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Background: Trypanosomatid parasites are widely distributed in nature, evolving monoxenous and dixenous cycles. These parasites thrive in a wide number of Insect Orders, some of them with an important economic and environmental value, such as bees. The objective of this work was to develop a robust and sensitive qPCR assay for detecting trypanosomatid parasites in any kind of parasitized insect sample. Methods: A TaqMan qPCR assay based on a trypanosomatid-conserved region of the α-tubulin gene was standardized and evaluated. The limits of detection, sensitivity, and versatility of the α-tub TaqMan assay have been tested and validated using field samples from honeybee workers, wild bees, bumblebees, and grasshoppers as well as in the human infective trypanosomatid Leishmania major. Results: This assay showed a detection limit of 1 parasite equivalent/µL and successfully detected trypanosomatids in 10 different hosts belonging to the Hymenoptera and Orthoptera. The methodology was also tested using honeybee samples from 4 apiaries (n= 224 worker honeybees) located in Alpujarra region (Granada, Spain). Trypanosomatids were detected in 2.7% of the honeybees, with an intra colony prevalence of 0 to 13%. Parasite loads in 4 different classes of insects ranged from 40.6 to up to 1.1 x 108 cell equivalents per host. Conclusions: These results showed that α-tubulin TaqMan qPCR assay is a versatile diagnostic tool for the accurate detection and quantification of trypanosomatid parasites in a wide range of environmental settings.

We implemented our method in R. We provide the codes and the bumblebee and black kite datasets in Dryad Digital Repository https://doi.org/10.5061/dryad.47d7wm390 (Pasquaretta et al., 2015). The roe deer dataset was obtained from MOVEBANK (Wikelski & Kays, 2020). Animal Identifier: Sandro (M06), from Cagnacci et al. (2011) (https://www.movebank.org/). The wolf dataset was obtained from MOVEBANK (Wikelski & Kays, 2020), Animal identifier: Zimzik, from Kaczensky et al. (2006) (https://www.movebank.org/).We implemented our method in R. We provide the codes and the bumblebee and black kite datasets in Dryad Digital Repository https://doi.org/10.5061/dryad.47d7wm390 (Pasquaretta et al., 2015). The roe deer dataset was obtained from MOVEBANK (Wikelski & Kays, 2020). Animal Identifier: Sandro (M06), from Cagnacci et al. (2011) (https://www.movebank.org/). The wolf dataset was obtained from MOVEBANK (Wikelski & Kays, 2020), Animal identifier: Zimzik, from Kaczensky et al. (2006) (https://www.movebank.org/).; International audience; 1. Understanding how animal movements change across space and time is a fundamental question in ecology. While classical analyses of trajectories give insightful descriptors of spatial patterns, a satisfying method for assessing the temporal succession of such patterns is lacking.2. Network analyses are increasingly used to capture properties of complex animal trajectories in simple graphical metrics. Here, building on this approach, we introduce a method that incorporates time into movement network analyses based on temporal sequences of network motifs.3. We illustrate our method using four example trajectories (bumblebee, black kite, roe deer, wolf) collected with different technologies (harmonic radar, platform terminal transmitter, global positioning system). First, we transformed each trajectory into a spatial network by defining the animal's coordinates as nodes and movements in between as edges. Second, we extracted temporal sequences of network motifs from each movement network and compared the resulting behavioural profiles to topological features of the original trajectory. Finally, we compared each sequence of motifs with simulated Brownian and Lévy random motions to statistically determine differences between trajectories and classical movement models.4. Our analysis of the temporal sequences of network motifs in individual movement networks revealed successions of spatial patterns corresponding to changes in behavioural modes that can be attributed to specific spatio‐temporal events of each animal trajectory. Future applications of our method to multi‐layered movement and social network analysis yield considerable promises for extending the study of complex movement patterns at the population level.

Environmental stressors have sublethal consequences on animals, often affecting the mean of phenotypic traits in a population. However, potential effects on variance are poorly understood. Since phenotypic variance is the basis for adaptation, any influence of stressors may have important implications for population resilience.Here we explored this possibility in insect pollinators by analysing raw datasets from 24 studies (6,913 bees) in which individuals were first exposed to stressors and then tested for cognitive tasks.While all types of stressors decreased the mean cognitive performance of bees, their effect on variance was complex. Focusing on 15 pesticide studies, we found that the dose and the mode of exposure to stressors were critical. At low pesticide doses, cognitive variance decreased following chronic exposures but not for acute exposures. Acute exposure to low doses thus seems less damaging at the population level. In all cases however, the variance decreased with increasing doses.Policy implications. Current guidelines for the authorization of plant protection products on the European market prioritize acute over chronic toxicity assessments on non-target organisms. By overlooking the consequences of a chronic exposure, regulatory authorities may register new products that are harmful to bee populations. Our findings thus call for more research on stress-induced phenotypic variance and its incorporation to policy guidelines to help identify levels and modes of exposure animals can cope with.

Nosema ceranae is the most prevalent endoparasite of Apis mellifera iberiensis and it is a major health problem for bees worldwide. The infective capacity of N. ceranae has been demonstrated experimentally in honey bee brood, however no data are available about its prevalence in brood under natural conditions. Thus, brood combs from 10 different hives were analyzed over two consecutive years, taking samples before and after winter. A total of 1433 larvae/pupae were analyzed individually and N. ceranae (3.53%) was the microsporidian most frequently detected, as opposed to Nosema apis (0.42%) which was more frequently detected in conjunction with N. ceranae (0.71%). The active multiplication of both microsporidians was confirmed by the expression (real-time-PCR) of the N. ceranae polar tube protein 3 gene and/or the N. apis RNA polymerase II gene in 24% of the brood samples positive for Nosema spp. Both genes are related to microsporidian multiplication. As such, N. ceranae multiplication was confirmed in 1.06% of the samples, while N. apis multiplication was only observed in co-infections with N. ceranae (0.07%). Brood cells were analyzed for the presence of Nosema spp., as those are the immediate environment where the brood stages develop. The brood samples infected by Nosema spp. were in brood cells in which that microsporidians were not detected, while brood cells positive for N. ceranae hosted brood stages that were not apparently infected, indicating that this is unlikely to be the main pathway of infection. Finally, the colonies with brood infected by N. ceranae showed higher levels (numbers) of infected adult bees, although the differences were not significant before (P = 0.260), during (P = 0.055) or after (P = 0.056) brood sampling. These results show that N. ceranae is a bee parasite ubiquitous to all members of the colony, irrespective of the age of the bee. It is also of veterinary interest and should be considered when studying the epidemiology of the disease.

Animals have evolved foraging strategies to acquire blends of nutrients that maximize fitness traits. In social insects, nutrient regulation is complicated by the fact that few individuals, the foragers, must address the divergent nutritional needs of all colony members simultaneously, including other workers, the reproductives, and the brood. Here we used 3D nutritional geometry design to examine how bumblebee workers regulate their collection of 3 major macronutrients in the presence and absence of brood. We provided small colonies artificial nectars (liquid diets) and pollens (solid diets) varying in their compositions of proteins, lipids, and carbohydrates during 2 weeks. Colonies given a choice between nutritionally complementary diets self-selected foods to reach a target ratio of 71% proteins, 6% carbohydrates, and 23% lipids, irrespective of the presence of brood. When confined to a single nutritionally imbalanced solid diet, colonies without brood regulated lipid collection and over-collected protein relative to this target ratio, whereas colonies with brood regulated both lipid and protein collection. This brood effect on the regulation of nutrient collection by workers suggests that protein levels are critical for larval development. Our results highlight the importance of considering bee nutrition as a multidimensional phenomenon to better assess the effects of environmental impoverishment and malnutrition on population declines.

International audience; Nutrition concerns physiological and behavioral processes involved in the acquisition of nutrients and other chemicals required for energy, growth, tissue maintenance, and reproduction. While some components are synthetized by the insects themselves, many are ingested with the food, which for social insects can be as diverse as plant leaves, flower pollen and nectar, honeydew, dead arthropods, or even wood. The challenge for insects is to ingest these foods in appropriate amounts and balance to avoid harmful excesses or deficits of nutrients or toxins. For an individual, nutritional regulation is achieved through a complex interplay between foraging decisions (e.g., based on learning and memory), preingestive processing of food (e.g., enzymatic degradation in the mouthparts), and postingestive assimilation (e.g., absorption and differential utilization of nutrients). For a colony, food collection, processing, assimilation, and excretion are decentralized processes achieved collectively by different individuals with divergent nutritional needs

Parasites alter the physiology and behaviour of their hosts. In domestic honey bees, the microsporidia Nosema ceranae induces an energetic stress and impairs the behaviour of foragers, potentially leading to colony collapse. Whether this emerging parasite similarly affects wild pollinators is little understood because of the low success rates of experimental infection protocols. Here we established a new apporach for infecting bumblebees (Bombus terrestris) with controlled amounts of N. ceranae, by briefly exposing individual bumblebees to a sucrose solution containing parasite spores, before feeding them with artificial diets. We validated our protocol by testing the effect of two spore dosages and two diets varying in their protein to carbohydrate ratio, on the prevalence of the parasite (proportion of infected bees), the intensity of infection (spore count in the gut), and the survival of bumblebees. Insects fed a low-protein high-carbohydrate diet showed highest parasite prevalence (up to 70%) but lived longest, suggesting that immunity and survival of bumblebees are maximised at different protein to carbohydrate ratios. Spore dosage had no effect on parasite infection rate and host survival. The identification of experimental conditions for successfully infecting bumblebees with N. ceranae in the lab will facilitate investigations of the sub-lethal effects of this parasite on the behaviour and cognition of wild pollinators.

International audience; Extended ground level structures like roads or field edges can be important cues for navigating animals, seen for example in road-following pigeons. In a landscape devoid of skyline cues but with a rectangular grid of pathways and roads, we used harmonic radar to track free-flying bumble bees, Bombus terrestris. Individual bees consistently used ground level linear features for navigation in a wide range of behavioural contexts. Bee exploration flights, search behaviour and foraging routes were shaped by linear features, with bees frequently flying along and parallel to pathways and roads. Comparisons of flight trajectories across these behavioural contexts show that individuals modulated their use of linear features strategically with respect to their individual goals and experience. Bees searching for a feeder used linear features to target their search, while foragers often followed pathways to return to their hive without overshooting. These findings on a major pollinator have important implications for the placements of bee colonies for agriculture and floral resources for conservation.

Parasites alter the physiology and behaviour of their hosts. In domestic honey bees, the microsporidia Nosema ceranae induces energetic stress that impairs the behaviour of foragers, potentially leading to colony collapse. Whether this parasite similarly affects wild pollinators is little understood because of the low success rates of experimental infection protocols. Here, we present a new approach for infecting bumblebees (Bombus terrestris) with controlled amounts of N. ceranae by briefly exposing individual bumblebees to parasite spores before feeding them with artificial diets. We validated our protocol by testing the effect of two spore dosages and two diets varying in their protein to carbohydrate ratio on the prevalence of the parasite (proportion of PCR-positive bumblebees), the intensity of parasites (spore count in the gut and the faeces), and the survival of bumblebees. Overall, insects fed a low-protein, high-carbohydrate diet showed the highest parasite prevalence (up to 70%) but lived the longest, suggesting that immunity and survival are maximised at different protein to carbohydrate ratios. Spore dosage did not affect parasite infection rate and host survival. The identification of experimental conditions for successfully infecting bumblebees with N. ceranae in the lab will facilitate future investigations of the sub-lethal effects of this parasite on the behaviour and cognition of wild pollinators.

The trypanosomatids Crithidia mellificae and Lotmaria passim are very prevalent in honey bee colonies and potentially contribute to colony losses that currently represent a serious threat to honey bees. However, potential pathogenicity of these trypanosomatids remains unclear and since studies of infection are scarce, there is little information about the virulence of their different morphotypes. Hence, we first cultured C. mellificae and L. passim (ATCC reference strains) in six different culture media to analyse their growth rates and to obtain potentially infective morphotypes. Both C. mellificae and L. passim grew in five of the media tested, with the exception of M199. These trypanosomatids multiplied fastest in BHI medium, in which they reached a stationary phase after around 96 h of growth. Honey bees inoculated with either Crithidia or Lotmaria died faster than control bees and their mortality was highest when they were inoculated with 96 h cultured L. passim. Histological and Electron Microscopy analyses revealed flagellated morphotypes of Crithidia and Lotmaria in the lumen of the ileum, and adherent non-flagellated L. passim morphotypes covering the epithelium, although no lesions were evident. These data indicate that parasitic forms of these trypanosomatids obtained from the early stationary growth phase infect honey bees. Therefore, efficient infection can be achieved to study their intra-host development and to assess the potential pathogenicity of these trypanosomatids.

International audience; AbstractA major difficulty in studying the behaviour of social insects, such as bees, is to collect quantitative data on large numbers of individuals and over long periods of time, in sometimes dark and not easily accessible nests. Over the past decade, connected hives equipped with large sets of sensors to monitor real-time data about bee colony health and environmental conditions have been increasingly used in fundamental research, precision beekeeping and outreach programs. Here, we argue that combining these connected hive systems with automated movement tracking devices to obtain long-term data about the behaviour of bees inside and outside the hive can lead to major breakthroughs by helping discover new behaviours and compare data across labs and species. First, we describe the main sensors and hive parameters commonly used in connected hives used for honey bee and bumblebee colonies. Next, we discuss how developing more integrated systems connecting bees, hives and their environment, will help ask novel fundamental questions on bee behaviour and ecology.

1. Bees are key pollinators and their widespread decline has raised considerable concerns regarding the sustainability of ecosystems and food production. Many environmental stressors do not directly kill bees, but they alter their physiology and behaviour, ultimately impacting colonies and populations. 2. This review considers the impact of parasites and pathogens on bee cognition. 3. First the main parasites and pathogens of bees are described, as well as how they modify the foraging behaviour, learning and memory of their hosts. 4. Next, the various defence mechanisms developed by bees to mitigate these effects at both the individual and collective levels are examined. 5. Finally, there is a discussion on how integrating research on host parasites, animal behaviour and cognition will provide a more detailed assessment of the contribution of parasites and pathogens to declines in the bee population and help to inform constructive ecological interventions.

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 ...

International audience; Over the past decades, research on insect cognition has made considerable advances in describing the ability of model species (in particular bees and fruit flies) to achieve cognitive tasks once thought to be unique to vertebrates, and investigating how these may be implemented in a miniature brain. While this lab-based research is critical to understand some fundamental mechanisms of insect brains and cognition, taking a more integrative and comparative view will help making sense of this rich behavioural repertoire and its evolution. Here we argue that there is a need to reconsider insect cognition into an ecological context in order to design experiments that address the cognitive challenges insects face in nature, identify competing hypotheses about the cognitive abilities driving the observed behavioural responses, and test them across different populations and species. Reconnecting with the tradition of naturalistic observations, by testing animals in the field or in ecologically-inspired setups and comparing their performances, is complementary to mechanistic research in the lab, and will greatly improve our understanding of the role of insect cognition, its diversity, and the influence of ecological factors in its evolution.

Nutrition is thought to be a major driver of social evolution, yet empirical support for this hypothesis is scarce. Here we illustrate how conceptual advances in nutritional ecology illuminate some of the mechanisms by which nutrition mediates social interactions in insects. We focus on experiments and models of nutritional geometry and argue that they provide a powerful means for comparing nutritional phenomena across species exhibiting various social ecologies. This approach, initially developed to study the nutritional behaviour of individual insects, has been increasingly used to study insect groups and societies, leading to the emerging field of social nutrition. We discuss future directions for exploring how these nutritional mechanisms may influence major social transitions in insects and other animals.

International audience; Animals often alter their food choices following a pathogen infection in order to increase immune function and combat the infection. Whether social animals that collect food for their brood or nestmates adjust their nutrient intake to the infection states of their social partners is virtually unexplored. Here we develop an individual-based model of nutritional geometry to examine the impact of collective nutrient balancing on pathogen spread in a social insect colony. The model simulates a hypothetical social insect colony infected by a horizontally transmitted parasite. Simulation experiments suggest that collective nutrition, by which foragers adjust their nutrient intake to simultaneously address their own nutritional needs as well as those of their infected nestmates, is an efficient social immunity mechanism to limit contamination when immune responses are short. Impaired foraging in infected workers can favour colony resilience when pathogen transmission rate is low (by reducing contacts with the few infected foragers) or trigger colony collapse when transmission rate is fast (by depleting the entire pool of foragers). Our theoretical examination of dietary collective medication in social insects suggests a new possible mechanism by which colonies can defend themselves against pathogens and provides a conceptual framework for experimental investigations of the nutritional immunology of social animals.

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.

International audience; The traveling salesman problem is the task of determining an optimal path through several points and return to the starting point.

Acarapisosis is a disease of the adult honey bee Apis mellifera L., caused by the tracheal mite Acarapis woodi (Rennie), that affects the prothoracic tracheas of worker honey bees. Although it is not usually considered a real problem for honey bee colonies in southern Europe (mainly Spain and Greece), where the majority of professional beekeepers are located in Europe, recent works have reported the constant presence of this mite in this area, making it a potential cofactor for colony losses. In this study, we developed a specific PCR diagnostic tool that improves the techniques used so far and allowed us to confirm the presence of this parasite in Spain, urging the need to monitor its prevalence and implications in the health of the colonies. Indeed, in a total of 635 apiaries analysed, the prevalence of A. woodi in 2010 was 8.3 and 4 % in 2011. The mite is present in bee colonies over time and should not be underestimated as a possible cofactor in the collapse of bee colonies. Additionally, some positive samples were cloned so a genetic analysis on the diversity within A. woodi isolates was also approached. This allowed us to identify different genetic variants within an isolate, even when they were present at low frequencies. And this genetic analysis revealed the existence of a different clade of Acarapis sequences that could represent a new species or subspecies, although more research is required to verify the identity of this novel lineage at genetic and morphological level.

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.

Summary 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.

SUMMARYNosema ceranaeis 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 onN. ceranaevariation 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, Hsp70andRPB1) in threeN. ceranaeisolates 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 inRPB1. 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 ofN. ceranaeinfecting honeybee colonies in the field which complicates, and must not be overlooked, further analysis of host–parasite interactions.

Crithidia mellificae, a trypanosomatid parasite of Apis mellifera, has been proposed to be one of the pathogens responsible for the serious honey bee colony losses produced worldwide in the last decade, either alone or in association with Nosema ceranae. Since this pathogenic effect contradicts the results of the experimental infections originally performed by Langridge and McGhee nearly 40 years ago, we investigated the potential linkage of this protozoan with colony decline under laboratory conditions. Nosema-free and trypanosomatid-free honey bees from three different colonies were experimentally infected with fresh C. mellificae spheroid forms (reference strain ATCC30254), with N. ceranae fresh spores and with both parasites at the same time. Replicate cages were kept at 27 °C and used to analyse survival. C. mellificae spheroid forms did not reduce significantly the survival of the worker bees (64.5% at 30 days post-infection vs. 77.8% for the uninfected bees used as controls; differences were non statistically significant) under these experimental conditions. In contrast, the cages infected with N. ceranae exhibited higher rates of mortality from the 20th day post-infection onwards, irrespective of the presence of C. mellificae, suggesting that the spheroid forms of the latter have no pathological effect on A. mellifera.

International audience; Microbes influence a wide range of host social behaviors and vice versa. So far, however, the mechanisms underpinning these complex interactions remain poorly understood. In social animals, where individuals share microbes and interact around foods, the gut microbiota may have considerable consequences on host social interactions by acting upon the nutritional behavior of individual animals. Here we illustrate how conceptual advances in nutritional ecology can help the study of these processes and allow the formulation of new empirically testable predictions. First, we review key evidence showing that gut microbes influence the nutrition of individual animals, through modifications of their nutritional state and feeding decisions. Next, we describe how these microbial influences and their social consequences can be studied by modelling populations of hosts and their gut microbiota into a single conceptual framework derived from nutritional geometry. Our approach raises new perspectives for the study of holobiont nutrition and will facilitate theoretical and experimental research on the role of the gut microbiota in the mechanisms and evolution of social behavior.

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. This study was supported by funds from the Instituto Nacional de Investigación y Tecnología Agraria (INIA; http://www.inia.es/; grant numbers RTA2013-00042-C10-05 and 06), the Regional Government of Murcia (Fundación Séneca; http://fseneca.es/; grant number 19908/GERM/2015) and the Ministerio de Agricultura, Alimentación y Medio Ambiente (MAGRAMA; Plan Apícola Nacional 2014; http://www.magrama.gob.es). PDR is presently a member and receives support from COST Action FA1307, Sustainable pollination in Europe: joint research on bees and other pollinators, SUPER-B (http://www.cost.eu/COST_Actions/fa/Actions/FA1307) SI

Background Here 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. Results Omnipresent 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. Conclusion The 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. Electronic supplementary material The online version of this article (doi:10.1186/1756-0500-7-649) contains supplementary material, which is available to authorized users.

Nosema ceranae has been found infecting Apis mellifera 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 Apis mellifera (8-16% of the total variance, p

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.

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.

International audience; 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.