Promastigote EPS secretion and haptomonad biofilm formation as evolutionary adaptations of trypanosomatid parasites for colonizing honeybee hosts.

Bees are major pollinators involved in the maintenance of all terrestrial ecosystems. Biotic and abiotic factors placing these insects at risk is a research priority for ecological and agricultural sustainability. Parasites are one of the key players of this global decline and the study of their mechanisms of action is essential to control honeybee colony losses. Trypanosomatid parasites and particularly the Lotmaria passim are widely spread in honeybees, however their lifestyle is poorly understood. In this work, we show how these parasites are able to differentiate into a new parasitic lifestyle: the trypanosomatid biofilms. Using different microscopic techniques, we demonstrated that the secretion of Extracellular Polymeric Substances by free-swimming unicellular promastigote forms is a prerequisite for the generation and adherence of multicellular biofilms to solid surfaces in vitro and in vivo. Moreover, compared to human-infective trypanosomatid parasites our study shows how trypanosomatid parasites of honeybees increases their resistance and thus resilience to drastic changes in environmental conditions such as ultralow temperatures and hypoosmotic shock, which would explain their success thriving within or outside their hosts. These results set up the basis for the understanding of the success of this group of parasites in nature and to unveil the impact of such pathogens in honeybees, a keystones species in most terrestrial ecosystems.

Development of a TaqMan qPCR assay for trypanosomatid multi-species detection and quantification in insects.

BACKGROUND: Trypanosomatid parasites are widely distributed in nature and can have a monoxenous or dixenous life-cycle. These parasites thrive in a wide number of insect orders, some of which have an important economic and environmental value, such as bees. The objective of this study was to develop a robust and sensitive real-time quantitative PCR (qPCR) assay for detecting trypanosomatid parasites in any type 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-tubulin TaqMan assay were tested and validated using field samples of honeybee workers, wild bees, bumblebees and grasshoppers, as well as in the human infective trypanosomatid Leishmania major. RESULTS: The assay showed a detection limit of 1 parasite equivalent/µl and successfully detected trypanosomatids in 10 different hosts belonging to the insect orders Hymenoptera and Orthoptera. The methodology was also tested using honeybee samples from four apiaries (n = 224 worker honeybees) located in the 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 the four different classes of insects ranged from 40.6 up to 1.1 × 108 cell equivalents per host. CONCLUSIONS: These results show that the α-tubulin TaqMan qPCR assay described here is a versatile diagnostic tool for the accurate detection and quantification of trypanosomatids in a wide range of environmental settings.

The gut parasite Nosema ceranae impairs olfactory learning in bumblebees.

Pollinators are exposed to numerous parasites and pathogens when foraging on flowers. These biological stressors may affect critical cognitive abilities required for foraging. Here, we tested whether exposure to Nosema ceranae, one of the most widespread parasites of honey bees also found in wild pollinators, impacts cognition in bumblebees. We investigated different forms of olfactory learning and memory using conditioning of the proboscis extension reflex. Seven days after being exposed to parasite spores, bumblebees showed lower performance in absolute, differential and reversal learning than controls. The consistent observations across different types of olfactory learning indicate a general negative effect of N. ceranae exposure that did not specifically target particular brain areas or neural processes. We discuss the potential mechanisms by which N. ceranae impairs bumblebee cognition and the broader consequences for populations of pollinators.

Artificial Diets Modulate Infection Rates by Nosema ceranae in Bumblebees.

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.

Experimental evidence of harmful effects of Crithidia mellificae and Lotmaria passim on honey bees.

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.

Bumblebees adjust protein and lipid collection rules to the presence of brood.

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.

The levels of natural Nosema spp. infection in Apis mellifera iberiensis brood stages.

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.

Social nutrition: an emerging field in insect science.

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.

Exploring Interactions between the Gut Microbiota and Social Behavior through Nutrition.

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.

A theoretical exploration of dietary collective medication in social insects.

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.

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.

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.

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

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.

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.

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.

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.