Prevalence and infection intensity of Nosema in honey bee (Apis mellifera L.) colonies in Virginia

Nosema ceranae is a recently described pathogen of Apis mellifera and Apis cerana. Relatively little is known about the distribution or prevalence of N. ceranae in the United States. To determine the prevalence and potential impact of this new pathogen on honey bee colonies in Virginia, over 300 hives were sampled across the state. The samples were analyzed microscopically for Nosema spores and for the presence of the pathogen using real-time PCR. Our studies indicate that N. ceranae is the dominant species in Virginia with an estimated 69.3% of hives infected. Nosema apis infections were only observed at very low levels (2.7%), and occurred only as co-infections with N. ceranae. Traditional diagnoses based on spore counts alone do not provide an accurate indication of colony infections. We found that 51.1% of colonies that did not have spores present in the sample were infected with N. ceranae when analyzed by real-time PCR. In hives that tested positive for N. ceranae, average C_T values were used to diagnose a hive as having a low, moderate, or a heavy infection intensity. Most infected colonies had low-level infections (73%), but 11% of colonies had high levels of infection and 16% had moderate level infections. The prevalence and mean levels of infection were similar in different regions of the state.     

The effect of induced queen replacement on Nosema spp. infection in honey bee (Apis mellifera iberiensis) colonies

Microsporidiosis of adult honeybees caused by Nosema apis and Nosema ceranae is a common worldwide disease with negative impacts on colony strength and productivity. Few options are available to control the disease at present. The role of the queen in bee population renewal and the replacement of bee losses due to Nosema infection is vital to maintain colony homeostasis. Younger queens have a greater egg laying potential and they produce a greater proportion of uninfected newly eclosed bees to compensate for adult bee losses; hence, a field study was performed to determine the effect of induced queen replacement on Nosema infection in honey bee colonies, focusing on colony strength and honey production. In addition, the impact of long-term Nosema infection of a colony on the ovaries and ventriculus of the queen was evaluated. Queen replacement resulted in a remarkable decrease in the rates of Nosema infection, comparable with that induced by fumagillin treatment. However, detrimental effects on the overall colony state were observed due to the combined effects of stressors such as the queenless condition, lack of brood and high infection rates. The ovaries and ventriculi of queens in infected colonies revealed no signs of Nosema infection and there were no lesions in ovarioles or epithelial ventricular cells.     

Comparison of within hive sampling and seasonal activity of Nosema ceranae in honey bee colonies

Nosema ceranae is a microsporidian parasite of the European honey bee, Apis mellifera, that is found worldwide and in multiple Apis spp.; however, little is known about the effects of N. ceranae on A. mellifera. Previous studies using spore counts suggest that there is no longer a seasonal cycle for N. ceranae and that it is found year round with little variation in infection intensity among months. Our goal was to determine whether infection levels differ in bees collected from different areas of the hive and if there may be seasonal differences in N. ceranae infections. A multiplex species-specific real-time PCR assay was used for the detection and quantification of N. ceranae. Colonies were sampled monthly from September 2009-2010 by collecting workers from honey supers, the fringe of the brood nest, and the brood nest. We found that all bees sampled were infected with N. ceranae and that there was no significant difference in infection levels among the different groups of bees sampled (P=0.74). However, significant differences in colony infection levels were found at different times of the year (P<0.01) with the highest levels in April-June and lower levels in the fall and winter. While our study was only performed for one year, it sheds light on the fact that there may be a seasonality to N. ceranae infections. Being able to predict future N. ceranae infections can be used to better advise beekeepers on N. ceranae management.     

Ultrastructure of the midgut of the worker honey bee Apis mellifera heavily infected with Nosema apis

Midgut epithelial cells from healthy bees possessed numerous mitochondria, strands of endoplasmic reticulum, evenly distributed ribosomes, zymogen granules, and two kinds of lipid inclusions. In heavily infected midguts of honey bees, Apis mellifera, all epithelial cells were observed to be infected with Nosema apis. Cells of the entire midgut were packed with mature spores and, in some cases, mixed with immature stages. Spores were not found among cells of the brush border and basal infolding. Muscle cells and tracheal end cells of the midgut were not infected. The cytoplasm of the infected cell contained a large number of vacuoles, numerous large inclusion bodies, and aggregated ribosomes. Signs of extensive lysis were observed within the heavily infected cells, although the cell membranes were intact.     

Spores of Paenibacillus larvae,Ascosphaera apis,Nosema ceranae and Nosema apis in bee products supervised by the Brazilian Federal Inspection Service

Due to their ecological and economic importance, honey bees have attracted much scientific attention, which has intensified due to the recent population decline of these insects in the several parts of the world. Among the factors related to these patterns, infection by pathogens are the most relevant, mainly because of the easy dissemination of these microorganisms. Although no zoonotic diseases are associated with these insects, the presence of infectious agents in bee products should still be considered because they play a role as disease dispersers, increasing the risk to animal health. Because of the possibility of dispersion of pathogens via bee products, this work aimed to identify the presence of spores of the pathogens Paenibacillus larvae, Ascosphaera apis and Nosema spp. in samples of honey, pollen and royal jelly that are registered with Brazil's Federal Inspection Service (S.I.F.) and commercially available in the state of São Paulo. Of the 41 samples of bee products analyzed, only one showed no contamination by any of these pathogens. N. ceranae and P. larvae had the highest prevalence considering all the samples analyzed (present in 87.80% and 85.37% of the total, respectively), with N. apis present in 26.83% and A. apis present in 73.17% of the samples. These results provide support for the formulation of government regulations for sanitary control of exotic diseases by preventing dispersion of pathogens, including through illegal importation, since local and international trade and the transfer of colonies between regions play important roles in the dispersion of these microorganisms.     

Pathogen webs in collapsing honey bee colonies

Recent losses in honey bee colonies are unusual in their severity, geographical distribution, and, in some cases, failure to present recognized characteristics of known disease. Domesticated honey bees face numerous pests and pathogens, tempting hypotheses that colony collapses arise from exposure to new or resurgent pathogens. Here we explore the incidence and abundance of currently known honey bee pathogens in colonies suffering from Colony Collapse Disorder (CCD), otherwise weak colonies, and strong colonies from across the United States. Although pathogen identities differed between the eastern and western United States, there was a greater incidence and abundance of pathogens in CCD colonies. Pathogen loads were highly covariant in CCD but not control hives, suggesting that CCD colonies rapidly become susceptible to a diverse set of pathogens, or that co-infections can act synergistically to produce the rapid depletion of workers that characterizes the disorder. We also tested workers from a CCD-free apiary to confirm that significant positive correlations among pathogen loads can develop at the level of individual bees and not merely as a secondary effect of CCD. This observation and other recent data highlight pathogen interactions as important components of bee disease. Finally, we used deep RNA sequencing to further characterize microbial diversity in CCD and non-CCD hives. We identified novel strains of the recently described Lake Sinai viruses (LSV) and found evidence of a shift in gut bacterial composition that may be a biomarker of CCD. The results are discussed with respect to host-parasite interactions and other environmental stressors of honey bees.     

Neric acid derivatives produced by the honey bee fungal entomopathogen Ascosphaera apis

Two yet unknown from natural sources monoterpenoids, 1 and 2, were isolated from a culture of the honey bee entomopathogenic fungus Ascosphaera apis and their structures were determined by spectroscopic methods. These compounds resembled certain pheromones of honey bees. Compounds 1 and 2 showed weak antifungal and antioxidant properties and 1 exhibited antibiotic activity. The compound p-hydroxyphenylethanol, showing antifungal properties, was also isolated and identified.     

Metabolic energy of intact honey bee colonies

• 1.1. In late winter, oxygen consumption of honey bee (Apis mellifera L.) clusters showed marked 24-hr periodicity, even when held under constant temperature conditions.
• 2.2. Minimal rates of metabolism (as low as 3.4 w kg ⁻¹) were usually reached at night (ca. 0500 hr), and maximum rates (as high as 33.5 w kg⁻¹) in midday (ca. 1400 hr).
• 3.3. Colonies with brood showed less excursion in daily metabolic rate, by maintaining higher night-time levels.
• 4.4. There is a pronounced decrease in metabolic rate for the intact cluster of 9480–23,394 bees from the rates reported for individuals or small groups of bees.

     

Host sharing by the honey bee parasites Lotmaria passim and Nosema ceranae

The trypanosome Lotmaria passim and the microsporidian Nosema ceranae are common parasites of the honey bee, Apis mellifera, intestine, but the nature of interactions between them is unknown. Here, we took advantage of naturally occurring infections and quantified infection loads of individual workers (N = 408) originating from three apiaries (four colonies per apiary) using PCR to test for interactions between these two parasites. For that purpose, we measured the frequency of single and double infections, estimated the parasite loads of single and double infections, and determined the type of correlation between both parasites in double infections. If interactions between both parasites are strong and antagonistic, single infections should be more frequent than double infections, double infections will have lower parasite loads than single infections, and double infections will present a negative correlation. Overall, a total of 88 workers were infected with N. ceranae, 53 with L. passim, and eight with both parasites. Although both parasites were found in all three apiaries, there were significant differences among apiaries in the proportions of infected bees. The data show no significant differences between the expected and observed frequencies of single‐ and double‐infected bees. While the infection loads of individual bees were significantly higher for L. passim compared to N. ceranae, there were no significant differences in infection loads between single‐ and double‐infected hosts for both parasites. These results suggest no strong interactions between the two parasites in honey bees, possibly due to spatial separation in the host. The significant positive correlation between L. passim and N. ceranae infection loads in double‐infected hosts therefore most likely results from differences among individual hosts rather than cooperation between parasites. Even if hosts are infected by multiple parasites, this does not necessarily imply that there are any significant interactions between them.     

Worker reproductive parasitism in naturally orphaned colonies of the Asian red dwarf honey bee, Apis florea

The truce between honey bee (Apis spp.) workers over reproduction is broken in the absence of their queen. Queenright workers generally abstain from personal reproduction, raising only the queen’s offspring. Queenless workers activate their ovaries, produce eggs, and reduce the rate at which they destroy worker-laid eggs, so that some eggs are reared to maturity. Reduced policing of worker-laid eggs renders queenless nests vulnerable to worker reproductive parasitism (WRP), and may result in the colony raising eggs of unrelated (non-natal) workers that parasitize it. Queenless colonies of A. florea are heavily parasitized with the eggs of non-natal workers. However, queenless colonies often abscond upon disturbance and build a small comb in which to rear their own male offspring. We investigated three naturally occurring orphaned colonies to determine if they are also parasitized. We show that WRP is present in orphaned colonies, and non-natal workers have significantly higher rates of ovary activation than natal workers. In contrast to experimentally manipulated colonies, in our samples, natal and non-natal workers had statistically equal reproductive success, but this may have been due to the small number of non-natals present.     

Polymorphic DNA sequences of the fungal honey bee pathogen Ascosphaera apis

The pathogenic fungus Ascosphaera apis is ubiquitous in honey bee populations. We used the draft genome assembly of this pathogen to search for polymorphic intergenic loci that could be used to differentiate haplotypes. Primers were developed for five such loci, and the species specificities were verified using DNA from nine closely related species. The sequence variation was compared among 12 A.?apis isolates at each of these loci, and two additional loci, the internal transcribed spacer of the ribosomal RNA (ITS) and a variable part of the elongation factor 1α (Ef1α). The degree of variation was then compared among the different loci, and three were found to have the greatest detection power for identifying A.?apis haplotypes. The described loci can help to resolve strain differences and population genetic structures, to elucidate host-pathogen interaction and to test evolutionary hypotheses for the world's most important pollinator: the honey bee and one of its most common pathogens.     

Biogenic amines and division of labor in honey bee colonies

Brain levels of dopamine, serotonin, and octopamine were measured in relation to both age-related division of labor and inter-individual differences in task specialization independent of age in honey bee colonies. The only differences among similarly aged bees performing different tasks were significantly lower levels of dopamine in food storers than comb builders and significantly lower levels of octopamine in soldiers than foragers, but soldiers also were slightly younger than foragers. Differences associated with age-related division of labor were stronger. Older bees, notably foragers, had significantly higher levels of all three amines than did younger bees working in the hive. Using social manipulations to unlink chronological age and behavioral status, octopamine was found to exhibit the most robust association between behavior and amine level, independent of age. Octopamine levels were significantly lower in normal-age nurses versus precocious foragers and overage nurses versus normal-age foragers, but not different in reverted nurses versus reversion colony foragers. Dopamine levels were significantly lower in normal-age nurses versus precocious foragers, but higher in reverted nurses versus reversion colony foragers. Serotonin levels did not differ in any of these comparisons. These correlative results suggest that octopamine is involved in the regulation of age-related division of labor in honey bees. Accepted: 10 February 1999     

Immune suppression in the honey bee (Apis mellifera) following infection by Nosema ceranae (Microsporidia)

Two microsporidia species have been shown to infect Apis mellifera , Nosema apis and Nosema ceranae . This work present evidence that N. ceranae infection significantly suppresses the honey bee immune response, although this effect was not observed following infection with N. apis . Immune suppression would also increase susceptibility to other bee pathogens and senescence. Despite the importance of both Nosema species in honey bee health, there is no information about their effect on the bees' immune system and present results can explain the different virulence between both microsporida infecting honeybees.     

Octopamine influences division of labor in honey bee colonies

Forager honey bees have higher brain levels of octopamine than do bees tending larvae in the hive. To test the hypothesis that octopamine influences honey bee division of labor we treated bees orally with octopamine or its immediate precursor tyramine and determined whether these treatments increased the probability of initiating foraging. Octopamine treatment significantly elevated levels of octopamine in the brain and caused a significant dose-dependent increase in the number of new foragers. This effect was seen for precocious foragers in single-cohort colonies and foragers in larger colonies with more typical age demographies. Tyramine treatment did not increase the number of new foragers, suggesting that octopamine was exerting a specific effect. Octopamine treatment was effective only when given to bees old enough to forage, i.e., older than 4 days of age. Treatment when bees were 1-3 days of age did not cause a significant increase in the number of new foragers when the bees reached the minimal foraging age. These results demonstrate that octopamine influences division of labor in honey bee colonies. We speculate that octopamine is acting in this context as a neuromodulator.     

Modelling the role of intracolonial genetic diversity on regulation of brood temperature in honey bee (Apis mellifera L.) colonies

In polyandrous social insects such as honey bees, a worker’s affinity for a particular task may be genetically infl uenced and so some patrilines may have lower stimulus thresholds for commencing a task than others. We used simulation models to investigate the effects of intracolonial diversity in the task thresholds that stimulate workers to engage in heating and cooling during nest thermoregulation. First, we simulated colonies comprised of one or 15 patrilines that were engaged in heating the brood nest, and observed that single patriline colonies maintained, on average, less stable brood nest temperatures than multiple patriline colonies. Second we simulated colonies with five patrilines that were engaged in cooling their nest, recording the proportions of bees of different patrilines that engaged in nest cooling in response to changing temperatures. Both of our simulations show remarkably similar qualitative patterns to those that we have previously observed empirically. This provides further support for the hypothesis that geneticallybased variability in task thresholds among patrilines within honey bee colonies is an important contributor to the ability of colonies to precisely thermoregulate their nests, and we suggest that diversity is important for optimal expression of a range of other colony-level phenotypes. Received 17 June 2005; revised 27 October 2005; accepted 23 December 2005.     

Exposure of the wild bee Osmia bicornis to the honey bee pathogen Nosema ceranae

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Sudden deaths and colony population decline in Greek honey bee colonies

During June and July of 2009, sudden deaths, tremulous movements and population declines of adult honey bees were reported by the beekeepers in the region of Peloponnesus (Mt. Mainalo), Greece. A preliminary study was carried out to investigate these unexplained phenomena in this region. In total, 37 bee samples, two brood frames containing honey bee brood of various ages, eight sugar samples and four sugar patties were collected from the affected colonies. The samples were tested for a range of pests, pathogens and pesticides. Symptomatic adult honey bees tested positive for Varroa destructor,Nosema ceranae, Chronic bee paralysis virus (CBPV), Acute paralysis virus (ABPV), Deformed wing virus (DWV), Sacbrood virus (SBV) and Black queen cell virus (BQCV), but negative for Acarapis woodi. American Foulbrood was absent from the brood samples. Chemical analysis revealed that amitraz, thiametoxan, clothianidin and acetamiprid were all absent from symptomatic adult bees, sugar and sugar patty samples. However, some bee samples, were contaminated with imidacloprid in concentrations between 14 ng/g and 39 ng/g tissue. We present: the infection of Greek honey bees by multiple viruses; the presence of N. ceranae in Greek honey bees and the first record of imidacloprid (neonicotonoid) residues in Greek honey bee tissues. The presence of multiple pathogens and pesticides made it difficult to associate a single specific cause to the depopulation phenomena observed in Greece, although we believe that viruses and N. ceranae synergistically played the most important role. A follow up in-depth survey across all Greek regions is required to provide context to these preliminary findings.     

Polar tube protein gene diversity among Nosema ceranae strains derived from a Greek honey bee health study

Honey bee samples from 54 apiaries originating from 37 geographic locations of Greece were screened for Nosema apis and Nosema ceranae. Furthermore 15 samples coming from 12 geographic locations were screened also for Paenibacilluslarvae and Melissococcus plutonius and seven honey bee virus species, for the first time on a nation-wide level. There was a tendency in finding proportionally higher spore counts in samples from apiaries that suffered important colony losses. P. larvae bacteria were identified in two samples and each of the tested bee viruses could be detected in at least one of the examined samples, with IAPV, CBPV and SBV being the least abundant and BQCV and DWV being the most abundant. In the study we focused on polymorphism of a N. ceranae gene encoding a polar tube protein (PTP) as similar genes were proven to be highly polymorphic in the microsporidian parasites Encephalitozoon cuniculi and Encephalitozoon hellem. The polymorphism observed in the PTP gene sequences from a single sample (bee hive) was unexpected and can thus be considered to be a major obstacle for genotyping.