Antibiotic-Induced Change of Bacterial Communities Associated with the Copepod Nitocra spinipes

Gorokhova E (2012) Antibiotic-Induced Change of Bacterial Communities Associated with the Copepod Nitocra spinipes. PLoS ONE 7(3): e33107. doi:10.1371/journal.pone.0033107 Antibiotic-Induced Change of Bacterial Communities Associated with the Copepod Nitocra spinipes Anna Edlund 0 Karin Ek 0 Magnus Breitholtz 0 Elena Gorokhova 0 A. Mark Ibekwe, U. S. Salinity Lab, United States of America 0 1 Department of Systems Ecology, Stockholm University, Stockholm, Sweden, 2 Department of Applied Environmental Science, Stockholm University , Stockholm , Sweden Environmental pressures, such as physical factors, diet and contaminants may affect interactions between microbial symbionts and their multicellular hosts. Despite obvious relevance, effects of antimicrobial contaminants on host-symbiont relations in non-target aquatic organisms are largely unknown. We show that exposure to antibiotics had negative effects on survival and juvenile development of the copepod Nitocra spinipes and caused significant alterations in copepodassociated bacterial communities. The significant positive correlations between indices of copepod development and bacterial diversity indicate that disruption of the microflora was likely to be an important factor behind retarded juvenile development in the experimental animals. Moreover, as evidenced by ribotype distribution in the bacterial clone libraries, the exposure to antibiotics caused a shift in dominance from Betaproteobacteria to Cardinium bacteria; the latter have been shown to cause reproductive manipulations in various terrestrial arthropods. Thus, in addition to providing evidence that the antibiotic-induced perturbation of the microbial community associates with reductions in fitness-related traits of the host, this study is the first record of a copepod serving as a host for endosymbiotic Cardinium. Taken together, our results suggest that (1) antimicrobial substances and possibly other stressors can affect micobiome and symbiont-mediated interactions in copepods and other hosts, and (2) Cardinium endosymbionts may occur in other copepods and affect reproduction of their hosts. - Funding: This research was supported by grants from the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS) to EG, Swedish Foundation for Strategic Environmental Research (MISTRA) through the MistraPharma programme (MB), and the Department of Systems Ecology, Stockholm University (AE). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Bacteria mediate a variety of interactions within and between organisms. During the past 500 million years they have evolved diverse mechanisms to gain entry and proliferate in multicellular eukaryotes [1], with their effects on hosts ranging from harmful to beneficial [1,2]. Examples of such effects include reproduction disorders caused by the parasite Wolbachia proliferating in arthropod ovaries and testes [3] and beneficial effects from the symbiont Buchnera providing essential amino acids to their aphid hosts [4]. The greatest impact of symbiotic bacteria upon hosts can be found in the highly diverse phylum Arthropoda, where obligate host-bacteria associations have been well studied from reproductive and nutrition perspectives, especially in insects [1,5]. A recently discovered bacterial symbiont causing reproductive disorders in terrestrial arthropods is the Cardinium bacteria [6,7]. Since its discovery, it has been found in four orders of insects and in 67% of arthropods, a small number compared to Wolbachia, which has been detected in all insects orders and in 66% of arthropods [79]; however the number of potential Cardinium host species tested so far is relatively small. Effects of Cardinium bacteria on host fitness are largely unknown, but reproduction disorders in infected populations have been reported for the parasitoid wasp Encarsia pergandiella and the spider mites, Eotetranychnus suginamensis and Bryobia sarothamni [8,1012]. Whereas the importance of endosymbionts is well appreciated in terrestrial arthropods, virtually nothing is known about their distribution and functioning in copepods, the most abundant aquatic arthropods. In both freshwater and marine environments, these crustaceans are often the key players in pelagic and benthic food webs [13]. They also serve as the main food source for fish larvae in the wild and in aquacultures [14]. In addition, copepods are being increasingly used as test species in ecotoxicology, as a sexually reproducing alternative to the parthenogenic cladoceran Daphnia [15]. Of particular interest, therefore, are copepodbacteria interactions that have a potential to affect growth and reproduction in these animals. Application of advanced microscopy techniques together with molecular and cultivation approaches has shown that diverse copepod-associated bacteria communities exist both inside copepod gut and on exoskeleton [1618]. Therefore, it is highly likely that similar to other arthropods, symbiotic bacteria are important for copepod nutrition, immune responses and reproduction. Environmental pressures, such as physical factors, diet and contaminants may affect interactions between microbial symbionts and their multicellular hosts. Antibiotics have become environmental contaminants of concern as they are biologically active, which obviously is a part of their nature. In order to be effective, they often have a low biodegradability: 3090% doses of antibiotics enter the environment in their original form [19]. Antibiotics have been detected in the ng/L to mg/L range in the effluent of sewage treatment plants, surface water and ground water [19]; however, despite obvious relevance, effects of these contaminants on host-symbiont relations in non-target aquatic organisms are largely unknown. To directly explore associations between bacterial diversity and development of a copepod host as well as effects of antibiotics on these associations, the harpacticoid copepod Nitocra spinipes exposed to antibiotics was used as a model system. Three commercially used antibiotics (ciprofloxacin, sulfomethoxazole and trimethoprim) at low concentrations were used to perturb composition and structure of bacterial communities. These antibiotics were selected because they: (1) represent three prominent classes of antibiotics with differing mechanisms of action against bacteria, (2) have been detected in surface waters [19,20], and (3) have been identified as posing a threat to aquatic environment by risk assessment analysis based on the ratio between predicted environmental concentration and predicted no effect concentration [20]. We hypothesized that this exposure would alter composition of bacterial assemblages associated with the copepods, and that the disrupted copepod-bacteria interactions would (...truncated)