INTRODUCTION
Bumblebees are responsible for the pollination of a variety of wild flowers and economically important crops throughout the world (Goulson, Reference Goulson2010). Recent evidence has highlighted declines of important pollinators in many areas worldwide and the potentially devastating consequences of its continuation (Potts et al. Reference Potts, Biesmeijer, Kremen, Neumann, Schweiger and Kunin2010). Bumblebee declines have so far been reported across Europe, North America, South America and Asia (Kosior et al. Reference Kosior, Celary, Olejniczak, Fijal, Król, Solarz and Plonka2007; Xie et al. Reference Xie, Williams and Tang2008; Martins and Melo, Reference Martins and Melo2010; Cameron et al. Reference Cameron, Lozier, Strange, Koch, Cordes, Solter and Griswold2011). Parasites are strongly implicated in the reduction of several bumblebee species (Williams and Osborne, Reference Williams and Osborne2009; Cameron et al. Reference Cameron, Lozier, Strange, Koch, Cordes, Solter and Griswold2011; Meeus et al. Reference Meeus, Brown, de Graaf and Smagghe2011). In addition, as bumblebee populations decline, the threat faced from parasites will increase due to bumblebee populations becoming fragmented and losing their genetic diversity (Whitehorn et al. Reference Whitehorn, Tinsley, Brown, Darvill and Goulson2011).
The development of bumblebees as a model system in host–parasite evolutionary ecology has allowed us to gain a very good understanding of 2 bumblebee parasites, Crithidia bombi and Nosema bombi (Schmid-Hempel, Reference Schmid-Hempel1998). Our knowledge of the biology of other bumblebee parasites, though, remains comparatively poor (Schmid-Hempel, Reference Schmid-Hempel1998; Meeus et al. Reference Meeus, Brown, de Graaf and Smagghe2011). Recent advances in the molecular detection of parasites have highlighted that bumblebees are in contact with a greater diversity of parasites than previously recognized (Singh et al. Reference Singh, Levitt, Rajotte, Holmes, Ostiguy, VanEngelsdorp, Lipkin, Depamphilis, Toth and Cox-Foster2010; Evison et al. Reference Evison, Roberts, Laurenson, Pietravalle, Hui, Biesmeijer, Smith, Budge and Hughes2012; Graystock et al. Reference Graystock, Yates, Evison, Darvill, Goulson and Hughes2013a ; Plischuk et al. Reference Plischuk, Sanscrainte, Becnel, Estep and Lange2015).
Apicystis bombi is a neogregarine parasite found infecting bumblebees and honey bees in Europe, North America and more recently, South America and Asia (Lipa and Triggiani, Reference Lipa and Triggiani1992; Colla et al. Reference Colla, Otterstatter, Gegear and Thomson2006; Plischuk and Lange, Reference Plischuk and Lange2009; Morimoto et al. Reference Morimoto, Kojima, Yoshiyama, Kimura, Yang, Peng and Kadowaki2013; Graystock et al. Reference Graystock, Goulson and Hughes2014). Apicystis bombi oocysts reside primarily in the hosts fatbody and are intermittently found in faeces and flowers suggesting an oral-faecal route of transmission, similar to that of other bee infecting protists though an additional transmission route via decomposing corpses has been suggested (Schmid-Hempel, Reference Schmid-Hempel1998; Graystock et al. Reference Graystock, Yates, Evison, Darvill, Goulson and Hughes2013a , Reference Graystock, Goulson and Hughes2015b ). Potential vertical transmission via eggs has also been suggested but not shown (Lipa and Triggiani, Reference Lipa and Triggiani1996). The prevalence of bumblebees with visually detectable A. bombi oocysts is often low, <7% of native UK bumblebees (Jones and Brown, Reference Jones and Brown2014), however sensitive molecular screening of this parasite suggests it is often much higher (>25%), with many infections being covert (Plischuk et al. Reference Plischuk, Martín-Hernández, Prieto, Lucía, Botías, Meana, Abrahamovich, Lange and Higes2009; Graystock et al. Reference Graystock, Yates, Evison, Darvill, Goulson and Hughes2013a , Reference Graystock, Goulson and Hughes2014; Murray et al. Reference Murray, Coffey, Kehoe and Horgan2013). A small number of infected queens have been shown to have a reduced likelihood to survive hibernation, possibly due to parasite effects on their stored fatbody (Rutrecht and Brown, Reference Rutrecht and Brown2008; Jones and Brown, Reference Jones and Brown2014). It has been argued that the emergence of this parasite is due to the spillover of non-native strains from introduced bumblebees and honey bees (Plischuk and Lange, Reference Plischuk and Lange2009; Maharramov et al. Reference Maharramov, Meeus, Maebe, Arbetman, Morales, Graystock, Hughes, Plischuk, Lange, de Graaf, Zapata, de la Rosa, Murray, Brown and Smagghe2013; Ravoet et al. Reference Ravoet, Maharramov, Meeus, De Smet, Wenseleers, Smagghe and de Graaf2013; Graystock et al. Reference Graystock, Goulson and Hughes2014, Reference Graystock, Blane, McFrederick, Goulson and Hughes2015a ). Recently there have been reports that A. bombi may be playing a role in declines in Argentinean bumblebees (Plischuk and Lange, Reference Plischuk and Lange2009; Plischuk et al. Reference Plischuk, Meeus, Smagghe and Lange2011), but currently there is insufficient knowledge of the biology or virulence of the parasite to assess what threat it poses to bumblebees.
Another parasite with an increased incidence of detection in bumblebees is the picornavirus, deformed wing virus (DWV) (Fürst et al. Reference Fürst, McMahon, Osborne, Paxton and Brown2014). This virus is ubiquitous in some honey bee populations, with little effect on the host unless in the presence of an additional parasite, Varroa destructor, which acts as a vector for viruses such as DWV, increasing their virulence and in the case of DWV, often leading to symptomatic honey bee wing deformities during their development as larvae to pupa (Ryabov et al. Reference Ryabov, Wood, Fannon, Moore, Bull, Chandler, Mead, Burroughs and Evans2014). Wild bumblebees with DWV have been seen to exhibit deformed wings, suggesting the virus can impose the same deformities to bumblebees as seen in honey bee hosts, even in the absence of V. destructor, with transmission likely to occur via shared flower use. The virulence of this virus in bumblebees is currently understudied though a laboratory based study showed single infection can cause mortality in bumblebees after just 6 days (Fürst et al. Reference Fürst, McMahon, Osborne, Paxton and Brown2014).
The detection of both DWV and A. bombi in wild and commercially reared bumblebee populations in combination with a shortage of knowledge regarding their virulence makes it imperative to understand the virulence of these parasites on bumblebee hosts. Studies often focus purely on a single parasite–host interaction, yet naturally, parasites often occur as mixed infections, having profound effects on the outcome of the interactions (Alizon et al. Reference Alizon, de Roode and Michalakis2013). Here, we investigate lethal and sublethal effects of single and mixed infections of these 2 little-studied parasites of bumblebees.
MATERIALS AND METHODS
Apicystis bombi extraction for ingestion treatments
The fatbody of 40 Bombus terrestris bumblebees from 2 colonies with single infections of A. bombi (screened as in Graystock et al. Reference Graystock, Yates, Evison, Darvill, Goulson and Hughes2013a for 5 parasites known to infect adult bumblebees: DWV, Nosema ceranae, N. bombi, C. bombi, A. bombi plus Nosema apis which has been found in bumblebees but with no evidence of active infection) were homogenized in 1000 µL of 30% sucrose solution and the resulting homogenate was slowly passed through a syringe filter to remove large tissue fragments. This solution was then centrifuged for 30 min at 9000 g and 15 °C and the resulting pellet of oocysts extracted with a pipette. Oocysts were washed by first centrifuging at 10 000 g for 20 min, removing the supernatant, and replacing with 30% sucrose solution before vortexing for 10 s. This wash process was repeated 3 times to eliminate any small particles. The resulting solution was confirmed with a compound microscope to be a suspension of A. bombi oocysts, free from bumblebee tissue membrane and other parasite infective forms. The suspended oocysts were visually identified as being A. bombi; this was then confirmed by polymerase chain reaction (PCR) amplification of the suspension with specific primers for A. bombi, and for the 2 Nosema species known to be found in bumblebee fatbody, N. bombi and N. ceranae (Macfarlane et al. Reference Macfarlane, Lipa and Liu1995; Klee et al. Reference Klee, Tek Tay and Paxton2006; Graystock et al. Reference Graystock, Yates, Darvill, Goulson and Hughes2013b ). The spore solution was then diluted in 30% sucrose solution to obtain a concentration of 5 × 105 oocysts mL−1. The control treatment was 30% sucrose solution without any oocysts added.
DWV extraction for injection treatments
The route of DWV transmission is currently undetermined in bumblebees but in honeybees multiple routes are known and the chosen route can have profound effects on the virulence of DWV (Ryabov et al. Reference Ryabov, Wood, Fannon, Moore, Bull, Chandler, Mead, Burroughs and Evans2014). Though once a bumblebee is infected, DWV is found in all bumblebee tissues except the eye (Li et al. Reference Li, Peng, Wu, Strange, Boncristiani and Chen2011). To avoid any inefficiencies in the study due to an inappropriately chosen transmission method, and to ensure successful inoculations throughout, virus was administered via injection into the haemolymph as performed in other viral studies on honey bee and bumblebees by Iqbal and Mueller (Reference Iqbal and Mueller2007) and Meeus et al. (Reference Meeus, Mosallanejad, Niu, de Graaf, Wäckers and Smagghe2014). The extraction protocol for DWV was adapted from Iqbal and Mueller (Reference Iqbal and Mueller2007). The fatbody of 50 B. terrestris bumblebees from a hive, singly infected with DWV (screened as above DWV, N. ceranae, N. bombi, C. bombi, A. bombi plus N. apis) were frozen in liquid nitrogen, and homogenized with 2·5 mL phosphate-buffered saline (PBS, pH 7·4), then centrifuged at 986 g for 30 min at 10 °C. The resulting solution was confirmed to be positive for DWV by reverse transcription polymerase chain reaction (RT–PCR), before being diluted down 1:1000 in PBS. Thus, the inoculant has been diluted from what occurs in naturally infected bumblebees down to a concentration of approximately 5%. Recent work has identified bumblebees can have multiple viruses (though their replication within bumblebees or presence in the fat body is still unclear) and while our inoculant was confirmed to contain DWV, it cannot be ruled out that other, unknown viruses may have been present (McMahon et al. Reference McMahon, Fürst, Caspar, Theodorou, Brown and Paxton2015). Control treatments were injections of just PBS (pH 7·4) which would prevent control bees from receiving any secondary, unknown viruses which may also be present in bumblebee fatbody.
Experimental infection
A total of 350 B. terrestris audax workers were collected from 5 colonies that had been obtained from Biobest NV in Westerlo (Belgium) and were confirmed to be parasite-free by screening 15 bees per colony by PCR and RT–PCR for C. bombi, A. bombi, N. bombi, N. ceranae and DWV by PCR and RT–PCR (see below). The 350 bees were placed as groups of 5 nestmate bees in 10 × 6 × 6 cm3 plastic boxes. Seven ingestion|injection treatment combinations were tested: A. bombi|DWV, A. bombi|control, control|DWV, control|control, A.bombi|no injection, no ingestion|DWV and no ingestion|no injection. Two groups of bees from each of the 5 colonies (50 bees in total) were tested with each of the 7 treatment combinations. For the A. bombi treatment, each bee was placed into a holding harness and individually fed a 5 µL dose of the parasite suspension, which contained 2500 A. bombi oocysts. This amount was chosen following oocyst quantification from 9 infected wild bumblebees from England found up to 2500 oocysts in a single bee gut (the presumed transmission route). In an entire bee, intensities of around 5 × 106 oocysts are common. Bees receiving the control ingestion treatment were treated similarly, but fed 5 µL of pure 30% sucrose solution, while bees in no ingestion combinations were not hand-fed at all. For the DWV treatment, a 5 µL dose of the DWV solution was injected into the ventral side of the abdomen, between the 2nd and 3rd sternites. Bees receiving the control injection were injected with 5 µL of PBS, while those in the no injection combinations were not injected with anything. The bees received their injection treatments first and were subsequently starved for 5 h before receiving their ingestion treatments. Following treatment the bees were replaced in their cohorts of 5 like-treated nestmates, provided with 50% sucrose solution ad libitum, and their survival checked daily for 15 days.
Sucrose sensitivity (SS)
The sensitivity of a bee to low sucrose concentrations has been linked to hunger and learning ability (Scheiner et al. Reference Scheiner, Page and Erber2001; Naug and Gibbs, Reference Naug and Gibbs2009) making it a good measure of sub-lethal effects of parasite infection. The SS of bumblebees to differing concentrations was therefore tested for every bee in the experiment using the proboscis extension response (PER). Every 5 days a PER experiment was performed, in which each bee was harnessed in a modified Eppendorf tube with moist cotton wool, under red-light conditions. While harnessed, bees were hand-fed to satiation with 30% sucrose solution before being left for a starvation period of 5 h. After starvation, each bee had its antenna touched with a drop of sucrose solution, the concentration of which was increased in 10% increments from 50 to 80%. Between each concentration trial, antennae were touched with H20 after 60 s to prevent bees becoming conditioned; the next sucrose concentration was then applied following a further 60 s interval. Individuals that were responsive to a particular concentration extended their proboscis, resulting in a SS score of 1, and, as each bee was individually presented with 5 different concentrations, each bee could therefore score a maximum SS of 5 (Riveros and Gronenberg, Reference Riveros and Gronenberg2009). The responses of each bee were measured, with high SS scores indicating bees responding to high and low sugar concentrations, and a low SS score indicating bees responding only to high sugar concentrations.
Lipid extraction
The leanness of each of the 350 bees was calculated by determining their lipid content relative to their body size (Brown et al. Reference Brown, Loosli and Schmid-Hempel2000). For this, each abdomen (minus 2 tergites which were removed for molecular screening) was dried at 70 °C for 5 days, weighed and then immersed in ether for 24 h to dissolve the lipids. After rinsing with fresh ether, the remaining abdominal tissues were dried for a further 5 days at 70 °C before being reweighed. Based on the resulting weight loss (mg) and taking the length of the left hind tibia (mm) as an index of body size, the worker lipid:body size ratio was calculated.
Molecular screening
Any bees that died during the experiment, and all those surviving to the end of the 15 days experimental period were placed in 100% ethanol. All 350 bumblebees were then removed from ethanol and each had their 5th and 6th tergites removed. These tergites are more posterior, and on the opposite side of the abdomen, to the site at which the injection treatments were administered. The fatbody attached to these 2 tergites was homogenized in 100 µL of 5% Chelex and incubated at 100 °C for 15 min to extract DNA and RNA. The fatbody extracts were then briefly vortexed before centrifuging at 2399 g for 15 min and collecting the supernatant.
Samples were first screened for 18S rDNA specific to Apidae as a host control to confirm DNA quality. A 10 µL reaction consisted of 0·4 mm dNTP, 1·5 mm MgCl2, 1·25 U Taq, 0·2 µ m each primer 3 µL Buffer and 1 µL template (Meeus et al. Reference Meeus, de Graaf, Jans and Smagghe2010). The PCR was then subjected to 2 min at 94 °C, 35 cycles of 30 s at 94 °C, 30 s at 56 °C, 45 s at 72 °C before a final elongation stage of 3 min at 72 °C. Samples were screened for A. bombi in 10 µL reactions consisting of 0·4 mm dNTP, 1·5 mm MgCl2, 1·25 U Taq, 0·5 µ m of each primer, 2 µL Buffer and 1 µL template. This was then subjected to 2 min at 94 °C, 35 cycles of 30 s at 94 °C, 30 s at 60 °C, 45 s at 72 °C before a final elongation stage of 3 min at 72 °C (Meeus et al. Reference Meeus, de Graaf, Jans and Smagghe2010). Samples were screened for DWV using reverse transcription PCR whereby 2 µL of sample was added to 5 µL TaqMan® Fast Virus 1-Step Master Mix, 650 nm of each primer and molecular grade water giving a total volume of 10 µL. The sample then underwent thermal cycling of 5 min at 50 °C, 20 s at 95 °C, 40 cycles of 3 s at 95 °C, 180 s at 60 °C before a final elongation stage of 10 min at 72 °C (Chen et al. Reference Chen, Higgins and Feldlaufer2005). PCR conditions for the other parasites that were screened for when preparing parasite suspensions or when confirming the parasite-free status of the experimental bees were as in Graystock et al (Reference Graystock, Yates, Evison, Darvill, Goulson and Hughes2013a ). PCR products were run on a 1% agarose gel stained with ethidium bromide to confirm amplicon size. Every assay included negative and positive controls.
Statistical analysis
Differences in bumblebee survival were analysed using a Cox proportional hazards regression model, with ingestion treatment, injection treatment and their interaction included in the model. Pairwise comparisons were made between individual treatments using Kaplan–Meier models with the Breslow χ 2 statistic. The interactive and singular effect of injection and ingestion treatments on lipid:body size ratio was compared using generalized linear models (GLM) with gamma distribution, log link function and the likelihood ratio χ 2 statistic. The interactive and singular effect of injection and ingestion treatments on SS was compared using a generalized linear mixed model (GLMM) with gamma distribution and log link function. Colony-of-origin and cohort were included in both the GLM and GLMM models. Non-significant interaction terms were removed stepwise in all models to obtain the minimum adequate models. Pairwise comparisons were made using Estimated Marginal Means. All analyses were carried out in SPSS 21(IBM, Armonk, NY, USA).
RESULTS
Infectivity
Treatment groups of A. bombi with either PBS, DWV or no injection had A. bombi infectivity of 80, 92 and 78%, respectively (Fig. 1). Treatment groups of DWV with either A. bombi, sucrose or no ingestion had DWV infectivity of 96, 98 and 96%, respectively (Fig. 1). All the following results include only those bees that were found to have systemic infections at the cessation of the experiment or upon death.
Fig. 1. Infectivity of Apicystis bombi (black bars, solid) and DWV (diagonal lines) in treated bees following death or the cessation of the experiment. Shown grouped by treatment combination of either fed Apicystis with PBS injection (AC), fed Apicystis and DWV injection(AD), fed sucrose control with PBS injection (CC), fed sucrose control and DWV injection (CD), fed Apicystis only (A), DWV injection only (-D), or no treatment at all (--).
Survival
There was a significant interaction between the effects of the ingestion and injection treatments on bumblebee survival (Wald = 16·3, D.F. = 2, P < 0·001). The bees which received either the control (sucrose)|control (PBS injection) or no ingestion|no injection treatment combinations had very high survival over the 15-day period, and the survival of bees was reduced significantly when they had received the A. bombi|no injection combination (Fig. 2a, Supplementary Table S1). Survival was significantly lower for bees which received either the control|DWV or, no ingestion|DWV combinations, and was very low for bees which received the A. bombi|control injection or A. bombi|DWV combinations (Fig. 2a, Supplementary Table S1). There was no interaction between injection event (weather or not they were injected, regardless of treatment) and ingestion treatment (Wald = 1·95, D.F. = 1, P = 0·16).
Fig. 2. Survival of bees over 15 days (a) and subsequent lipid: body ratio (b) following hand feeding with either Apicystis bombi oocysts (black points/bars), sucrose control (white points/bars) or no hand feeding (grey points/bars) while also being injected with DWV (solid lines/diagonal bar pattern), PBS control (dashed lines/horizontal pattern) or no injection (dotted lines/no pattern). Treatment combinations were either fed Apicystis with PBS injection (AC), fed Apicystis and DWV injection(AD), fed sucrose control with PBS injection (CC), fed sucrose control and DWV injection (CD), fed Apicystis only (A), DWV injection only (-D), or no treatment at all (--). Whilst different italicized letters indicate treatments which differed significantly from one another, the same italicized letters indicate non-significant in pairwise comparisons.
Leanness
The lipid:body size ratio was affected by the ingestion treatments (Wald = 16·5, D.F. = 2, P = 0·001), but not by the injection treatments and there was also no interaction between ingestion and injection treatments (Wald = 1·3, D.F. = 2, P = 0·5; Wald = 5·6, D.F. = 2, P = 0·06, respectively). Pairwise comparisons between the treatment groups find that bees in the control|control treatment had the highest average lipid:body size ratio and none of the treatments that included DWV differed significantly from this (Fig. 2b, Supplementary Table S2). The 3 treatments that included A. bombi ingestion had the lowest mean lipid:body ratios. The A. bombi|Control group was significantly lower than any of the treatments where A. bombi was not ingested unless DWV was also present (Fig. 2b; Supplementary Table S2).
Sucrose sensitivity
On day 0, 322 bumblebees were tested for their responsiveness to sucrose and no differences were observed between the treatment groups (χ 2 = 2·56, D.F. = 6, P = 0·9; Fig. 3). On day 5, however, the remaining 313 bumblebees displayed a significant response to treatment (χ 2 = 26·4, D.F. = 6, P < 0·001; Fig. 3). Pairwise analysis between all 7 treatment types on day 5 show bees infected with DWV tended to have higher SS (Fig. 3, Supplementary Table S3). On day 10 and 15, the remaining 202 and 182 bumblebees still displayed a significant response to treatment (χ 2 = 21·6, D.F. = 6, P = 0·001; χ 2 = 14·5, D.F. = 6, P = 0·024, respectively) while maintaining the general trend of DWV infected bees being more sensitive for sucrose except on the 15th day when dual infected bees have reduced sensitivity than DWV infected bees (Fig. 3, Supplementary Tables S4 and S5). By the 15th day, however, the experiment had become unbalanced with bees in different treatment groups dying (and so being unable to be part of the sucrose assay) at different rates in later days. Treatment group sizes ranged from 36 to 50 on day 5, 11 to 50 on day 10 and 4 to 48 on day 15 (Supplementary Table S6).
Fig. 3. Bumblebee responsiveness to sucrose over 15 days following either hand feeding with Apicystis bombi oocysts (black bars), sucrose control (white bars) or no hand feeding (grey bars) whilst also being injected with DWV (bars patterned with diagonal lines), PBS control (bars patterned with horizontal lines) or no injection (bars not patterned). The same italicized letters indicate non-significant in pairwise comparisons. Error bars represent ± standard error.
DISCUSSION
Apicystis bombi and DWV infections were virulent to bumblebees, with infections causing mortality to increase by 18 and 50%, respectively. Both A. bombi and DWV infections altered bumblebee sensitivity to sucrose, while A. bombi also significantly reduced the amounts of stored fat in infected bumblebees.
It had previously been noted anecdotally that the fatbody of bumblebees infected with A. bombi appeared ‘much reduced’ (Liu et al. Reference Liu, Macfarlane and Pengelly1974). The results here provide the first empirical evidence of this, with fat/lipid content being reduced by 17% on average by A. bombi infections. The fatbody is essential for overwintering queen bumblebees and any reduction in their fatbody would lower their chances of founding successful colonies. Workers would also be negatively affected by reduced fatbody as it is the site for many biochemical reactions that are important for their immunity and metabolism (Arrese and Soulages, Reference Arrese and Soulages2010). After 5 days, the SS of A. bombi infected bumblebees was elevated, presumably due to an increased demand for carbohydrates to compensate for the reduced fatbody. In the wild this would likely increase the workers need to forage for nectar, reducing their pollen foraging efficiency for the colony and developing larvae. Apicystis bombi infections caused high mortality over 15 days. This mortality increased 3-fold if bees had been injected in their abdomen with either PBS or DWV prior to spore ingestion, suggesting that A. bombi infected bees also have reduced ability to cope with the effects of wounding. While DWV was detectable in fatbody tissue, we found no change in the lipid mass of DWV-infected bumblebees, with bumblebees co-infected by DWV and A. bombi having an intermediate lipid:body mass ratio. In honey bees, DWV infection increases the hosts SS (Iqbal and Mueller, Reference Iqbal and Mueller2007), but we found here the opposite effect in bumblebees with infected workers having decreased SS. Interestingly, co-infected bees maintain SS similar to that of control bees suggesting co-infected bees do not increase sensitivity in response to A. bombi. These results suggest there may be a negative interaction between DWV and A. bombi within the host whereby DWV infection reduces the sub lethal impacts of A. bombi infection. This would be a reverse of what is found in honeybees infected with N. ceranae which can inhibit DWV infection, though weather DWV infected honeybees would also benefit from reduced A. bombi virulence remains to be shown (Doublet et al. Reference Doublet, Natsopoulou, Zschiesche and Paxton2015).
The results demonstrate that both A. bombi and DWV can have significant negative effects on infected bumblebees. While single infections of A. bombi cause only moderate mortality, its sublethal effects may be more important, particularly for hibernating queens whose survival depends on their fat reserves. This supports recent correlative evidence that the emergence of A. bombi in South America may be responsible for rapid declines in some native bumblebee species (Arbetman et al. Reference Arbetman, Meeus, Morales, Aizen and Smagghe2013). In addition, the high mortality of bumblebees infected with DWV makes it clear that both these parasites should be monitored more frequently in bumblebee populations. Both A. bombi and DWV are significantly understudied given the virulence they exhibit in bumblebees. In addition, the rates of transmission within bumblebee populations, and between honey bees and bumblebees, are still unknown. DWV is almost ubiquitous in honey bees and is being increasingly found in bumblebees (Tentcheva et al. Reference Tentcheva, Gauthier, Zappulla, Dainat, Cousserans, Colin and Bergoin2004; Manley et al. Reference Manley, Boots and Wilfert2015; McMahon et al. Reference McMahon, Fürst, Caspar, Theodorou, Brown and Paxton2015), while A. bombi is found mostly in bumblebees (Plischuk et al. Reference Plischuk, Meeus, Smagghe and Lange2011). A recent study has also found more ‘honey bee viruses’ in screened bumblebees but as yet, these have not been shown to be active infections (McMahon et al. Reference McMahon, Fürst, Caspar, Theodorou, Brown and Paxton2015). The negative fitness effects from viral and A. bombi infections shown here in B. terrestris highlights the pressing need to understand more about the effects of these parasites, in bumblebees and their potential role in bumblebee declines.
SUPPLEMENTARY MATERIAL
To view supplementary material for this article, please visit http://dx.doi.org/10.1017/S0031182015001614.
ACKNOWLEDGEMENTS
We wish to thank Adam Smith and Sophie Evison for laboratory assistance, Katherine Roberts, Rowena Mitchell, Eamonn Mallon and Alison Dunn for constructive comments and the Super-B COST Action for facilitating fruitful discussion of the work. We also wish to thank two anonymous reviewers for their helpful and constructive comments on the manuscript.
FINANCIAL SUPPORT
This work was funded by NERC (Natural Environment Research Council, UK; grant no. NE/G012113/1), the Bumblebee Conservation Trust and a Philip Leverhulme Prize, all awarded to WOHH.
