Genetic modification of the bee parasite Crithidia bombi for improved visualization and protein localization.

Crithidia bombi is a trypanosomatid parasite that infects several species of bumble bees (Bombus spp.), by adhering to their intestinal tract. Crithidia bombi infection impairs learning and reduces survival of workers and the fitness of overwintering queens. Although there is extensive research on the ecology of this host-pathogen system, we understand far less about the mechanisms that mediate internal infection dynamics. Crithidia bombi infects hosts by attaching to the hindgut via the flagellum, and one previous study found that a nectar secondary compound removed the flagellum, preventing attachment. However, approaches that allow more detailed observation of parasite attachment and growth would allow us to better understand factors mediating this host-pathogen relationship. We established techniques for genetic manipulation and visualization of cultured C. bombi. Using constructs established for Crithidia fasciculata, we successfully generated C. bombi cells expressing ectopic fluorescent transgenes using two different selectable markers. To our knowledge, this is the first genetic modification of this species. We also introduced constructs that label the mitochondrion and nucleus of the parasite, showing that subcellular targeting signals can function across parasite species to highlight specific organelles. Finally, we visualized fluorescently tagged parasites in vitro in both their swimming and attached forms, and in vivo in bumble bee (Bombus impatiens) hosts. Expanding our cell and molecular toolkit for C. bombi will help us better understand how factors such as host diet, immune system, and physiology mediate outcomes of infection by these common parasites.

Differential bumble bee gene expression associated with pathogen infection and pollen diet.

BACKGROUND: Diet and parasitism can have powerful effects on host gene expression. However, how specific dietary components affect host gene expression that could feed back to affect parasitism is relatively unexplored in many wild species. Recently, it was discovered that consumption of sunflower (Helianthus annuus) pollen reduced severity of gut protozoan pathogen Crithidia bombi infection in Bombus impatiens bumble bees. Despite the dramatic and consistent medicinal effect of sunflower pollen, very little is known about the mechanism(s) underlying this effect. However, sunflower pollen extract increases rather than suppresses C. bombi growth in vitro, suggesting that sunflower pollen reduces C. bombi infection indirectly via changes in the host. Here, we analyzed whole transcriptomes of B. impatiens workers to characterize the physiological response to sunflower pollen consumption and C. bombi infection to isolate the mechanisms underlying the medicinal effect. B. impatiens workers were inoculated with either C. bombi cells (infected) or a sham control (un-infected) and fed either sunflower or wildflower pollen ad libitum. Whole abdominal gene expression profiles were then sequenced with Illumina NextSeq 500 technology. RESULTS: Among infected bees, sunflower pollen upregulated immune transcripts, including the anti-microbial peptide hymenoptaecin, Toll receptors and serine proteases. In both infected and un-infected bees, sunflower pollen upregulated putative detoxification transcripts and transcripts associated with the repair and maintenance of gut epithelial cells. Among wildflower-fed bees, infected bees downregulated immune transcripts associated with phagocytosis and the phenoloxidase cascade. CONCLUSIONS: Taken together, these results indicate dissimilar immune responses between sunflower- and wildflower-fed bumble bees infected with C. bombi, a response to physical damage to gut epithelial cells caused by sunflower pollen, and a strong detoxification response to sunflower pollen consumption. Identifying host responses that drive the medicinal effect of sunflower pollen in infected bumble bees may broaden our understanding of plant-pollinator interactions and provide opportunities for effective management of bee pathogens.

Sunflower plantings reduce a common gut pathogen and increase queen production in common eastern bumblebee colonies.

Community diversity can reduce the prevalence and spread of disease, but certain species may play a disproportionate role in diluting or amplifying pathogens. Flowers act as both sources of nutrition and sites of pathogen transmission, but the effects of specific plant species in shaping bee disease dynamics are not well understood. We evaluated whether plantings of sunflower (Helianthus annuus), whose pollen reduces infection by some pathogens when fed to bees in captivity, lowered pathogen levels and increased reproduction in free-foraging bumblebee colonies (Bombus impatiens). Sunflower abundance reduced the prevalence of a common gut pathogen, Crithidia bombi, and reduced infection intensity, with an order of magnitude lower infection intensity at high sunflower sites compared with sites with little to no sunflower. Sunflower abundance was also positively associated with greater queen production in colonies. Sunflower did not affect prevalence of other detected pathogens. This work demonstrates that a single plant species can drive disease dynamics in foraging B. impatiens, and that sunflower plantings can be used as a tool for mitigating a prevalent pathogen while also increasing reproduction of an agriculturally important bee species.

A comparison of the chemiluminescence immunoassay and Crithidia luciliae immunofluorescence test in detecting anti-dsDNA antibodies and assessing the activity of systemic lupus erythematosus.

OBJECTIVES: This study aimed to compare the YHLO chemiluminescence immunoassay (CLIA) with the Crithidia luciliae immunofluorescence test (CLIFT) to detect anti-dsDNA antibodies and its correlation with disease activity in systemic lupus erythematosus (SLE). METHOD: In total, 208 patients diagnosed with SLE, 110 other autoimmune patients, 70 infectious disorders patients, and 105 healthy people were enrolled in this study. Serum samples were tested using CLIA in a YHLO chemiluminescence system and CLIFT. RESULTS: The overall agreement between YHLO CLIA and CLIFT was 76.9% (160/208), with a moderate correlation (kappa = 0.530, p < 0.001). The sensitivity of YHLO CLIA and CLIFT were 58.2% and 55.3%, respectively. The specificity of YHLO CLIA and CLIFT were 95.1% and 99.3%, respectively. The sensitivity of YHLO CLIA was increased to 66.8% with a specificity of 93.6% when the cut-off value was set at 24 IU/mL. Spearman's correlation coefficient between the quantitative results of YHLO CLIA and the titers of CLIFT was 0.59 (p < .01). A significant correlation was found between the anti-dsDNA results detected by YHLO CLIA and the SLE Disease Activity Index 2000 (SLEDAI-2K). Spearman's correlation coefficient between YHLO CLIA and SLEDAI-2K (r = 0.66, p < .01) was higher than that of CLIFT (r = 0.60, p < .01). CONCLUSIONS: Good correlation and agreement were found between YHLO CLIA and CLIFT. In addition, there was a significant correlation between YHLO CLIA and the SLE Disease Activity Index, which was superior to that of CLIFT. The YHLO chemiluminescence system is recommended for the assessment of disease activity.

Frequent Parasitism of Apis mellifera by Trypanosomatids in Geographically Isolated Areas with Restricted Beekeeping Movements.

Trypanosomatids form a group of high prevalence protozoa that parasitise honey bees, with Lotmaria passim as the predominant species worldwide. However, the knowledge about the ecology of trypanosomatids in isolated areas is limited. The Portuguese archipelagos of Madeira and Azores provide an interesting setting to investigate these parasites because of their geographic isolation, and because they harbour honey bee populations devoid of two major enemies: Varroa destructor and Nosema ceranae. Hence, a total of 661 honey bee colonies from Madeira and the Azores were analysed using different molecular techniques, through which we found a high prevalence of trypanosomatids despite the isolation of these islands. L. passim was the predominant species and, in most colonies, was the only one found, even on islands free of V. destructor and/or N. ceranae with severe restrictions on colony movements to prevent the spread of them. However, islands with V. destructor had a significantly higher prevalence of L. passim and, conversely, islands with N. ceranae did not shown any significant correlation with the trypanosomatid. Crithidia bombi was detected in Madeira and on three islands of the Azores, almost always coincident with L. passim. By contrast, Crithidia mellificae was not detected in any sample. A high-throughput sequencing analysis distinguished two main haplotypes of L. passim, which accounted for 98% of the total sequence reads. This work suggests that L. passim and C. bombi are parasites that have been associated with honey bees predating the spread of V. destructor and N. ceranae.

Gut Transplants from Bees Fed an Antipathogenic Pollen Diet Do Not Confer Pathogen Resistance to Recipients.

Pollinators are threatened by diverse stressors, including microbial pathogens such as Crithidia bombi. Consuming sunflower pollen dramatically reduces C. bombi infection in the bumble bee Bombus impatiens, but the mechanism behind this medicinal effect is unclear. We asked whether diet mediates resistance to C. bombi through changes in the gut microbiome. We hypothesized that sunflower pollen changes the gut microbiome, which in turn reduces Crithidia infection. To test this, we performed a gut transplant experiment. We fed donor bees either a sunflower pollen treatment or buckwheat pollen as a control treatment and then inoculated recipient bees with homogenized guts from either sunflower-fed or buckwheat-fed donor bees. All recipient bees were then fed a wildflower pollen diet. Two days after the transplant, we infected recipients with C. bombi, and 2 days later, we provided another donor gut transplant. To quantify infection, we performed both fecal screens and dissections of the recipient bees. We found no significant differences in C. bombi infection intensity or presence between bees that received sunflower-fed microbiomes versus buckwheat-fed microbiomes. This suggests that sunflower pollen's effects on pathogen resistance are not mediated by gut microbiota.

Methods matter: the influence of method on infection estimates of the bumblebee parasite Crithidia bombi.

The bumblebee gut parasite, Crithidia bombi, is widespread and prevalent in the field. Its interaction with Bombus spp. is a well-established epidemiological model. It is spread faecal-orally between colonies via the shared use of flowers when foraging. Accurately measuring the level of infection in bumblebees is important for assessing its distribution in the field, and also when conducting epidemiological experiments. Studies generally use 1 of 2 methods for measuring infection. One approach measures infection in faeces whereas the other method measures infection in guts. We tested whether the method of measuring infection affected the estimation of infection. Bumblebees were inoculated with a standardized inoculum and infection was measured 1 week later using either the faecal or gut method. We found that when the gut method was used to measure infection intensity estimates were significantly different to and approximately double those from the faecal method. These results have implications for the interpretation of previous study results and for the planning of future studies. Given the importance of bumblebees as pollinators, the impact of C. bombi on bumblebee health, and its use as an epidemiological model, we call on researchers to move towards consistent quantification of infections to enable future comparisons and meta-analyses of studies.

A case study of the diet-microbiota-parasite interplay in bumble bees.

AIMS: Diets and parasites influence the gut bacterial symbionts of bumble bees, but potential interactive effects remain overlooked. The main objective of this study was to assess the isolated and interactive effects of sunflower pollen, its phenolamides, and the widespread trypanosomatid Crithidia sp. on the gut bacterial symbionts of Bombus terrestris males. METHODS AND RESULTS: Bumble bee males emerged in microcolonies fed on either (i) willow pollen (control), (ii) sunflower pollen, or (iii) willow pollen spiked with phenolamide extracts from sunflower pollen. These microcolonies were infected by Crithidia sp. or were pathogen-free. Using 16S rRNA amplicon sequencing (V3-V4 region), we observed a significant alteration of the beta diversity but not of the alpha diversity in the gut microbial communities of males fed on sunflower pollen compared to males fed on control pollen. Similarly, infection by the gut parasite Crithidia sp. altered the beta diversity but not the alpha diversity in the gut microbial communities of males, irrespective of the diet. By contrast, we did not observe any significant alteration of the beta or alpha diversity in the gut microbial communities of males fed on phenolamide-enriched pollen compared to males fed on control pollen. Changes in the beta diversity indicate significant dissimilarities of the bacterial taxa between the treatment groups, while the lack of difference in alpha diversity demonstrates no significant changes within each treatment group. CONCLUSIONS: Bumble bees harbour consistent gut microbiota worldwide, but our results suggest that the gut bacterial communities of bumble bees are somewhat shaped by their diets and gut parasites as well as by the interaction of these two factors. This study confirms that bumble bees are suitable biological surrogates to assess the effect of diet and parasite infections on gut microbial communities.

Minimal sharing of nosematid and trypanosomatid parasites between honey bees and other bees, but extensive sharing of Crithidia between bumble and mason bees.

We document gut parasites in co-occurring Apis, Bombus, and Osmia spp. in the Northern Virginia region, USA. Trypanosomatidea occurred in sixty percent of specimens and 13% carried Nosematidae. We found strong host partitioning: Lotmaria passim and Vairimorpha (Nosema) ceranae predominated in Apis, and Crithidia bombi and V. bombi in Bombus. We did not detect pathogen spread from Apis to Bombus but did detect sharing of C. bombi between Bombus and Osmia, higher parasite levels in Apis at sites with apiaries, and clustering of Vairimopha infection. Given the presence of C. bombi in Osmia, we suggest disease sharing across taxa be monitored.

Trypanosomatids in honey bee colonies in Spain: A new specific qPCR method for specific quantification of Lotmaria passim, Crithidia mellificae and Crithidia bombi.

Bee trypanosomatids have not been widely studied due to the original belief that these organisms were not pathogenic to honey bees. However, trypanosomatids have been linked to increased winter mortality in honey bee colonies in recent years and it has been shown that these pathogens can shorten a honey bee worker's lifespan in laboratory conditions. These studies found that this mortality corresponded to dose-dependent infection. Although Lotmaria passim is the most prevalent species worldwide, the natural load in colonies remains poorly investigated. Here we describe a new highly specific and sensitive qPCR method that allows the differentiation and quantification of the parasitic load of each of the three most common trypanosomatid species described to date in honey bee colonies: L. passim, Crithidia mellificae, and Crithidia bombi. We have used this new method to analyze honey bee colonies in central Spain and confirm that L. passim is the most common species and the one with higher parasitic loads in the colonies, which increased over the years, being higher in spring than in autumn. Crithidia mellificae was present along the study, with the highest prevalence in autumn 2019 and lately it was only found in non-quantifiable loads. Crithidia bombi was not detected in any of the colonies analyzed.

RNA viruses of Crithidia bombi, a parasite of bumblebees.

Leishbuviridae (Bunyavirales) are a diverse monophyletic group of negative-sense single-stranded RNA virus infecting parasitic flagellates of the family Trypanosomatidae. The presence of RNA viruses in trypanosomatids can influence the virulence of the latter. Here, we performed a screening for viruses in Crithidia bombi - a common parasite of important pollinators Bombus spp. (bumblebees) that negatively affects its host in stressful conditions. The majority (8/10) of C. bombi isolates collected in Europe and North America were positive for a virus that we named Crithidia bombi leishbuvirus 1 with high conservation of amino acid sequences between isolates. The results of our comparative phylogenetic analyses of the trypanosomatids and their viruses suggest that the high mobility of bumblebees and frequent coinfections by different strains of C. bombi determine an extensive viral exchange between the latter.

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.

Determination of specific autoantibodies in patients with systemic lupus erythematosus by Line immunoassay, ELISA and CLIF assay.

BACKGROUND: Detection of specific antinuclear-antibodies is very importance in term of diagnosis, prognosis and management of patients with systemic lupus erythematosus (SLE). To date, Line immunoassay (LIA), enzyme-linked immunosorbent assay (ELISA) and Crithidia luciliae indirect immunofluorescence (CLIF) assay are commonly used for detection of specific antinuclear-antibodies. OBJECTIVE: To determine the performance of LIA, ELISA and CLIF for the detection of anti-double-stranded DNA (dsDNA), anti-nucleosome, and anti-extractable nuclear antigens (ENA) antibodies in patients with SLE. METHODS: A total 100 sera from 50 patients with SLE, 25 patients with disease control and 25 healthy control subjects were tested for anti-dsDNA, anti-nucleosome, and anti-ENA antibodies by LIA, ELISA, and CLIF assay. Agreement and diagnostic performance of each assay were analyzed using Cohen's kappa coefficient and receiver operating characteristic curve analysis. RESULTS: For the detection of anti-dsDNA antibody, ELISA had a substantial agreement with CLIF assay (? = 0.74) but LIA had a fair agreement with ELISA and CLIF assay (? = 0.37, and 0.35 respectively). For the detection of anti-nucleosome, anti-nRNP/Sm, anti-Sm, anti-SSA, and anti-SSB antibodies, LIA had a substantial to perfect agreement with ELISA (? = 0.64, 0.78, 0.68, 0.91, and 0.74, respectively). Anti-dsDNA-NcX ELISA and anti-dsDNA CLIF assay had equally diagnostic performance (sensitivity, 66% vs. 68%, and specificity, 96% vs. 94%, respectively) whereas, anti-dsDNA LIA has low sensitivity (22%) but high specificity (100%). CONCLUSIONS: LIA, ELISA, and CLIF demonstrated comparable performance for the detection of specific antinuclear-antibodies. However, there were some discrepancy between assays particularly in the detection of anti-dsDNA antibody.

Sunflower pollen reduces a gut pathogen in the model bee species, Bombus impatiens, but has weaker effects in three wild congeners.

Commercial bumblebees have become popular models to understand stressors and solutions for pollinator health, but few studies test whether results translate to other pollinators. Consuming sunflower pollen dramatically reduces infection by the gut parasite Crithidia bombi in commercially reared Bombus impatiens. We assessed the effect of sunflower pollen on infection in wild B. impatiens, Bombus griseocollis, Bombus bimaculatus and Bombus vagans. We also asked how pollen diet (50% sunflower pollen versus wildflower pollen) and infection (yes/no) affected performance in wild B. impatiens microcolonies. Compared to controls, sunflower pollen dramatically reduced Crithidia infection in commercial and wild B. impatiens, had similar but less dramatic effects in B. bimaculatus and B. vagans, and no effect in B. griseocollis. Bombus impatiens, B. bimaculatus and B. vagans are in the same subgenus, suggesting that responses to sunflower pollen may be phylogenetically conserved. In microcolonies, 50% sunflower pollen reduced infection compared to wildflower pollen, but also reduced reproduction. Sunflower pollen could control Crithidia infections in B. impatiens and potentially close relatives, but may hinder reproduction if other resources are scarce. We caution that research using managed bee species, such as B. impatiens, be interpreted carefully as findings may not relate to all bee species.

Bee Trypanosomatids: First Steps in the Analysis of the Genetic Variation and Population Structure of Lotmaria passim, Crithidia bombi and Crithidia mellificae.

Trypanosomatids are among the most prevalent parasites in bees but, despite the fact that their impact on the colonies can be quite important and that their infectivity may potentially depend on their genotypes, little is known about the population diversity of these pathogens. Here we cloned and sequenced three non-repetitive single copy loci (DNA topoisomerase II, glyceraldehyde-3-phosphate dehydrogenase and RNA polymerase II large subunit, RPB1) to produce new genetic data from Crithidia bombi, C. mellificae and Lotmaria passim isolated from honeybees and bumblebees. These were analysed by applying population genetic tools in order to quantify and compare their variability within and between species, and to obtain information on their demography and population structure. The general pattern for the three species was that (1) they were subject to the action of purifying selection on nonsynonymous variants, (2) the levels of within species diversity were similar irrespective of the host, (3) there was evidence of recombination among haplotypes and (4) they showed no haplotype structuring according to the host. C. bombi exhibited the lowest levels of synonymous variation (πS= 0.06 ± 0.04 %) - and a mutation frequency distribution compatible with a population expansion after a bottleneck - that contrasted with the extensive polymorphism displayed by C. mellificae (πS= 2.24 ± 1.00 %), which likely has a more ancient origin. L. passim showed intermediate values (πS= 0.40 ± 0.28 %) and an excess of variants a low frequencies probably linked to the spread of this species to new geographical areas.

Effects of an alternative host on the prevalence and intensity of infection of a bumble bee parasite.

Several bee parasites are transmitted through flowers, and some of them can infect multiple host species. Given the shared use of flowers by bee species, parasites can potentially encounter multiple host species, which could affect the evolution of parasite virulence. We used the trypanosomatid parasite Crithidia bombi and its host, the common eastern bumble bee (Bombus impatiens), to explore the effect of infecting an alternative host, the alfalfa leaf-cutter bee (Megachile rotundata), on parasite infectivity and ability to replicate. We conducted a serial passage experiment on primary and alternative hosts, assessing infectivity and intensity of infection during five passes. Parasite cells from each pass through the alternative host were also used to infect a group of primary hosts. We found that serial passes through the alternative host increased infectivity, but there was no effect on intensity of infection. Interestingly, both the probability and intensity of infection on the primary host increased after serial passage through the alternative host. This increase in intensity of infection could be due to maladaptation after selection of new C. bombi strains has occurred in the alternative host. This study suggests that host switching has the potential to affect the adaptation of bee parasites to their hosts.

Antiparasitic effects of three floral volatiles on trypanosomatid infection in honey bees.

Trypanosomatid gut parasites are common in pollinators and costly for social bees. The recently described honey bee trypanosomatid Lotmaria passim is widespread, abundant, and correlated with colony losses in some studies. The potential for amelioration of infection by antimicrobial plant compounds has been thoroughly studied for closely related trypanosomatids of humans and is an area of active research in bumble bees, but remains relatively unexplored in honey bees. We recently identified several floral volatiles that inhibited growth of L. passim in vitro. Here, we tested the dose-dependent effects of four such compounds on infection, mortality, and food consumption in parasite-inoculated honey bees. We found that diets containing the monoterpenoid carvacrol and the phenylpropanoids cinnamaldehyde and eugenol at > 10-fold the inhibitory concentrations for cell cultures reduced infection, with parasite numbers decreased by > 90 % for carvacrol and cinnamaldehyde and > 99 % for eugenol; effects of the carvacrol isomer thymol were non-significant. However, both carvacrol and eugenol also reduced bee survival, whereas parasite inoculation did not, indicating costs of phytochemical exposure that could exceed those of infection itself. To our knowledge, this is the first controlled screening of phytochemicals for effects on honey bee trypanosomatid infection, identifying potential treatments for managed bees afflicted with a newly characterized, cosmopolitan intestinal parasite.

Wide diversity of parasites in Bombus terrestris (Linnaeus, 1758) revealed by a high-throughput sequencing approach.

Assessing the extent of parasite diversity requires the application of appropriate molecular tools, especially given the growing evidence of multiple parasite co-occurrence. Here, we compared the performance of a next-generation sequencing technology (Ion PGM ™ System) in 12 Bombus terrestris specimens that were PCR-identified as positive for trypanosomatids (Leishmaniinae) in a previous study. These bumblebees were also screened for the occurrence of Nosematidae and Neogregarinorida parasites using both classical protocols (either specific PCR amplification or amplification with broad-range primers plus Sanger sequencing) and Ion PGM sequencing. The latter revealed higher parasite diversity within individuals, especially among Leishmaniinae (which were present as a combination of Lotmaria passim, Crithidia mellificae and Crithidia bombi), and the occurrence of taxa never reported in these hosts: Crithidia acanthocephali and a novel neogregarinorida species. Furthermore, the complementary results produced by the different sets of primers highlighted the convenience of using multiple markers to minimize the chance of some target organisms going unnoticed. Altogether, the deep sequencing methodology offered a more comprehensive way to investigate parasite diversity than the usual identification methods and provided new insights whose importance for bumblebee health should be further analysed.

Hot and sour: parasite adaptations to honeybee body temperature and pH.

Host temperature and gut chemistry can shape resistance to parasite infection. Heat and acidity can limit trypanosomatid infection in warm-blooded hosts and could shape infection resistance in insects as well. The colony-level endothermy and acidic guts of social bees provide unique opportunities to study how temperature and acidity shape insect-parasite associations. We compared temperature and pH tolerance between three trypanosomatid parasites from social bees and a related trypanosomatid from poikilothermic mosquitoes, which have alkaline guts. Relative to the mosquito parasites, all three bee parasites had higher heat tolerance that reflected body temperatures of hosts. Heat tolerance of the honeybee parasite Crithidia mellificae was exceptional for its genus, implicating honeybee endothermy as a plausible filter of parasite establishment. The lesser heat tolerance of the emerging Lotmaria passim suggests possible spillover from a less endothermic host. Whereas both honeybee parasites tolerated the acidic pH found in bee intestines, mosquito parasites tolerated the alkaline conditions found in mosquito midguts, suggesting that both gut pH and temperature could structure host-parasite specificity. Elucidating how host temperature and gut pH affect infection-and corresponding parasite adaptations to these factors-could help explain trypanosomatids' distribution among insects and invasion of mammals.

Crithidia bombi can infect two solitary bee species while host survivorship depends on diet.

Pathogens and lack of floral resources interactively impair global pollinator health. However, epidemiological and nutritional studies aimed at understanding bee declines have historically focused on social species, with limited evaluations of solitary bees. Here, we asked whether Crithidia bombi, a trypanosomatid gut pathogen known to infect bumble bees, could infect the solitary bees Osmia lignaria (females) and Megachile rotundata (males), and whether nutritional stress influenced infection patterns and bee survival. We found that C. bombi was able to infect both solitary bee species, with 59% of O. lignaria and 29% of M. rotundata bees experiencing pathogen replication 5­11 days following inoculation. Moreover, access to pollen resulted in O. lignaria living longer, although it did not influence M. rotundata survival. Access to pollen did not affect infection probability or resulting pathogen load in either species. Similarly, inoculating with the pathogen did not drive survival patterns in either species during the 5­11-day laboratory assays. Our results demonstrate that solitary bees can be hosts of a known bumble bee pathogen, and that access to pollen is an important contributing factor for bee survival, thus expanding our understanding of factors contributing to solitary bee health.