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- Materials and methods
1. Animal hosts harbour diverse and often specific bacterial communities (microbiota) in their gut. These microbiota can provide crucial services to the host such as aiding in digestion of food and immune defence. However, the ecological factors correlating with and eventually shaping these microbiota under natural conditions are poorly understood.
2. Bumblebees have recently been shown to possess simple and highly specific microbiota. We here examine the dynamics of these microbiota in field colonies of the bumblebee Bombus terrestris over one season. The gut bacteria were assessed with culture-independent methods, that is, with terminal restriction fragment length profiles of the 16S rRNA gene.
3. To further understand the factors that affect the microbiota, we experimentally manipulated field-placed colonies in a fully factorial experiment by providing additional food or by priming the workers’ immune system by injecting heat-killed bacteria. We furthermore looked at possible correlates of diversity and composition of the microbiota for (i) natural infections with the microbial parasites Crithidia bombi and Nosema bombi, (ii) bumblebee worker size, (iii) colony identity, and (iv) colony age.
4. We found an increase in diversity of the microbiota in individuals naturally infected with either C. bombi or N. bombi. Crithidia bombi infections, however, appear to be only indirectly linked with higher microbial diversity when comparing colonies. The treatments of priming the immune system with heat-killed bacteria and additional food supply, as well as host body size, had no effect on the diversity or composition of the microbiota. Host colony identity had only a weak effect on the composition of the microbiota at the level of resolution of our method. We found both significant increases and decreases in the relative abundance of selected bacterial taxa over the season.
5. We present the first study on the ecological dynamics of gut microbiota in bumblebees and identify parasite infections, colony identity and colony age as important factors influencing the diversity and composition of the bacterial communities. The absence of an effect of our otherwise effective experimental treatments suggests a remarkable ability of the host to maintain a homoeostasis in this community under widely different environments.
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- Materials and methods
Specific microbial communities (microbiota) colonizing animal hosts are ubiquitous in nature and can play important roles, such as for digestive functions, immune defence and organ formation (Fraune & Bosch 2010). However, we know little about the dynamics and the ecological factors shaping these communities in the field. The field situation is of course especially interesting, as it can differ fundamentally from the situation in laboratory-reared hosts (e.g. Xiang et al. 2006). As a case study, we here investigate the bacterial microbiota of important pollinators in temperate regions, the bumblebees (Bombus spp.) (Bingham & Orthner 1998). The case is likely of practical relevance, as bumblebee populations have been declining world-wide (Goulson, Lye & Darvill 2008; Williams & Osborne 2009), and this has been linked to pathogens (Williams & Osborne 2009; Cameron et al. 2011). With regard to their microbiota, recent evidence shows that bumblebees and honeybees have a distinct and relatively species-poor microbiota in the gut (Koch & Schmid-Hempel 2011a; Martinson et al. 2011). These bacteria might play an important role in defence against pathogens (Forsgren et al. 2010; Koch & Schmid-Hempel 2011b) and the digestion of food (Gilliam 1997; Vásquez & Olofsson 2009). But the field situation, where different ecological factors affect the microbiota, and the respective functions remain poorly studied (Hamdi et al. 2011).
Plausible ecological factors that affect the microbiota include nutritional status of the host (Dillon et al. 2010) and the activation of the immune system upon parasitic infections (Ryu et al. 2008; Lazzaro & Rolff 2011). We tested these factors by experimentally manipulating colonies of B. terrestris (Linnaeus) either by stimulating the immune system with heat-killed bacteria or by providing additional food (sugar water) to field-placed colonies in a full-factorial design. We also looked at the following questions:
At the individual level: (a) Does the microbiota change when natural infections by the two common microbial parasites C. bombi
Lipa & Triggiani and N. bombi
Fantham & Porter are present?
An infection could alter the gut microbiota, either through direct interaction or through activation of the immune system by parasites, disturbing the homoeostasis with resident microbiota (Ryu et al. 2008
; Lazzaro & Rolff 2011
). (b) Does host body size influence the diversity of gut microbiota (allometry)? For macroscopic parasites, host size tends to be correlated with higher parasite diversity (Poulin & Morand 2000
). Hosts size can be expected to be related to gut volume (Yang & Joern 1994
) and therefore to the size of the potential habitat for gut microbiota within a host. The diversity of free-living bacteria shows a species–area relationship comparable to that found in macroscopic organisms (Bell et al. 2005
). However, whether such a relationship also exists for microbial communities within hosts of different sizes has so far not been examined. Bumblebees present a good model to study this question, as highly related workers within one colony can vary up to 10-fold in body mass (Couvillon et al. 2010
At the colony level: (a) Do different colonies harbour distinct microbiota? The microbiota may be colony-specific as has been observed in termites (Minkley et al. 2006
). Colony specificity of microbiota could arise through predominantly vertical transmission within the nest in contrast to environmental transmission in the field. If the host genotype can influence the microbiota (Ley, Peterson & Gordon 2006
), microbiota should differ between colonies. (b) Does the microbiota change over the colony cycle? While the microbiota of isolated bumblebee individuals have been characterized (Koch & Schmid-Hempel 2011a
; Martinson et al. 2011
), there is no information on a possible change in microbiota throughout the colony cycle. We looked both at changes in overall microbial diversity and at changes in relative abundance of specific bacterial taxa.
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- Materials and methods
This study investigated the dynamics of bacterial gut communities in bumblebee host colonies in the field. Remarkably, neither experimentally altering the food supply nor immune priming of the host had a significant effect on the diversity and composition of the gut microbiota. As shown in a companion study, the food supply treatment strongly affected the development of our experimental colonies as a whole though. Non-food-supplied colonies mostly starved after several weeks in the field, indicating harsh conditions that were ameliorated with food supplementation (Cisarovsky, Koch & Schmid-Hempel 2012). In this analysis, we also found a significant increase in antimicrobial activity of the haemolymph of workers 7 days after the experimental immune challenge in comparison with the control group (Cisarovsky, Koch & Schmid-Hempel 2012). Therefore, both of our experimental treatments can be assumed to have had a significant effect on individual worker condition. Because we here report no change, this indicates an astonishing resilience of the gut microbiota in the face of different host environments. Similarly, laboratory-based studies with Drosophila melanogaster have shown a sophisticated tuning of the immune system towards different gut bacterial burdens and thereby the maintenance of an immune homoeostasis in the gut (Leulier & Royet 2009). Our results suggest that bumblebees may also be able to keep up a high degree of gut homoeostasis under different conditions in the field. Perhaps, they thereby maintain a ‘healthy’ relationship with their specific resident gut flora (Koch & Schmid-Hempel 2011a,b), in the sense of maintaining a functionally competent microbiota. This homoeostasis might be of potentially great importance for functionalities such as the defence against natural pathogens (Koch & Schmid-Hempel 2011b; Lazzaro & Rolff 2011).
Looking at the potential effects of food addition, it seems plausible that the gut of better-fed individuals may represent an environment richer in nutrients and therefore a more productive one for bacterial growth. Extrapolating from observations on free-living microbial communities (Abrams 1995; Horner-Devine et al. 2003), such higher productivity may increase the diversity of microbes able to coexist in the gut. However, diversity–productivity relationships are often found to be hump-shaped (Smith 2007), which could lead to the observation of both positive and negative relationships in nature if only two states are compared (as was the case in our factorial experiment). In addition, a better nutritional status may also positively influence the host immune system (Tyler, Adams & Mallon 2006), which in turn may alter the community of gut bacteria (Ryu et al. 2008). Regardless, we here found no significant effect (Fig. 1). Our results contrast the study by Dillon et al. (2010) who found an increase in gut bacterial diversity for starving desert locusts as compared to normally fed individuals. Clearly, more experimental work is needed to understand the relationship of microbial diversity within hosts and nutritional status. As a caveat, we did not investigate the population size of the microbiota quantitatively, but rather focused on the community composition and diversity regardless of the overall population sizes. Thus, a change in the population size owing to our experimental treatments would have gone unnoticed if it were not also linked to a change in the diversity or composition of the microbiota.
Infection with the parasites N. bombi and C. bombi were inevitable and happened naturally in our field experiment. These infections were correlated with an increase in bacterial diversity in the gut. On closer examination, the two parasitic infections showed different patterns, however. Infections with the intracellular parasite N. bombi may be directly linked to a higher diversity of bacterial taxa in the gut of an individual (though marginally non-significant with P = 0·06, Table 1 and Fig. 1). Nosema bombi infects various body tissues but is only found in the gut in the form of inactive spores. This may point towards an indirect interaction, perhaps mediated through the host immune system. For example, infection could start an immune response, which in turn may disrupt the homoeostasis of the commensal bacteria in the gut and alter the diversity of the community. In line with this hypothesis, an overexpression of antimicrobial peptides has previously shown to lead to a disturbance of the mutualistic gut microbial community in Drosophila (Ryu et al. 2008). Whatever the potential mechanisms, our experimental challenge of the host immune system with heat-killed bacteria (treatment immune-primed) did not significantly affect the microbiota (Fig. 1) even though it had previously been shown that such priming upregulates the bumblebee immune response for up to 14 days (Korner & Schmid-Hempel 2004) under laboratory conditions. Yet, it may not present a strong enough stimulus under field conditions in contrast to an active infection with a highly virulent parasite such as N. bombi (Otti & Schmid-Hempel 2007).
Crithidia bombi, by contrast, is an intestinal parasite residing in the hindgut and that therefore may potentially directly interact with the gut microbiota. Surprisingly, however, we only find an indirect link between higher diversity of gut microbiota and C. bombi infections, as colonies with higher C. bombi prevalence had – on average – more diverse gut bacterial communities. No such trend was observed when comparing C. bombi-infected and uninfected single individuals within a given colony by, at the same time, correcting for the colony effect (Table 1). This points towards variation among colonies in the ability to both limit the infections with higher numbers of bacterial taxa and contain the parasite C. bombi in the gut. Note that this view may suggest that the bacterial species colonize and grow to the best of their capacities, rather than forming a tightly regulated community. By implication, the observed homoeostasis in the bacterial community would result from a dynamic process that reflects the – not necessarily convergent – interests of hosts and microbiota.
The diversity of macroscopic organisms generally follows a species–area relationship with habitats of greater size supporting a greater number of species (MacArthur & Wilson 1967). This relationship has also been found for free-living microorganisms, for example for bacteria in water-filled tree holes (Bell et al. 2005). If one considers the gut of a host as a similar ‘island’ habitat, one might predict bigger individuals of the same species (with larger gut volume) to harbour a greater diversity of gut microbiota. We did, however, not find a significant effect of bumblebee worker size (correlated with gut size) on bacterial diversity despite a nearly sixfold difference in the body mass among adult workers. This suggests that other factors are more important in shaping the diversity of the gut microbiota in the current case.
In contrast to a study on termites that also looked at colonies from the same population under identical environmental conditions (Minkley et al. 2006), we did not find a clear separation of the gut microbiota between colonies. We did, nevertheless, find significant differences for pairwise comparisons between some of the colonies. This is remarkable as colonies foraged in the same area under identical environmental conditions. The distinct microbiota of some colonies may be due to the close proximity of bees within a nest, which would facilitate within-nest transmission over transmission between individuals of different colonies. Workers are probably colonized after pupal eclosion with gut bacteria from their nest mates (Koch & Schmid-Hempel 2011b; Martinson, Moy & Moran 2012). Generally though, most bacterial taxa seem to be shared between colonies, at least at the level of resolution offered by our method, which cannot distinguish between different strains of the same bacterial species. These results are in line with previous findings (Koch & Schmid-Hempel 2011a) showing the most abundant bacterial taxa to be shared even between different bumblebee species and separate geographical localities. Future studies should try to disentangle the effects of host genotype and social transmission mode on microbiota composition.
An increase has been observed within a colony over the season for the diversity of strains of the intestinal parasite C. bombi (Imhoof & Schmid-Hempel 1998), which is horizontally transmitted at flowers (Durrer & Schmid-Hempel 1994). In this study, we observed an overall slight trend towards a decrease in gut bacterial diversity over the season, but this trend only became apparent in the last weeks. This decrease should be treated with caution, as the low number of individuals sampled at the end of the season makes this observation less reliable. The observed change in the relative abundance of certain bacterial taxa may be related to an accumulation of environmentally transmitted bacteria over the season, which would increase the relative contribution of these taxa over the bacteria preferentially vertically transmitted and potentially originating from the founder queen. We did observe this general pattern, as on the one hand the peak representing the probably unspecific acidophilic Fructobacillus sp. found on flowers (Endo & Okada 2008) significantly increased in relative abundance in the gut over the season. This finding is in line with the results of McFrederick et al. (2012), suggesting that a number of acidophilic bacteria may be transmitted between flowers and bees. On the other hand, the highly specific and potentially vertically transmitted ‘Ca. Snodgrassella alvi’ (Koch & Schmid-Hempel 2011a,b; Martinson, Moy & Moran 2012) decreased in relative abundance over the season.
In summary, bumblebees appear to be able to maintain a homoeostasis of their gut microbiota even in the face of strong environmental disturbances such as immune priming by non-specific bacteria and drastically altered nutritional states of the colony. Furthermore, even a sixfold difference in host size was not associated with changes in the specific microbiota. In contrast to these factors, parasite infections were associated with a higher diversity of the gut microbiota. Because these are correlations, the causal relationship remains unclear and may differ between C. bombi and N. bombi. Surprisingly, only a weak effect of colony affiliation on the taxonomic composition of the gut microbiota was detected, but a better resolving method may be needed to distinguish between different strains of the dominant bacteria in the bumblebee gut. Colonies however widely differed in the diversity of the microbiota associated with them. Finally, we observed that the composition of the microbiota changes through the colony cycle, potentially caused by a stronger representation of environmentally acquired bacteria in contrast to vertically transmitted bumblebee-specific bacteria later in the season.