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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. [Display omitted] • First described method for genetic modification of the bee parasite Crithidia bombi. • Ectopic transgenes can be introduced using multiple selectable markers. • Cytoplasmic and organelle markers can be visualized in swimming and attached developmental forms. • Simplified method for culture adaptation of parasites from bees for subsequent modification. • Imaging of fluorescent C. bombi parasites in vivo in the bumble bee Bombus impatiens. [ABSTRACT FROM AUTHOR]

This study evaluates the efficacy of a cryopreservation protocol for spermatozoa derived from the accessory testis of male Bombus impatiens. It is also the first report of successful cryopreservation of bumble bee spermatozoa. The spermatozoa viability was compared with the similarly treated honey bee spermatozoa derived from its accessory testis. The semen was frozen using a yolk-free non-activating buffer containing dimethyl sulphoxide and stored in liquid nitrogen for 24 h to ~14 days. Thereafter, the frozen samples were thawed rapidly and assessed by staining with live/dead differentiating fluorescent dyes. Semen viability in cryopreserved samples (55.8 ± 14.0%) was significantly different than controls (96.2 ± 10.5%). Similar assessment with A. mellifera resulted in 82.2 ± 7.0% viable cryopreserved spermatozoa versus 99.4 ± 0.1% in controls. A similar proportion of the sperm cells were also capable of motility upon dilution of the extender medium with phosphate buffered saline. The proportion of viable accessory testis derived sperm cells obtained post-cryopreservation was estimated to be sufficient to initiate long term storage and artificial insemination programs. [ABSTRACT FROM AUTHOR]

[Display omitted] • New highly prevalent virus is described in Crithidia bombi , a parasite of bumblebees. • C. bombi and viruses show the same deep split between European and American strains. • No geographic or coevolutionary patterns detected in European C. bombi and viruses. 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 Bombu s 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. [ABSTRACT FROM AUTHOR]

Individuals sometimes exhibit striking constancy to a single behaviour even when they are capable of short-term behavioural flexibility. Constancy enables animals to avoid costs such as memory constraints, but can also inflict significant opportunity costs through behaviour–environment mismatch. It is unclear when individuals should exhibit behavioural constancy and which types of costs most strongly influence such behaviour. We use a case in which individuals within a population exhibit more than one handling tactic for a single food type to investigate whether costs associated with switching among tactics constrain expression of intra-individual variation. Using wild bumble bees (Bombus spp.) that feed on nectar through flower openings (legitimate visits) or through holes at the base of flowers (robbing), we asked three questions. (1) Do individual bees exhibit tactic constancy within and across foraging bouts? (2) Are individuals willing to switch their food-handling tactics? (3) Is constancy in food-handling tactics maintained by costs associated with switching tactics? We measured energetic costs in addition to handling times. We found that bees freely foraging in meadows were highly constant to a single food-handling tactic both within and across bouts. However, experiments with individual captive bees showed that these bees were willing to switch tactics and experienced minimal costs in doing so. Thus, switching costs do not drive the observed constancy in food-handling tactics of bumble bees within and across foraging bouts. • We examined factors influencing constancy of foraging tactics in bumble bees. • Bees forage via floral openings (legitimate) or via holes at the flower base (rob). • Bee foraging was highly constant: feeding legitimately or robbing nectar. • Bees readily switched tactics, and there was no cost to switching. [ABSTRACT FROM AUTHOR]

Foraging bees expend considerable time and energy handling flowers that are morphologically complex (with concealed food rewards) while simple flowers (with readily available rewards) bloom simultaneously in their foraging environment. Previous studies have investigated the consequences of floral morphology for both foragers and plants, often treating 'complex' and 'simple' morphologies as dichotomous traits. How pollinators' foraging choices and success vary along a floral complexity gradient has received less attention. Here we investigated, using real flowers of increasing morphological complexity, how complex flowers are chosen and handled by naïve and experienced bumblebees when presented along with simple ones. Intact flowers of Tecoma x 'Orange Jubilee' (Bignoniaceae), Antirrhinum majus (Plantaginaceae) and Lupinus pilosus (Fabaceae) represented a gradient of increasing morphological complexity. We manipulated some flowers of each species to look simple with a readily accessible food reward, while keeping their colour and odour unchanged. Bombus terrestris workers were given four simple and four complex flowers of a single species with equal rewards in choice assays in a flight room. Sixty per cent of naïve foragers chose a complex flower on their first visit to all three flower species arrays. Experienced bees visited complex flower types of all three species but had lower feeding success and longer handling times on the more complex species. Thus, the bees' foraging efficiency on the complex option decreased with increasing complexity of the flowers, while individual variation in feeding success on the complex option was increased. These results suggest that inexperienced foragers and unsuccessful feeding attempts increasingly contribute to floral pollination along the morphological complexity gradient. • Naïve bumblebees exhibited mild preferences for complex shapes. • Bees fed from complex shapes when presented with equally rewarding simple flowers. • Highly complex flowers benefit from bees' exploratory/unsuccessful attempts. [ABSTRACT FROM AUTHOR]

There is a pressing need to better understand the factors contributing to declines of wild pollinators such as bumblebees. Many different contributors have been postulated including: loss of flower-rich habitats and nesting sites; monotonous diets; impacts of invasive pathogens; exposure to pesticides such as neonicotinoids. Past research has tended to investigate the impacts of these stressors in isolation, despite the increasing recognition that bees are simultaneously exposed to a combination of stressors, with potentially additive or synergistic effects. No studies to date have investigated the combined effects of a monotonous diet and exposure to pesticides. Using queenless micro-colonies of Bombus terrestris audax , we examined this interaction by providing bees with monofloral or polyfloral pollen that was either contaminated with field-realistic levels of thiamethoxam, a commonly used neonicotinoid, or not contaminated. Both treatments were found to have a significant effect on various parameters relating to micro-colony performance. Specifically, both pesticide-treated micro-colonies and those fed monofloral pollen grew more slowly than those given polyfloral pollen or pollen without pesticides. The two factors appeared to act additively. Micro-colonies given monofloral pollens also exhibited lower reproductive efforts and produced smaller drones. Although further research is needed to examine whether similar effects are found in whole colonies, these findings increase our understanding of the likely effects of multiple stressors associated with agricultural intensification on bee declines. [ABSTRACT FROM AUTHOR]

• Trypanosomes, Vairimorpha apis and Spiroplasma apis are prevalent in Eucera pruinosa. • Eucera pruinosa , Bombus impatiens and Apis mellifera host different trypanosomes. • Novel trypanosome species in the Leptomonas - Crithidia complex detected in bees. The squash bee Eucera (Peponapis) pruinosa is emerging as a model species to study how stressors impact solitary wild bees in North America. Here, we describe the prevalence of trypanosomes, microsporidians and mollicute bacteria in E. pruinosa and two other species, Bombus impatiens and Apis mellifera , that together comprise over 97% of the pollinator visitors of Cucurbita agroecosystems in Pennsylvania (United States). Our results indicate that all three parasite groups are commonly detected in these bee species, but E. pruinosa often exhibit higher prevalences. We further describe novel trypanosome parasites detected in E. pruinosa , however it is unknown how these parasites impact these bees. We suggest future work investigates parasite replication and infection outcomes. [ABSTRACT FROM AUTHOR]

Honeybees and bumblebees are the most important pollinators of agricultural crops. For this purpose honeybees and bumblebees are reared and transported. A pathogen-free status of bees in general, is crucial. Indeed anthropogenic transports of hosts carrying parasites could alter the natural host/pathogen association, inducing an extra pathogenic stress. Therefore the creation of a pathogen-free rearing environment is needed. For bumblebees this is possible, as these species are reared in a closed environment. Although, a link remains between reared bumblebees and the outside bee community, as honeybee-collected pollen is essential food for bumblebee mass rearing. Here we evaluated if gamma irradiation can minimize the risk of this potential route of exposure and can inactivate viral particles present in honeybee-collected pollen. We show that 16.9 kGy gamma irradiation induced a 100–1000 fold reduction on the ability of IAPV to cause mortality after injections. This result opens avenues toward rearing pathogen-free bumblebees and towards eliminating the risks of pathogen spillover to native wild bee species. [ABSTRACT FROM AUTHOR]

Israeli acute paralysis virus (IAPV) together with Acute bee paralysis virus (ABPV) and Kashmir bee virus (KBV) constitute a complex of closely related dicistroviruses. They are infamous for their high mortality after injection in honeybees. These viruses have also been reported in non- Apis hymenopteran pollinators such as bumblebees, which got infected with IAPV when placed in the same greenhouse with IAPV infected honeybee hives. Here we orally infected Bombus terrestris workers with different doses of either IAPV or KBV viral particles. The success of the infection was established by analysis of the bumblebees after the impact studies: 50 days after infection. Doses of 0.5 × 10 7 and 1 × 10 7 virus particles per bee were infectious over this period, for IAPV and KBV respectively, while a dose of 0.5 × 10 6 IAPV particles per bee was not infectious. The impact of virus infection was studied in micro-colonies consisting of 5 bumblebees, one of which becomes a pseudo-queen which proceeds to lay unfertilized (drone) eggs. The impact parameters studied were: the establishment of a laying pseudo-queen, the timing of egg-laying, the number of drones produced, the weight of these drones and worker mortality. In this setup KBV infection resulted in a significant slower colony startup and offspring production, while only the latter can be reported for IAPV. Neither virus increased worker mortality, at the oral doses used. We recommend further studies on how these viruses transmit between different pollinator species. It is also vital to understand how viral prevalence can affect wild bee populations because disturbance of the natural host-virus association may deteriorate the already critically endangered status of many bumblebee species. [ABSTRACT FROM AUTHOR]

Highlights: [•] We investigate the status of the emergent honeybee parasite, Nosema ceranae, in bumblebees. [•] We molecularly identify the presence of N. ceranae in multiple species of wild bumblebee across sites in England. [•] We inoculate Bombus terrestris bumblebees with N. ceranae spores to test its infectivity. [•] We find, following the ingestion of N. ceranae spores, bumblebees suffer from altered behaviour and reduced survival. [•] Comparatively, N. ceranae has higher virulence in bumblebees than in its natural honey bee host. [ABSTRACT FROM AUTHOR]

Abstract: Bumblebees are of profound ecological importance because of the pollination services they provide in natural and agricultural ecosystems. Any decline of these pollinators is therefore of great concern for ecosystem functioning. Increased parasite pressures have been discussed as a major factor for the loss of pollinators. One of the main parasites of bumblebees is Nosema bombi, an intracellular microsporidian parasite with considerable impact on the vitality of the host. Here we study the effect of host colony density and host genetic variability on N. bombi infections in natural populations of the bumblebee Bombus terrestris. We sampled males and workers from six B. terrestris populations located in an agricultural landscape in Middle Sweden to determine the prevalence and degree of N. bombi infections. All individuals were genotyped with five microsatellite markers to infer the colony densities in the sampled populations and the genetic variability of the host population. We confirmed that genetic variability and sex significantly correlate with the degree of infection with N. bombi. Males and workers with lower genetic variability had significantly higher infection levels than average. Also colony density had a significant impact on the degree of infection, with high density populations having higher infected individuals. [Copyright &y& Elsevier]

Pollinators are confronted with spatial heterogeneity in floral resources. Acquiring information about their environment enables pollinators to exploit floral resources optimally, and hence strongly affects their fitness. Despite their evolutionary importance, spatial resource distribution and social information use have never been addressed in a plant–pollinator framework. We experimentally tested the effects of inadvertent social information (ISI) use (without or with an experienced demonstrator) in relation to spatial flower distribution (random or patchy) in foraging Bombus terrestris. We studied naïve bees'' associative learning when confronted with rewarding and nonrewarding flowers that differed slightly in colour, reproducing the natural mimicry of deceptive species. Naïve bees were more efficient foragers with than without a demonstrator in the patchy flower distribution, suggesting that they used ISI from conspecifics, probably in the form of social attraction at the patch scale. In the random flower distribution, however, ISI did not improve performance of naïve bees, and could not replace individual sampling. Analyses of nonrewarding intraspecific transitions indicate that the efficiency of naïve bees in deceptive species pollination was lowered when rewarding and nonrewarding inflorescences were patchily distributed and especially when bees relied on ISI. Thus, spatial flower distribution and ISI availability interact in determining pollinator behaviour, which has important consequences for plant community pollination. This suggests that bumblebees are adapted to exploit patchily distributed resources socially, a characteristic that might be exploited by deceptive plant species. By coupling behavioural and community ecology, this study offers promising perspectives for cognitive ecology studies in plant–pollinator interactions. [Copyright &y& Elsevier]

[Display omitted] • We quantified the prevalence and intensity of honey bee viruses in wild bees. • We tested if bee floral visitation predicts viral titers in managed and wild bees. • We detected three out of the five viruses tested in managed honey bees and wild bees. • DWV was the most common virus and had the highest titers in all bee species screened. • Floral bee visitation was not correlated with DWV intensity in bees. Managed and wild bee populations are in decline around the globe due to several biotic and abiotic stressors. Pathogenic viruses associated with the Western honey bee (Apis mellifera) have been identified as key contributors to losses of managed honey bee colonies, and are known to be transmitted to wild bee populations through shared floral resources. However, little is known about the prevalence and intensity of these viruses in wild bee populations, or how bee visitation to flowers impacts viral transmission in agroecosystems. This study surveyed honey bee, bumble bee (Bombus impatiens) and wild squash bee (Eucera (Peponapis) pruinosa) populations in Cucurbita agroecosystems across Pennsylvania (USA) for the prevalence and intensity of five honey bee viruses: acute bee paralysis virus (ABPV), deformed wing virus (DWV), Israeli acute paralysis virus (IAPV), Kashmir bee virus (KBV), and slow bee paralysis virus (SBPV). We investigated the potential role of bee visitation rate to flowers on DWV intensity among species in the pollinator community, with the expectation that increased bee visitation to flowers would increase the opportunity for transmission events between host species. We found that honey bee viruses are highly prevalent but in lower titers in wild E. pruinosa and B. impatiens than in A. mellifera populations throughout Pennsylvania (USA). DWV was detected in 88% of B. impatiens , 48% of E. pruinosa , and 95% of A. mellifera. IAPV was detected in 5% of B. impatiens and 4% of E. pruinosa , compared to 9% in A. mellifera. KBV was detected in 1% of B. impatiens and 5% of E. pruinosa , compared to 32% in A. mellifera. Our results indicate that DWV titers are not correlated with bee visitation in Cucurbita fields. The potential fitness impacts of these low viral titers detected in E. pruinosa remain to be investigated. [ABSTRACT FROM AUTHOR]

Gonadotropic hormones coordinate processes in diverse tissues regulating animal reproductive physiology and behavior. Juvenile hormone (JH) is the ancient and most common gonadotropin in insects, but not in advanced eusocial honey bees and some ants. To start probing the evolutionary basis of this change, we combined endocrine manipulations, transcriptomics, and behavioral analyses to study JH regulated processes in a bumble bee showing a relatively simple level of eusociality. We found that in worker fat body, more JH-regulated genes were up- rather than down-regulated, and enriched for metabolic and biosynthetic pathways. This transcriptomic pattern is consistent with earlier evidence that JH is the major gonadotropin in bumble bees. In the brain, more JH-regulated genes were down- rather than up-regulated and enriched for protein turnover pathways. Brain ribosomal protein gene expression shows a similar trend of downregulation in dominant workers, which naturally have high JH titers. In other species, similar downregulation of protein turnover is found in aging brains or under stress, associated with compromised long-term memory and health. These findings suggest a previously unknown gonadotropin-mediated tradeoff. Analysis of published data reveals no such downregulation of protein turnover pathways in the brain of honey bee workers, which exhibit more complex eusociality and in which JH is not a gonadotropin but rather regulates division of labor. These results suggest that the evolution of complex eusociality in honey bees was associated with modifications in hormonal signalling supporting extended and extremely high fertility while reducing the ancient costs of high gonadotropin titers to the brain. • JH manipulation affected the expression of many genes in bumble bees • JH up-regulated mitochondria and metabolism pathways genes in the fat body • JH down-regulated genes involved in protein biosynthesis and turnover in the brain • Similar differences were found in bumble bees that naturally differ in JH levels • Different JH effects were found in honey bees, in which JH is not a gonadotropin [ABSTRACT FROM AUTHOR]

• Two out of six honey bee viruses were detected in tropical bee species. • DWV and BQCV infect and multiply in several tropical bee species. • Honey bee viruses could be detrimental to the health of multiple bee species. We determined the presence of six viruses in different bee species collected in subtropical environments. Deformed wing virus (DWV) and black queen cell virus (BQCV) were detected in >90% of honey bee samples and in 50–100% of four stingless bee, two bumble bee and one solitary bee species. Additionally, minus DWV and BQCV RNA strands were detected, indicating that the viruses replicate in several hosts. This is the first report of honey bee viruses replicating in six wild bee species in the tropics. If pathogenic to them, viral infections could result in negative impacts in agricultural and unmanaged ecosystems. [ABSTRACT FROM AUTHOR]