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.Euprymna scolopes | microarray | mutualism | Vibrio fischeri | cytoskeleton

SummaryIn 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.

bacterial resistant stages ͉ doxycycline ͉ medical symbiotics ͉ multiple sclerosis ͉ spirochete cysts

SummaryPeptidoglycan 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 apoptosismediated 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.

Extraordinary preservation in amber of the Miocene termite Mastotermes electrodominicus has led to the discovery of fossil symbiotic microbes. Spirochete bacteria and wood-digesting protists were identified in the intestinal tissue of the insect. Fossil wood (xylem: developing vessel-element cells, fibers, pit connections), protists (most likely xylophagic amitochondriates), an endospore (probably of the filamentous intestinal bacterium Arthromitus ‫؍‬ Bacillus), and large spirochetes were seen in thin section by light and transmission electron microscopy. The intestinal microbiota of the living termite Mastotermes darwiniensis, a genus now restricted to northern Australia, markedly resembles that preserved in amber. This is a direct observation of a 20-million-year-old xylophagus termite fossil microbial community. Mastotermes, a genus of large tropical wood-ingesting termites, is of evolutionary importance for two reasons: (i) they harbor Mixotricha paradoxa and other amitochondriate many-genomed protists considered key to the early history of nucleated cells, and (ii) they are phylogenetically proximal to cockroaches (1). Both morphological and molecular features consistently place the single extant species (Mastotermes darwiniensis, family Mastotermitidae) as an early taxon ancestral to other termites. For example, Mastotermes is the only termite that, like cockroaches, oviposits an ootheca (egg case), although it is rudimentary (2). Numerous mastotermitid fossil specimens, corresponding to four extinct genera and about 20 species, occur from the Eocene [40 million years ago (mya)] to the Miocene (20-5 mya) of Australia, Brazil, the Caribbean, Central America, and especially Europe (3, 4). The two finest preserved fossil species, extremely similar to M. darwiniensis, occur in Oligocene and Miocene amber from southern Mexico (Mastotermes electromexicus) (5) and the Dominican Republic (Mastotermes electrodominicus) (6). The notoriously polyphagous ''living fossil'' M. darwiniensis is limited to northern Australia.Termites exhibit a complex and unique symbiosis with prokaryotic (eubacterial and archaebacterial) and eukaryotic microorganisms that live in their hindgut. The symbiotic microbiota digest cellulose to sugars and acetate and produce hydrogen, methane, and carbon dioxide (7). In addition to Mixotricha paradoxa, a giant trichomonad motile by means of a unique set of attached surface spirochetes (8, 9), M. darwiniensis harbors other Archaeprotista (class Parabasalia, those with distinctive parabasal bodies, a type of Golgi apparatus), including the two hypermastigote genera (Koruga and Deltotrichonympha), a devescovinid (Metadevescovina extranea), and a trichomonad (Pentatrichomonoides scroa) that harbors endosymbiotic methanogenic bacteria (10).Where and when did this symbiosis evolve? Here we use the remarkable preservative properties of termites in amber to seek direct paleontological evidence for the evolution of termite intestinal symbionts. Cellular and subcellular structures of amber-preserved tis...

A high frequency of dieback and mortality was found in Rhizophora mangle (red mangrove) in coastal mangrove forests of southwest Puerto Rico. Eight plots were established in a variety of mangrove environments to assess the extent and severity of the dieback and mortality. The imperfect fungus Cytorpora rhizophorae was isolated consistently from stem dieback and associated canker tissues. No other pathogenic microorganisms were isolated. Pathogenicity was demonstrated in greenhouse and field inoculation experiments with C. rhizophorae using young R. mangle seedlings. In field studies of seedlings inoculated, mortality was 33 percent, and greenhouse mortality was as high as 50 percent. Cytospora rhizophorae was isolated consistently from diseased mangrove tissue; the fungus was grown in pure culture and then inoculated into healthy red mangroves, causing cankers. Koch'~. postulates were fulfilled by infecting R. mangh with C. rhizophorae, producing disease symptoms, and successfully re-isolating C. rhizophorue from the diseased tissue. This is the first documentation of the pathogenicity of C. rhizophorae associated with mortality in red mangrove. In the coastal mangrove forests of southwest Puerto Rico, C. rhizophorae may play an important role in the frequency of dieback and mortality in R. mangle, producing slow stem diameter growth and causing frequent stem and root wounds.

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