Tamara Gómez-Moracho

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.

Holistic screening of collapsing honey bee colonies in Spain: a case study

BackgroundHere we present a holistic screening of collapsing colonies from three professional apiaries in Spain. Colonies with typical honey bee depopulation symptoms were selected for multiple possible factors to reveal the causes of collapse.ResultsOmnipresent were Nosema ceranae and Lake Sinai Virus. Moderate prevalences were found for Black Queen Cell Virus and trypanosomatids, whereas Deformed Wing Virus, Aphid Lethal Paralysis Virus strain Brookings and neogregarines were rarely detected. Other viruses, Nosema apis, Acarapis woodi and Varroa destructor were not detected. Palinologic study of pollen demonstrated that all colonies were foraging on wild vegetation. Consequently, the pesticide residue analysis was negative for neonicotinoids. The genetic analysis of trypanosomatids GAPDH gene, showed that there is a large genetic distance between Crithidia mellificae ATCC30254, an authenticated cell strain since 1974, and the rest of the presumed C. mellificae sequences obtained in our study or published. This means that the latter group corresponds to a highly differentiated taxon that should be renamed accordingly.ConclusionThe results of this study demonstrate that the drivers of colony collapse may differ between geographic regions with different environmental conditions, or with different beekeeping and agricultural practices. The role of other pathogens in colony collapse has to bee studied in future, especially trypanosomatids and neogregarines. Beside their pathological effect on honey bees, classification and taxonomy of these protozoan parasites should also be clarified.

Recent worldwide expansion of Nosema ceranae (Microsporidia) in Apis mellifera populations inferred from multilocus patterns of genetic variation.

Nosema ceranae has been found infecting Apismellifera colonies with increasing frequency and it now represents a major threat to the health and long-term survival of these honeybees worldwide. However, so far little is known about the population genetics of this parasite. Here, we describe the patterns of genetic variation at three genomic loci in a collection of isolates from all over the world. Our main findings are: (i) the levels of genetic polymorphism (πS≈1%) do not vary significantly across its distribution range, (ii) there is substantial evidence for recombination among haplotypes, (iii) the best part of the observed genetic variance corresponds to differences within bee colonies (up to 88% of the total variance), (iv) parasites collected from Asian honeybees (Apis cerana and Apis florea) display significant differentiation from those obtained from Apismellifera (8-16% of the total variance, p<0.01) and (v) there is a significant excess of low frequency variants over neutral expectations among samples obtained from A. mellifera, but not from Asian honeybees. Overall these results are consistent with a recent colonization and rapid expansion of N. ceranae throughout A. mellifera colonies.

High levels of genetic diversity in Nosema ceranae within Apis mellifera colonies

Nosema ceranae is a widespread honeybee parasite, considered to be one of the pathogens involved in the colony losses phenomenon. To date, little is known about its intraspecific genetic variability. The few studies on N. ceranae variation have focused on the subunits of ribosomal DNA, which are not ideal for this purpose and have limited resolution. Here we characterized three single copy loci (Actin, Hsp70 and RPB1) in three N. ceranae isolates from Hungary and Hawaii. Our results provide evidence of unexpectedly high levels of intraspecific polymorphism, the coexistence of a wide variety of haplotypes within each bee colony, and the occurrence of genetic recombination in RPB1. Most haplotypes are not shared across isolates and derive from a few frequent haplotypes by a reduced number of singletons (mutations that appear usually just once in the sample), which suggest that they have a fairly recent origin. Overall, our data indicate that this pathogen has experienced a recent population expansion. The presence of multiple haplotypes within individual isolates could be explained by the existence of different strains of N. ceranae infecting honeybee colonies in the field which complicates, and must not be overlooked, further analysis of host-parasite interactions.

Virulence and polar tube protein genetic diversity of Nosema ceranae (Microsporidia) field isolates from Northern and Southern Europe in honeybees (Apis mellifera iberiensis)

Infection of honeybees by the microsporidian Nosema ceranae is considered to be one of the factors underlying the increased colony losses and decreased honey production seen in recent years. However, these effects appear to differ in function of the climatic zone, the distinct beekeeping practices and the honeybee species employed. Here, we compared the response of Apis mellifera iberiensis worker bees to experimental infection with field isolates of N. ceranae from an Oceanic climate zone in Northern Europe (Netherlands) and from a Mediterranean region of Southern Europe (Spain). We found a notable but non-significant trend (P = 0.097) towards higher honeybee survival for bees infected with N. ceranae from the Netherlands, although no differences were found between the two isolates in terms of anatomopathological lesions in infected ventricular cells or the morphology of the mature and immature stages of the parasite. In addition, the population genetic survey of the N. ceranae PTP3 locus revealed high levels of genetic diversity within each isolate, evidence for meiotic recombination, and no signs of differentiation between the Dutch and Spanish populations. A cross-infection study is needed to further explore the differences in virulence observed between the two N. ceranae populations in field conditions.

Population Genetics of Nosema apis and Nosema ceranae: One Host (Apis mellifera) and Two Different Histories

Two microsporidians are known to infect honey bees: Nosema apis and Nosema ceranae. Whereas population genetics data for the latter have been released in the last few years, such information is still missing for N. apis. Here we analyze the patterns of nucleotide polymorphism at three single-copy loci (PTP2, PTP3 and RPB1) in a collection of Apis mellifera isolates from all over the world, naturally infected either with N. apis (N = 22) or N. ceranae (N = 23), to provide new insights into the genetic diversity, demography and evolution of N. apis, as well as to compare them with evidence from N. ceranae. Neutral variation in N. apis and N. ceranae is of the order of 1%. This amount of diversity suggests that there is no substantial differentiation between the genetic content of the two nuclei present in these parasites, and evidence for genetic recombination provides a putative mechanism for the flow of genetic information between chromosomes. The analysis of the frequency spectrum of neutral variants reveals a significant surplus of low frequency variants, particularly in N. ceranae, and suggests that the populations of the two pathogens are not in mutation-drift equilibrium and that they have experienced a population expansion. Most of the variation in both species occurs within honey bee colonies (between 62%-90% of the total genetic variance), although in N. apis there is evidence for differentiation between parasites isolated from distinct A. mellifera lineages (20%-34% of the total variance), specifically between those collected from lineages A and C (or M). This scenario is consistent with a long-term host-parasite relationship and contrasts with the lack of differentiation observed among host-lineages in N. ceranae (< 4% of the variance), which suggests that the spread of this emergent pathogen throughout the A. mellifera worldwide population is a recent event.

Evidence for weak genetic recombination at the PTP2 locus of Nosema ceranae.

The microsporidian Nosema ceranae is an emergent pathogen that threatens the health of honeybees and other pollinators all over the world. Its recent rapid spread across a wide variety of host species and environments demonstrated an enhanced ability of adaptation, which seems to contradict the lack of evidence for genetic recombination and the absence of a sexual stage in its life cycle. Here we retrieved fresh data of the patterns of genetic variation at the PTP2 locus in naturally infected Apis mellifera colonies, by means of single genome amplification. This technique, designed to prevent the formation of chimeric haplotypes during polymerase chain reaction (PCR), provides more reliable estimates of the diversity levels and haplotype structure than standard PCR-cloning methods. Our results are consistent with low but significant rates of recombination in the history of the haplotypes detected: estimates of the population recombination rate are of the order of 30 and support recent evidence for unexpectedly high levels of variation of the parasites within honeybee colonies. These observations suggest the existence of a diploid stage at some point in the life cycle of this parasite and are relevant for our understanding of the dynamics of its expanding population.

Passive laboratory surveillance in Spain: pathogens as risk factors for honey bee colony collapse

Many European and North American countries have reported losses in honey bee populations over recent years. Although multiple factors appear to be involved in this decline, the influence of pathogens may be particularly important. In the present study, we analyzed the presence of the major honey bee pathogens and neonicotinoid insecticides in ten professional apiaries (five affected by high honey bee colony mortality and five asymptomatic apiaries). Our results confirm that Nosema ceranae and trypanosomatids are very prevalent in the apiaries affected by depopulation and high colony loss. By contrast, and while they have been demonstrated to be highly toxic to bees, neonicotinoid insecticides do not seem to play an important role in colony mortality in these apiaries. This kind of passive surveillance system is important to collect information about pathogen incidence, particularly when supplemented with a strong system of active disease surveillance that enables emergent diseases to be detected.

Any role for the dissemination of Nosema spores by the blue-tailed bee-eater Merops philippinus?

Pathogens are major suspects for the current massive losses of honey bee colonies worldwide. Information about circulation and modes of transmission of Nosema ceranae, an emergent microsporidium parasite of Apis mellifera, is currently lacking. Here we examine the dispersion of N. ceranae and N. apis by the blue-tailed bee-eater Merops philippinus, a migratory bird whose distribution along Southeast Asia overlaps with the original distribution of N. ceranae. We investigate the presence of Nosema spp. spores in bee-eater pellets, feces and nest material collected on the isolated Kinmen island (Taiwan). We found N. ceranae in all types of samples, whilst N. apis was not detected. We describe for the first time the occurrence of N. ceranae spores in birds’ feces, which increases the potential of this bird species as a dispersive agent of the parasite. We molecularly assessed the occurrence of A. cerana and A. mellifera remains in infected and non-infected samples. We discuss the low prevalence of N. ceranae in fomites with Apis spp. in relation to the ecological characteristics of the study area (isolation, low abundance of bees) and their implications on the origin and dispersal of the pathogen. Our results allow us to generalize the likely role of avian bee predators as potential disseminators of this pathogen all over the world.

Preliminary effect of an experimental treatment with “Nozevit®”, (a phyto-pharmacological preparation) for Nosema ceranae control

Mariano Higes, Tamara Gómez-Moracho, Cristina Rodriguez-García, Cristina Botias, Raquel Martín-Hernández Centro Apícola, Laboratorio de Patología, Consejería de Agricultura, Junta de Comunidades de Castilla-La Mancha, 19180 Marchamalo, Guadalajara, Spain. Instituto de Recursos Humanos para la Ciencia y la Tecnología (INCRECYT), Fundación Parque Científico y Tecnológico de Albacete, Spain. Present address: School of Life Sciences, University of Sussex, Falmer, Brighton, BN1 9QG, UK.