Dimethyl sulfoxide, an alternative for control of Nosema ceranae infection in honey bees (Apis mellifera).

Nosema ceranae is a microsporidian parasite that threatens current apiculture. N. ceranae-infected honey bees (Apis mellifera) exhibit morbid physiological impairments and reduced honey production, malnutrition, shorter life span, and higher mortality than healthy honey bees. In this study, we found that dimethyl sulfoxide (DMSO) could enhance the survival rate of N. ceranae-infected honey bees. Therefore, we investigated the effect of DMSO on N. ceranae-infected honey bees using comparative RNA sequencing analysis. Our results revealed that DMSO was able to affect several biochemical pathways, especially the metabolic-related pathways in N. ceranae-infected honey bees. Based on these findings, we conclude that DMSO may be a useful alternative for treating N. ceranae infection in apiculture.

Caterpillar-parasitoid interactions: species-specific influences on host microbiome composition.

There is increasing evidence that host-parasitoid interactions can have a pronounced impact on the microbiome of host insects, but it is unclear to what extent this is caused by the host and/or parasitoid. Here, we compared the internal and external microbiome of caterpillars of Pieris brassicae and Pieris rapae parasitized by Cotesia glomerata or Cotesia rubecula with nonparasitized caterpillars. Additionally, we investigated the internal and external microbiome of the parasitoid larvae. Both internal and external bacterial densities were significantly higher for P. brassicae than P. rapae, while no differences were found between parasitized and nonparasitized caterpillars. In contrast, parasitism significantly affected the composition of the internal and external microbiome of the caterpillars and the parasitoid larvae, but the effects were dependent on the host and parasitoid species. Irrespective of host species, a Wolbachia species was exclusively found inside caterpillars parasitized by C. glomerata, as well as in the corresponding developing parasitoid larvae. Similarly, a Nosema species was abundantly present inside parasitized caterpillars and the parasitoid larvae, but this was independent of the host and the parasitoid species. We conclude that parasitism has pronounced effects on host microbiomes, but the effects depend on both the host and parasitoid species.

Prevalence and Phylogenetic Network Analysis of Nosema apis and Nosema ceranae Isolates from Honeybee Colonies in Türkiye.

PURPOSE: Nosemosis is a disease that infects both Western honeybees (Apis mellifera L.) and Asian honeybees (Apis cerana) and causes colony losses and low productivity worldwide. In order to control nosemosis, it is important to determine the distribution and prevalence of this disease agent in a particular region. For this purpose, a national study was conducted to assess the prevalence of Nosema ceranae and N. apis throughout Türkiye, to perform network analyses of the parasites, and to determine the presence of nosemosis. METHODS: In this study which aimed to assess the prevalence of N. apis and N. ceranae in different colony types and regions where beekeeping is intensive in Türkiye, specimens were collected from hives with no clinical signs. RESULTS: A total of 1194 Western honeybee colonies in 400 apiaries from 40 provinces of Türkiye were examined by microscopic and molecular techniques. Nosemosis was found in all of 40 provinces. The mean prevalence ratio was 64.3 ± 3.0, with 95% CI in apiaries and 40.5 ± 2.9, 95% CI in hives. Nosema ceranae DNA was detected in all of positive hives, while N. ceranae and N. apis co-infection was detected in only four colonies. CONCLUSION: This study showed that nosemosis has spread to all provinces, and it is common in every region of Türkiye. All of the N. ceranae or N. apis samples examined were 100% identical within themselves. Network analysis showed that they were within largest haplotype reported worldwide.

Exploring the interactions between Nosema ceranae infection and the honey bee gut microbiome.

Managed colonies of the European honey bee, Apis mellifera, have faced considerable losses in recent years. A widespread contributing factor is a microsporidian pathogen, Nosema ceranae, which occurs worldwide, is increasingly resistant to antibiotic treatment, and can alter the host's immune response and nutritional uptake. These obligate gut pathogens share their environment with a natural honey bee microbiome whose composition can affect pathogen resistance. We tested the effect of N. ceranae infection on this microbiome by feeding 5 day-old adult bees that had natural, fully developed microbiomes with live N. ceranae spores (40,000 per bee) or a sham inoculation, sterile 2.0 M sucrose solution. We caged and reared these bees in a controlled lab environment and tracked their mortality over 12 d, after which we dissected them, measured their infection levels (gut spore counts), and analyzed their microbiomes. Bees fed live spores had two-fold higher mortality by 12 d and 36.5-fold more spores per bee than controls. There were also strong colony effects on infection levels, and 9% of spore-inoculated bees had no spore counts at all (defined as fed-spores-but-not-infected). Nosema ceranae infection had significant but subtle effects on the gut microbiomes of experimentally infected bees, bees with different infection levels, and fed-spores-but-not-infected vs. bees with gut spores. Specific bacteria, including Gilliamella ASVs, were positively associated with infection, indicating that multiple strains of core gut microbes either facilitate or resist N. ceranae infection. Future studies on the interactions between bacterial, pathogen, and host genotypes would be illuminating.

The effects of queen mandibular pheromone on Nosema (Vairimorpha) ceranae infections in caged honey bees.

Honey bees utilize queen mandibular pheromone (QMP) for maintaining social hierarchy and colony development. In controlled cage studies, synthetic QMP is often introduced to mimic natural conditions. However, questions have arisen about the effects of QMP on nosema disease studies. This short report identifies significant early-stage suppression effects of QMP on Nosema (Vairimorpha) ceranae infections. QMP was found to significantly lower infection rates below the reported infectious dose for 50 % infectivity (ID50) and to slow disease development in a dose-independent manner. These effects diminished at doses exceeding ID100. We recommend that studies investigating treatment effects using caged bees avoid QMP to ensure unambiguous results. Additionally, employing multiple infectious doses with shorter incubation times would be useful for evaluating other treatments that may have subtle effects. Furthermore, our findings support previous field studies suggesting that queen replacement reduces nosema disease at levels similar to treatment with fumagillin.

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%).

Construction of a Full-Length Transcriptome of Western Honeybee Midgut Tissue and Improved Genome Annotation.

Honeybees are an indispensable pollinator in nature with pivotal ecological, economic, and scientific value. However, a full-length transcriptome for Apis mellifera, assembled with the advanced third-generation nanopore sequencing technology, has yet to be reported. Here, nanopore sequencing of the midgut tissues of uninoculated and Nosema ceranae-inoculated A. mellifera workers was conducted, and the full-length transcriptome was then constructed and annotated based on high-quality long reads. Next followed improvement of sequences and annotations of the current reference genome of A. mellifera. A total of 5,942,745 and 6,664,923 raw reads were produced from midguts of workers at 7 days post-inoculation (dpi) with N. ceranae and 10 dpi, while 7,100,161 and 6,506,665 raw reads were generated from the midguts of corresponding uninoculated workers. After strict quality control, 6,928,170, 6,353,066, 5,745,048, and 6,416,987 clean reads were obtained, with a length distribution ranging from 1 kb to 10 kb. Additionally, 16,824, 17,708, 15,744, and 18,246 full-length transcripts were respectively detected, including 28,019 nonredundant ones. Among these, 43,666, 30,945, 41,771, 26,442, and 24,532 full-length transcripts could be annotated to the Nr, KOG, eggNOG, GO, and KEGG databases, respectively. Additionally, 501 novel genes (20,326 novel transcripts) were identified for the first time, among which 401 (20,255), 193 (13,365), 414 (19,186), 228 (12,093), and 202 (11,703) were respectively annotated to each of the aforementioned five databases. The expression and sequences of three randomly selected novel transcripts were confirmed by RT-PCR and Sanger sequencing. The 5' UTR of 2082 genes, the 3' UTR of 2029 genes, and both the 5' and 3' UTRs of 730 genes were extended. Moreover, 17,345 SSRs, 14,789 complete ORFs, 1224 long non-coding RNAs (lncRNAs), and 650 transcription factors (TFs) from 37 families were detected. Findings from this work not only refine the annotation of the A. mellifera reference genome, but also provide a valuable resource and basis for relevant molecular and -omics studies.

The recent revision of the genera Nosema and Vairimorpha (Microsporidia: Nosematidae) was flawed and misleads the bee scientific community.

The genus Vairimorpha was proposed for several species of Nosema in 1976 (Pilley, 1976), almost 70 years after Nosema apis Zander (Zander, 1909). Tokarev and colleagues proposed the redefinition of 17 microsporidian species in four genera, Nosema, Vairimorpha, Rugispora, and Oligosporidium, based on phylogenetic trees of two genetic markers (SSU rRNA and RPB1) (Tokarev et al., 2020). Several issues should invalidate this new classification, leading to the synonymization of Vairimorpha within Nosema.

Transcriptomic analysis of Bombyx mori in its early larval stage (2nd instar) of development upon Nosema bombycis transovarial infection.

The infection caused by Nosema bombycis often known as pebrine, is a devastating sericulture disease. The infection can be transmitted to the next generation through eggs laid by infected female Bombyx mori moths (transovarial) as well as with N. bombycis contaminated food (horizontal). Most diagnoses were carried out in the advanced stages of infection until the time that infection might spread to other healthy insects. Hence, early diagnosis of pebrine is of utmost importance to quarantine infected larvae from uninfected silkworm batches and stop further spread of the infection. The findings of our study provide an insight into how the silkworm larval host defence system was activated against early N. bombycis transovarial infection. The results obtained from transcriptome analysis of infected 2nd instar larvae revealed significant (adjusted P-value < 0.05) expression of 1888 genes of which 801 genes were found to be upregulated and 1087 genes were downregulated when compared with the control. Pathway analysis indicated activation of the immune deficiency (IMD) pathway, which shows a potential immune defence response against pebrine infection as well as suppression of the melanin synthesis pathway due to lower expression of prophenoloxidase activating enzyme (PPAE). Liquid chromatography mass spectrometry (LC-MS/MS) analysis of haemolymph from infected larvae shows the secretion of serpin binding protein of N. bombycis which might be involved in the suppression of the melanization pathway. Moreover, among the differentially expressed genes, we found that LPMC-61, yellow-y, gasp and osiris 9 can be utilised as potential markers for early diagnosis of transovarial pebrine infection in B. mori. Physiological as well as biochemical roles and functions of many of the essential genes are yet to be established, and enlightened research will be required to characterize the products of these genes.

Vairimorpha (Nosema) ceranae can promote Serratia development in honeybee gut: an underrated threat for bees?

The genus Serratia harbors opportunistic pathogenic species, among which Serratia marcescens is pathogenic for honeybees although little studied. Recently, virulent strains of S. marcescens colonizing the Varroa destructor mite's mouth were found vectored into the honeybee body, leading to septicemia and death. Serratia also occurs as an opportunistic pathogen in the honeybee's gut with a low absolute abundance. The Serratia population seems controlled by the host immune system, but its presence may represent a hidden threat, ready to arise when honeybees are weakened by biotic and abiotic stressors. To shed light on the Serratia pathogen, this research aims at studying Serratia's development dynamics in the honeybee body and its interactions with the co-occurring fungal pathogen Vairimorpha ceranae. Firstly, the degree of pathogenicity and the ability to permeate the gut epithelial barrier of three Serratia strains, isolated from honeybees and belonging to different species (S. marcescens, Serratia liquefaciens, and Serratia nematodiphila), were assessed by artificial inoculation of newborn honeybees with different Serratia doses (104, 106, and 108 cells/mL). The absolute abundance of Serratia in the gut and in the hemocoel was assessed in qPCR with primers targeting the luxS gene. Moreover, the absolute abundance of Serratia was assessed in the gut of honeybees infected with V. ceranae at different development stages and supplied with beneficial microorganisms and fumagillin. Our results showed that all tested Serratia strains could pass through the gut epithelial barrier and proliferate in the hemocoel, with S. marcescens being the most pathogenic. Moreover, under cage conditions, Serratia better proliferates when a V. ceranae infection is co-occurring, with a positive and significant correlation. Finally, fumagillin and some of the tested beneficial microorganisms could control both Serratia and Vairimorpha development. Our findings suggest a correlation between the two pathogens under laboratory conditions, a co-occurring infection that should be taken into consideration by researches when testing antimicrobial compounds active against V. ceranae, and the related honeybees survival rate. Moreover, our findings suggest a positive control of Serratia by the environmental microorganism Apilactobacillus kunkeei in a in vivo model, confirming the potential of this specie as beneficial bacteria for honeybees.

Detrimental effects of amitraz exposure in honey bees (Apis mellifera) infected with Nosema ceranae.

In recent years, there has been growing concern on the potential weakening of honey bees and their increased susceptibility to pathogens due to chronic exposure to xenobiotics. The present work aimed to study the effects on bees undergoing an infection by Nosema ceranae and being exposed to a frequently used in-hive acaricide, amitraz. To achieve this, newly emerged bees were individually infected with N. ceranae spores and/or received a sublethal concentration of amitraz in their diets under laboratory conditions. Mortality, food intake, total volume excrement, body appearance, and parasite development were registered. Bees exposed to both stressors jointly had higher mortality rates compared to bees exposed separately, with no difference in the parasite development. An increase in sugar syrup consumption was observed for all treated bees while infected bees fed with amitraz also showed a diminishment in pollen intake. These results coupled with an increase in the total number of excretion events, alterations in behavior and body surface on individuals that received amitraz could evidence the detrimental action of this molecule. To corroborate these findings under semi-field conditions, worker bees were artificially infected, marked, and released into colonies. Then, they were exposed to a commercial amitraz-based product by contact. The recovered bees showed no differences in the parasite development due to amitraz exposure. This study provides evidence to which extent a honey bee infected with N. ceranae could potentially be weakened by chronic exposure to amitraz treatment.

Establishment of a Secretory Protein-Inducible CRISPR/Cas9 System for Nosema bombycis in Insect Cells.

Gene editing techniques are widely and effectively used for the control of pathogens, but it is difficult to directly edit the genes of Microsporidia due to its unique spore wall structure. Innovative technologies and methods are urgently needed to break through this limitation of microsporidia therapies. Here, we establish a microsporidia-inducible gene editing system through core components of microsporidia secreted proteins, which could edit target genes after infection with microsporidia. We identified that Nosema bombycis NB29 is a secretory protein and found to interact with itself. The NB29-N3, which lacked the nuclear localization signal, was localized in the cytoplasm, and could be tracked into the nucleus after interacting with NB29-B. Furthermore, the gene editing system was constructed with the Cas9 protein expressed in fusion with the NB29-N3. The system could edit the exogenous gene EGFP and the endogenous gene BmRpn3 after overexpression of NB29 or infection with N. bombycis.

Identification of long non-coding RNAs in response to microsporidia infection in Silkworm, Bombyx mori.

Microsporidia Nosema bombycis (Nb) is a cellular parasite responsible for pébrine disease in silkworms, significantly impacting the sericulture industry. Long non-coding RNAs (lncRNAs), which are RNA fragments longer than 200 nucleotides, are pivotal in a range of cellular and physiological functions. However, the potential role of silkworm lncRNAs in response to Nb infection remains unknown. This study conducted transcriptome sequencing on both larvae and Nb-infected midguts of silkworms, identifying 1,440 lncRNAs across all examined midgut samples. Within the Nb-infected group, 42 differentially expressed lncRNAs (DElncRNAs) and 305 differentially expressed mRNAs (DEmRNAs) were detected. Functional annotation and pathway analysis showed that these DEmRNAs are mostly involved in metabolism, apoptosis, autophagy, and other key pathways. The co-expression network of DEmRNAs and DElncRNAs illustrates that 1 gene could be regulated by multiple lncRNAs and 1 lncRNA may target multiple genes, indicating that the regulation of lncRNA is intricate and networked. In addition, the DElncRNA-miRNA-mRNA network showed that some DElncRNAs may be involved in the immune response and metabolism through miRNA. Notably, the study observed an increase in lncRNA MSTRG857.1 following Nb infection, which may promote Nb proliferation. These findings offer insights into the complex interplay between insects and microsporidia.

Perilipin1 inhibits Nosema bombycis proliferation by promoting Domeless- and Hop-mediated JAK-STAT pathway activation in Bombyx mori.

Lipid droplets (LDs) are dynamic organelles that participate in the regulation of lipid metabolism and cellular homeostasis inside of cells. LD-associated proteins, also known as perilipins (PLINs), are a family of proteins found on the surface of LDs that regulate lipid metabolism, immunity, and other functions. In silkworms, pébrine disease caused by infection by the microsporidian Nosema bombycis (Nb) is a severe threat to the sericultural industry. Although we found that Nb relies on lipids from silkworms to facilitate its proliferation, the relationship between PLINs and Nb proliferation remains unknown. Here, we found Nb infection caused the accumulation of LDs in the fat bodies of silkworm larvae. The characterized perilipin1 gene (plin1) promotes the accumulation of intracellular LDs and is involved in Nb proliferation. plin1 is similar to perilipin1 in humans and is conserved in all insects. The expression of plin1 was mostly enriched in the fat body rather than in other tissues. Knockdown of plin1 enhanced Nb proliferation, whereas overexpression of plin1 inhibited its proliferation. Furthermore, we confirmed that plin1 increased the expression of the Domeless and Hop in the JAK-STAT immune pathway and inhibited Nb proliferation. Taken together, our current findings demonstrate that plin1 inhibits Nb proliferation by promoting the JAK-STAT pathway through increased expression of Domeless and Hop. This study provides new insights into the complicated connections among microsporidia pathogens, LD surface proteins, and insect immunity.IMPORTANCELipid droplets (LDs) are lipid storage sites in cells and are present in almost all animals. Many studies have found that LDs may play a role in host resistance to pathogens and are closely related to innate immunity. The present study found that a surface protein of insect lipid droplets could not only regulate the morphological changes of lipid droplets but also inhibit the proliferation of a microsporidian pathogen Nosema bombycis (Nb) by activating the JAK-STAT signaling pathway. This is the first discovery of the relationship between microsporidian pathogen and insect lipid surface protein perilipin and insect immunity.

Current status of Nosema spp. infection cases in apis mellifera in eurasian countries and Ptp3 gene haplotypes in the Republic of Tatarstan, Russia.

The current status of Nosema spp. infections in A. mellifera throughout Eurasia was characterized using electronic databases. Although N. ceranae was predominantly detected in southwestern and south-central regions and N. apis in northwestern and north-central areas, most studies reported the occurrence of both species in Eurasia. In addition, the occurrence of Nosema spp. and Ptp3 gene haplotypes was investigated in the Republic of Tatarstan, Russia. Most of the examined honey bees were infected with both N. apis and N. ceranae. N. apis and N. ceranae isolates were either heterozygous or belonged to different strains and showed infection with more than one strain. New haplotypes were found for N. apis and N. ceranae in the Republic of Tatarstan, Russia. This study expands the data regarding existing haplotypes of Nosema species: there are currently 9 shared and 56 unique Ptp3 nucleotide sequence haplotypes of N. ceranae, and 2 shared and 7 unique haplotypes of N. apis, respectively.

Interaction of acetamiprid, Varroa destructor, and Nosema ceranae in honey bees.

Health of honey bees is threatened by a variety of stressors, including pesticides and parasites. Here, we investigated effects of acetamiprid, Varroa destructor, and Nosema ceranae, which act either alone or in combination. Our results suggested that interaction between the three factors was additive, with survival risk increasing as the number of stressors increased. Although exposure to 150 µg/L acetamiprid alone did not negatively impact honey bee survival, it caused severe damage to midgut tissue. Among the three stressors, V. destructor posed the greatest threat to honey bee survival, and N. ceranae exacerbated intestinal damage and increased thickness of the midgut wall. Transcriptomic analysis indicated that different combinations of stressors elicited specific gene expression responses in honey bees, and genes involved in energy metabolism, immunity, and detoxification were altered in response to multiple stressor combinations. Additionally, genes associated with Toll and Imd signalling, tyrosine metabolism, and phototransduction pathway were significantly suppressed in response to different combinations of multiple stressors. This study enhances our understanding of the adaptation mechanisms to multiple stressors and aids in development of suitable protective measures for honey bees. ENVIRONMENTAL IMPLICATION: We believe our study is environmentally relevant for the following reasons: This study investigates combined effects of pesticide, Varroa destructor, and Nosema ceranae. These stressors are known to pose a threat to long-term survival of honey bees (Apis mellifera) and stability of the ecosystems. The research provides valuable insights into the adaptive mechanisms of honey bees in response to multiple stressors and developing effective conservation strategies. Further research can identify traits that promote honey bee survival in the face of future challenges from multiple stressors to maintain the overall stability of environment.

Transcriptomic changes in the microsporidia proliferation and host responses in congenitally infected embryos and larvae.

Congenital infection caused by vertical transmission of microsporidia N. bombycis can result in severe economic losses in the silkworm-rearing industry. Whole-transcriptome analyses have revealed non-coding RNAs and their regulatory networks in N. bombycis infected embryos and larvae. However, transcriptomic changes in the microsporidia proliferation and host responses in congenitally infected embryos and larvae remains unclear. Here, we simultaneously compared the transcriptomes of N. bombycis and its host B. mori embryos of 5-day and larvae of 1-, 5- and 10-day during congenital infection. For the transcriptome of N. bombycis, a comparison of parasite expression patterns between congenital-infected embryos and larva showed most genes related to parasite central carbon metabolism were down-regulated in larvae during infection, whereas the majority of genes involved in parasite proliferation and growth were up-regulated. Interestingly, a large number of distinct or shared differentially expressed genes (DEGs) were revealed by the Venn diagram and heat map, many of them were connected to infection related factors such as Ricin B lectin, spore wall protein, polar tube protein, and polysaccharide deacetylase. For the transcriptome of B. mori infected with N. bombycis, beyond numerous DEGs related to DNA replication and repair, mRNA surveillance pathway, RNA transport, protein biosynthesis, and proteolysis, with the progression of infection, a large number of DEGs related to immune and infection pathways, including phagocytosis, apoptosis, TNF, Toll-like receptor, NF-kappa B, Fc epsilon RI, and some diseases, were successively identified. In contrast, most genes associated with the insulin signaling pathway, 2-oxacarboxylic acid metabolism, amino acid biosynthesis, and lipid metabolisms were up-regulated in larvae compared to those in embryos. Furthermore, dozens of distinct and three shared DEGs that were involved in the epigenetic regulations, such as polycomb, histone-lysine-specific demethylases, and histone-lysine-N-methyltransferases, were identified via the Venn diagram and heat maps. Notably, many DEGs of host and parasite associated with lipid-related metabolisms were verified by RT-qPCR. Taken together, simultaneous transcriptomic analyses of both host and parasite genes lead to a better understanding of changes in the microsporidia proliferation and host responses in embryos and larvae in N. bombycis congenital infection.

Interactive effects of dinotefuran and Nosema ceranae on the survival status and gut microbial community of honey bees.

Growing evidences have shown that the decline in honey bee populations is mainly caused by the combination of multiple stressors. However, the impacts of parasitic Nosema ceranae to host fitness during long-term pesticide exposure-induced stress is largely unknown. In this study, the effects of chronic exposure to a sublethal dose of dinotefuran, in the presence or absence of N. ceranae, was examined in terms of survival, food consumption, detoxification enzyme activities and gut microbial community. The interaction between dinotefuran and Nosema ceranae on the survival of honey bee was synergistic. Co-exposure to dinotefuran and N. ceranae led to less food consumption and greater changes of enzyme activities involved in defenses against oxidative stress. Particularly, N. ceranae and dinotefuran-N. ceranae co-exposure significantly impacted the gut microbiota structure and richness in adult honey bees, while dinotefuran alone did not show significant alternation of core gut microbiota compared to the control group. We herein demonstrated that chronical exposure to dinotefuran decreases honey bee's survival but is not steadily associated with the gut microbiota dysbiosis; by contrast, N. ceranae parasitism plays a dominant role in the combination in influencing the gut microbial community of the host honey bee. Our findings provide a comprehensive understanding of combinatorial effects between biotic and abiotic stressors on one of the most important pollinators, honey bees.

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.

Production, characterization and therapeutic efficacy of egg yolk antibodies specific to Nosema ceranae.

Nosema disease, caused by Nosema ceranae, one of the single-celled fungal microsporidian parasites, is one of the most important and common diseases of adult honey bees. Since fumagillin, which has been used for decades in the control of Nosema disease in honey bees (Apis mellifera), poses a toxic threat and its efficacy against N. ceranae is uncertain, there is an urgent need to develop alternative prophylactic and curative strategies for the treatment of this disease. The main aim of this study was to investigate the therapeutic potential of specific egg yolk immunoglobulins (IgY) on Nosema disease. For this purpose, the presence of N. ceranae was determined by microscopic and PCR methods in honey bees collected from Nosema suspicious colonies by conducting a field survey. Layered Ataks chickens, divided into four groups each containing 20 animals, were vaccinated with live and inactivated vaccines prepared from field isolates of N. ceranae. Eggs were collected weekly for 10 weeks following the last vaccination. IgY extraction was performed using the PEG precipitation method from egg yolks collected from each group, and the purity of the antibodies was determined by SDS-PAGE and Western Blot. The presence of N. ceranae-specific IgYs was investigated by Western Blot and indirect ELISA methods. It was determined that specific IgYs showed high therapeutic efficacy on Nosema disease in naturally infected bee colonies. In addition, honey bees collected from infected colonies were brought to the laboratory and placed in cages with 30 bees each, and the effectiveness of IgYs was investigated under controlled conditions. It was detected that specific IgY reduced the Nosema spore load and the number of infected bees significantly in both the field and experimental study groups treated for seven days. It was concluded that chicken IgYs, an innovative and eco-friendly method, had a significant potential for use as an alternative to antifungal drugs.