Bacterial epibionts of Daphnia: a potential route for the transfer of dissolved organic carbon in freshwater food webs

Abstract

The identification of interacting species and elucidation of their mode of interaction may be crucial to understand ecosystem-level processes. We analysed the activity and identity of bacterial epibionts in cultures of Daphnia galeata and of natural daphnid populations. Epibiotic bacteria incorporated considerable amounts of dissolved organic carbon (DOC), as estimated via uptake of tritiated leucine: three times more tracer was consumed by microbes on a single Daphnia than in 1 ml of lake water. However, there was virtually no incorporation if daphnids were anaesthetised, suggesting that their filtration activity was essential for this process. Microbial DOC uptake could predominantly be assigned to microbes that were located on the filter combs of daphnids, where the passage of water would ensure a continuously high DOC supply. Most of these bacteria were Betaproteobacteria from the genus Limnohabitans. Specifically, we identified a monophyletic cluster harbouring Limnohabitans planktonicus that encompassed sequence types from D. galeata cultures, from the gut of Daphnia magna and from daphnids of Lake Zurich. Our results suggest that the epibiotic growth of bacteria related to Limnohabitans on Daphnia spp. may be a widespread and rather common phenomenon. Moreover, most of the observed DOC flux to Daphnia in fact does not seem to be associated with the crustacean biomass itself but with its epibiotic microflora. The unexplored physical association of daphnids with heterotrophic bacteria may have considerable implications for our understanding of carbon transfer in freshwater food webs, that is, a trophic 'shortcut' between microbial DOC uptake and predation by fish.

Figure 1

Schematic depiction of the setup of experiment I. For the raw water (Rw) and filtrate (F) treatments, active D. galeata individuals were incubated for 1 h together with the lake water microbial assemblages (Rw) or in lake water filtrates (F). Both Rw and F treatments had been preincubated with tritiated leucine for 1 h before the addition of daphnids. In the anaesthetised treatment (A), daphnids were placed in CO₂-enriched mineral water before incubation in the lake water filtrates (negative control). Subsequently, daphnids were either dissected for microautoradiography (MAR-FISH, one animal), or total leucine uptake per individual was determined by scintillation counting (three animals). All treatments were done in triplicates.

Figure 2

Leucine uptake by the heterotrophic bacterial community (B) after 1 h of labelling by active D. galeata after 1 h of incubation in the labelled raw water (Rw), and by active (F) and anaesthetised (A) D. galeata maintained for 1 h in tracer containing 0.2 μm prefiltered lake water. Error bars represent the standard errors of triplicate water samples (B) or the standard errors of measurements from three replicates (Rw, F and A). Different lowercase letters above the bars of Rw, F and A indicate significant differences between treatments (analysis of variance (ANOVA), P<0.5).

Figure 3

Confocal photomicrographs of Daphnia spp. epibionts (left), and localisation of the depicted structures in a schematic drawing of a daphnid (right). Green cells are hybridised with probe R-Bt065, targeting Lhb bacteria. Depicted in red are other DNA-containing objects, that is, bacterial cells that are not Lhb and nuclei of Daphnia. (a, b) D. galeata feeding appendages with hybridised Lhb cells. (c) D. galeata feeding combs with hybridised Lhb cells surrounded by black halos from microautoradiography staining that indicates the uptake of tritiated leucine by these bacteria. (d) Feeding appendage of daphnid from Lake Zurich with hybridised Lhb and numerous other bacteria. TL3 and TL4 indicate trunk limbs 3 and 4.

Figure 4

The left panel indicates shared and unique OTUs (99% identity cutoff) of 16S rRNA gene sequences of Lhb bacteria from cultured Daphnia galeata, from Lake Zurich daphnids, and from cultured D. magna (Freese et al., 2009). The right panel indicates phylogenetic analysis (Maximum Likelihood method) of Limnohabitans spp. including sequences from cultured strains (Kasalický et al., 2013) and Daphnia metagenome (Qi et al., 2009). The individual OTUs are depicted as grey boxes, the broken line links sets of sequences from a single OTU. Values in brackets refer to the numbers of sequences in ‘collapsed' clusters depicted as wedges. Only bootstrap values of >50% (1000 replications) are reported. Scale bar, 10% estimated sequence divergence.

Figure 5

Development of chlorophyll a, temperature and of the populations of Daphnia sp. and pelagic Lhb bacteria in Lake Zurich from 17 April to 31 May 2013. Upper panel indicates Chlorophyll a concentrations and temperature between 0 and 15 m depth. Grey circles indicate the dates and depth of samplings for the incubations with radiolabelled tracers, and for DNA extraction to identify Lhb epibionts (last time point). Lower panel indicates abundances of juvenile (juv) and adult (adlt) daphnids and proportions of pelagic Lhb bacteria of all bacterioplankton cells.

Figure 6

Uptake of tritiated leucine (upper panels) and NAG (lower panels) by the heterotrophic bacterial community (B) after 1 h of incubation, by lake daphnids after 1 h of incubation in this labelled raw water (Rw), and by active (F) and anaesthetised lake daphnids (A) maintained for 1 h in tracer containing 0.2 μm prefiltered lake water. The experimental dates are indicated in each panel. Error bars represent the standard errors of triplicate water samples (B) or the standard errors of measurements from three replicates (Rw, F and A). Different lowercase letters on the bars of Rw, F and A indicate significant differences between the treatments (P<0.5).