Nosema Spores Research Articles

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%).

Inactivation of Nosema spp. with zinc phthalocyanine

Most honey bee pathogens, such as Vairimorpha (Nosema), cannot be rapidly and definitively diagnosed in a natural setting, consequently there is typically the spread of these diseases through shared and re-use of beekeeping equipment. Furthermore, there are no viable treatment options available for Nosema spores to aid in managing the spread of this bee disease. We therefore aimed to develop a new method using novel Zinc Phthalocyanine (ZnPc) as a photosensitizer for the photodynamic inactivation of Nosema spores that could be used for the decontamination of beekeeping equipment. Nosema spores were propagated for in vitro testing using four caged Apis mellifera honey bees. The ZnPc treatment was characterized, encapsulated with a liposome, and then used as either a 10 or 100 µM treatment for the freshly harvested Nosema spores, for either a 30 and or 60-minute time period, under either light or dark conditions, in-vitro, in 96-well plates. In the dark treatment, after 30-min, the ZnPc 100 µM treatment, caused a 30 % Nosema mortality, while this increased to 80 % at the same concentration after the light treatment. The high rate of anti-spore effects, in a short period of time, supports the notion that this could be an effective treatment for managing honey bee Nosema infections in the future. Our results also suggest that the photo activation of the treatment could be applied in the field setting and this would increase the sterilization of beekeeping equipment against Nosema.

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.

Prevalence and distribution of Varroa destructor and Nosema spp. in symptomatic honey bee colonies across the USA from 2015 to 2022

USDA-ARS Bee Research Laboratory received symptomatic honey bee (Apis mellifera L.) samples across the United States for disease diagnosis. Here, we present a retrospective study and cartography of ectoparasite Varroa destructor and intracellular microsporidia parasite Nosema spp. These two major parasites were identified in the diseased honey bee samples between 2015 and 2022. Varroa infestation level (VIL) was examined by a wash technique (Mites/100 bees) and calculated as a percentage, while Nosema infection was quantified by microscopical spore count (Million Spores/Bee). Data were analyzed by month, year, state, and by nine geographical climate regions described in the U.S. Of adult bee samples (n = 4039) that were analyzed for Varroa mite infestation, the overall VIL in the U.S. ranged between 0.4 and 30.85%, with an overall national VIL and Varroa prevalence of 8.21% and 85.14%, respectively. Overall monthly data showed VIL constantly exceeded the critical level of 4% except from June to September and reached a maximum of 15% in January and December. Nationwide, VIL significantly (p < 0.001) increased from 2015 to 2018 (1.1–4.7%), plateaued from 2018 to 2021 (4.7–4.5%), followed by a significant decrease in 2022 (3.6%). Significant VIL differences (p < 0.001) were recorded among climate regions, with the highest mite infestation levels in the Upper Midwest region (13.9%) and the lowest in the West region (5.1%). Of adult bee samples (n = 2,994) that were analyzed for Nosema infection, Nosema spore count ranged between (1–16.8) million spores per bee among states, with a national average of 6.8 and a prevalence of 99.7%. The lowest and highest Nosema loads were respectively recorded in the South region (3.1) and Upper Midwest (10.5), a significant difference (p < 0.001). No statistical differences were recorded among the six other climate regions. Overall, VIL and Nosema infection correlated significantly (p < 0.001) with a regression coefficient of (R2 = 0.6). Our data, which originated from ailing bee colonies, showed significantly higher rates of maladies compared to data from healthy colonies obtained by the USDA-APHIS National Honey Bee Survey, demonstrating the role of bee diseases caused by Varroa mite and Nosema in honey bee population declines.

Sulfoxaflor effects depend on the interaction with other pesticides and Nosema ceranae infection in the honey bee (Apis mellifera)

Honey bees health is compromised by many factors such as the use of agrochemicals in agriculture and the various diseases that can affect them. Multiple studies have shown that these factors can interact, producing a synergistic effect that can compromise the viability of honey bees. This study analyses the interactions between different pesticides and the microsporidium Nosema ceranae and their effect on immune and detoxification gene expression, sugar consumption and mortality in the Iberian western honey bee (Apis mellifera iberiensis). For this purpose, workers were infected with N. ceranae and subjected to a sugar-water diet with field concentrations of the pesticides sulfoxaflor, azoxystrobin and glyphosate. Increased sugar intake and altered immune and cytochrome P450 gene expression were observed in workers exposed to sulfoxaflor and infected with N. ceranae. None of the pesticides affected Nosema spore production in honey bee gut. Of the three pesticides tested (alone or in combination) only sulfoxaflor increased mortality in honey bees. Taken together, our results suggest that the effects of sulfoxaflor were attenuated in contact with other pesticides, and that Nosema infection leads to increase sugar intake in sulfoxaflor-exposed bees. Overall, this underlines the importance of studying the interaction between different stressors to understand their overall impact not only on honey bee but also on wild bees health.

Screening of Entomopathogenic Fungal Culture Extracts with Honeybee Nosemosis Inhibitory Activity.

This study aimed to select the most effective culture extracts for controlling honeybee nosemosis using 342 entomopathogenic fungi of 24 species from 18 genera. The germination inhibitory activity of the fungal culture extract on Nosema ceranae spores was evaluated using an in vitro germination assay method. Among 89 fungal culture extracts showing germination inhibitory activity of approximately 80% or more, 44 fungal culture extracts that maintained their inhibitory activity even at a concentration of 1% were selected. Finally, the honeybee nosemosis inhibitory activity was evaluated using the cultured extracts of five fungal isolates having a Nosema inhibitory activity of approximately 60% or more, even when the extract was removed after treatment. As a result, the proliferation of Nosema spores was reduced by all fungal culture extract treatments. However, only the treatment of the culture extracts from Paecilomyces marquandii 364 and Pochonia bulbillosa 60 showed a reduction in honeybee mortality due to nosemosis. In particular, the extracts of these two fungal isolates also increased the survival of honeybees.

Potential of Fumagillin Against &lt;i&gt;Nosema&lt;/i&gt; SP., a Microsporidian Endoparasite of Honey Bee &lt;i&gt;Apis mellifera&lt;/i&gt; L

Nosema apis and Nosema ceranae are two kinds of microsporidian parasites that cause the recently identified illness known as nosemosis in European honey bees (Apis mellifera). 10 samples of adult honey bees from each infected colony of a single apiary were collected, located in the village of Handia, district of Prayagraj, India, from September 2022 to January 2023 to investigate the frequency of intestinal infections and the effectiveness of fumagillin antibiotic against nosemosis in A. mellifera with the use of monocular LED optical microscopy and a haemocytometer, the mean and median spore count of each composite sample was calculated. It was observed that 5% of the infected gut samples contained Nosema spores. The results show that 10% of colonies tested positive for Nosema, and fumagillin is effective against nosemosis.

The effect of different feeding strategies on honey bee gut microbiota and the presence of Nosema

Colony development in honey bees depends on both the environmental conditions and their genetic structure. Nutrition is the one of the most important factors in the honey bee’s health. This study was carried out in 48 colonies and included six groups (a control and five feeding groups). In the experiment, the effect of different feeding strategies on intestinal flora and the presence of Nosema was investigated. Pantoea agglomerans, Pseudomonas luteola, Burkholderia cepacia, Brevibacillus nitrificans, Sphingomonas paucimobilis, Aeromonas hydrophila, and P. alcalidimonas were detected in the intestinal microflora of the bee samples by morphological and phenotypic identification. The results of phenotypic identification were confirmed using 16S rRNA sequence analysis for P. agglomerans and B. nitrificans strains. The presence of Nosema was simultaneously investigated in all groups. Only Nosema cerenae was detected using DNA analysis in the positive Nosema spore samples.

Effect of bee bread on Africanized honey bees infected with spores of Nosema spp.

Floral diversity allows honey bees, Apis mellifera L. (including its hybrids between subspecies) (Hymenoptera: Apidae), to meet their nutritional requirements. However, infection by Nosema spp. (Microsporidia: Nosematidae) destroys a bee's digestive epithelium, hindering the ability to absorb nutrients. In some subtropical regions, such as the Yucatan Peninsula, Mexico, nosemosis is one of the most widespread diseases in commercial apiaries. To date, there have been no reports of colony deaths attributable to the disease. It is possible that the naturally occurring diet in the Yucatan Peninsula sustains honey bee health. We tested this hypothesis by evaluating the effect of four diets – bee bread collected in the Yucatan's rainy and dry seasons, sucrose syrup, and a pollen substitute – on the response of caged Africanized honey bees (A. m. scutellata × A. m. adansonii × A. mellifera) to nosemosis infection (treatment with spores of Nosema apis Zander and Nosema ceranae Fries). We found that infected and uninfected bees consumed food in similar amounts regardless of the diet, although, overall, they fed at significantly lower rates when provided the pollen-substitute diet. Bees fed either of the bee bread diets had relatively low fat-body weight and expression of Vg, proPO, and GOx genes, three genes related to immune function. Bees fed sucrose syrup had low fat-body weight but relatively high gene expression. Infection from Nosema spores only reduced the individual survival probability for bees fed the bee bread collected in the dry season, yet bees fed the dry-season bee bread had the lowest infection rates. Our results indicate that natural stored bee bread in honey bee colonies from the Yucatan Peninsula could be one of the main factors influencing their survival and reducing Nosema spp. spore load.

In Vivo Inhibitory Assessment of Potential Antifungal Agents on Nosema ceranae Proliferation in Honey Bees.

Nosema&amp;nbsp;ceranae Fries, 1996, causes contagious fungal nosemosis disease in managed honey bees, Apis&amp;nbsp;mellifera L. It is associated around the world with winter losses and colony collapse disorder. We used a laboratory in vivo screening assay to test curcumin, fenbendazole, nitrofurazone and ornidazole against N. ceranae in honey bees to identify novel compounds with anti-nosemosis activity compared to the commercially available medication Fumagilin-B®. Over a 20-day period, Nosema-inoculated bees in Plexiglas cages were orally treated with subsequent dilutions of candidate compounds, or Fumagilin-B® at the recommended dose, with three replicates per treatment. Outcomes indicated that fenbendazole suppressed Nosema spore proliferation, resulting in lower spore abundance in live bees (0.36 ± 1.18 million spores per bee) and dead bees (0.03 ± 0.25 million spores per bee), in comparison to Fumagilin-B®-treated live bees (3.21 ± 2.19 million spores per bee) and dead bees (3.5 ± 0.6 million spores per bee). Our findings suggest that Fumagilin-B® at the recommended dose suppressed Nosema. However, it was also likely responsible for killing Nosema-infected bees (24% mortality). Bees treated with fenbendazole experienced a greater survival probability (71%), followed by ornidazole (69%), compared to Nosema-infected non-treated control bees (20%). This research revealed that among screened compounds, fenbendazole, along with ornidazole, has potential effective antifungal activities against N. ceranae in a controlled laboratory environment.

Detection of Varroa destructor Mite and Nosema spp. in Bee Samples From Bulgaria

This study is focused on the investigation of honey bee samples for the presence of the two most common and widely distributed honey bee parasites. In a two-year period during 2020-2021, 185 bee samples were tested. All samples were examined by morphological and light microscopic methods. The obtained results showed that 32.43% of bee samples were infested with Varroa destructor. The degree of infection in the bees ranged from 0.5% to 60%. Spores of Nosema spp. were established in 25.40% of samples with a degree of invasion in the range from 3x105 to 26x106 per bee. Mixed infections of both parasites were observed in 32.43% of the samples. Negative samples were with the lowest value of 9.74%.

Use of Thymol in Nosema ceranae Control and Health Improvement of Infected Honey Bees.

Simple SummaryIn the European Union, there is no registered product for the control of the honey bee endoparasite Nosema ceranae. Thus, researchers are looking for options for Nosema treatment. The aim of this study was to investigate the effect of a natural essential-oil ingredient (thymol) derived from Thymus vulgaris on honey bees infected with N. ceranae. Thymol exerted certain positive effects (increasing bee survival, immunity, and antioxidative protection), as well as positively affecting the spore loads in Nosema-infected bees. However, when applied to Nosema-free bees, thymol caused certain health disorders; therefore, beekeepers should be careful with its use.Nosema ceranae is the most widespread microsporidian species which infects the honey bees of Apis mellifera by causing the weakening of their colonies and a decline in their productive and reproductive capacities. The only registered product for its control is the antibiotic fumagillin; however, in the European Union, there is no formulation registered for use in beekeeping. Thymol (3-hydroxy-p-cymene) is a natural essential-oil ingredient derived from Thymus vulgaris, which has been used in Varroa control for decades. The aim of this study was to investigate the effect of thymol supplementation on the expression of immune-related genes and the parameters of oxidative stress and bee survival, as well as spore loads in bees infected with the microsporidian parasite N. ceranae. The results reveal mostly positive effects of thymol on health (increasing levels of immune-related genes and values of oxidative stress parameters, and decreasing Nosema spore loads) when applied to Nosema-infected bees. Moreover, supplementation with thymol did not induce negative effects in Nosema-infected bees. However, our results indicate that in Nosema-free bees, thymol itself could cause certain disorders (affecting bee survival, decreasing oxidative capacity, and downregulation of some immune-related gene expressions), showing that one should be careful with preventive, uncontrolled, and excessive use of thymol. Thus, further research is needed to reveal the effect of this phytogenic supplement on the immunity of uninfected bees.

Mathematical modelling of between hive transmission of Nosemosis by drifting

Nosema ceranae is a microsporidian parasite of the Western honeybee (Apis mellifera). Building on a previous model of within-hive transmission we formulate a mathematical model for the spread of this disease between hives in an apiary. The underlying honeybee population model considers hive and forager bees separately; to account for seasonal variations in honeybee and disease biology, the model parameters vary in time with a period of one year. In the hive, two transmission routes are considered, one is indirect (infected hive bees deposit Nosema spores that are ingested by other bees), the other one is direct and follows the usual mass action kinetics of SIR models. Inter colony transmission is assumed to be caused by drifting. The resulting ecoepidemiological metapopulation model is studied numerically. Our simulations suggest that in the case of indirect within-hive transmission the longterm behaviour is not very sensitive to drifting intensity, and that across the apiary the individual hives synchronise and stratify quickly. In the case of direct within-hive transmission very irregular and complex behaviour can be observed, for example some colonies might survive in working order whereas neighbouring hives might fail. This is due to the underlying single hive model. If both within-hive transmission routes are combined the solutions of the metapopulation model, depending on parameters, can show facets of both transmission routes.

A new duplex qPCR assay for the quantification of honey bee (Apis mellifera) parasites Nosema ceranae and Nosema apis tested with low dose experimental exposure

Nosema ceranae and Nosema apis are microsporidian parasites that cause disease in the European honey bee. Nosema infection is identified as a potential cause of colony loss by beekeepers. Given the importance of Nosema infection in colony mortality and productivity, developing new and improved ways of detecting and quantifying infection loads is vital. We designed and tested a new duplex qPCR assay for the accurate quantification of both N. ceranae and N. apis utilising TaqMan chemistry and the new gBlock® method for the standards. The assay showed good linearity with natural Nosema infection, and a strong correlation with microscopic spore counts. This new assay has high sensitivity and repeatability and was used to investigate Nosema infection in hive surveys and following low dose experimental exposure. In local hives, we found relativity low levels of N. ceranae and very little N. apis across three sites in Blacksburg, VA. A survey of two bee yards in West Virginia showed much higher levels of N. ceranae, but consistent low levels of N. apis. For the experiment, caged bees from two different hives were fed 100 Nosema spores (or a control solution). Exposed bees were collected after two or five days, and infection was quantified using the new assay. Given the low dose, infection levels were not 100%, with some exposed bees remaining N. ceranae free, while others only developed low level infection. This low dose exposure and subsequent infection status (low level infection or infection free) could provide a new understanding of Nosema infection establishment.

Identification of the Nosema spp., a microsporidian parasite isolated from the honey bees (Apis mellifera) and its association with honey bee colony losses in apiaries of Iran

The aim of this study was to determine the Nosema species by microscopic and molecular method and its association with honeybee colony losses (Colony Collapse Disorder) in apiaries of Urmia, Northwest of Iran. For this purpose, honeybee samples were collected from 840 colonies kept in 120 apiaries in five different location of Urmia. The specimens were examined for the presence of Nosema spores. After DNA isolation, the 16S rRNA gene was evaluated using multiplex PCR. Total infection prevalence with the microscopic evaluation was 32% while in PCR test was 58.2%. Nosema positive samples were evaluated by PCR sequencing. Based on the results of PCR, all identified cases were N. ceranae. The obtained sequences were transferred to GenBank/NCBI (samples accession numbers MT001887 and MT001893). The results showed the prevalence of Colony Collapse Disorder like symptoms in the studied honeybee colonies were 13.33%. N. ceranae was detected by PCR in 20.28 % of honeybee colonies with Colony Collapse Disorder like signs. Our findings showed that there was a significant relation between Colony Collapse Disorder and presence of N. ceranae. The results of this study concluded that N. ceranae is the only specie that affects the honeybees which may have an important role in the occurrence of collapse of bee families and depopulation of hives in this area.

Reproductive Potential Impacts Body Maintenance Parameters and Global DNA Methylation in Honeybee Workers (Apis mellifera L.)

Simple SummaryThe queens and sterile workers arise from genetically identical eggs but as imagoes, they differ in their life span, DNA methylation, and their functions. In the absence of the queen, the larvae develop into rebels, i.e., workers with increased reproductive potential. We assumed that since rebels are similar to the queen in many anatomical and behavioral features, they live longer and have lower levels of global DNA methylation, even when infected, e.g., by Nosema spp. Rebels always lived longer in comparison in normal workers and unexpectedly extended longevity of normal workers when they were together, similarly as the presence of a queen did. Rebels became infected more easily but tolerated the infection better. They also had lower level of global DNA methylation than normal workers. These features expand possibilities of the use of honeybees as a model for studies on senescence, nosemosis, eusocial evolution, and epigenetics.The widely accepted hypothesis in life history evolution about the trade-off between fecundity and longevity is not confirmed by long-living and highly fecund queens in eusocial insects. The fact that the queens and facultatively sterile workers usually arise from genetically identical eggs but differ in DNA methylation makes them a good model for studies on senescence, eusocial evolution, and epigenetics. Therefore, honeybees seem to be especially useful here because of long living rebel-workers (RW) with high reproductive potential recently described. Longevity, ovariole number, nosema tolerance, and global DNA methylation have been assayed in normal workers (NW) versus RW in hives and cages. RW always lived longer than NW and unexpectedly extended longevity of NW when they were together, similarly as the presence of a queen did. RW lived longer despite the fact that they had higher Nosema spore load; surprisingly they became infected more easily but tolerated the infection better. Global DNA methylation increased with age, being lower in RW than in NW. Therefore, RW are queen-like considering global DNA methylation and the link between fecundity, longevity, and body maintenance. Presented features of RW expands possibilities of the use of honeybees as a model for studies on senescence, nosemosis, eusocial evolution, and epigenetics.

Natural extracts as potential control agents for Nosema ceranae infection in honeybees, Apis mellifera

Nosema disease is one factor that can cause colony decline in honeybees (Apis mellifera L.) worldwide. Nosema ceranae has outcompeted Nosema apis in the Western honeybee (A. mellifera) which is its original host. Fumagilin is an effective antibiotic treatment to control Nosema infection but currently it is forbidden in many countries. In this study, 12 plant extracts were evaluated for their toxicity to adult bees and antimicrosporidian activity under laboratory and field conditions. N. ceranae-infected adult bees were fed ad libitum with 50% sucrose solution containing 1% and 5% (w/v) of each plant extract. Bee mortality in N. ceranae-infected groups fed with plant extracts was higher than that in the control group treated with fumagilin. The results demonstrated that 9 of 12 extracts had high antimicrosporidian activity against N. ceranae and their efficacies were comparable to fumagilin. Spore reduction in infected bees was 4–6 fold less after extract treatment. Following laboratory screening, Annona squamosa, Ocimum basilicum, Psidium guajava and Syzygium jambos were tested in honeybee colonies. Plant extracts of 2% concentration (w/v) inhibited the development of Nosema spores after 30 days of treatment. At the end of experiment (90 days), spores in the plant extract treated groups were lower than in group treated with fumagilin but there was no significant difference. Although, extracts tested in this study showed high toxicity to bee in laboratory cages, they did not show negative affects on bees under whole colony conditions. Therefore, the effectiveness of plant extracts tested in this study was notable and warrants further study as potential Nosema control agents in honey bees. Plant extracts would offer a non-antibiotic alternative for Nosema control and help reduce the overuse of antibiotics in livestock.

The Effects of Nosema apis and Nosema ceranae Infection on Survival and Phenoloxidase Gene Expression in Galleria mellonella (Lepidoptera: Galleriidae) Compared to Apis mellifera.

Simple SummaryThe greater wax moth is known as one of the pests in the beekeeping sector. Nosema ceranae and Nosema apis, which are the most significant microsporidia for adult honeybees, are present in the greater wax moth grown at laboratory conditions and collected from apiaries. In this study, single N. apis and N. ceranae infection and mixed infection groups have been created for each insect to detect the colonization capability of Nosema species and the effect of microsporidial infection on survival and phenoloxidase gene expression. For this purpose, greater wax moth and honeybee experimental groups were fed with 106 single- and mixed-Nosema-spore-containing diet, and survival rates of wax moths and bees were recorded. In addition, changes in the level of expression of the phenoloxidase-related gene, which plays a significant role in invertebrates’ immune reaction, was also observed. Honeybees have some mortality rates in mixed and single Nosema infection cases, the absence of death in greater wax moth shows that the greater wax moth exhibits a resistance towards Nosema species. While the expression of phenoloxidase gene increased in honeybees, the level of phenoloxidase mRNA did not change in the greater wax moth. This shows that the infection remains stable in the wax moth, unlike honeybees.The study aims to prove the possibility of colonization of N. apis and N. ceranae to the intestine of the greater wax moth, detect the differences of greater wax moth based on the presence of Nosema species and examine the effect of Nosema species on the phenoloxidase level of greater wax moth compared with honeybees. Each group was fed on the 1st day of the experiment with its appropriate diet containing 106 Nosema spores per insect. Each group was checked daily, and dead insects were counted. Furthermore, changes in the level of expression of the phenoloxidase-related gene after Nosema spp. treatment on the 6th, 9th and 12th days, which was detected by Q-PCR, and the mRNA level of phenoloxidase gene were measured in all experiment groups with the CFX Connect Real-Time PCR Detection System. This study shows that Apis mellifera L. has a 66.7% mortality rate in mixed Nosema infections, a 50% mortality rate in N. ceranae infection, a 40% mortality rate in N. apis infection, while there is no death in G. mellonella. A significant difference was found in the mixed Nosema infection group compared to the single Nosema infection groups by means of A. mellifera and G. mellonella (Duncan, p < 0.05). G. mellonella histopathology also shows that Nosema spores multiply in the epithelial cells of greater wax moth without causing any death. The increase in the mRNA level of Phenoloxidase gene in A. mellifera was detected (Kruskal–Wallis, p < 0.05), while the mRNA level of the Phenoloxidase gene did not change in G. mellonella (Kruskal–Wallis, p > 0.05). These findings prove that the Nosema species can colonize into the greater wax moth, which contributes to the dissemination of these Nosema species between beehives.

Potential of Fumagillin and Agaricus blazei Mushroom Extract to Reduce Nosema ceranae in Honey Bees.

Simple SummaryNosemosis is a disease in bees that causes severe problems for their vitality, reproduction and productivity. Traditionally, the treatment involves the application of fumagillin, an antibiotic with proven effect. Recently, fumagillin production and registration has faced problems worldwide, leading to the absence of adequate treatment. Motivated by the reported health issues and the occurrence of residues after fumagillin application, scientists around the world have sought a medication or a supplement that could help beekeepers to control Nosema. Current trends include the search for alternative non-antibiotic treatments. In a laboratory (cage) experiment, we studied the effect of fumagillin and natural extract of mushroom Agaricus blazei on the survival of Nosema infected bees, Nosema spore loads and levels of immune-related gene expression and oxidative stress markers. The results undoubtedly confirmed the anti-Nosema effect of fumagillin, as seen in better bee survival and monitored parameters; its application without Nosema infection (preventive) caused disturbance in some of the parameters. The application of A. blazei extract, however, showed positive effects in both preventive and curative applications. These beneficial properties of A. blazei extract indicate a potential that needs to be further investigated.Depending on the infection level and colony strength, Nosema ceranae, a microsporidian endoparasite of the honey bee may have significant consequences on the health, reproduction and productivity of bee colonies. Despite exerting some side effects, fumagillin is most often used for Nosema control. In this study, in a cage experiment, N. ceranae infected bees were treated with fumagillin or the extract of Agaricus blazei mushroom, a possible alternative for Nosema control. Bee survival, Nosema spore loads, the expression levels of immune-related genes and parameters of oxidative stress were observed. Fumagillin treatment showed a negative effect on monitored parameters when applied preventively to non-infected bees, while a noticeable anti-Nosema effect and protection from Nosema-induced immunosuppression and oxidative stress were proven in Nosema-infected bees. However, a protective effect of the natural A. blazei extract was detected, without any side effects but with immunostimulatory activity in the preventive application. The results of this research suggest the potential of A. blazei extract for Nosema control, which needs to be further investigated.

An international inter-laboratory study on Nosema spp. spore detection and quantification through microscopic examination of crushed honey bee abdomens

Nosemosis is a microsporidian disease causing mortality and weakening of honey bee colonies, especially in the event of co-exposure to other sources of stress. As a result, the disease is regulated in some countries. Reliable and harmonised diagnosis is crucial to ensure the quality of surveillance and research results. For this reason, the first European Interlaboratory Comparison (ILC) was organised in 2017 in order to assess both the methods and the results obtained by National Reference Laboratories (NRLs) in counting Nosema spp. spores by microscopy. Implementing their own routine conditions of analysis, the 23 participants were asked to perform an assay on a panel of ten positive and negative samples of crushed honey bee abdomens. They were asked to report results from a qualitative and quantitative standpoint. The assessment covered specificity, sensitivity, trueness and precision. Quantitative results were analysed in compliance with international standards NF ISO 13528 (2015) and NF ISO 5725-2 (1994). Three results showed a lack of precision and five a lack of trueness. However, overall results indicated a global specificity of 98% and a global sensitivity of 100%, thus demonstrating the advanced performance of the microscopic methods applied to Nosema spores by the NRLs. Therefore, the study concluded that using microscopy to detect and quantify spores of Nosema spp. was reliable and valid.