Pilar García-Palencia

Honeybee colony collapse due to Nosema ceranae in professional apiaries.

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.

Natural infections of Nosema ceranae in European honey bees

The classification of the large and heterogeneous group of microorganisms called protozoans has been recently updated as increased knowledge of the biology and phylogenetics is acquired. The Committee on Systematics and Evolution of the Society of Protozoologists established seven phyla in the elevated subkingdom Protozoa under the Kingdom Protista more than two decades ago (Levin, et al., 1980). Later, it was suggested that the protists should be reorganized into the broader category protoctists that would also include fungi and algae besides the small eukaryotes consisting of a single or a few cells (Margulis et al., 1990). However, with the rapidly increasing phylogenetic data accumulating, this higher order taxonomy is no longer accurate. Based on molecular phylogenies, microsporidia are actually included into the cluster Fungi, rank Opisthokonta which comprises the animals, the fungi and others eukaryotes (Sina et al., 2005). Microsporidia are, thus, to be regarded as highly specialized parasitic fungi. Only two microsporidian parasites are described so far from honey bees (Nosema apis Zander 1909 and Nosema ceranae Fries et al. 1996). Nosema apis was detected in the European honey bee (Apis mellifera L 1758) and is one of the first microsporidia to be described (Zander, 1909). Although the parasite and its life cycle have been described by many authors (see Gray et al., 1969), vegetative stages are difficult to recognize and identify by light microscopy. These early descriptions have later been complemented with ultrastructural features of the parasite (Liu, 1984; Fries, 1989) and also with a molecular characterization (Gatehouse & Malone, 1998). Nosema ceranae, isolated from the Asian honey bee (Apis cerana Fabricius 1793) in China is a more recent description (Fries et al., 1996). There are good reasons to assume that other microsporidia species are also present in honey bees and await full descriptions (i.e. Buys, 1977; Clark 1980). Prior to the description of N. ceranae, observations of microsporidian infections in A. cerana had already been made (Sing, 1975). Yakobson (1992) observed microsporidia infections in both A. cerana and A. mellifera in apiaries with both honey bee species mixed and suggested that cross infection experiments using N. apis spores could perhaps elucidate the question of host specificity in N. apis. Many species of microsporidia cannot be distinguished using light microscopy and only with difficulty using electron microscopy (Larsson, 1986; Rice, 2001) and it cannot be excluded that some earlier observations of microsporidia infections in A. cerana, and possibly also in A. mellifera, may in fact have been observations of N. ceranae. Reports in the past of damage to A. cerana colonies attributed to N. apis infections (Lian, 1980) may actually be reports of N. ceranae, since differences between N. ceranae and N. apis may have gone unnoticed when investigated under the light microscope (Fries et al., 1996). Cross infections between the two host species have demonstrated that N. apis is in fact infective for A. cerana, but also that this parasite develops less well in the Asian host compared to the European host (Fries & Feng, 1995). It has also been stated that N. ceranae is infective for A. mellifera and multiplies more readily in A. mellifera than N. apis does in A. cerana (Fries, 1997), although detailed data were never published. At the annual meeting of Society for Invertebrate Pathology in Anchorage, Alaska, 2005 it was reported that N. ceranae had been found in natural infections in A. mellifera in Taiwan (Huang et al., 2005). The apiary where the infection was detected had harboured both A. mellifera and A. cerana. Thus, it was apparent that N. ceranae could cross the host species barrier, although no data on bee pathological repercussions due to N. ceranae in Apis mellifera were mentioned by the authors. Almost at the same time and following progressively increased incidences of problems with nosema disease in Spain (Martin et al., 2005), the laboratory of Centro Apícola Regional, involved in Journal of Apicultural Research 45(3): 230–233 (2006)

Effect of Temperature on the Biotic Potential of Honeybee Microsporidia

ABSTRACT 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.

Tumorigenic activity of p21Waf1/Cip1 in thymic lymphoma

The cell cycle inhibitor p21Waf1/Cip1 is among the most important mediators of the tumor suppressor p53. However, there is increasing evidence indicating that p21 could favor tumorigenesis in specific cell types. In particular, the absence of p21 delays the development of thymic lymphomas induced either by ataxia-telangiectasia mutated deficiency or by ionizing irradiation. Here, we extend these observations to the context of p53-deficient mice. The absence of p21 results in a significant extension of the lifespan of p53-null and p53-haploinsufficient mice, and this effect can be attributed exclusively to a decrease in the incidence of spontaneous thymic lymphomas. Specifically, despite the occurrence of a variety of tumor types in the context of p53 deficiency, the only tumors that were significantly impaired by the absence of p21 were thymic lymphomas. Moreover, the absence of p21 also delays the incidence of radiation-induced thymic lymphomas in p53-deficient mice. Interestingly, p21-deficient lymphomas have a higher apoptotic rate than p21-proficient lymphomas, and this could be on the basis of the delayed incidence of thymic lymphomas in the absence of p21. Together, our results indicate that p21 plays an oncogenic role restricted to thymic lymphomas that is mechanistically independent of p53 and associated to a lower tumor apoptotic rate.

Horizontal transmission of Nosema ceranae (Microsporidia) from worker honeybees to queens (Apis mellifera)

Horizontal transmission from worker honeybees to queens is confirmed in a laboratory essay as a possible route of Nosema ceranae infection in field colonies and pathological repercussions on honeybee queens are described. Lesions are only detected in the epithelial ventricular layer of the infected queens and death occurs within 3 weeks when the nurse workers are experimentally and collectively infected with approximately 5000 viable spores per bee. These data suggest that the higher number of infected house bees, the higher risk of transmission to queens. The presented data may explain the role of house honeybees in natural queen infection, although it is probably that a high proportion of infected house bees must be required to infect the queen.

Regurgitated pellets of Merops apiaster as fomites of infective Nosema ceranae (Microsporidia) spores

The importance of transmission factor identification is of great epidemiological significance. The bee-eater (Merops apiaster) is a widely distributed insectivorous bird, locally abundant mainly in arid and semi-arid areas of southern Europe, northern Africa and western Asia but recently has been seen breeding in central Europe and Great Britain. Bee-eaters predominantly eat insects, especially bees, wasps and hornets. On the other hand, Nosema ceranae is a Microsporidia recently described as a parasite in Apis mellifera honeybees in Europe. Due to the short time since its description scarce epidemiological data are available. In this study we investigate the role of the regurgitated pellets of the European bee-eater as fomites of infective spores of N. ceranae. Spore detection in regurgitated pellets of M. apiaster is described [phase-contrast microscopy (PCM) and polymerase chain reaction (PCR) methods]. Eighteen days after collection N. ceranae spores still remain viable and their infectivity is shown after artificial infection of Nosema-free 8-day-old adult bees. The epidemiological consequences of the presence of Nosema spores in this fomites are discussed.

Apoptosis in the pathogenesis of Nosema ceranae (Microsporidia: Nosematidae) in honey bees (Apis mellifera)

Nosema ceranae is a parasite of the epithelial ventricular cells of the honey bee that belongs to the microsporidian phylum, a biological group of single-cell, spore-forming obligate intracellular parasites found in all major animal lineages. The ability of host cells to accommodate a large parasitic burden for several days suggests that these parasites subvert the normal host cells to ensure optimal environmental conditions for growth and development. Once infected, cells can counteract the invasive pathogen by initiating their own death by apoptosis as a defence strategy. To determine whether N. ceranae blocks apoptosis in infected ventricular cells, cell death was assessed in sections of the ventriculum from experimentally infected honey bees using the TUNEL assay and by immunohistochemistry for caspase-3. Ventricular epithelial cells from infected bees were larger than those in the uninfected control bees, and they contained N. ceranae at both mature and immature stages in the cytoplasm. Apoptotic nuclei were only observed in some restricted areas of the ventriculum, whereas apoptosis was typically observed throughout the epithelium in uninfected bees. Indeed, the apoptotic index was higher in uninfected versus infected ventriculi. Our results suggested that N. ceranae prevents apoptosis in epithelial cells of infected ventriculi, a mechanism possible designed to enhance parasite development.

Comparative study of Nosema ceranae (Microsporidia) isolates from two different geographic origins

The intestinal honey bee parasite Nosema ceranae (Microsporidia) is at the root of colony losses in some regions while in others its presence causes no direct mortality. This is the case for Spain and France, respectively. It is hypothesized that differences in honey bee responses to N. ceranae infection could be due to the degree of virulence of N. ceranae strains from different geographic origins. To test this hypothesis, we first performed a study to compare the genetic variability of an rDNA fragment that could reveal differences between two N. ceranae isolates, one from Spain and one from France. Then we compared the infection capacity of both isolates in Apis mellifera iberiensis, based on the anatomopathological lesions due to N. ceranae development in the honey bee midgut, N. ceranae spore-load in the midgut and the honey bee survival rate. Our results suggest that there is no specific genetic background of the two N. ceranae isolates, from Spain or France, used in this study. These results agree with the infection development, honey bee survival and spore-loads that were similar between honey bees infected with both N. ceranae isolates. Probably, differences in honey bee response to infection are more related to the degree of tolerance of honey bee subspecies or local hybrids to N. ceranae, or experimental conditions in the case of laboratory trials, than to differences between N. ceranae isolates. Further studies should be done to estimate the contribution of each of these factors on the response of the honey bees to infection.

Different cooperating effect of p21 or p27 deficiency in combination with INK4a/ARF deletion in mice

The control exerted by the INK4a/ARF locus on cellular proliferation is crucial to restrict tumor development. In agreement with this, mice with defects in this locus are highly tumor prone. However, the potential contribution of other pathways in modulating tumorigenesis in the absence of INK4a/ARF is largely unexplored. In the present study, we investigated the consequences of the combined loss of either of two cyclin-dependent kinase inhibitors, p21 and p27, in cooperation with deletion of the INK4a/ARF locus. Our results show a clear differential effect in tumorigenesis depending on the CKI that is absent. The absence of p21 produced no overt alteration of the lifespan of the INK4a/ARF-null mice, although it modified their tumor spectrum, causing a significant increase in the incidence of fibrosarcomas and the appearance of a small number of rhabdomyosarcomas. In contrast, deficiency of p27 resulted in a significant increase in lethality due to accelerated tumor development, especially in the case of T-cell lymphomas. Finally, combined deficiency of INK4a/ARF and p27 resulted in a significant increase in the number of metastatic tumors. These results demonstrate genetically the oncogenic cooperation between defects on INK4a/ARF and p27, which are common alterations in human cancer.

Combined loss of p21 waf1/cip1 and p27 kip1 enhances tumorigenesis in mice

The cell cycle inhibitors p21Waf1/Cip1 and p27Kip1 are frequently downregulated in many human cancers, and correlate with a worse prognosis. We show here that combined deficiency in p21 and p27 proteins in mice is linked to more aggressive spontaneous tumorigenesis, resulting in a decreased lifespan. The most common tumors developed in p21p27 double-null mice were endocrine, with a higher incidence of pituitary adenomas, pheochromocytomas and thyroid adenomas. The combined absence of p21 and p27 proteins delays the incidence of radiation-induced thymic lymphomas with a higher apoptotic rate, measured by active caspase-3 and cleaved PARP-1 immunoexpresion. These results provide experimental evidence for a cooperation of both cyclin-dependent kinase inhibitors in tumorigenesis in mice.