Hydrothermal vent gastropods from the same family (Provannidae) harbour epsilon- and gamma-proteobacterial endosymbionts

The discovery of new hydrothermal vent systems in the back-arc basins of the Western Pacific revealed chemosynthesis-based faunal communities distinct from those of other vents. These vents are dominated by two related gastropods (Alviniconcha spp. and Ifremeria nautilei) that harbour symbiotic bacteria in their gills. We used comparative 16S ribosomal RNA (rRNA) gene sequencing and in situ hybridization with rRNA-targeted probes to characterize the bacterial symbionts of Alviniconcha sp. and I. nautilei from the Manus Basin in the Western Pacific. The analyses revealed that these two gastropod species, although affiliated with the same family, harbour phylogenetically distant chemosymbionts, suggesting independent origins of these endosymbioses. The I. nautilei endosymbiont clusters with sulfur-oxidizing bacteria within the gamma-Proteobacteria, as is the case for all previously characterized endosymbionts from a wide diversity of host taxa harbouring thioautotrophic prokaryotes. In contrast, the Alviniconcha endosymbiont is affiliated with sulfur-oxidizing bacteria within the epsilon-Proteobacteria. These results show that bacteria from the epsilon-Proteobacteria are also capable of forming endosymbiotic associations with marine invertebrates from chemosynthetic environments. More generally, the endosymbiotic lifestyle is now shown to be distributed throughout all recognized classes of the Proteobacteria.     

Trypanosoma cruzi: ultrastructural studies of adhesion, lysis and biofilm formation by Serratia marcescens

A few days after blood meal the number of bacteria in the anterior midgut (stomach) of Rhodnius prolixus, a vector of Trypanosoma cruzi, the causative agent of Chagas' disease, increases dramatically. Many of the bloodstream trypomastigotes of the pathogenic protozoan as well as ingested erythrocytes are lysed in the stomach. Incubation of T. cruzi with Serratia marcescens variant SM365, lead to parasite lysis. In the present study, this bacterium rapidly adhered to the protozoan surface through d-mannose recognizing fimbriae and rapidly induced its complete lysis. In contrast, the DB11 variant of the same bacterial species did not adhere and did not induce protozoan lysis. Scanning and transmission electron microscopy revealed that following bacteria-protozoan attachment there is an assembly of long filamentous structures, identified as a biofilm, which connect the protozoan to the bacteria forming bacterial clusters. We conclude that parasite lysis and biofilm formation mechanisms are important for understanding parasite-microbiota interactions in the gut of insect vectors of trypanosomatids.     

Metagenomic analysis reveals diverse polyketide synthase gene clusters in microorganisms associated with the marine sponge Discodermia dissoluta

Sponge-associated bacteria are thought to produce many novel bioactive compounds, including polyketides. PCR amplification of ketosynthase domains of type I modular polyketide synthases (PKS) from the microbial community of the marine sponge Discodermia dissoluta revealed great diversity and a novel group of sponge-specific PKS ketosynthase domains. Metagenomic libraries totaling more than four gigabases of bacterial genomes associated with this sponge were screened for type I modular PKS gene clusters. More than 90% of the clones in total sponge DNA libraries represented bacterial DNA inserts, and 0.7% harbored PKS genes. The majority of the PKS hybridizing clones carried small PKS clusters of one to three modules, although some clones encoded large multimodular PKSs (more than five modules). The most abundant large modular PKS appeared to be encoded by a bacterial symbiont that made up < 1% of the sponge community. Sequencing of this PKS revealed 14 modules that, if expressed and active, is predicted to produce a multimethyl-branched fatty acid reminiscent of mycobacterial lipid components. Metagenomic libraries made from fractions enriched for unicellular or filamentous bacteria differed significantly, with the latter containing numerous nonribosomal peptide synthetase (NRPS) and mixed NRPS-PKS gene clusters. The filamentous bacterial community of D. dissoluta consists mainly of Entotheonella spp., an unculturable sponge-specific taxon previously implicated in the biosynthesis of bioactive peptides.     

A topographical and typological comparison of the rodlike setae of ambulatory dactylopodites in decapod crustaceans

The arrangement and external morphology of the rodlike setae and associated structures located on the dactylopodites of the walking legs of six species of decapod crustaceans are compared. The dactyls of littoral species, represented by the rock crab, Cancer antennarius, and the spiny lobster, Panulirus interruptus, have dense tufts and bands of rodlike setae, as is typical of many decapods, and additionally only a few small plumed setae. The arrangement of setae on the dactyls of the recently discovered Galapagos vent crab, Bythograea thermydron, closely resembles that of C. antennarius. Rodlike and long plumed setae occur in about equal numbers on the dactyls of the pelagic anomuran, Pleuroncodes planipes. The dactyls having the fewest rodlike setae are those of the terrestrial hermit crab, Coenobita perlatus, and those of the kelp crab, Pugettia producta, where flat setae typical of Majidae have replaced most rodlike setae. The presence and structures of the terminal pores in rodlike setae vary intra- and interspecifically, possibly as a function of molt stage. Variations in some features of rodlike setae, such as tip acuity and presence of microsetae and surface sculpting, appear to be related to development. Serrated setae occur on the dactyls of megalopal P. producta but not in later stages. The topography and typology of setae located on the ambulatory dactyls of decapod crustaceans are considered in light of recent interest in using setal characteristics to determine the sensory functions of sensilla and to clarify the phylogeny of arthropod groups.     

Metagenomic Analysis Reveals Diverse Polyketide Synthase Gene Clusters in Microorganisms Associated with the Marine Sponge Discodermia dissoluta

Sponge-associated bacteria are thought to produce many novel bioactive compounds, including polyketides. PCR amplification of ketosynthase domains of type I modular polyketide synthases (PKS) from the microbial community of the marine sponge Discodermia dissoluta revealed great diversity and a novel group of sponge-specific PKS ketosynthase domains. Metagenomic libraries totaling more than four gigabases of bacterial genomes associated with this sponge were screened for type I modular PKS gene clusters. More than 90% of the clones in total sponge DNA libraries represented bacterial DNA inserts, and 0.7% harbored PKS genes. The majority of the PKS hybridizing clones carried small PKS clusters of one to three modules, although some clones encoded large multimodular PKSs (more than five modules). The most abundant large modular PKS appeared to be encoded by a bacterial symbiont that made up <1% of the sponge community. Sequencing of this PKS revealed 14 modules that, if expressed and active, is predicted to produce a multimethyl-branched fatty acid reminiscent of mycobacterial lipid components. Metagenomic libraries made from fractions enriched for unicellular or filamentous bacteria differed significantly, with the latter containing numerous nonribosomal peptide synthetase (NRPS) and mixed NRPS-PKS gene clusters. The filamentous bacterial community of D. dissoluta consists mainly of Entotheonella spp., an unculturable sponge-specific taxon previously implicated in the biosynthesis of bioactive peptides.     

Microstructure of the tarsal attachment devices in the earwig Timomenus komarovi

The biological attachment device on the tarsal appendage of the earwig, Timomenus komarovi (Insecta: Dermaptera: Forficulidae) was investigated using field emission scanning electron microscopy to reveal the fine structural characteristics of its biological attachment devices to move on smooth and rough surfaces. They attach to rough substrates using their pretarsal claws; however, attachment to smooth surfaces is achieved by means of two groups of hairy tarsal pads. This biological attachment device consists of fine hairy setae with various contact sizes. Three different groups of tenent setae were distinguished depending on the cuticular substructure of the endplates. Two groups of setae commonly had flattened surfaces, and they were covered with either spoon‐shaped or spatula‐shaped endplates, respectively. While the flattened tip setae were distributed at the central region, the pointed tip setae were characteristically found along the marginal region. There were no obvious gender‐specific differences between fibrillar adhesive pads in this insect mainly because the forceps‐like pincers are used during copulation to grasp the partner.     

The design of the fly adhesive pad: distal tenent setae are adapted to the delivery of an adhesive secretion

Flies (Brachycera) have adhesive pads called pulvilli at the terminal tarsomere. The pulvilli are covered by tenent setae, sometimes termed tenent hairs, which serve to increase the actual area of attachment to the surface. By using transmission and scanning electron microscopy it is shown that proximal and distal tenent setae have different ultrastructures. The design of distal adhesive setae is adapted for the release of adhesive substances close to the area of contact. It is concluded that secretion injection is precisely targeted under the distal tip of a single seta.     

Leishmania (Leishmania) chagasi interactions with Serratia marcescens: ultrastructural studies, lysis and carbohydrate effects

Studies on the lysis of L. chagasi caused by the bacteria Serratia marcescens were carried out. In vitro experiments demonstrated that S. marcescens variant SM 365, a prodigiosin pigment producer, lysed this species of Leishmania but variant DB11, a nonpigmented bacteria, was unable to lyse the parasite. High concentrations of d-mannose were found to protect L. chagasi markedly diminishing the lysis by S. marcescens SM 365. Promastigotes of L. chagasi bound the lectin Concanavalin A conjugated with FITC, the fluorescence was intensely found at the base of the flagellum (flagellar pocket). Scanning electron microscopy revealed that the bacteria adherence occurred mainly in the flagellar pocket. S. marcescens SM 365 formed filamentous structures, identified as biofilms, which connect the protozoan to the developing bacterial clusters, in low concentrations of bacteria after 30 min incubation time. We suggest that bacterial mannose-sensitive (MS) fimbriae are relevant to S. marcescens SM 365 in the lysis of L. chagasi.     

Scanning Electron Microscope Study of Bacteria Associated with the Rumen Epithelium of Sheep

Examination of the rumen epithelium of sheep by scanning electron microscopy revealed bacteria associated with the epithelial surface. Comparison of epithelial surfaces from 10 sheep revealed areas that were consistently densely covered with bacteria and other areas where the cover was consistently light. The bacterial populations were frequently of mixed morphological types, but areas populated with a single type were also observed. This finding, together with the discovery of bacterial forms not previously described in rumen contents, suggests that a specific flora may exist on the rumen epithelial surface. The functional significance of such a population is discussed.     

Bacteria Associated with the Gut Tract of Larval Stages of the Aquatic Cranefly Tipula abdominalis (Diptera; Tipulidae)

Scanning electron microscopy, light microscopy, and direct isolations were used to examine the distribution and diversity of bacteria in the gut tracts of larval stages of Tipula abdominalis. The animal had an enlarged hindgut which housed a diverse bacterial community in the lumen and directly attached to the gut wall. Distinct localization was noted, with the most dense and most diverse community anterior to the rectum. A distinct architecture of bacteria occurred in this region, characterized by a layering or a “weblike” array of filamentous bacteria overlying mats of bacteria closely associated with the gut wall. Although morphological diversity was high in the hindgut, filamentous bacteria were the dominant morphology observed. The attached microbiota, sloughed during ecdysis, recolonized to the same density and diversity observed before the molt. The majority of the isolatable bacterial types were facultatively anaerobic. The distinct localization and attached nature of the hindgut bacteria and the recolonization after each molt suggest they are indigenous to this region of the gut tract.     

Phylogenetic analysis of intestinal bacteria in the Chinese mitten crab (Eriocheir sinensis)

AIMS: To identify the dominant intestinal bacteria in the Chinese mitten crab, and to investigate the differences in the intestinal bacteria between pond-raised and wild crabs. METHODS AND RESULTS: The diversity of intestinal bacteria in the Chinese mitten crabs was investigated by denaturing gradient gel electrophoresis (DGGE) fingerprinting, 16S rRNA gene clone library analysis and real-time quantitative PCR. The principal component analysis of DGGE profiles indicated that substantial intersubject variations existed in intestinal bacteria in pond-raised crab. The sequencing of 16S rRNA genes revealed that 90-95% of the phylotypes in the clone libraries were affiliated with Proteobacteria and Bacteroidetes. Some genera were identified as unique in wild crabs and in pond-raised crabs, whereas Bacteroidetes was found to be common in all sampled crab groups. Real-time quantitative PCR indicated that the abundance of Bacteroides and the total bacterial load were approximately four-to-10 times higher in pond-raised crabs than in wild crabs. A significant portion of the phylotypes shared low similarity with previously sequenced organisms, indicating that the bacteria in the gut of Chinese mitten crabs are yet to be described. CONCLUSIONS: The intestinal bacteria of pond-raised crabs showed higher intersubject variation, total diversity and abundance than that observed in wild crabs. The high proportion of the clones of Proteobacteria and Bacteroidetes in the clone library is an indication that these bacteria may be the dominant population in the gut of the Chinese mitten crab. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated obvious differences in the intestinal bacterial composition of pond-raised crabs and wild crabs. This knowledge will increase our understanding of the effects of aquaculture operations on bacterial community composition in the crab gut and provide necessary data for the development of probiotic products for crab cultivation.     

Explaining Bacterial Dispersion on Leaf Surfaces with an Individual-Based Model (PHYLLOSIM)

We developed the individual-based model PHYLLOSIM to explain observed variation in the size of bacterial clusters on plant leaf surfaces (the phyllosphere). Specifically, we tested how different ‘waterscapes’ impacted the diffusion of nutrients from the leaf interior to the surface and the growth of individual bacteria on these nutrients. In the ‘null’ model or more complex ‘patchy’ models, the surface was covered with a continuous water film or with water drops of equal or different volumes, respectively. While these models predicted the growth of individual bacterial immigrants into clusters of variable sizes, they were unable to reproduce experimentally derived, previously published patterns of dispersion which were characterized by a much larger variation in cluster sizes and a disproportionate occurrence of clusters consisting of only one or two bacteria. The fit of model predictions to experimental data was about equally poor (<5%) regardless of whether the water films were continuous or patchy. Only by allowing individual bacteria to detach from developing clusters and re-attach elsewhere to start a new cluster, did PHYLLOSIM come much closer to reproducing experimental observations. The goodness of fit including detachment increased to about 70–80% for all waterscapes. Predictions of this ‘detachment’ model were further supported by the visualization and quantification of bacterial detachment and attachment events at an agarose-water interface. Thus, both model and experiment suggest that detachment of bacterial cells from clusters is an important mechanism underlying bacterial exploration of the phyllosphere.     

A thin-section and freeze-fracture study of microfilament-membrane attachments in choroid plexus and intestinal microvilli

Choroid plexus and intestinal microvilli in thin sections have microfilaments in the cytoplasm adjacent to the membranes, and in replicas have broken strands of filaments in both cytoplasm and on E faces of plasm membranes. The microfilaments contain actin as indicated by their binding of heavy meromyosin (HMM). In sections of choroid plexus, the microfilaments are 7-8 nm in diameter and form a loose meshwork which lies parallel to the membrane and which is connected to the membranes both by short, connecting filaments (8 times 30 nm) and dense globules (approximately 15-20 nm). The filamentous strands seen in replicas are approximately 8 nm in diameter. Because they are similar in diameter and are connected to the membrane, these filamentous strands seen in replicas apparently represent the connecting structures, portions of the microfilaments, or both. The filamentous strands attached to the membrane are usually associated with the E face and appear to be pulled through the P half-membrane. In replicas of intestinal brush border microvilli, the connecting strands attaching core microfilaments to the membrane are readily visualized. In contrast, regions of attachment of core microfilaments to dense material at the tips of microvilli are associated with few particles on P faces and with few filamentous strands on the E faces of the membranes. Freeze-fracture replicas suggest a morphologically similar type of connecting strand attachment for microfilament-membrane binding in both choroid plexus and intestinal microvilli, despite the lack of a prominent core bundle of microfilaments in choroid plexus microvilli.     

Resolving the nanoscale adhesion of individual gecko spatulae by atomic force microscopy

Animals that cling to walls and walk on ceilings owe this ability to micrometre and nanoscale attachment elements. The highest adhesion forces are encountered in geckoes, which have developed intricate hierarchical structures consisting of toes (millimetre dimensions), lamella (400-600microm size), setae (micrometre dimensions) and spatulae ( approximately 200nm size). Adhesion forces of setae on different substrates have previously been measured by a micro-electromechanical system technique. Here we report the first successful experiments in which the force-displacement curves were determined for individual spatulae by atomic force microscopy. The adhesion force for these smallest elements of the gecko's attachment system is reproducibly found to be about 10nN. This method sheds new light on the nanomechanisms of attachment and will help in the rational design of artificial attachment systems.     

中华绒螯蟹不同生理阶段腹肢的结构变化及粘液腺的发育特征

用显微及亚显微方法研究了中华绒螯蟹雌体不同生理阶段(幼蟹、未成熟蟹、成熟蟹、抱卵蟹和流产蟹)腹肢的体壁结构变化和粘液腺发育特征。体壁的上皮细胞层常与粘液腺相连,粘液腺分泌物经导管穿过各层角膜排出。不同生理状况下中华绒螯蟹的腹肢体壁各角膜层结构的比例及致密度有明显的差异,粘液腺细胞及导管的数量不同,粘液腺与上皮细胞的连接程度也不同。粘液腺是否正常分泌、刚毛囊的开闭均与中华绒螯蟹的胚胎附着有关。比较研究发现,太湖抱卵蟹腹肢体腔内腺体比温州本地抱卵蟹腹肢内腺体发达,分泌的粘液也特别多。分析认为中华绒螯蟹腹肢组织结构、粘液腺的发育和分泌状况与胚胎流产有密切的关系。     

The attachment of Enteromorpha zoospores to a bacterial biofilm assemblage

This study has investigated the relationship between bacterial biofilms and the attachment of zoospores of the green macroalga Enteromorpha. Zoospore attachment to glass slides was enhanced in the presence of a bacterial biofilm assemblage, and the number attaching increased with the number of bacteria present. Zoospores also attached to control surfaces, but at lower numbers; glass surfaces conditioned in autoclaved seawater had the same number of zoospores attached as new glass surfaces. The spatial relationship between bacterial cells and attached zoospores was quantified by image analysis. The hypothesis tested was that zoospores attached preferentially to, or in the very close vicinity of, bacterial cells. Spatial microscopic analysis showed that more bacteria were covered by zoospores than would be expected if zoospore attachment was a random process and zoospores appeared to attach to bacterial clusters. The most likely explanation is that zoospores are attracted to bacterial cells growing on surfaces and the presence of a bacterial biofilm enhances their settlement. The possibility is discussed that Enteromorpha zoospores respond to a chemical signal produced by bacteria, i.e. that there may be prokaryote‐eukaryote cell signalling.     

High-frequency interconversion of turbid and clear plaque strains of bacteriophage f1 and associated host cell death

Under normal cultivation conditions, a mixture of turbid and clear plaques is often apparent in cultures of bacterial cells infected with filamentous bacteriophages. Beginning with a culture of wild-type filamentous phage f1, which itself produces turbid plaques, a clear plaque strain (c1) was isolated. From c1, the turbid plaque strain t1 was isolated; from t1, the clear plaque strain c2 was isolated; and from c2, the turbid plaque strain t2 was isolated. Each of these strains was generated with a frequency of approximately 1 x 10(-4). Although filamentous phages have been thought not to induce host cell death, both turbid and clear plaque strains of f1 killed host bacteria. Plating of bacterial cells 1 h after infection revealed that colonies produced by cells infected with either wild-type f1 or strain c2 were smaller than those derived from uninfected cells, and that colony formation by infected cells was reduced by 15% and 38%, respectively. The time course of bacterial growth revealed that, at 4 h after infection, the number of CFU per milliliter of culture of cells infected with wild-type f1 or with strain c2 was reduced by 27% and 95%, respectively, compared with that for uninfected cells. Microculture analysis also revealed that the percentages of nondividing cells in f1 or c2 infected were 19% and 52%, respectively, 4 h after infection with wild-type f1 or with strain c2; no such cells were detected in cultures of uninfected cells. Negative staining and electron microscopy showed that 20% and 61% of cells infected with wild-type f1 or with strain c2 were dead 4 h postinfection. Finally, although the rates of DNA synthesis were similar for infected and uninfected cells, the rates of RNA and protein synthesis were markedly reduced in infected cells.     

Contact behaviour of tenent setae in attachment pads of the blowfly Calliphora vicina (Diptera, Calliphoridae)

To enable strong attachment forces between pad and substrata, a high proximity between contacting surfaces is required. One of the mechanisms, which can provide an intimate contact of solids, is a high flexibility of both materials. It has been previously presumed that setae of hairy attachment pads of insects are composed of flexible cuticle, and are able to replicate the surface profile. The aim of this work was to visualise the contact behaviour of the setae by freezing-substitution technique to understand setal mechanics while adhering to a smooth surface. This approach revealed considerable differences in the area of the setal tips between contacting and non-contacting pulvilli. Based on the assumption that setae behave like a spring pushed by the tip, a spring constant of 1.31 N m(-1) was calculated from direct measurements of single setae by atomic force microscopy. In order to explain the relationship between the behaviour of the attachment setae at a microscale and leg movements, high-speed video recordings were made of walking flies. This data show that some proximal movement of the leg is present during contact formation with the substrate.     

Dancing for food in the deep sea: bacterial farming by a new species of Yeti crab

Vent and seep animals harness chemosynthetic energy to thrive far from the sun's energy. While symbiont-derived energy fuels many taxa, vent crustaceans have remained an enigma; these shrimps, crabs, and barnacles possess a phylogenetically distinct group of chemosynthetic bacterial epibionts, yet the role of these bacteria has remained unclear. We test whether a new species of Yeti crab, which we describe as Kiwa puravida n. sp, farms the epibiotic bacteria that it grows on its chelipeds (claws), chelipeds that the crab waves in fluid escaping from a deep-sea methane seep. Lipid and isotope analyses provide evidence that epibiotic bacteria are the crab's main food source and K. puravida n. sp. has highly-modified setae (hairs) on its 3(rd) maxilliped (a mouth appendage) which it uses to harvest these bacteria. The ε- and γ- proteobacteria that this methane-seep species farms are closely related to hydrothermal-vent decapod epibionts. We hypothesize that this species waves its arm in reducing fluid to increase the productivity of its epibionts by removing boundary layers which may otherwise limit carbon fixation. The discovery of this new species, only the second within a family described in 2005, stresses how much remains undiscovered on our continental margins.