

Spatiotemporal evolution of the distribution of Chronic bee paralysis virus (CBPV) in honey bee colonies.

Chronic bee paralysis virus (CBPV) is a Apis mellifera viral infectious disease, exhibiting dark and hairless abdomen in workers with tremors and ataxita. Clinical signs are also typically linked to adverse weather conditions and overcrowding in the hive. The disease occurs in spring but recently it has been observed cases increase and seasonality loss of the disease incidence. This study analyses the evolution of CBPV in Italy, through data collected from 2009 to 2023 within three monitoring projects comprising nationwide extended detection networks, aimed to investigate the evolution of the CBPV spatial distribution, identifying high-risk areas for the virus spread. This study highlights an increased risk over years. Prevalence increased from 4.3% during 2009-2010 to 84.7% during 2021-2023 monitoring years. CBPV outbreaks were irregular between investigated seasons, highlighting Spring and Autumn as the most susceptible seasons. Risk of CBPV infection has increased, reaching high-risk in last years of monitoring. Sequence analysis showed a high similarity to other isolated Italian CBPVs. The study offers an epidemiological insight into the aetiology of this disease. CBPV distribution is a prerequisite to predict its future spread and factors involved in its propagation not only in honey bees but also in other pollinators and environments.

Phenotypic and genotypic antimicrobial resistance patterns in honey bee (Apis mellifera L.) bacterial symbionts.

Antimicrobial resistance (AMR) is a major global public health problem. Nevertheless, the knowledge of the factors driving the spread of resistance among environmental microorganisms is limited, and few studies have been performed worldwide. Honey bees (Apis mellifera L.) have long been considered bioindicators of environmental pollution and more recently also of AMR. In this study, 53 bacterial strains isolated from the body surface of honey bees at three ontogenetic stages, collected from ten different geographic locations, were tested for their phenotypic and genotypic resistance to eight classes of the most widely used antimicrobials in human and veterinary medicine. Results showed that 83% of the strains were resistant to at least one antimicrobial and 62% were multidrug-resistant bacteria, with a prevalence of resistance to nalidixic acid, cefotaxime, and aztreonam. A high percentage of isolates harbouring at least one antimicrobial gene was also observed (85%). The gene encoding resistance to colistin mcr-1 was the most abundant, followed by those for tetracycline tetM and tetC. Geographical features influenced the distribution of these traits more than bacterial species or bee stage, supporting the use of honey bee colonies and their associated bacteria as indicators to monitor environmental resistance. This approach can improve the scientific understanding of this global threat by increasing data collection capacity.

Pathogen occurrence in different developmental stages of the invasive Vespa velutina nigrithorax (Buysson, 1905).

BACKGROUND: The yellow-legged hornet (Vespa velutina nigrithorax) is a predatory species native to South-East Asia. The hornet is invasive in Europe, spreading to several countries and becoming a pest for Apis mellifera due to its behaviour of preying in front of apiaries. The aim of this study was (i) to investigate the presence of honey bee pathogens within the developmental stages of V. velutina after neutralizing a nest in Bologna province (Emilia-Romagna, Italy) and (ii) to analyze the mitochondrial DNA to determine if the population derived from the population initially introduced in Europe. RESULTS: The results indicated that deformed wing virus (82.76%) and Nosema ceranae (67.28%) were the most prevalent pathogens. Deformed wing virus, N. ceranae and sacbrood virus were found in all investigated stages, while chronic bee paralysis virus and Kashmir bee virus were exclusively found in foraging adults. All detected viruses were found to be replicative, highlighting active infection in the hosts. The mtDNA analysis demonstrated that the origin derived from the invasive population arrived in France. CONCLUSION: This study underscores the importance of further research to understand the effect of interspecific transmission, especially concerning the potential role of these pathogens as a biocontrol for the invasive V. velutina nigrithorax. © 2024 Society of Chemical Industry.

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.

Taxonomic revision of the Stenodynerus fastidiosissimus species-group in Western Europe and North Africa (Hymenoptera: Vespidae: Eumeninae).

The fastidiosissimus species-group of Stenodynerus de Saussure, 1863 is revised in Western Europe and North Africa, combining morphological data and DNA barcoding. Six species are recognized: S. difficilis (Morawitz, 1867) stat. resurr. (= S. fastidiosissimus auct.), S. fastidiosissimus (de Saussure, 1855), S. laborans (Costa, 1882) stat. resurr., S. montanus Selis, sp. nov., S. muelleri (Dusmet, 1917) (= Stenodynerus gusenleitneri Giordani Soika, 1986 syn. nov.), and S. rufescens Giordani Soika, 1977 stat. nov. Lectotypes are designated for Odynerus fastidiosissimus de Saussure, 1855 and Odynerus insularis Andr, 1883 non Smith, 1859. A key for the identification of members of this species-group is provided. DNA barcodes are published for every species, representing the first available sequences for the fastidiosissimus species-group.

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.

The use of honey bee (Apis mellifera L.) as biological monitors for pathogenic bacteria and antimicrobial resistance: A systematic review.

The phenomenon of antimicrobial resistance (AMR) is an increasingly real and relevant health problem. It is essential to verify the spread of this phenomenon in the environment. The European honey bee, Apis mellifera L., is a globally managed pollinator continuously used for biomonitoring thanks to its morphological and behavioural characteristics. During their foraging activities, a large number of honey bees move in the area surrounding the hive within a 1.5 km of radius. Besides, their body covered with hair and bristles are able to intercept pollen and minute particles, such as atmospheric particles, contaminants and microorganisms. For these reasons, A. mellifera L. is widely used as an environmental sentinel, especially for detecting pollutants, pesticides, microorganisms, and AMR. This systematic review aimed to collect and summarize the role of honey bee colonies as a biological monitor of AMR pathogenic bacteria and the environmental spread of antimicrobial resistance genes (ARGs). From honey bees were isolated a wide range of pathogenic and environmental bacteria strains, harbouring AMR and ARGs. However, AMR and ARGs were detected not only in environmental bacteria but also in symbiotic bacteria colonizing the bee gut. This systematic review highlights the employment of potential use of honey bees as AMR sentinel helpful for ecosystem health to implement possible control measures for humans, animals and plants, in the context of the "One-Health" approach.

Challenges and Advances in Bee Health and Diseases.

Understanding the health status of bees is crucial in assessing the epidemiology of pathogens that cause diseases in honey bee (Apis mellifera) colonies and wild bees [...].

The Epidemiological Situation of the Managed Honey Bee (Apis mellifera) Colonies in the Italian Region Emilia-Romagna.

The recent decades witnessed the collapse of honey bee colonies at a global level. The major drivers of this collapse include both individual and synergic pathogen actions, threatening the colonies' survival. The need to define the epidemiological pattern of the pathogens that are involved has led to the establishment of monitoring programs in many countries, Italy included. In this framework, the health status of managed honey bees in the Emilia-Romagna region (northern Italy) was assessed, throughout the year 2021, on workers from 31 apiaries to investigate the presence of major known and emerging honey bee pathogens. The prevalence and abundance of DWV, KBV, ABPV, CBPV, Nosema ceranae, and trypanosomatids (Lotmaria passim, Crithidia mellificae, Crithidia bombi) were assessed by molecular methods. The most prevalent pathogen was DWV, followed by CBPV and N. ceranae. Trypanosomatids were not found in any of the samples. Pathogens had different peaks in abundance over the months, showing seasonal trends that were related to the dynamics of both bee colonies and Varroa destructor infestation. For some of the pathogens, a weak but significant correlation was observed between abundance and geographical longitude. The information obtained in this study increases our understanding of the epidemiological situation of bee colonies in Emilia-Romagna and helps us to implement better disease prevention and improved territorial management of honey bee health.

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.

A novel and non-invasive method for DNA extraction from dry bee specimens.

In recent years molecular techniques have been used on museum material as integrative support for classic taxonomy. This cumulative systematics approach is especially for rare or extinct specimens, and genetic analysis may be useful to discern information that is not possible to glean from live materials or morphology. To date, the extraction of DNA required at least a partial destruction of the specimens, which is not possible for all individuals, especially the types. In this study, we described a novel method to extract mitochondrial DNA (mtDNA) from pinned museum bee individuals to avoid any external morphological damage. This method was able to amplify the mtDNA Cytochrome C oxidase subunit I (COI) gene in bee samples collected up to 27 years ago. We tested the efficacy of this method on 72 preserved be specimens belonging to nine species among four families, it could be used on many museums' rare and/or extinct bee species because it does not provide external morphological damages. The method could be helpful for providing ecological, taxonomic, and phylogenetic information about specimens preserved in museum collections.

Special Issue: "Infection in Honey Bees: Host-Pathogen Interaction and Spillover".

Honey bee health is a very important topic that has recently raised the interest of researchers [...].

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.

Glucosinolate Bioactivation by Apis mellifera Workers and Its Impact on Nosema ceranae Infection at the Colony Level.

The microsporidian fungus Nosema ceranae represents one of the primary bee infection threats worldwide and the antibiotic fumagillin is the only registered product for nosemosis disease control, while few alternatives are, at present, available. Natural bioactive compounds deriving from the glucosinolate-myrosinase system (GSL-MYR) in Brassicaceae plants, mainly isothiocyanates (ITCs), are known for their antimicrobial activity against numerous pathogens and for their health-protective effects in humans. This work explored the use of Brassica nigra and Eruca sativa defatted seed meal (DSM) GSL-containing diets against natural Nosema infection in Apis mellifera colonies. DSM patties from each plant species were obtained by adding DSMs to sugar candy at the concentration of 4% (w/w). The feeding was administered in May to mildly N. ceranae-infected honey bee colonies for four weeks at the dose of 250 g/week. In the treated groups, no significant effects on colony development and bee mortality were observed compared to the negative controls. The N. ceranae abundance showed a slight but significant decrease. Furthermore, the GSL metabolism in bees was investigated, and MYR hydrolytic activity was qualitatively searched in isolated bee midgut and hindgut. Interestingly, MYR activity was detected both in the bees fed DSMs and in the control group where the bees did not receive DSMs. In parallel, ITCs were found in gut tissues from the bees treated with DSMs, corroborating the presence of a MYR-like enzyme capable of hydrolyzing ingested GSLs. On the other hand, GSLs and other GSL hydrolysis products other than ITCs, such as nitriles, were found in honey produced by the treated bees, potentially increasing the health value of the final product for human consumption. The results are indicative of a specific effect on the N. ceranae infection in managed honey bee colonies depending on the GSL activation within the target organ.

Honey Bee Health.

Honey bee health is a crucial issue that has recently received increased interest from researchers, stakeholders, and citizens [...].