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.

Cross-infectivity of honey and bumble bee-associated parasites across three bee families.

Recent declines of wild pollinators and infections in honey, bumble and other bee species have raised concerns about pathogen spillover from managed honey and bumble bees to other pollinators. Parasites of honey and bumble bees include trypanosomatids and microsporidia that often exhibit low host specificity, suggesting potential for spillover to co-occurring bees via shared floral resources. However, experimental tests of trypanosomatid and microsporidial cross-infectivity outside of managed honey and bumble bees are scarce. To characterize potential cross-infectivity of honey and bumble bee-associated parasites, we inoculated three trypanosomatids and one microsporidian into five potential hosts - including four managed species - from the apid, halictid and megachilid bee families. We found evidence of cross-infection by the trypanosomatids Crithidia bombi and C. mellificae, with evidence for replication in 3/5 and 3/4 host species, respectively. These include the first reports of experimental C. bombi infection in Megachile rotundata and Osmia lignaria, and C. mellificae infection in O. lignaria and Halictus ligatus. Although inability to control amounts inoculated in O. lignaria and H. ligatus hindered estimates of parasite replication, our findings suggest a broad host range in these trypanosomatids, and underscore the need to quantify disease-mediated threats of managed social bees to sympatric pollinators.

Flowering plant composition shapes pathogen infection intensity and reproduction in bumble bee colonies.

Pathogens pose significant threats to pollinator health and food security. Pollinators can transmit diseases during foraging, but the consequences of plant species composition for infection is unknown. In agroecosystems, flowering strips or hedgerows are often used to augment pollinator habitat. We used canola as a focal crop in tents and manipulated flowering strip composition using plant species we had previously shown to result in higher or lower bee infection in short-term trials. We also manipulated initial colony infection to assess impacts on foraging behavior. Flowering strips using high-infection plant species nearly doubled bumble bee colony infection intensity compared to low-infection plant species, with intermediate infection in canola-only tents. Both infection treatment and flowering strips reduced visits to canola, but we saw no evidence that infection treatment shifted foraging preferences. Although high-infection flowering strips increased colony infection intensity, colony reproduction was improved with any flowering strips compared to canola alone. Effects of flowering strips on colony reproduction were explained by nectar availability, but effects of flowering strips on infection intensity were not. Thus, flowering strips benefited colony reproduction by adding floral resources, but certain plant species also come with a risk of increased pathogen infection intensity.

Habitat-specific variation in gut microbial communities and pathogen prevalence in bumblebee queens (Bombus terrestris).

Gut microbial communities are critical for the health of many insect species. However, little is known about how gut microbial communities respond to anthropogenic changes and how such changes affect host-pathogen interactions. In this study, we used deep sequencing to investigate and compare the composition of gut microbial communities within the midgut and ileum (both bacteria and fungi) in Bombus terrestris queens collected from natural (forest) and urbanized habitats. Additionally, we investigated whether the variation in gut microbial communities under each habitat affected the prevalence of two important bumblebee pathogens that have recently been associated with Bombus declines (Crithidia bombi and Nosema bombi). Microbial community composition differed strongly among habitat types, both for fungi and bacteria. Fungi were almost exclusively associated with bumblebee queens from the forest habitats, and were not commonly detected in bumblebee queens from the urban sites. Further, gut bacterial communities of urban B. terrestris specimens were strongly dominated by bee-specific core bacteria like Snodgrassella (Betaproteobacteria) and Gilliamella (Gammaproteobacteria), whereas specimens from the forest sites contained a huge fraction of environmental bacteria. Pathogen infection was very low in urban populations and infection by Nosema was only observed in specimens collected from forest habitats. No significant relationship was found between pathogen prevalence and microbial gut diversity. However, there was a significant and negative relationship between prevalence of Nosema and relative abundance of the core resident Snodgrassella, supporting its role in pathogen defense. Overall, our results indicate that land-use change may lead to different microbial gut communities in bumblebees, which may have implications for bumblebee health, survival and overall fitness.

Effects of the floral phytochemical eugenol on parasite evolution and bumble bee infection and preference.

Ecological and evolutionary pressures on hosts and parasites jointly determine infection success. In pollinators, parasite exposure to floral phytochemicals may influence between-host transmission and within-host replication. In the bumble bee parasite Crithidia bombi, strains vary in phytochemical resistance, and resistance increases under in vitro selection, implying that resistance/infectivity trade-offs could maintain intraspecific variation in resistance. We assessed costs and benefits of in vitro selection for resistance to the floral phytochemical eugenol on C. bombi infection in Bombus impatiens fed eugenol-rich and eugenol-free diets. We also assessed infection-induced changes in host preferences for eugenol. In vitro, eugenol-exposed cells initially increased in size, but normalized during adaptation. Selection for eugenol resistance resulted in considerable (55%) but non-significant reductions in infection intensity; bee colony and body size were the strongest predictors of infection. Dietary eugenol did not alter infection, and infected bees preferred eugenol-free over eugenol-containing solutions. Although direct effects of eugenol exposure could influence between-host transmission at flowers, dietary eugenol did not ameliorate infection in bees. Limited within-host benefits of resistance, and possible trade-offs between resistance and infectivity, may relax selection for eugenol resistance and promote inter-strain variation in resistance. However, infection-induced dietary shifts could influence pollinator-mediated selection on floral traits.

The genomes of Crithidia bombi and C. expoeki, common parasites of bumblebees.

Trypanosomatids (Trypanosomatidae, Kinetoplastida) are flagellated protozoa containing many parasites of medical or agricultural importance. Among those, Crithidia bombi and C. expoeki, are common parasites in bumble bees around the world, and phylogenetically close to Leishmania and Leptomonas. They have a simple and direct life cycle with one host, and partially castrate the founding queens greatly reducing their fitness. Here, we report the nuclear genome sequences of one clone of each species, extracted from a field-collected infection. Using a combination of Roche 454 FLX Titanium, Pacific Biosciences PacBio RS, and Illumina GA2 instruments for C. bombi, and PacBio for C. expoeki, we could produce high-quality and well resolved sequences. We find that these genomes are around 32 and 34 MB, with 7,808 and 7,851 annotated genes for C. bombi and C. expoeki, respectively-which is somewhat less than reported from other trypanosomatids, with few introns, and organized in polycistronic units. A large fraction of genes received plausible functional support in comparison primarily with Leishmania and Trypanosoma. Comparing the annotated genes of the two species with those of six other trypanosomatids (C. fasciculata, L. pyrrhocoris, L. seymouri, B. ayalai, L. major, and T. brucei) shows similar gene repertoires and many orthologs. Similar to other trypanosomatids, we also find signs of concerted evolution in genes putatively involved in the interaction with the host, a high degree of synteny between C. bombi and C. expoeki, and considerable overlap with several other species in the set. A total of 86 orthologous gene groups show signatures of positive selection in the branch leading to the two Crithidia under study, mostly of unknown function. As an example, we examined the initiating glycosylation pathway of surface components in C. bombi, finding it deviates from most other eukaryotes and also from other kinetoplastids, which may indicate rapid evolution in the extracellular matrix that is involved in interactions with the host. Bumble bees are important pollinators and Crithidia-infections are suspected to cause substantial selection pressure on their host populations. These newly sequenced genomes provide tools that should help better understand host-parasite interactions in these pollinator pathogens.

Global decline of bumblebees is phylogenetically structured and inversely related to species range size and pathogen incidence.

Conservation biology can profit greatly from incorporating a phylogenetic perspective into analyses of patterns and drivers of species extinction risk. We applied such an approach to analyse patterns of bumblebee (Bombus) decline. We assembled a database representing approximately 43% of the circa 260 globally known species, which included species extinction risk assessments following the International Union fo Conservation of Nature Red List categories and criteria, and information on species traits presumably associated with bumblebee decline. We quantified the strength of phylogenetic signal in decline, range size, tongue length and parasite presence. Overall, about one-third of the assessed bumblebees are declining and declining species are not randomly distributed across the Bombus phylogeny. Susceptible species were over-represented in the subgenus Thoracobombus (approx. 64%) and under-represented in the subgenus Pyrobombus (approx. 6%). Phylogenetic logistic regressions revealed that species with small geographical ranges and those in which none of three internal parasites were reported (i.e. Crithidia bombi, Nosema spp. or Locustacarus buchneri) were particularly vulnerable. Bumblebee evolutionary history will be deeply eroded if most species from threatened clades, particularly those stemming from basal nodes, become finally extinct. The habitat of species with restricted distribution should be protected and the importance of pathogen tolerance/resistance as mechanisms to deal with pathogens needs urgent research.

Larvae act as a transient transmission hub for the prevalent bumblebee parasite Crithidia bombi.

Disease transmission networks are key for understanding parasite epidemiology. Within the social insects, structured contact networks have been suggested to limit the spread of diseases to vulnerable members of their society, such as the queen or brood. However, even these complex social structures do not provide complete protection, as some diseases, which are transmitted by workers during brood care, can still infect the brood. Given the high rate of feeding interactions that occur in a social insect colony, larvae may act as disease transmission hubs. Here we use the bumblebee Bombus terrestris and its parasite Crithidia bombi to determine the role of brood in bumblebee disease transmission networks. Larvae that were artificially inoculated with C. bombi showed no signs of infection seven days after inoculation. However, larvae that received either an artificial inoculation or a contaminated feed from brood-caring workers were able to transmit the parasite to naive workers. These results suggest that the developing brood is a potential route of intracolonial disease transmission and should be included when considering social insect disease transmission networks.

Evolution of resistance to single and combined floral phytochemicals by a bumble bee parasite.

Repeated exposure to inhibitory compounds can drive the evolution of resistance, which weakens chemical defence against antagonists. Floral phytochemicals in nectar and pollen have antimicrobial properties that can ameliorate infection in pollinators, but evolved resistance among parasites could diminish the medicinal efficacy of phytochemicals. However, multicompound blends, which occur in nectar and pollen, present simultaneous chemical challenges that may slow resistance evolution. We assessed evolution of resistance by the common bumble bee gut parasite Crithidia bombi to two floral phytochemicals, singly and combined, over 6 weeks (~100 generations) of chronic exposure. Resistance of C. bombi increased under single and combined phytochemical exposure, without any associated costs of reduced growth under phytochemical-free conditions. After 6 weeks' exposure, phytochemical concentrations that initially inhibited growth by > 50%, and exceeded concentrations in floral nectar, had minimal effects on evolved parasite lines. Unexpectedly, the phytochemical combination did not impede resistance evolution compared to single compounds. These results demonstrate that repeated phytochemical exposure, which could occur in homogeneous floral landscapes or with therapeutic phytochemical treatment of managed hives, can cause rapid evolution of resistance in pollinator parasites. We discuss possible explanations for submaximal phytochemical resistance in natural populations. Evolved resistance could diminish the antiparasitic value of phytochemical ingestion, weakening an important natural defence against infection.

Bumble bee parasite strains vary in resistance to phytochemicals.

Nectar and pollen contain diverse phytochemicals that can reduce disease in pollinators. However, prior studies showed variable effects of nectar chemicals on infection, which could reflect variable phytochemical resistance among parasite strains. Inter-strain variation in resistance could influence evolutionary interactions between plants, pollinators, and pollinator disease, but testing direct effects of phytochemicals on parasites requires elimination of variation between bees. Using cell cultures of the bumble bee parasite Crithidia bombi, we determined (1) growth-inhibiting effects of nine floral phytochemicals and (2) variation in phytochemical resistance among four parasite strains. C. bombi growth was unaffected by naturally occurring concentrations of the known antitrypanosomal phenolics gallic acid, caffeic acid, and chlorogenic acid. However, C. bombi growth was inhibited by anabasine, eugenol, and thymol. Strains varied >3-fold in phytochemical resistance, suggesting that selection for phytochemical resistance could drive parasite evolution. Inhibitory concentrations of thymol (4.53-22.2 ppm) were similar to concentrations in Thymus vulgaris nectar (mean 5.2 ppm). Exposure of C. bombi to naturally occurring levels of phytochemicals-either within bees or during parasite transmission via flowers-could influence infection in nature. Flowers that produce antiparasitic phytochemicals, including thymol, could potentially reduce infection in Bombus populations, thereby counteracting a possible contributor to pollinator decline.

Testing Dose-Dependent Effects of the Nectar Alkaloid Anabasine on Trypanosome Parasite Loads in Adult Bumble Bees.

The impact of consuming biologically active compounds is often dose-dependent, where small quantities can be medicinal while larger doses are toxic. The consumption of plant secondary compounds can be toxic to herbivores in large doses, but can also improve survival in parasitized herbivores. In addition, recent studies have found that consuming nectar secondary compounds may decrease parasite loads in pollinators. However, the effect of compound dose on bee survival and parasite loads has not been assessed. To determine how secondary compound consumption affects survival and pathogen load in Bombus impatiens, we manipulated the presence of a common gut parasite, Crithidia bombi, and dietary concentration of anabasine, a nectar alkaloid produced by Nicotiana spp. using four concentrations naturally observed in floral nectar. We hypothesized that increased consumption of secondary compounds at concentrations found in nature would decrease survival of uninfected bees, but improve survival and ameliorate parasite loads in infected bees. We found medicinal effects of anabasine in infected bees; the high-anabasine diet decreased parasite loads and increased the probability of clearing the infection entirely. However, survival time was not affected by any level of anabasine concentration, or by interactive effects of anabasine concentration and infection. Crithidia infection reduced survival time by more than two days, but this effect was not significant. Our results support a medicinal role for anabasine at the highest concentration; moreover, we found no evidence for a survival-related cost of anabasine consumption across the concentration range found in nectar. Our results suggest that consuming anabasine at the higher levels of the natural range could reduce or clear pathogen loads without incurring costs for healthy bees.

Comprehensive bee pathogen screening in Belgium reveals Crithidia mellificae as a new contributory factor to winter mortality.

Since the last decade, unusually high honey bee colony losses have been reported mainly in North-America and Europe. Here, we report on a comprehensive bee pathogen screening in Belgium covering 363 bee colonies that were screened for 18 known disease-causing pathogens and correlate their incidence in summer with subsequent winter mortality. Our analyses demonstrate that, in addition to Varroa destructor, the presence of the trypanosomatid parasite Crithidia mellificae and the microsporidian parasite Nosema ceranae in summer are also predictive markers of winter mortality, with a negative synergy being observed between the two in terms of their effects on colony mortality. Furthermore, we document the first occurrence of a parasitizing phorid fly in Europe, identify a new fourth strain of Lake Sinai Virus (LSV), and confirm the presence of other little reported pathogens such as Apicystis bombi, Aphid Lethal Paralysis Virus (ALPV), Spiroplasma apis, Spiroplasma melliferum and Varroa destructor Macula-like Virus (VdMLV). Finally, we provide evidence that ALPV and VdMLV replicate in honey bees and show that viruses of the LSV complex and Black Queen Cell Virus tend to non-randomly co-occur together. We also noticed a significant correlation between the number of pathogen species and colony losses. Overall, our results contribute significantly to our understanding of honey bee diseases and the likely causes of their current decline in Europe.

Barrier immune effectors are maintained during transition from nurse to forager in the honey bee.

Foragers facilitate horizontal pathogen transmission in honey bee colonies, yet their systemic immune function wanes during transition to this life stage. In general, the insect immune system can be categorized into mechanisms operating at both the barrier epithelial surfaces and at the systemic level. As proposed by the intergenerational transfer theory of aging, such immunosenescence may result from changes in group resource allocation. Yet, the relative influence of pathogen transmission and resource allocation on immune function in bees from different stages has not been examined in the context of barrier immunity. We find that expression levels of antimicrobial peptides (AMPs) in honey bee barrier epithelia of the digestive tract do not follow a life stage-dependent decrease. In addition, correlation of AMP transcript abundance with microbe levels reveals a number of microbe-associated changes in AMPs levels that are equivalent between nurses and foragers. These results favor a model in which barrier effectors are maintained in foragers as a first line of defense, while systemic immune effectors are dismantled to optimize hive-level resources. These findings have important implications for our understanding of immunosenescence in honey bees and other social insects.

Probing mixed-genotype infections II: high multiplicity in natural infections of the trypanosomatid, Crithidia bombi, in its host, Bombus spp.

Mixed-genotype infections have major consequences for many essential elements of host-parasite interactions. With genetic exchange between co-infecting parasite genotypes increased diversity among parasite offspring and the emergence of novel genotypes from infected hosts is possible. We here investigated mixed- genotype infections using the host, Bombus spp. and its trypanosome parasite Crithidia bombi as our study case. The natural infections of C. bombi were genotyped with a novel method for a representative sample of workers and spring queens in Switzerland. We found that around 60% of all infected hosts showed mixed-genotype infections with an average of 2.47±0.22 (S.E.) and 3.65±1.02 genotypes per worker or queen, respectively. Queens, however, harboured up to 29 different genotypes. Based on the genotypes of co-infecting strains, these could be putatively assigned to either 'primary' and 'derived' genotypes - the latter resulting from genetic exchange among the primary genotypes. High genetic relatedness among co-infecting derived but not primary genotypes supported this scenario. Co-infection in queens seems to be a major driver for the diversity of genotypes circulating in host populations.

Gut microbiota instead of host genotype drive the specificity in the interaction of a natural host-parasite system.

Specific interactions between parasite genotypes and host genotypes (G(p) × G(h)) are commonly found in invertebrate systems, but are largely lacking a mechanistic explanation. The genotype of invertebrate hosts can be complemented by the genomes of microorganisms living on or within the host ('microbiota'). We investigated whether the bacterial gut microbiota of bumble bees (Bombus terrestris) can account for the specificity of interactions between individuals from different colonies (previously taken as host genotype proxy) and genotypes of the parasite Crithidia bombi. For this, we transplanted the microbiota between individuals of six colonies. Both the general infection load and the specific success of different C. bombi genotypes were mostly driven by the microbiota, rather than by worker genotype. Variation in gut microbiota can therefore be responsible for specific immune phenotypes and the evolution of gut parasites may be driven by interactions with 'microbiota types' as well as with host genotypes.

Host modulation of parasite competition in multiple infections.

Parasite diversity is a constant challenge to host immune systems and has important clinical implications, but factors underpinning its emergence and maintenance are still poorly understood. Hosts typically harbour multiple parasite genotypes that share both host resources and immune responses. Parasite diversity is thus shaped not only by resource competition between co-infecting parasites but also by host-driven immune-mediated competition. We investigated these effects in an insect-trypanosome system, combining in vivo and in vitro single and double inoculations. In vivo, a non-pathogenic, general immune challenge was used to manipulate host immune condition and resulted in a reduced ability of hosts to defend against a subsequent exposure to the trypanosome parasites, illustrating the costs of immune activation. The associated increase in available host space benefited the weaker parasite strains of each pair as much as the otherwise more competitive strains, resulting in more frequent multiple infections in immune-challenged hosts. In vitro assays showed that in the absence of a host, overall parasite diversity was minimal because the outcome of competition was virtually fixed and resulted in strain extinction. Altogether, this shows that parasite competition is largely host-mediated and suggests a role for host immune condition in the maintenance of parasite diversity.

Robustness of the outcome of adult bumblebee infection with a trypanosome parasite after varied parasite exposures during larval development.

The outcome of defence by the invertebrate immunity has recently been shown to be more complex than previously thought. In particular, the outcome is affected by biotic and abiotic environmental variation, host genotype, parasite genotype and their interaction. Knowledge of conditions under which environmental variation affects the outcome of an infection is one important question that relates to this complexity. We here use the model system of the bumblebee, Bombus terrestris, infected by the trypanosome, Crithidia bombi, combined with a split-colony design to test the influence of the parasite environment during larval rearing on adult resistance. We find that genotype-specific interactions are maintained and adult resistance is not influenced. This demonstrates that environmental dependence of bumblebee-trypanosome interactions is not ubiquitous, and yet unknown constraints will maintain standard coevolutionary dynamics under such environmental deviations.

Interspecific geographic distribution and variation of the pathogens Nosema bombi and Crithidia species in United States bumble bee populations.

Several bumble bee (Bombus) species in North America have undergone range reductions and rapid declines in relative abundance. Pathogens have been suggested as causal factors, however, baseline data on pathogen distributions in a large number of bumble bee species have not been available to test this hypothesis. In a nationwide survey of the US, nearly 10,000 specimens of 36 bumble bee species collected at 284 sites were evaluated for the presence and prevalence of two known Bombus pathogens, the microsporidium Nosema bombi and trypanosomes in the genus Crithidia. Prevalence of Crithidia was ≤10% for all host species examined but was recorded from 21% of surveyed sites. Crithidia was isolated from 15 of the 36 Bombus species screened, and were most commonly recovered from Bombus bifarius, Bombus bimaculatus, Bombus impatiens and Bombus mixtus. Nosema bombi was isolated from 22 of the 36 US Bombus species collected. Only one species with more than 50 sampled bees, Bombus appositus, was free of the pathogen; whereas, prevalence was highest in Bombus occidentalis and Bombus pensylvanicus, two species that are reportedly undergoing population declines in North America. A variant of a tetranucleotide repeat in the internal transcribed spacer (ITS) of the N. bombi rRNA gene, thus far not reported from European isolates, was isolated from ten US Bombus hosts, appearing in varying ratios in different host species. Given the genetic similarity of the rRNA gene of N. bombi sampled in Europe and North America to date, the presence of a unique isolate in US bumble could reveal one or more native North American strains and indicate that N. bombi is enzootic across the Holarctic Region, exhibiting some genetic isolation.

Natural variation in the genetic architecture of a host-parasite interaction in the bumblebee Bombus terrestris.

The genetic architecture of fitness-relevant traits in natural populations is a topic that has remained almost untouched by quantitative genetics. Given the importance of parasitism for the host's fitness, we used QTL mapping to study the genetic architecture of traits relevant for host-parasite interactions in the trypanosome parasite, Crithidia bombi and its host, Bombus terrestris. The three traits analysed were the parasite's infection intensity, the strength of the general immune response (measured as the encapsulation of a novel antigen) and body size. The genetic architecture of these traits was examined in three natural, unmanipulated mapping populations of B. terrestris. Our results indicate that the intracolonial phenotypic variation of all three traits is based on a network of QTLs and epistatic interactions. While these networks are similar between mapping populations in complexity and number of QTLs, as well as in their epistatic interactions, the variability in the position of QTL and the interacting loci was high. Only one QTL for body size was plausibly found in at least two populations. QTLs for encapsulation and Crithidia infection intensity were located on the same linkage groups.

Within-host dynamics of an intestinal pathogen of bumble bees.

The success of a pathogen depends not only on its transmission to new hosts, but also on its ability to colonize and persist within its current host. Studies of within-host dynamics have focused on only a few diseases of humans, whereas little is known about the factors that influence pathogen populations as they develop inside non-human hosts. Here, we investigate pathogen dynamics occurring within bumble bees (Bombus impatiens) infected by the gut trypanosome Crithidia bombi. Infection by C. bombi showed several features characteristic of vertebrate diseases, including a rapid initial increase in infection intensity, marked oscillations in parasitaemia, and the stimulation of a systemic immune response in infected bees. Within-host dynamics generated substantial variation in the infectiousness and flower-visiting behaviour of bumble bees. Changes in bee foraging that arise from infection may influence the probability of C. bombi transmission between bees at flowers.