Hauke Koch

Bacterial gut symbiont communities are critical for the health of many insect species. However, little is known about how microbial communities vary among host species or how they respond to anthropogenic disturbances. Bacterial communities that differ in richness or composition may vary in their ability to provide nutrients or defenses. We used deep sequencing to investigate gut microbiota of three species in the genus Bombus (bumble bees). Bombus are among the most economically and ecologically important non-managed pollinators. Some species have experienced dramatic declines, probably due to pathogens and land-use change. We examined variation within and across bee species and between semi-natural and conventional agricultural habitats. We categorized as ‘core bacteria’ any operational taxonomic units (OTUs) with closest hits to sequences previously found exclusively or primarily in the guts of honey bees and bumble bees (genera Apis and Bombus). Microbial community composition differed among bee species. Richness, defined as number of bacterial OTUs, was highest for B. bimaculatus and B. impatiens. For B. bimaculatus, this was due to high richness of non-core bacteria. We found little effect of habitat on microbial communities. Richness of non-core bacteria was negatively associated with bacterial abundance in individual bees, possibly due to deeper sampling of non-core bacteria in bees with low populations of core bacteria. Infection by the gut parasite Crithidia was negatively associated with abundance of the core bacterium Gilliamella and positively associated with richness of non-core bacteria. Our results indicate that Bombus species have distinctive gut communities, and community-level variation is associated with pathogen infection.

Here we report the genome of one gammaproteobacterial member of the gut microbiota, for which we propose the name Candidatus Schmidhempelia bombi, that was inadvertently sequenced alongside the genome of its host, the bumble bee, Bombus impatiens. This symbiont is a member of the recently described bacterial order Orbales, which has been collected from the guts of diverse insect species, however, Schmidhempelia has been identified exclusively with bumble bees. Metabolic reconstruction reveals that Schmidhempelia lacks many genes for a functioning NADH dehydrogenase I, all genes for the high-oxygen cytochrome o, and most genes in the TCA cycle. Schmidhempelia has retained NADH dehydrogenase II, the low-oxygen specific cytochrome bd, anaerobic nitrate respiration, mixed acid fermentation pathways, and citrate fermentation, which may be important for survival in low-oxygen or anaerobic environments found in the bee hindgut. Additionally, a type-6 secretion system, a Flp pilus, and many antibiotic/multidrug transporters suggest complex interactions with its host and other gut commensals or pathogens. This genome has signatures of reduction (2.0 megabase pairs) and rearrangement, as previously observed for genomes of host-associated bacteria. A survey of wild and laboratory B. impatiens revealed that Schmidhempelia is present in 90% of individuals and, therefore, may provide benefits to its host.

Several floral microbes are known to be pathogenic to plants or floral visitors such as pollinators.  Despite the ecological and economic importance of pathogens deposited in flowers, we often lack a basic understanding of how floral traits influence disease transmission.  Here we provide the first systematic review regarding how floral traits attract vectors (for plant pathogens) or hosts (for animal pathogens), mediate disease establishment, and evolve under complex interactions with plant mutualists that can be vectors for microbial antagonists.  Attraction of floral visitors is influenced by numerous phenological, morphological, and chemical traits, and several plant pathogens manipulate floral traits to attract vectors.  There is rapidly growing interest in how floral secondary compounds and antimicrobial enzymes influence disease establishment in plant hosts.  Similarly, new research suggests that consumption of floral secondary compounds can reduce pathogen loads in animal pollinators.  Given recent concerns about pollinator declines caused in part by pathogens, the role of floral traits in mediating pathogen transmission is a key area for further research.  We conclude by discussing important implications of floral transmission of pathogens for agriculture, conservation, and human health, suggesting promising avenues for future research in both basic and applied biology.

Zusammenfassung Diese Doktorarbeit untersucht die Artenvielfalt, Ökologie und Evolution der bakteriellen Gemeinschaften im Darm von Hummeln und ihre Interaktion mit Parasiten. In einer ersten Bestandsaufnahme der Darmbakterien von mitteleuropäischen Hummelarten wurde eine vergleichsweise artenarme und noch unbekannte bakterielle Gemeinschaft, dominiert von je einem Beta-und Gammaproteobacterium, Milchsäurebakterien, Bacteroidetes und Bifidobakterien gefunden.

The animal gut is a habitat for diverse communities of microorganisms (microbiota). Honeybees and bumblebees have recently been shown to harbour a distinct and species poor microbiota, which may confer protection against parasites. Here, we investigate diversity, host specificity and transmission mode of two of the most common, yet poorly known, gut bacteria of honeybees and bumblebees: Snodgrassella alvi (Betaproteobacteria) and Gilliamella apicola (Gammaproteobacteria). We analysed 16S rRNA gene sequences of these bacteria from diverse bee host species across most of the honeybee and bumblebee phylogenetic diversity from North America, Europe and Asia. These focal bacteria were present in 92% of bumblebee species and all honeybee species but were found to be absent in the two related corbiculate bee tribes, the stingless bees (Meliponini) and orchid bees (Euglossini). Both Snodgrassella alvi and Gilliamella apicola phylogenies show significant topological congruence with the phylogeny of their bee hosts, albeit with a considerable degree of putative host switches. Furthermore, we found that phylogenetic distances between Gilliamella apicola samples correlated with the geographical distance between sampling locations. This tentatively suggests that the environmental transmission rate, as set by geographical distance, affects the distribution of G. apicola infections. We show experimentally that both bacterial taxa can be vertically transmitted from the mother colony to daughter queens, and social contact with nest mates after emergence from the pupa greatly facilitates this transmission. Therefore, sociality may play an important role in vertical transmission and opens up the potential for co-evolution or at least a close association of gut bacteria with their hosts.

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

Laboratory experiments are often preferred over field experiments because they allow the control of confounding factors that would otherwise influence the causal effect of a particular focal experimental factor. These confounding factors can, however, significantly alter the response of an organism confronted with a particular situation, which can have great implications. In a field experiment with a bumblebee host–parasite system, we looked at the influence of additional food supply and immune challenge on various colony fitness values and parasite traits. We could confirm the importance of food on the colony fitness, but not on parasite infection probability or parasite genetic diversity. In contrast to the findings of laboratory experiments of this system, challenge of the immune system had no significant effect on colony fitness or parasite infections. These results likely reflect an overriding effect of environmental variation without disproving the concept of a cost of defence per se. But the results also demonstrate that confounding factors purposely controlled for in the laboratory have to be weighed against their ecological relevance, and stress the need for careful analysis before any direct transfer is made of laboratory results to field situations.

"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. Bumble bees have recently been shown to possess simple and highly specific microbiota. We here examine the dynamics of these microbiota in field colonies of the bumble bee Bombus terrestris over one season. The gut bacteria were assessed with culture-independent methods, i.e. with terminal restriction fragment length profiles (TRFLPs) 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 a) natural infections with the microbial parasites Crithidia bombi and Nosema bombi; b) bumble bee worker size; c) colony identity, and d) colony age.
4. We found an increase in diversity of the microbiota in individuals naturally infected with either C. bombi or N. bombi. C. 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 bumble bees 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 homeostasis in this community under widely different environments."

Populations of important pollinators, such as bumble bees and
honey bees, are declining at alarming rates worldwide. Parasites
are likely contributing to this phenomenon. A distinct resident
community of bacteria has recently been identified in bumble bees and honey bees that is not shared with related solitary bee species. We now show that the presence of these microbiota protects bee hosts against a widespread and highly virulent natural parasite (Crithidia bombi) in an experimental setting. We add further support to this antagonistic relationship from patterns found in field data. For the successful establishment of these microbiota and a protective effect, exposure to feces from nest mates was needed after pupal eclosion. Transmission of beneficial gut bacteria could therefore represent an important benefit of sociality. Our results stress the importance of considering the host microbiota as an “extended immune phenotype” in addition to the host immune system itself and provide a unique perspective to understanding bees in health and disease.

Recent studies on the microbial flora of the honeybee gut have revealed an apparently highly specific community of resident bacteria that might play a role in immune defence and food preservation for their hosts. However, at present very little is known about the diversity and ecology of bacteria occurring in non-domesticated bees like bumblebees, which are of similar importance as honeybees for the pollination of agricultural and wild flowers. To fill this gap in knowledge, we examined six of the most common bumblebee species in central Europe from three locations in Germany and Switzerland for their bacterial communities. We used a culture independent molecular approach based on sequencing the 16S rRNA gene from a selection of individuals and examining a larger number of samples by terminal restriction fragment length polymorphism profiles (TRFLPs). The gut flora was dominated by very few and mostly undescribed groups of bacteria belonging to the Proteobacteria, Bacteroidetes, Firmicutes and Actinobacteria. This core set of bacteria was present in all of the examined bumblebee species. These bacteria are similar to, but distinct from bacteria previously described from the honeybee gut. Significant differences were observed between the communities of bacteria in the different bumblebee species, the effect of sampling location was less strong. A novel group of Betaproteobacteria additionally shows evidence for host species specific genotypes. The gut flora of bumblebees therefore is apparently composed of relatively few, highly specialized bacteria, indicating a strong interaction and possibly important functions to their hosts.

We present the first record of honeydew feeding in Malagasy stingless bees. Two species of stingless bees, Liotrigona mahafalya and L. madecassa, collected honeydew produced by mealybugs on an Albizia perrieri (Fabaceae) tree in the dry deciduous forest of Kirindy, Madagascar. Honeydew might represent an important part of the diet of Malagasy stingless bees, especially in times of scarce floral resources in the highly seasonal environment of western Madagascar. The interaction between the bees and two species of invasive ants, Monomorium destructor and Paratrechina longicornis, in competition for the honeydew resource, was studied. Numbers of stingless bees and ants on the honeydew source were negatively correlated, with ants decreasing in density distally from the main trunk of the tree and bees showing the opposite trend. Invasive ants could therefore potentially threaten the native bees by displacing them from this resource.

The taxonomy of the western Malagasy stingless bees (Liotrigona spp.) has been complicated by the high degree of morphological similarity between the described species. This group includes one of the smallest bee species of the world, Liotrigona bitika (Brooks & Michener, 1988). However, the description of this species is solely based on size differences, and observation of individuals intermediate in size between Liotrigona bitika and Liotrigona madecassa (Saussure, 1890) have cast doubt on the distinctness of the former taxon. I examined specimens, collected in the dry deciduous forest of Kirindy, representing all three of the described Liotrigona species of western Madagascar using a combined approach of size measures and DNA sequence analysis of the cytochrome oxidase I barcoding region. The analysis revealed all described taxa to be valid and furthermore uncovered the existence of a fourth taxon, described as Liotrigona kinzelbachi sp. n. The sympatric species exhibit non-overlapping body size ranges, indicating a strong influence of body size in niche differentiation of this otherwise morphological highly similar clade.

Tropical dry deciduous forest is an endangered ecosystem whose plant-pollinator relationships are little known. We characterised a portion of the web of interactions between flowering plants and flower visitors in the Kirindy Forest of the Menabe region of west-central Madagascar. Taking a plant-centered approach, we observed individuals of the 5 most abundant native plant species that were coming into flower at the end of the annual dry season, and recorded all identifiable flower-visitors. Taking a visitor-centered approach, we walked a network of established trails and listened for distinctive calls of a common flower-visiting bird, noting the plant species visited. The former approach revealed connections among the early-flowering species via birds and insects, whereas the latter confirmed these connections and added an additional plant species. Flowers of the 6 plant species were visited on average by 5.5 animal species, while 10 visitor species for which we had reasonable samples frequented on average the flowers of 3.3 plant species. These qualitative results resemble those reported from other temperate and tropical webs, in that interactions appeared to be relatively generalised by pollinator species and body plan (e.g., birds vs. bees). Also in agreement, the visitation web was significantly nested, with more-specialised species tending to interact with mutualistic partners that were themselves more generalised. In addition to documenting previously-unreported interactions, therefore, this preliminary web conforms to more widespread patterns emerging for pollination systems at the community level.