Molecular Detection of Nosema spp. in Three Eco Regions of Slovakia.

Microsporidia are unicellular obligate intracellular parasitic fungi that infect a wide range of vertebrates and invertebrates. There are two known species of microsporidia infecting honey bees in Slovakia- first Nosema apis and also Nosema ceranae. Our aim was to examine samples of honey bees collected from bee queen breeders in three ecoregions of the Slovak Republic in 2021 and 2022. First, microscopic diagnostics were used, and then randomly selected samples were examined using molecular methods. There were 4018 samples examined using microscopic diagnostics and the positivity was demonstrated in 922 samples. From the microscopically diagnosed positive samples, 507 samples were randomly selected, and using molecular methods, the positivity was proved in 488 samples. After sequencing the positive PCR products and comparing the sequences (BLAST) with the sequences stored in the gene bank, the Nosema ceranae species was detected in all positive samples.

Detection of Microsporidia in Pollinator Communities of a Mediterranean Biodiversity Hotspot for Wild Bees.

Insect pollination is crucial for the maintenance of natural and managed ecosystems but the functioning of this ecosystem service is threatened by a worldwide decline of pollinators. Key factors in this situation include the spread and interspecific transmission of pathogens worldwide through the movement of managed pollinators. Research on this field has been mainly conducted in some particular species, while studies assessing the interspecific transmission of pathogens at a community level are scarce. However, this information is pivotal to design strategies to protect pollinators. Herein, we analysed the prevalence of two common microsporidia pathogens of managed honey bees (Nosema ceranae and N. apis) in bee communities of semiarid Mediterranean areas from the Southeast of the Iberian Peninsula. Our results confirm the ability of N. ceranae to disperse across wild bee communities in semiarid Mediterranean ecosystems since it was detected in 36 Apoidea species (39% of the sampling; for the first time in nine genera). The prevalence of the pathogen did not show any phylogenetic signal which suggests a superfamily host range of the pathogen or that wild bees may be acting only as vectors of N. ceranae. In addition, N. apis was detected in an Eucera species, which is the second time it has been detected by molecular techniques in a host other than the honey bee. Our study represents the primary assessment of the prevalence of microsporidia at community level in Mediterranean areas and provides outstanding results on the ability of Nosema pathogens to spread across the landscape.

Nosema apis and Nosema ceranae Tissue Tropism in Worker Honey Bees (Apis mellifera).

The microsporidia Nosema apis and Nosema ceranae are major honey bee pathogens that possess different characteristics in terms of the signs they produce, as well as disease development and transmission. Although the ventricular epithelium is generally considered the target tissue, indirect observations led to speculation that N. ceranae may also target other structures, possibly explaining at least some of the differences between these 2 species. To investigate the tropism of Nosema for honey bee tissues, we performed controlled laboratory infections by orally administering doses of 50 000 or 100 000 fresh mature spores of either species. The fat body was isolated from the infected bees, as well as organs from the digestive (esophagus, ventriculus, ileum, rectum), excretory (Malpighian tubules), circulatory (aorta, heart), respiratory (thoracic tracheas), exocrine (hypopharyngeal, mandibular and labial, cephalic, thoracic salivary glands), and sensory/nervous (brain, eyes and associated nerve structures, thoracic nerve ganglia) systems. Tissues were examined by light and electron microscopy at 7, 10, and 15 days postinfection. Both Nosema species were found to infect epithelial cells and clusters of regenerative cells in the ventriculus, and while the ileum and rectum contained spores of the microsporidia in the lumen, these structures did not show overt lesions. No stages of the parasites or cellular lesions were detected in the other organs tested, confirming the high tropism of both species for the ventricular epithelium cells. Thus, these direct histopathological observations indicate that neither of these 2 Nosema species exhibit tropism for honey bee organs other than the ventriculus.

Development of a loop-mediated isothermal amplification (LAMP) and a direct LAMP for the specific detection of Nosema ceranae, a parasite of honey bees.

Nosema ceranae is a ubiquitous microsporidian pathogen infecting the midgut of honey bees. The infection causes bee nosemosis, a disease associated with malnutrition, dysentery, and lethargic behavior, and results in considerable economic losses in apiculture. The use of a rapid, sensitive, and inexpensive DNA-based molecular detection method assists in the surveillance and eventual control of this pathogen. To this end, a loop-mediated isothermal amplification (LAMP) assay targeting the single-copy gene encoding the polar tube protein 3 (PTP3) has been developed. Genomic DNA of N. ceranae-infected forager bees sampled from distant geographic regions could be reliably amplified using the established LAMP assay. The N. ceranae-LAMP showed higher sensitivity than a classical reference PCR (98.6 vs 95.7%), when both approaches were applied to the detection of N. ceranae. LAMP detected a ten-fold lower infection rate than the reference PCR (1 pg vs 10 pg genomic DNA, respectively). In addition, we show highly specific and sensitive detection of N. ceranae from spore preparations in a direct LAMP format. No cross-reactions with genomic DNA and/or spores from N. apis, often co-infecting A. mellifera, or from N. bombi, infecting bumble bees, were observed. This low-cost and time-saving molecular detection method can be easily applied in simple laboratory settings, facilitating a rapid detection of N. ceranae in honey bees in epidemiological studies, surveillance and control, as well as evaluation of therapeutic measures against nosemosis.

Synergistic effects of pathogen and pesticide exposure on honey bee (Apis mellifera) survival and immunity.

Declines in native insect pollinator populations and substantial losses in managed honey bees have been reported on a global scale and become a widespread concern because of the importance of these insects for human food production and ecosystem stability. Several potential factors have been studied as possible causes of declining pollinator health, such as parasites and pathogens, exposure to agricultural pesticides, habitat loss and/or climate change. More recently, a combination of these factors rather than a single cause have been blamed for observed pollinator losses, but field studies of such interactions are challenging, especially in the presence of confounding environmental stressors. We therefore examined the impact of single and combined stressors on the honey bee (Apis mellifera) in a generally healthy Australian population. We exposed workers during their larval development and drones until they reached sexual maturity to the neonicotinoid pesticide Thiamethoxam, at concentrations more than 20 times lower than we initially measured in the field, the microsporidian gut pathogen Nosema apis or both stressors at the same time. We found that simultaneous exposure significantly reduced bee health. We observed a substantial increase in mortality and a reduction of immunocompetence in workers exposed to both the pathogen and the pesticide. We conclude that the exposure of generally healthy bees to multiple environmental stressors results in synergistic effects where the effects are expected to negatively impact performance and could be sufficient to trigger colony collapse. We found that the vast majority of males did not survive to sexual maturity after exposure to very low levels of Thiamethoxam. This would not only reduce the reproductive success of individual colonies, but can also impact gene flow and genetic diversity at the population level, which are both known as key components of honey bee health.

Molecular, physiological and behavioral responses of honey bee (Apis mellifera) drones to infection with microsporidian parasites.

Susceptibility to pathogens and parasites often varies between sexes due to differences in life history traits and selective pressures. Nosema apis and Nosema ceranae are damaging intestinal pathogens of European honey bees (Apis mellifera). Nosema pathology has primarily been characterized in female workers where infection is energetically costly and accelerates worker behavioral maturation. Few studies, however, have examined infection costs in male honey bees (drones) to determine if Nosema similarly affects male energetic status and sexual maturation. We infected newly emerged adult drones with Nosema spores and conducted a series of molecular, physiological, and behavioral assays to characterize Nosema etiology in drones. We found that infected drones starved faster than controls and exhibited altered patterns of flight activity in the field, consistent with energetic distress or altered rates of sexual maturation. Moreover, expression of candidate genes with metabolic and/or hormonal functions, including members of the insulin signaling pathway, differed by infection status. Of note, while drone molecular responses generally tracked predictions based on worker studies, several aspects of infected drone flight behavior contrasted with previous observations of infected workers. While Nosema infection clearly imposed energetic costs in males, infection had no impact on drone sperm numbers and had only limited effects on antennal responsiveness to a major queen sex pheromone component (9-ODA). We compare Nosema pathology in drones with previous studies describing symptoms in workers and discuss ramifications for drone and colony fitness.

Nosema ceranae in South American Native Stingless Bees and Social Wasp.

Besides the incipient research effort, the role of parasites as drivers of the reduction affecting pollinator populations is mostly unknown. Given the worldwide extension of the beekeeping practice and the diversity of pathogens affecting Apis mellifera populations, honey bee colonies are a certain source of parasite dispersion to other species. Here, we communicate the detection of the microsporidium Nosema ceranae, a relatively new parasite of honey bees, in stingless bees (Meliponini) and the social wasp Polybia scutellaris (Vespidae) samples from Argentina and Brazil by means of duplex PCR. Beyond the geographic location of the nests, N. ceranae was detected in seven from the eight Meliponini species analyzed, while Nosema apis, another common parasite of A. mellifera, was absent in all samples tested. Further research is necessary to determine if the presence of the parasite is also associated with established infection in host tissues. The obtained information enriches the current knowledge about pathologies that can infect or, at least, be vectored by native wild pollinators from South America.

Changes in the Bacteriome of Honey Bees Associated with the Parasite Varroa destructor, and Pathogens Nosema and Lotmaria passim.

The honey bee, Apis mellifera, is a globally important species that suffers from a variety of pathogens and parasites. These parasites and pathogens may have sublethal effects on their bee hosts via an array of mechanisms, including through a change in symbiotic bacterial taxa. Our aim was to assess the influence of four globally widespread parasites and pathogens on the honey bee bacteriome. We examined the effects of the ectoparasitic mite Varroa destructor, the fungal pathogens Nosema apis and Nosema ceranae, and the trypanosome Lotmaria passim. Varroa was detected by acaricidal treatment, Nosema and L. passim by PCR, and the bacteriome using MiSeq 16S rRNA gene sequencing. Overall, the 1,858,850 obtained sequences formed 86 operational taxonomic units (OTUs) at 3 % dissimilarity. Location, time of year, and degree of infestation by Varroa had significant effects on the composition of the bacteriome of honey bee workers. Based on statistical correlations, we found varroosis more important factor than N. ceranae, N. apis, and L. passim infestation influencing the honey bee bacteriome and contributing to the changes in the composition of the bacterial community in adult bees. At the population level, Varroa appeared to modify 20 OTUs. In the colonies with high Varroa infestation levels (varroosis), the relative abundance of the bacteria Bartonella apis and Lactobacillus apis decreased. In contrast, an increase in relative abundance was observed for several taxa including Lactobacillus helsingborgensis, Lactobacillus mellis, Commensalibacter intestini, and Snodgrassella alvi. The results showed that the "normal" bacterial community is altered by eukaryotic parasites as well as displaying temporal changes and changes associated with the geographical origin of the beehive.

Distribution and prevalence of Nosema apis and N. ceranae in temperate and subtropical eco-regions of Argentina.

A total of 361 colonies from 59 apiaries located in two temperate and three subtropical eco-regions were examined during the post-harvest period to determine distribution and prevalence of Nosema spp. Apiaries from subtropical eco-regions showed a lower spore count than those from temperate eco-regions. Pure N. ceranae and co-infection were detected in apiaries from all regions. In contrast, pure N. apis infection was exclusively observed in the subtropical study region. The predominant detection of N. apis in a subtropical region joining a southern temperate region where mainly co-infected apiaries were identified is in contrast to previous reports.

Higher prevalence and levels of Nosema ceranae than Nosema apis infections in Canadian honey bee colonies.

This study was conducted to determine the prevalence and infection levels of the microsporidia fungi Nosema apis and/or Nosema ceranae in honey bee colonies of two Canadian provinces. Three surveys were conducted in the springs of 2008, 2010 and 2012 and PCR identification of Nosema species were performed in samples from 169 and 181 Ontario colonies and from 76 Alberta colonies that tested positive to Nosema spp. Infection levels of positive colonies were determined by microscopy and analyzed by Nosema spp. Results showed that N. ceranae was the dominant species in all three surveys (prevalence range of 41-91 vs. 4-34 % for N. apis), whereas mixed infections were less frequent than single infections (5-25 %). Infection levels of colonies parasitized by N. ceranae were three to five times higher than those of colonies parasitized by N. apis in the three surveys whereas mixed infections showed the highest spore counts. This is the first field study demonstrating significantly higher infection levels in colonies parasitized with either N. ceranae only or with both, N. ceranae and N. apis, than in colonies parasitized with N. apis only. Taken together, these results suggest that N. ceranae may be more virulent and better adapted than N. apis in cold climates such as Canadian environments.

Infectivity and virulence of Nosema ceranae and Nosema apis in commercially available North American honey bees.

Nosema ceranae infection is ubiquitous in western honey bees, Apis mellifera, in the United States and the pathogen has apparently replaced Nosema apis in colonies nationwide. Displacement of N. apis suggests that N. ceranae has competitive advantages but N. ceranae was significantly less infective and less virulent than N. apis in commercially available lineages of honey bees in studies conducted in Illinois and Texas. At 5 days post eclosion, the most susceptible age of adult bees tested, the mean ID50 for N. apis was 359 spores compared to 3217 N. ceranae spores, a nearly 9-fold difference. Infectivity of N. ceranae was also lower than N. apis for 24-h and 14-day worker bees. N. ceranae was less infective than reported in studies using European strains of honey bees, while N. apis infectivity, tested in the same cohort of honey bees, corresponded to results reported globally from 1972 to 2010. Mortality of worker bees was similar for both pathogens at a dosage of 50 spores and was not different from the uninfected controls, but was significantly higher for N. apis than N. ceranae at dosages ⩾500 spores. Our results provide comparisons for evaluating research using different ages of bees and pathogen dosages and clarify some controversies. In addition, comparisons among studies suggest that the mixed lineages of US honey bees may be less susceptible to N. ceranae infections than are European bees or that the US isolates of the pathogen are less infective and less virulent than European isolates.

Parasitized honey bees are less likely to forage and carry less pollen.

Research into loss of pollination capacity has focused primarily on documenting pollinator declines and their causes with comparatively little attention paid to how stressors may affect pollinating behavior of surviving pollinators. The European honey bee, Apis mellifera is one of the world's most important generalist pollinators, and Nosema apis is a widespread microsporidian gut parasite of adult A. mellifera. We individually fed 960 newly eclosed A. mellifera workers either a sucrose solution or 400 N. apis spores in a sucrose solution and tagged them with a unique radio frequency identification (RFID) tag to monitor their foraging behavior. We found spore-fed bees were less likely to forage than those fed sugar only. Those that did forage started foraging when they were older and stopped foraging when they were younger than bees fed sugar only. However, inoculated and non-inoculated bees did not significantly differ in the number of foraging trips taken per day, the total hours foraged over their lifetime, or homing ability. Inoculated returning foragers were 4.3 times less likely to be carrying available pollen than non-inoculated returning foragers and the number of pollen grains carried was negatively correlated with the number of N. apis spores. In an arena of artificial flowers, inoculated bees had a tendency (p=0.061) to choose sugar flowers over pollen flowers, compared to non-inoculated bees which visited pollen and sugar flowers equally. These results demonstrate that even a relatively low dose of a widespread disease of A. mellifera may adversely affect bees' ability to pollinate.

Comparative virulence and competition between Nosema apis and Nosema ceranae in honey bees (Apis mellifera).

Honey bees (Apis mellifera) are infected by two species of microsporidia: Nosema apis and Nosemaceranae. Epidemiological evidence indicates that N. ceranae may be replacing N. apis globally in A. mellifera populations, suggesting a potential competitive advantage of N. ceranae. Mixed infections of the two species occur, and little is known about the interactions among the host and the two pathogens that have allowed N. ceranae to become dominant in most geographical areas. We demonstrated that mixed Nosema species infections negatively affected honey bee survival (median survival=15-17days) more than single species infections (median survival=21days and 20days for N. apis and N. ceranae, respectively), with median survival of control bees of 27days. We found similar rates of infection (percentage of bees with active infections after inoculation) for both species in mixed infections, with N. apis having a slightly higher rate (91% compared to 86% for N. ceranae). We observed slightly higher spore counts in bees infected with N. ceranae than in bees infected with N. apis in single microsporidia infections, especially at the midpoint of infection (day 10). Bees with mixed infections of both species had higher spore counts than bees with single infections, but spore counts in mixed infections were highly variable. We did not see a competitive advantage for N. ceranae in mixed infections; N. apis spore counts were either higher or counts were similar for both species and more N. apis spores were produced in 62% of bees inoculated with equal dosages of the two microsporidian species. N. ceranae does not, therefore, appear to have a strong within-host advantage for either infectivity or spore growth, suggesting that direct competition in these worker bee mid-guts is not responsible for its apparent replacement of N. apis.

Quantifying spore viability of the honey bee pathogen Nosema apis using flow cytometry.

Honey bees are hosts to more than 80 different parasites, some of them being highly virulent and responsible for substantial losses in managed honey bee populations. The study of honey bee pathogens and their interactions with the bees' immune system has therefore become a research area of major interest. Here we developed a fast, accurate and reliable method to quantify the viability of spores of the honey bee gut parasite Nosema apis. To verify this method, a dilution series with 0, 25, 50, 75, and 100% live N. apis was made and SYTO 16 and Propidium Iodide (n = 35) were used to distinguish dead from live spores. The viability of spores in each sample was determined by flow cytometry and compared with the current method based on fluorescence microscopy. Results show that N. apis viability counts using flow cytometry produced very similar results when compared with fluorescence microscopy. However, we found that fluorescence microscopy underestimates N. apis viability in samples with higher percentages of viable spores, the latter typically being what is found in biological samples. A series of experiments were conducted to confirm that flow cytometry allows the use of additional fluorescent dyes such as SYBR 14 and SYTOX Red (used in combination with SYTO 16 or Propidium Iodide) to distinguish dead from live spores. We also show that spore viability quantification with flow cytometry can be undertaken using substantially lower dye concentrations than fluorescence microscopy. In conclusion, our data show flow cytometry to be a fast, reliable method to quantify N. apis spore viabilities, which has a number of advantages compared with existing methods.

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.

A Systematic Review of Fumagillin Field Trials for the Treatment of Nosema Disease in Honeybee Colonies.

This article systematically reviews controlled field trials of fumagillin dicyclohexylamine in honeybee colonies to determine whether fumagillin effectively controls nosema and whether it is beneficial to colonies. Fifty publications were found that described controlled field trials of fumagillin in honeybee colonies between 1952 and 2023. Fumagillin consistently reduced the prevalence and severity of nosema infections. Doses applied in recent studies were similar to or below those recommended historically. Furthermore, our study showed no negative effects on colony health. Improvements in colony survival, size, and honey production have been demonstrated frequently, though not consistently, in both historic and recent studies. Nevertheless, some practices are not optimal. Treatment decision thresholds based on the number of spores per bee are not well supported by evidence and may be no better than calendar-based prophylactic treatments. In addition, reasonable recommendations to employ quarantine and disinfection procedures together with fumagillin treatment do not appear to have been widely adopted. When used as stand-alone treatments, both the fall- and spring-label doses provide benefits but may be too low and short-term to ensure full control of the disease.

Health status of honeybee colonies (Apis mellifera) and disease-associated risk factors in different agroecological zones of Southwest Ethiopia.

A cross-sectional study design was conducted in different agroecological zones of southwest Ethiopia from October 2019 to October 2021. The study aimed to determine the prevalence and associated risk factors for honeybee diseases and pests, as well as the impact of these issues on honeybee colonies and their products. To identify potential risk factors for honeybee disease and pests, a multivariate random effects logistic regression analysis was used. Adult honeybee and brood samples from a total of 384 honeybee colonies were collected and tested using standard laboratory diagnostic methods. The highest prevalence (55.8%) of ants was recorded, followed by wax moths (22.5%) and hive beetles (23.3%). In the current study, the main honeybee diseases observed in the study areas were varroosis (36.5%), bee lice (5.2%), nosemosis (39.6%), amoeba (56%), and chalkbrood (4.5%). However, tracheal mites, sachbrood, and American and European foul brood, were not detected. The agroecological zone (OR = 5.2, 95% CI: 1.75-14.85), type of hive (OR = 2.9, 95% CI: 1.17-17.03), management system (OR = 4.3, 95% CI: 1.23-14.70), and the management of the colony (OR = 3.5, 95% CI: 1.31-9.14) were identified as risk factors for varroosis in these areas. The occurrence of nosemosis in colonies was also influenced by the agroecological zone (OR = 12.2, 95% CI: 3.06-48.54) and colony management (OR = 3.4, 95% CI: 1.59-7.23). The agroecological zone (OR = 10.5, 95% CI: 12.76-22.63) and hive type (OR = 3.0, 95% CI: 1.39-6.36) were the primary risk factors for the occurrence of amoeba in honeybee colonies. However, the occurrence of bee lice (OR = 34.7, 95% CI: 3.96-104.93) and chalkbrood (OR = 4.8, 95% CI: 1.44-13.16) in honeybee colonies was only influenced by the agroecological zone in the study areas. This study demonstrated that losses in honey production in the area are significantly attributed to honeybee disease and pests. Therefore, it is essential to increase public awareness of how honeybee diseases and pests affect honey production and to develop and implement appropriate control measures for these diseases and pests. Furthermore, more studies should be conducted to characterize and isolate other causes of honeybee diseases and pests in various locations.

Nosema ceranae has been present in Brazil for more than three decades infecting Africanized honey bees.

Until the mid-1990s, the only microsporidium known to infect bees of the genus Apis was Nosema apis. A second species, Nosema ceranae, was first identified in 1996 from Asian honey bees; it is postulated that this parasite was transmitted from the Asian honey bee, Apis cerana, to the European honey bee, Apis mellifera. Currently, N. ceranae is found on all continents and has often been associated with honey bee colony collapse and other reports of high bee losses. Samples of Africanized drones collected in 1979, preserved in alcohol, were analyzed by light microscopy to count spores and were subjected to DNA extraction, after which duplex PCR was conducted. All molecular analyses (triplicate) indicated that the drones were infected with both N. ceranae and N. apis. PCR products were sequenced and matched to sequences reported in the GenBank (Acc. Nos. JQ639316.1 and JQ639301.1). The venation pattern of the wings of these males was compared to those of the current population living in the same area and with the pattern of drones collected in 1968 from Ribeirão Preto, SP, Brazil, from a location close to where African swarms first escaped in 1956. The morphometric results indicated that the population collected in 1979 was significantly different from the current living population, confirming its antiquity. Considering that the use of molecular tools for identifying Nosema species is relatively recent, it is possible that previous reports of infections (which used only light microscopy, without ultrastructural analysis) wrongly identified N. ceranae as N. apis. Although we can conclude that N. ceranae has been affecting Africanized honeybees in Brazil for at least 34 years, the impact of this pathogen remains unclear.

Natural Substances, Probiotics, and Synthetic Agents in the Treatment and Prevention of Honeybee Nosemosis.

Honeybees are important pollinators, but they are continuously exposed to a variety of fungal and bacterial diseases. One of the various diseases affecting honeybees is nosemosis caused by microsporidia from the Nosema genus. Honeybees are mainly infected through consumption of infected food or faeces containing Nosema spp. spores. Nosemosis causes damage to the middle intestine epithelium, which leads to food absorption disorders and honeybee malnutrition. Fumagillin, i.e., the antibiotic used to treat nosemosis, was withdrawn in 2016 from EU countries. Therefore, researchers have been looking for compounds of both natural and synthetic origin to fight nosemosis. Such compounds should not have a negative impact on bees but is expected to inhibit the disease. Natural compounds tested against nosemosis include, e.g., essential oils (EOs), plant extracts, propolis, and bacterial metabolites, while synthetic substances tested as anti-nosemosis agents are represented by porphyrins, vitamins, antibiotics, phenolic, ascorbic acids, and others. This publication presents an 18-year overview of various studies of a number of natural and synthetic compounds used in the treatment and prevention of nosemosis cited in PubMed, GoogleScholar, and CrossRef.

Specific detection and quantification of three microsporidia infecting bees, Nosema apis, Nosema ceranae, and Nosema bombi, using probe-based real-time PCR.

Among stressors affecting bee health, Nosema microsporidia are prevalent intracellular parasites. Nosema apis and Nosema ceranae have been described in honey bees (Apis spp.), while Nosema bombi has been described in bumble bees (Bombus spp.). Although available molecular methods serve as a complement to microscopic diagnosis of nosemosis, they do not enable accurate quantification of these three Nosema species. We developed three quantitative real-time PCRs (qPCRs) starting from in silico design of specific primers, probes, and recombinant plasmids, to target the RNA polymerase II subunit B1 (RPB1) gene in the three species. The complete methods, including bee grinding, DNA purification, and qPCR, were validated in honey bee (Apis mellifera) homogenate. Specificity was assessed in silico and in vitro with several types of bee samples. The limit of detection was estimated at 4 log10 copies/honey bee. A small, systematic method bias was corrected for accurate quantification up to 10 log10 copies/honey bee. Method accuracy was also verified in bumble bee (Bombus terrestris) and mason bee (Osmia bicornis) homogenates in the range of 5 to 10 log10 copies/bee. These validated qPCR methods open perspectives in nosemosis diagnosis and in the study of the parasite's eco-dynamics in managed and wild bees.