Aránzazu Meana

How natural infection by Nosema ceranae causes honeybee colony collapse.

In recent years, honeybees (Apis mellifera) have been strangely disappearing from their hives, and strong colonies have suddenly become weak and died. The precise aetiology underlying the disappearance of the bees remains a mystery. However, during the same period, Nosema ceranae, a microsporidium of the Asian bee Apis cerana, seems to have colonized A. mellifera, and its now frequently detected all over the world in both healthy and weak honeybee colonies. For first time, we show that natural N. ceranae infection can cause the sudden collapse of bee colonies, establishing a direct correlation between N. ceranae infection and the death of honeybee colonies under field conditions. Signs of colony weakness were not evident until the queen could no longer replace the loss of the infected bees. The long asymptomatic incubation period can explain the absence of evident symptoms prior to colony collapse. Furthermore, our results demonstrate that healthy colonies near to an infected one can also become infected, and that N. ceranae infection can be controlled with a specific antibiotic, fumagillin. Moreover, the administration of 120 mg of fumagillin has proven to eliminate the infection, but it cannot avoid reinfection after 6 months. We provide Kochs postulates between N. ceranae infection and a syndrome with a long incubation period involving continuous death of adult bees, non-stop brood rearing by the bees and colony loss in winter or early spring despite the presence of sufficient remaining pollen and honey.

Outcome of Colonization of Apis mellifera by Nosema ceranae

ABSTRACT 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. From the year 2000 onward, statistically significant differences have been found in the proportions of Nosema spp. spore-positive samples collected between and within years. In the first period examined (1999 to 2002), the smallest number of samples diagnosed as Nosema positive was found during the summer months, showing clear seasonality in the diagnosis, which is characteristic of N. apis. From 2003 onward a change in the tendency resulted in an increase in Nosema-positive samples in all months until 2005, when a total absence of seasonality was detected. A significant causative association between the presence of N. ceranae and hive depopulation clearly indicates that the colonization of Apis mellifera by N. ceranae is related to bee losses.

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.

Nosema ceranae in Europe: an emergent type C nosemosis

AbstractIn this review, relevant data is presented on an emerging disease of the 21th century in European countries, caused by Nosema ceranae. Within a few years after it was detected in Spain in 2005, the rest of European countries that had technical capacity to differentiate Nosema apis from N. ceranae reported its presence. In a similar way as the initial detection of Varroa in Europe, active scientific work is raising many questions due to the absence of clinical symptoms in infected colonies and a long incubation period of the pathogen. N. ceranae presents a different epidemiological pattern and pathology compared to N. apis. The disease caused by N. ceranae is now named nosemosis type C (COLOSS workshop, 2009) and is characterized by the ability to detect the disease-causing agent throughout the year. The continuous death of highly infected bees, mostly foragers, has a clear effect on colony population and productivity. Although there has been a huge effort in the last years to increase knowledge about this disease, significant research is still needed on epidemiology, pathology, prophyllaxis and treatment.ZusammenfassungIm Jahr 2005 wurde sowohl aus Asien als auch aus Europa über Nachweise von Nosema ceranae in der Honigbiene berichtet. Bis dahin wurde angenommen, dass die Nosemose der Bienen durch eine Infektion der Ventrikelzellen von erwachsenen Bienen mit Nosema apis verursacht wird. Allerdings weicht die durch N. ceranae verursachte, neu auftretende Krankheit in ihrer Epidemiologie, sowie ihrer Symptomatik und Pathologie ab. Daher ist es erforderlich, zwei verschiedene klinische Verläufe zu differenzieren: Nosemose Typ A, verursacht durch N. apis, und Nosemose Typ C, verursacht durch N. ceranae. Die Infektion der Bienen geschieht durch das Aufnehmen von reifen Sporen. Der Lebenszyklus von N. ceranae wird in weniger als 3 Tagen komplett durchlaufen; die intrazelluläre Keimung der Sporen wurde beobachtet. N. ceranae kann während des ganzen Jahres in Bienen nachgewiesen werden, die Übertragungsmechanismen sind jedoch noch nicht vollständig aufgeklärt. Sowohl Königinnen als auch Arbeiterinnen können sich mit N. ceranae infizieren. Pollen aus Pollenhöschen, von Bienenfressern ausgewürgte Pellets, sowie Imkereigeräte wurden als Reservoir von infektiösen Sporen beschrieben. Experimentelle Infektionen von A. mellifera mit N. ceranae zeigten eine höhere Pathogenität in diesem Wirt, die im Vergleich zu N. apis eine höhere Sterblichkeit zur Folge hatte. Jedoch wurden von anderen Autoren Unterschiede in den Sterblichkeitsraten beschrieben, möglicherweise beeinflussen bisher noch unbekannte Faktoren die Ergebnisse. Infizierte Arbeiterinnen wiesen eine deutliche Degeneration der Epithelzellen des Ventrikulums auf, wobei in histologischen Schnitten aus anderen Geweben keine Anzeichen einer Infektion mit Sporen gefunden wurden. N. ceranae kann die humoralen und zellulären Abwehrmechanismen der Bienen teilweise unterdrücken, was bei Infektionen mit N. apis nicht der Fall ist.Es wurde nachgewisen, dass die Postulate von Koch sowohl für Völker als auch für Einzelbienen gelten. Die Parasitierung einzelner Bienen durch N. ceranae hat einen deutlichen Effekt auf der Volksebene, was zu einem kontinuierlichen Totenfall hochinfizierter Bienen führt. Die lange und symptomfreie Inkubationszeit auf der Volksebene kann die Abwesenheit von sichtbaren Symptomen vor dem Zusammenbruch des Volkes erklären. Der Erreger wurde auch als Schlüsselfaktor für die Völkerverluste in Berufsimkereien in Spanien diskutiert. Es gibt jedoch einander widersprechende Berichte über die Folgen einer Infektion von Völkern mit N. ceranae aus verschiedenen Teilen Europas.Der Nachweis von N. ceranae erfordert den Einsatz von molekularbiologischen Methoden. Das Antibiotikum Fumagillin wirkt gegen beide Arten von Nosema, obwohl sein Einsatz in der Europäischen Union verboten ist. Einige neue potenzielle Bekämpfungsmittel sind Thymol und Resveratrol, sowie ApiHerb oder Nonosz®. Eine gute imkerliche Praxis ist entscheidend für die Vermeidung und Kontrolle dieser Krankheit.Es fehlt noch an Wissen über die epidemiologischen Faktoren und klinischen Symptome in verschiedenen Regionen Europas und anderer Teile der Welt, wo unterschiedliche klimatische Bedingungen herrschen und verschiedene Formen der Imkerei ausgeübt werden. Zukünftige Studien über die Nosemose Typ C werden zweifellos ihre Rolle bei Völkerzusammenbrüchen aufklären und werden den Wissensstand über viele noch unbekannte Faktoren im Zusammenhang mit dieser neuen Krankheit verbessern.

South American native bumblebees (Hymenoptera: Apidae) infected by Nosema ceranae (Microsporidia), an emerging pathogen of honeybees (Apis mellifera)

As pollination is a critical process in both human-managed and natural terrestrial ecosystems, pollinators provide essential services to both nature and humans. Pollination is mainly due to the action of different insects, such as the bumblebee and the honeybee. These important ecological and economic roles have led to widespread concern over the recent decline in pollinator populations that has been detected in many regions of the world. While this decline has been attributed in some cases to changes in the use of agricultural land, the effects of parasites could play a significant role in the reduction of these populations. For the first time, we describe here the presence of Nosema ceranae, an emerging honeybee pathogen, in three species of Argentine native bumblebees. A total of 455 bumblebees belonging to six species of genus Bombus were examined. PCR results showed that three of the species are positive to N. ceranae (Bombus atratus, Bombus morio and Bombus bellicosus). We discuss the appearance of this pathogen in the context of the population decline of this pollinators.

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)

Nosema ceranae (Microsporidia), a controversial 21st century honey bee pathogen

The worldwide beekeeping sector has been facing a grave threat, with losses up to 100-1000 times greater than those previously reported. Despite the scale of this honey bee mortality, the causes underlying this phenomenon remain unclear, yet they are thought to be multifactorial processes. Nosema ceranae, a microsporidium recently detected in the European bee all over the world, has been implicated in the global phenomenon of colony loss, although its role remains controversial. A review of the current knowledge about this pathogen is presented focussing on discussion related with divergent results, trying to analyse the differences specially based on different methodologies applied and divisive aspects on pathology while considering a biological or veterinarian point of view. For authors, the disease produced by N. ceranae infection cannot be considered a regional problem but rather a global one, as indicated by the wide prevalence of this parasite in multiple hosts. Not only does this type of nosemosis causes a clear pathology on honeybees at both the individual and colony levels, but it also has significant effects on the production of honeybee products.

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.

Microsporidia infecting Apis mellifera: coexistence or competition. Is Nosema ceranae replacing Nosema apis?

Nosema ceranae has been suggested to be replacing Nosema apis in some populations of Apis mellifera honeybees. However, this replacement from one to the other is not supported when studying the distribution and prevalence of both microsporidia in professional apiaries in Spanish territories (transverse study), their seasonal pattern in experimental hives with co-infection or their prevalence at individual level (either in worker bees or drones). Nevertheless, N.ceranae has shown to present a higher prevalence at all the studied levels that could indicate any advantage for its development over N.apis or that it is more adapted to Spanish conditions. Also, both microsporidia show a different pattern of preference for its development according to the prevalence in the different Spanish bioclimatic belts studied. Finally, the fact that all analyses were carried out using an Internal PCR Control (IPC) newly developed guarantees the confidence of the data extracted from the PCR analyses. This IPC provides a useful tool for laboratory detection of honeybee pathogens.

A preliminary study of the epidemiological factors related to honey bee colony loss in Spain

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.