A multiplex PCR-based method, in which two small-subunit rRNA regions are simultaneously amplified in a single reaction, was designed for parallel detection of honeybee microsporidians (Nosema apis and Nosema ceranae). Each of two pairs of primers exclusively amplified the 16S rRNA targeted gene of a specific microsporidian. The multiplex PCR assay was useful for specific detection of the two species of microsporidians related to bee nosemosis, not only in purified spores but also in honeybee homogenates and in naturally infected bees. The multiplex PCR assay was also able to detect coinfections by the two species. Screening of bee samples from Spain, Switzerland, France, and Germany using the PCR technique revealed a greater presence of N. ceranae than of N. apis in Europe, although both species are widely distributed.

Honeybee colony collapse is a sanitary and ecological worldwide problem. The features of this syndrome are an unexplained disappearance of adult bees, a lack of brood attention, reduced colony strength, and heavy winter mortality without any previous evident pathological disturbances. To date there has not been a consensus about its origins. This report describes the clinical features of two professional bee-keepers affecting by this syndrome. Anamnesis, clinical examination and analyses support that the depopulation in both cases was due to the infection by Nosema ceranae (Microsporidia), an emerging pathogen of Apis mellifera. No other significant pathogens or pesticides (neonicotinoids) were detected and the bees had not been foraging in corn or sunflower crops. The treatment with fumagillin avoided the loss of surviving weak colonies. This is the first case report of honeybee colony collapse due to N. ceranae in professional apiaries in field conditions reported worldwide.

The biological cycle of Nosema spp. in honeybees depends on temperature. When expressed as total spore counts per day after infection, the biotic potentials of Nosema apis and N. ceranae at 33°C were similar, but a higher proportion of immature stages of N. ceranae than of N. apis were seen. At 25 and 37°C, the biotic potential of N. ceranae was higher than that of N. apis. The better adaptation of N. ceranae to complete its endogenous cycle at different temperatures clearly supports the observation of the different epidemiological patterns.Biotic potential represents the maximum reproductive capacity of a population under optimum environmental conditions. Thus, a species fulfilling its biotic potential would exhibit maximal exponential population growth, thereby augmenting the possibilities of transmission of the species. A wide range of factors affects the biotic potential of each species, and among the external factors, temperature clearly influences the life cycles of most parasitic species (4).Nosemosis is a common worldwide disease of adult honeybees (Apis mellifera) that is caused by microsporidia (19). Nosema apis was the only agent known to produce this disease in A. mellifera until N. ceranae was identified in this host in 2005, in Europe (11) and Taiwan (12). Both these microsporidia infect and multiply in the ventricular cells of A. mellifera, and they can be found under different environmental conditions in both the northern and southern hemispheres (17, 13). Significantly, N. ceranae seems to be more pathogenic than N. apis in caged worker bees (10, 18), and it has recently been related to significant losses of bees and colony collapses under field conditions (17,8).Due to the lack of comparative studies of the factors affecting parasite virulence, trials were designed to determine the influence of temperature on the biotic potentials of both microsporidia. Only the deleterious effect of high exogenous temperature on spores of N. apis has been checked previously (16).In this work, purified N. apis and N. ceranae spores with a minimum viability of 99% (tested with 4% trypan blue) were obtained from experimentally infected honeybees always maintained at 33°C as described previously (9). The spores were counted using a hemocytometer chamber (19), while the Nosema species identification was confirmed by PCR (17).The experimental infection of bees was carried out as described previously (10). Briefly, young Nosema-free honeybees were starved for 2 h and fed 2 l of 50% sucrose solution containing 100,000 viable N. ceranae or N. apis spores. Honeybees were anesthetized with CO 2 , and later, a droplet of the spore solution was administered to each honeybee by touching a micropipette to its mouthparts until the entire droplet was consumed. The bees that did not consume the entire droplet were discarded. Uninfected control bees were fed 2 l of 50% sucrose solution alone.Three trials were carried out for 1 week each at three different temperatures (25, 33, and 37°C). Each trial included four replicate cages ...

In recent years, a worldwide decline in the Apis mellifera populations has been detected in many regions, including Spain. This decline is thought to be related to the effects of pathogens or pesticides, although to what extent these factors are implicated is still not clear. In this study, we estimated the prevalence of honey bee colony depopulation symptoms in a random selected sample (n = 61) and we explored the implication of different pathogens, pesticides and the flora visited in the area under study. The prevalence of colony depopulation symptoms in the professional apiaries studied was 67.2% [95% confidence interval (CI) = 54.6-79.8; P < 0.0001]. The most prevalent pathogen found in the worker honey bee samples was Nosema ceranae[65.6%; 95% CI = 52.8-78.3; P < 0.0001], followed by Varroa destructor[32.7%; 95% CI = 20.2-45.4; P < 0.0001] and 97.5% of the colonies infected by N. ceranae were unhealthy (depopulated). Co-infection by V. destructor and N. ceranae was evident in 22.9% (95% CI = 11.6-34.3; P < 0.0001) of the samples and only in unhealthy colonies. Of the 40 pesticides studied, only nine were detected in 49% of the stored pollen samples analysed. Fipronil was detected in only three of 61 stored pollen samples and imidacloprid was not detected in any. Acaricides like fluvalinate, and chlorfenvinphos used to control Varroa mite were the most predominant residues in the stored pollen, probably as a result of their application in homemade formulae. None of the pesticides identified were statistically associated to colony depopulated. This preliminary study of epidemiological factors suggests that N. ceranae is a key factor in the colony losses detected over recent years in Spain. However, more detailed studies that permit subgroup analyses will be necessary to contrast these findings.

In the last decade, an increase in honey bee (Apis mellifera L.) colony losses has been reported in several countries. The causes of this decline are still not clear. This study was set out to evaluate the pesticide residues in stored pollen from honey bee colonies and their possible impact on honey bee losses in Spain. In total, 1,021 professional apiaries were randomly selected. All pollen samples were subjected to multiresidue analysis by gas chromatography-mass spectrometry (MS) and liquid chromatography-MS; moreover, specific methods were applied for neonicotinoids and fipronil. A palynological analysis also was carried out to confirm the type of foraging crop. Pesticide residues were detected in 42% of samples collected in spring, and only in 31% of samples collected in autumn. Fluvalinate and chlorfenvinphos were the most frequently detected pesticides in the analyzed samples. Fipronil was detected in 3.7% of all the spring samples but never in autumn samples, and neonicotinoid residues were not detected. More than 47.8% of stored pollen samples belonged to wild vegetation, and sunflower (Heliantus spp.) pollen was only detected in 10.4% of the samples. A direct relation between pesticide residues found in stored pollen samples and colony losses was not evident accordingly to the obtained results. Further studies are necessary to determine the possible role of the most frequent and abundant pesticides (such as acaricides) and the synergism among them and with other pathogens more prevalent in Spain.