Genetically divergent Symbiodinium sp. display distinct molecular responses to pathogenic Vibrio and thermal stress.

Global climate change and anthropogenic activities are threatening the future survival of coral reef ecosystems. The ability of reef-building zooxanthellate coral to survive these stressors may be determined through fundamental differences within their symbiotic dinoflagellates (Symbiodinium sp.). We define the in vitro apoptotic response of 2 evolutionarily distant Symbiodinium sp., subtypes B2 and C1, to determine the synergistic effects of disease and temperature on cell viability using flow cytometry. The putative yellow band disease (YBD) consortium of Vibrio spp. bacteria and temperature (33°C) had a positive synergistic effect on C1 apoptosis, while B2 displayed increased apoptosis to elevated temperature (29 and 33°C), the Vibrio consortium, and a lone virulent strain of V. alginolyticus, but no synergistic effects. Additionally, heat shock protein 60 expression revealed differential cell-mediated temperature sensitivity between subtypes via western blotting. This result marks the first evidence of Symbiodinium sp. apoptotic variations to YBD pathogens and emphasizes the potential impact of synergistic stress on globally distributed coral-Symbiodinium symbioses.

Ulcerated yellow spot syndrome: implications of aquaculture-related pathogens associated with soft coral Sarcophyton ehrenbergi tissue lesions.

We introduce a new marine syndrome called ulcerated yellow spot, affecting the soft coral Sarcophyton ehrenbergi. To identify bacteria associated with tissue lesions, tissue and mucus samples were taken during a 2009 Indo-Pacific research expedition near the Wakatobi Island chain, Indonesia. Polymerase chain reaction targeting the 16S rDNA gene indicated associations with the known fish-disease-causing bacterium Photobacterium damselae, as well as multiple Vibrio species. Results indicate a shift toward decreasing diversity of bacteria in lesioned samples. Photobacterium damselae ssp. piscicida, formerly known as Pasteurella piscicida, is known as the causative agent of fish pasteurellosis and in this study, was isolated solely in lesioned tissues. Globally, fish pasteurellosis is one of the most damaging fish diseases in marine aquaculture. Vibrio alginolyticus, a putative pathogen associated with yellow band disease in scleractinian coral, was also isolated from lesioned tissues. Lesions appear to be inflicting damage on symbiotic zooxanthellae (Symbiodinium sp.), measurable by decreases in mitotic index, cell density and photosynthetic efficiency. Mitotic index of zooxanthellae within infected tissue samples was decreased by ~80%, while zooxanthellae densities were decreased by ~40% in lesioned tissue samples compared with healthy coral. These results provide evidence for the presence of known aquaculture pathogens in lesioned soft coral and may be a concern with respect to cross-species epizootics in the tropics.

Spirochete attachment ultrastructure: Implications for the origin and evolution of cilia.

The fine structure of spirochete attachments to the plasma membrane of anaerobic protists displays variations here interpreted as legacies of an evolutionary sequence analogous to that from free-living spirochetes to undulipodia (eukaryotic "flagella" and homologous structures). Attached spirochetes form a vestment, a wriggling fringe of motile cells at the edge of the plasma membrane of unidentified cellulolytic protist cells in the hypertrophied hindgut of the digestive system of Mastotermes darwiniensis, the large wood-feeding termite from northern Australia. From the membrane extend both undulipodia and a complex of comparably sized (10-12 microm x 0.2-0.3 microm) ectosymbiotic spirochetes that resembles unruly ciliated epithelium. In the intestines are helical (swimming) and round-body morphotypes. Round bodies (RBs) are slow or immotile spirochetes, propagules known to revert to typical swimming helices under culture conditions favorable for growth. The surfaces of both the spirochete gram-negative eubacteria and the parabasalid protists display distinctive attachment structures. The attached hypertrophied structures, some of which resemble ciliate kinetids, are found consistently at sites where the spirochete termini contact the protist plasma membranes.

Transcriptional patterns in both host and bacterium underlie a daily rhythm of anatomical and metabolic change in a beneficial symbiosis.

Mechanisms for controlling symbiont populations are critical for maintaining the associations that exist between a host and its microbial partners. We describe here the transcriptional, metabolic, and ultrastructural characteristics of a diel rhythm that occurs in the symbiosis between the squid Euprymna scolopes and the luminous bacterium Vibrio fischeri. The rhythm is driven by the host's expulsion from its light-emitting organ of most of the symbiont population each day at dawn. The transcriptomes of both the host epithelium that supports the symbionts and the symbiont population itself were characterized and compared at four times over this daily cycle. The greatest fluctuation in gene expression of both partners occurred as the day began. Most notable was an up-regulation in the host of >50 cytoskeleton-related genes just before dawn and their subsequent down-regulation within 6 h. Examination of the epithelium by TEM revealed a corresponding restructuring, characterized by effacement and blebbing of its apical surface. After the dawn expulsion, the epithelium reestablished its polarity, and the residual symbionts began growing, repopulating the light organ. Analysis of the symbiont transcriptome suggested that the bacteria respond to the effacement by up-regulating genes associated with anaerobic respiration of glycerol; supporting this finding, lipid analysis of the symbionts' membranes indicated a direct incorporation of host-derived fatty acids. After 12 h, the metabolic signature of the symbiont population shifted to one characteristic of chitin fermentation, which continued until the following dawn. Thus, the persistent maintenance of the squid-vibrio symbiosis is tied to a dynamic diel rhythm that involves both partners.

Taming the symbiont for coexistence: a host PGRP neutralizes a bacterial symbiont toxin.

In horizontally transmitted mutualisms between marine animals and their bacterial partners, the host environment promotes the initial colonization by specific symbionts that it harvests from the surrounding bacterioplankton. Subsequently, the host must develop long-term tolerance to immunogenic bacterial molecules, such as peptidoglycan and lipopolysaccaride derivatives. We describe the characterization of the activity of a host peptidoglycan recognition protein (EsPGRP2) during establishment of the symbiosis between the squid Euprymna scolopes and its luminous bacterial symbiont Vibrio fischeri. Using confocal immunocytochemistry, we localized EsPGRP2 to all epithelial surfaces of the animal, and determined that it is exported in association with mucus shedding. Most notably, EsPGRP2 was released by the crypt epithelia into the extracellular spaces housing the symbionts. This translocation occurred only after the symbionts had triggered host morphogenesis, a process that is induced by exposure to the peptidoglycan monomer tracheal cytotoxin (TCT), a bacterial 'toxin' that is constitutively exported by V. fischeri. Enzymatic analyses demonstrated that, like many described PGRPs, EsPGRP2 has a TCT-degrading amidase activity. The timing of EsPGRP2 export into the crypts provides evidence that the host does not export this protein until after TCT induces morphogenesis, and thereafter EsPGRP2 is constantly present in the crypts ameliorating the effects of V. fischeri TCT.

Peptidoglycan induces loss of a nuclear peptidoglycan recognition protein during host tissue development in a beneficial animal-bacterial symbiosis.

Peptidoglycan recognition proteins (PGRPs) are mediators of innate immunity and recently have been implicated in developmental regulation. To explore the interplay between these two roles, we characterized a PGRP in the host squid Euprymna scolopes (EsPGRP1) during colonization by the mutualistic bacterium Vibrio fischeri. Previous research on the squid-vibrio symbiosis had shown that, upon colonization of deep epithelium-lined crypts of the host light organ, symbiont-derived peptidoglycan monomers induce apoptosis-mediated regression of remote epithelial fields involved in the inoculation process. In this study, immunofluorescence microscopy revealed that EsPGRP1 localizes to the nuclei of epithelial cells, and symbiont colonization induces the loss of EsPGRP1 from apoptotic nuclei. The loss of nuclear EsPGRP1 occurred prior to DNA cleavage and breakdown of the nuclear membrane, but followed chromatin condensation, suggesting that it occurs during late-stage apoptosis. Experiments with purified peptidoglycan monomers and with V. fischeri mutants defective in peptidoglycan-monomer release provided evidence that these molecules trigger nuclear loss of EsPGRP1 and apoptosis. The demonstration of a nuclear PGRP is unprecedented, and the dynamics of EsPGRP1 during apoptosis provide a striking example of a connection between microbial recognition and developmental responses in the establishment of symbiosis.