How symbioses between bacteria and aquatic animals influence food webs in freshwater ecosystems is a fundamental question in ecology. We investigated symbiosis between a crustacean zooplankton Daphnia magna and its dominant bacterial symbiont Limnohabitans, an abundant and globally distributed freshwater Betaproteobacteria. Aposymbiotic juvenile Daphnia were prepared and exposed to any of four Limnohabitans sp. - Limnohabitans strains DM1, 2KL-3, 2KL-7 and Limnohabitans planktonicus strain II-D5, all previously found in D. magna digestive tract or culture. Re-infected Daphnia were cultured until they produced the first clutch of juveniles. Limnohabitans strain DM1 and L. planktonicus strain II-D5 successfully re-infected Daphnia through single exposure at the first instar juvenile stage. In contrast to aposymbiotic Daphnia that produced non-viable juveniles, re-infected Daphnia produced viable juveniles and increased fecundity to levels of that of symbiotic Daphnia. Re-infected Daphnia did not increase their number of eggs nor growth rates. Limnohabitans strains 2KL-7 and 2KL-3 could not recover fecundity even in multiple exposures during culture. This study shows the functional evidence demonstrating that a single bacterium Limnohabitans regulates fecundity of the consumer Daphnia through symbiosis. Our results indicated that symbiotic relationship between major bacterioplankton and zooplankton is important for maintaining the population of zooplankton in freshwater ecosystems.

The filter-feeding crustacean Daphnia is a key organism in freshwater ecosystems. Here, we report the effect of symbiotic bacteria on ecologically important life history traits, such as population dynamics and longevity, in Daphnia magna. By disinfection of the daphniid embryos with glutaraldehyde, aposymbiotic daphniids were prepared and cultured under bacteria-free conditions. Removal of bacteria from the daphniids was monitored by quantitative polymerase chain reaction for bacterial 16S rRNA gene. The population of aposymbiotic daphniids was reduced 10-folds compared with that of the control daphniids. Importantly, re-infection with symbiotic bacteria caused daphniids to regain bacteria and increase their fecundity to the level of the control daphniids, suggesting that symbiotic bacteria regulate Daphnia fecundity. To identify the species of symbiotic bacteria, 16S rRNA genes of bacteria in daphniids were sequenced. This revealed that 50% of sequences belonged to the Limnohabitans sp. of the Betaproteobacteria class and that the diversity of bacterial taxa was relatively low. These results suggested that symbiotic bacteria have a beneficial effect on D. magna, and that aposymbiotic Daphnia are useful tools in understanding the role of symbiotic bacteria in the environmental responses and evolution of their hosts.

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