Suppressive subtractive hybridisation transcriptomics provides a novel insight into the functional role of the hypobranchial gland in a marine mollusc

The hypobranchial gland present in gastropods is an organ whose function is not clearly understood. Involved in mucus production, within members of the family Muricidae it is also the source of the ancient dye Tyrian purple and its bioactive precursors. To gain further insights into hypobranchial gland biology, suppressive subtractive hybridisation was performed on hypobranchial gland and mantle tissue from the marine snail Dicathais orbita creating a differentially expressed cDNA library. 437 clones were randomly sequenced, analysed and annotated and 110 sequences had their functions putatively identified. Importantly this approach identified a putative gene involved in Tyrian purple biosynthesis, an arylsulphatase gene. Confirmation of the upregulation of arylsulphatase in the hypobranchial gland compared to the mantle was demonstrated using quantitative real-time PCR. Other genes identified as playing an important role in the hypobranchial gland were those involved in mucus protein synthesis, choline ester regulation, protein and energy production. This study confirms that the hypobranchial gland is involved in the production of mucus secretion and also identifies it as a site of chemical interaction and biosynthesis. This study lays the foundation for a better understanding of the enzymatic production of Tyrian purple precursors within the gland.     

Observations on the production of purple pigments in the egg capsules,hypobranchial and reproductive glands from seven species of Muricidae (Gastropoda: Mollusca)

Summary

Tyrian purple is a well-known colourant that can be obtained from the hypobranchial glands of muricids. Here we investigate the presence of purple and related pigments in the hypobranchial glands, reproductive glands and egg capsules of three Rapaninae, three Ocenebrinae and one Muricinae species. Observations on the dissected specimens revealed the presence of purple pigmentation in the hypobranchial glands of all species. All of the Rapaninae and the Muricinae, but only some species of Ocenebrinae, appear to transfer the pigment precursors to their egg capsules. This provides evidence that the precursors of Tyrian purple are not produced for the sole purpose of protecting the egg masses as has been previously suggested. In all the subfamilies, the hypobranchial and reproductive (capsule and prostate) glands lie adjacent to one another. Colour changes in the reproductive glands, indicating the presence of dye precursors, were observed in two species of Rapaninae. In Dicathais orbita, colour changes could be seen in both the capsule glands of females and prostrate glands of males. The final colour was more red in the reproductive glands than the purple observed in the hypobranchial glands. Preliminary observations of detached hypobranchial gland sections in both D. orbita and Pterynotus triformis indicate a different suite of colour reactions occur when compared to sections that are intact and attached to the reproductive glands. This demonstrates that the reproductive glands can influence the chemical conversions of dye precursors synthesized by these species. Further studies on the secondary metabolism occurring within the hypobranchial glands and reproductive organs of Muricidae are ongoing, including histological sectioning and confirmation of the chemistry behind the colours observed and functional analysis of Tyrian purple precursors.     

Sulfur bacteria in wastewater stabilization ponds periodically affected by the ‘red-water’ phenomenon

Several wastewater stabilization ponds (WSP) in Tunisia suffer periodically from the ‘red-water’ phenomenon due to blooming of purple sulfur bacteria, indicating that sulfur cycle is one of the main element cycles in these ponds. In this study, we investigated the microbial diversity of the El Menzeh WSP and focused in particular on the different functional groups of sulfur bacteria. For this purpose, we used denaturing gradient gel electrophoresis of PCR-amplified fragments of the 16S rRNA gene and of different functional genes involved in microbial sulfur metabolism (dsrB, aprA, and pufM). Analyses of the 16S rRNA revealed a relatively high microbial diversity where Proteobacteria, Chlorobi, Bacteroidetes, and Cyanobacteria constitute the major bacterial groups. The dsrB and aprA gene analysis revealed the presence of deltaproteobacterial sulfate-reducing bacteria (i.e., Desulfobacter and Desulfobulbus), while the analysis of 16S rRNA, aprA, and pufM genes assigned the sulfur-oxidizing bacteria community to the photosynthetic representatives belonging to the Chlorobi (green sulfur bacteria) and the Proteobacteria (purple sulfur and non sulfur bacteria) phyla. These results point on the diversity of the metabolic processes within this wastewater plant and/or the availability of sulfate and diverse electron donors.     

Characterization of a novel gastropod toxin (6-bromo-2-mercaptotryptamine) that inhibits shaker K channel activity

A novel potassium channel antagonist has been purified from the defensive mucus secreted by Calliostoma canaliculatum, a marine snail found in the temperate coastal waters of the western Pacific. The toxin is expelled from the hypobranchial gland as part of a defensive response and is contained within a viscous matrix that minimizes dilution and degradation. The active compound was isolated by multistage microbore HPLC separations followed by bioactivity assays. Nuclear magnetic resonance, combined with electrospray ionization Fourier-transform ion cyclotron resonance and electrospray ionization ion trap mass spectrometry indicate that the active component is a heretofore unknown indole-derivative, a disulfide-linked dimer of 6-bromo-2-mercaptotryptamine (BrMT). Exudates from the hypobranchial glands of various marine mollusks have been sources for dye compounds such as 6-6 dibromoindigo, the ancient dye Tyrian purple. BrMT represents the first correlation of a hypobranchial gland exudate with a molecular response. Voltage clamp experiments with a number of K channel subtypes indicate that BrMT inhibits certain voltage-gated K channels of the Kv1 subfamily.     

Application of targeted metagenomics to explore abundance and diversity of CO2-fixing bacterial community using cbbL gene from the rhizosphere of Arachis hypogaea

Sequestration of CO₂ by autotrophic bacteria is a key process of biogeochemical carbon cycling in soil ecosystem. Rhizosphere is a rich niche of microbial activity and diversity, influenced by change in atmospheric CO₂. Structural changes in rhizosphere composition influence microbial communities and the nutrient cycling. In the present study, the bacterial diversity and population dynamics were established using cbbL and 16S rRNA gene targeted metagenomics approach from the rhizosphere of Arachis hypogaea. A total of 108 cbbL clones were obtained from the rhizospheric soil which revealed predominance of cbbL sequences affiliated to Rhizobium leguminosarum, Bradyrhizobium sp., Sinorhizobium meliloti, Ochrobactrum anthropi and a variety of uncultured cbbL harboring bacteria. The 16S rRNA gene clone library exhibited the dominance of Firmicutes (34.4%), Proteobacteria (18.3%), Actinobacteria (17.2%) and Bacteroidetes (16.1%). About 43% nucleotide sequences of 16S rRNA gene clone library were novel genera which showed < 95% homology with published sequences. Gene copy number of cbbL and 16S rRNA genes, determined by quantitative real‐time PCR (qRT PCR), was 9.38 ± 0.75 × 10⁷ and 5.43 ± 0.79 × 10⁸ (per g dry soil), respectively. The results exhibited bacterial community structure with high bacterial diversity and abundance of CO₂‐fixing bacteria, which can be explored further for their role in carbon cycling, sustainable agriculture and environment management.     

Vibrio harveyi Adheres to and Penetrates Tissues of the European Abalone Haliotis tuberculata within the First Hours of Contact

Vibrio harveyi is a marine bacterial pathogen responsible for episodic epidemics generally associated with massive mortalities in many marine organisms, including the European abalone Haliotis tuberculata. The aim of this study was to identify the portal of entry and the dynamics of infection of V. harveyi in the European abalone. The results indicate that the duration of contact between V. harveyi and the European abalone influences the mortality rate and precocity. Immediately after contact, the epithelial and mucosal area situated between the gills and the hypobranchial gland was colonized by V. harveyi. Real-time PCR analyses and culture quantification of a green fluorescent protein-tagged strain of V. harveyi in abalone tissues revealed a high density of bacteria adhering to and then penetrating the whole gill-hypobranchial gland tissue after 1 h of contact. V. harveyi was also detected in the hemolymph of a significant number of European abalones after 3 h of contact. In conclusion, this article shows that a TaqMan real-time PCR assay is a powerful and useful technique for the detection of a marine pathogen such as V. harveyi in mollusk tissue and for the study of its infection dynamics. Thus, we have revealed that the adhesion and then the penetration of V. harveyi in European abalone organs begin in the first hours of contact. We also hypothesize that the portal of entry of V. harveyi in the European abalone is the area situated between the gills and the hypobranchial gland.     

Biases in community structures of ammonia/ammonium-oxidizing microorganisms caused by insufficient DNA extractions from Baijiang soil revealed by comparative analysis of coastal wetland sediment and rice paddy soil

Repetitive extraction of DNAs from surface sediments of a coastal wetland in Mai Po Nature Reserve (MP) of Hong Kong and surface Baijiang soils from a rice paddy (RP) in Northeast China was conducted to compare the microbial diversity in this study. Community structures of ammonia/ammonium-oxidizing microorganisms in these samples were analyzed by PCR-DGGE technique. The diversity and abundance of ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), and anaerobic ammonium-oxidizing (anammox) bacteria were also analyzed based on archaeal and bacterial ammonia monooxygenase subunit A encoding (amoA) and anammox bacterial 16S rRNA genes, respectively. DGGE profiles of archaeal and bacterial amoA and anammox bacterial 16S rRNA genes showed a similar pattern among all five repetitively extracted DNA fractions from both MP and RP, except the anammox bacteria in RP, indicating a more diverse anammox community retrieved in the second to the fifth fractions than the first one. Both soil and marine group AOA were detected while soil and coastal group AOB and Scalindua-anammox bacteria were dominant in MP. Soil group AOA and marine group AOB were dominant in RP, while both Scalindua and Kuenenia species were detected in RP. Pearson correlation analysis showed that the abundance of archaeal and bacterial amoA and anammox bacterial 16S rRNA genes was significantly correlated with the DNA concentrations of the five DNA fractions from MP, but not from RP (except the archaeal amoA gene). Results suggest that anammox bacteria diversity may be biased by insufficient DNA extraction of rice paddy soil samples.     

Microbial epibionts of the colonial ascidians Didemnum galacteum and Cystodytes sp.

Symbiosis with microorganisms has been well documented for many marine invertebrate taxa. However, knowledge of the diversity of microorganisms associated with ascidians is still limited. This study assessed the microbial epibionts of Didemnum galacteum and Cystodytes sp., two ascidian species collected from the western coast of Ceará state (Brazil), at Dois Coqueiros beach and the port of Pecém, respectively. The microbiota were examined using optical microscopy, followed by subsequent analysis of fingerprinting profiles obtained by denaturing gradient gel electrophoresis (DGGE) and 16S rRNA clone libraries. The microscopy analysis showed for both ascidians a community comprising cyanobacteria, mainly Prochloron-like species, and diatoms. The DGGE results indicated that D. galacteum hosts a more diverse microbiota when compared to Cystodytes sp. The same analysis also suggested that the diversity of the seawater microbiota was higher than the diversity of the ascidian-associated microbiota. The analysis of the 16S rRNA clone libraries revealed the dominance of Proteobacteria symbionts associated with both ascidians, with Alphaproteobacteria as the major component in D. galacteum and Gammaproteobacteria the major component in Cystodytes sp. The analysis of the clone libraries also revealed the presence of other taxa such as Bacteroidetes, Planctomycetes, Actinobacteria, Cyanobacteria, and uncultured bacteria in D. galacteum, but not in Cystodytes sp. Among the bacteria found to be exclusively associated with the ascidians, none were shared by the two studied hosts. The combined results point to a diverse microbiota associated with the external surface of the ascidians, with a mixed composition including organisms typically found in the surrounding seawater, but also a more specific set of taxa.     

Small core communities and high variability in bacteria associated with the introduced ascidian Styela plicata

The solitary ascidian Styela plicata is an introduced species in harbors of temperate and tropical oceans around the world. The invasive potential of this species has been studied through reproductive biology and population genetics but no study has yet examined the microbial diversity associated with this ascidian and its potential role in host ecology and invasiveness. Here, we used 16S rRNA gene tag pyrosequencing and transmission electron microscopy to characterize the abundance, diversity and host-specificity of bacteria associated with 3 Mediterranean individuals of S. plicata. Microscopy revealed low bacterial abundance in the inner tunic and their absence from gonad tissues, while pyrosequencing revealed a high diversity of S. plicata-associated bacteria (284 OTUs from 16 microbial phyla) in the inner tunic. The core symbiont community was small and consisted of 16 OTUs present in all S. plicata hosts. This core community included a recently described ascidian symbiont (Hasllibacter halocynthiae) and several known sponge and coral symbionts, including a strictly anaerobic Chloroflexi lineage. Most recovered bacterial OTUs (79.6 %) were present in single S. plicata individuals and statistical analyses of genetic diversity and community structure confirmed high variability of bacterial communities among host individuals. These results suggest that diverse and variable bacterial communities inhabit the tunic of S. plicata, including environmental and host-associated bacterial lineages that appear to be re-established each host generation. We hypothesize that bacterial communities in S. plicata are dynamic and have the potential to aid host acclimation to new habitats by establishing relationships with beneficial, locally sourced bacteria.     

Photosynthetic and Phylogenetic Primers for Detection of Anoxygenic Phototrophs in Natural Environments

Primer sets were designed to target specific 16S ribosomal DNA (rDNA) sequences of photosynthetic bacteria, including the green sulfur bacteria, the green nonsulfur bacteria, and the members of the Heliobacteriaceae (a gram-positive phylum). Due to the phylogenetic diversity of purple sulfur and purple nonsulfur phototrophs, the 16S rDNA gene was not an appropriate target for phylogenetic rDNA primers. Thus, a primer set was designed that targets the pufM gene, encoding the M subunit of the photosynthetic reaction center, which is universally distributed among purple phototrophic bacteria. The pufM primer set amplified DNAs not only from purple sulfur and purple nonsulfur phototrophs but also from Chloroflexus species, which also produce a reaction center like that of the purple bacteria. Although the purple bacterial reaction center structurally resembles green plant photosystem II, the pufM primers did not amplify cyanobacterial DNA, further indicating their specificity for purple anoxyphototrophs. This combination of phylogenetic- and photosynthesis-specific primers covers all groups of known anoxygenic phototrophs and as such shows promise as a molecular tool for the rapid assessment of natural samples in ecological studies of these organisms.     

Intergenerational Transfer of Specific Bacteria in Corals and Possible Implications for Offspring Fitness

Diverse and abundant bacterial populations play important functional roles in the multi-partite association of the coral holobiont. The specificity of coral-associated assemblages remains unclear, and little is known about the inheritance of specific bacteria from the parent colony to their offspring. This study investigated if broadcast spawning and brooding corals release specific and potentially beneficial bacteria with their offspring to secure maintenance across generations. Two coral species, Acropora tenuis and Pocillopora damicornis, were maintained in 0.2 μm filtered seawater during the release of their gametes and planulae, respectively. Water samples, excluding gametes and planulae, were subsequently collected, and bacterial diversity was assessed through a pyrosequencing approach amplifying a 470-bp region of the 16S rRNA gene including the variable regions 1–3. Compared to the high bacterial diversity harboured by corals, only a few taxa of bacteria were released by adult corals. Both A. tenuis and P. damicornis released similar bacteria, and the genera Alteromonas and Roseobacter were abundant in large proportions in the seawater of both species after reproduction. This study suggests that adult corals may release bacteria with their offspring to benefit the fitness in early coral life stages.     

Molecular Characterization of Sulfate-Reducing Bacteria in the Guaymas Basin

The Guaymas Basin (Gulf of California) is a hydrothermal vent site where thermal alteration of deposited planktonic and terrestrial organic matter forms petroliferous material which supports diverse sulfate-reducing bacteria. We explored the phylogenetic and functional diversity of the sulfate-reducing bacteria by characterizing PCR-amplified dissimilatory sulfite reductase (dsrAB) and 16S rRNA genes from the upper 4 cm of the Guaymas sediment. The dsrAB sequences revealed that there was a major clade closely related to the acetate-oxidizing delta-proteobacterial genus Desulfobacter and a clade of novel, deeply branching dsr sequences related to environmental dsr sequences from marine sediments in Aarhus Bay and Kysing Fjord (Denmark). Other dsr clones were affiliated with gram-positive thermophilic sulfate reducers (genus Desulfotomaculum) and the delta-proteobacterial species Desulforhabdus amnigena and Thermodesulforhabdus norvegica. Phylogenetic analysis of 16S rRNAs from the same environmental samples resulted in identification of four clones affiliated with Desulfobacterium niacini, a member of the acetate-oxidizing, nutritionally versatile genus Desulfobacterium, and one clone related to Desulfobacula toluolica and Desulfotignum balticum. Other bacterial 16S rRNA bacterial phylotypes were represented by non-sulfate reducers and uncultured lineages with unknown physiology, like OP9, OP8, as well as a group with no clear affiliation. In summary, analyses of both 16S rRNA and dsrAB clone libraries resulted in identification of members of the Desulfobacteriales in the Guaymas sediments. In addition, the dsrAB sequencing approach revealed a novel group of sulfate-reducing prokaryotes that could not be identified by 16S rRNA sequencing.     

Distribution and Diversity of Symbiotic Thermophiles, Symbiobacterium thermophilum and Related Bacteria, in Natural Environments

Symbiobacterium thermophilum is a tryptophanase-positive thermophile which shows normal growth only in coculture with its supporting bacteria. Analysis of the 16S rRNA gene (rDNA) indicated that the bacterium belongs to a novel phylogenetic branch at the outermost position of the gram-positive bacterial group without clustering to any other known genus. Here we describe the distribution and diversity of S. thermophilum and related bacteria in the environment. Thermostable tryptophanase activity and amplification of the specific 16S rDNA fragment were effectively employed to detect the presence of Symbiobacterium. Enrichment with kanamycin raised detection sensitivity. Mixed cultures of thermophiles containing Symbiobacterium species were frequently obtained from compost, soil, animal feces, and contents in the intestinal tracts, as well as feeds. Phylogenetic analysis and denaturing gradient gel electrophoresis of the specific 16S rDNA amplicons revealed a diversity of this group of bacteria in the environment.     

Culture-Dependent and Independent Studies of Microbial Diversity in Highly Copper-Contaminated Chilean Marine Sediments

Cultivation and molecular-based approaches were used to study microbial diversity in two Chilean marine sediments contaminated with high (835 ppm) and very high concentrations of copper (1,533 ppm). The diversity of cultivable bacteria resistant to copper was studied at oxic and anoxic conditions, focusing on sulfate-, thiosulfate-, and iron-reducing bacteria. For both sediments, the cultivable bacteria isolated at oxic conditions were mostly affiliated to the genus Bacillus, while at anoxic conditions the majority of the cultivable bacteria found were closely related to members of the genera Desulfovibrio, Sphingomonas, and Virgibacillus. Copper resistance was between 100 and 400 ppm, with the exception of a strain affiliated to members of the genus Desulfuromonas, which was resistant up to 1,000 ppm of copper. In parallel, cloning and sequencing of 16S rRNA was performed to study the total bacterial diversity in the sediments. A weak correlation was observed between the isolated strains and the 16S rRNA operational taxonomic units detected. The presence of copper resistance genes (copA, cusA, and pcoA) was tested for all the strains isolated; only copA was detected in a few isolates, suggesting that other copper resistance mechanisms could be used by the bacteria in those highly copper-contaminated sediments.     

Diversity of the Intestinal Bacteria of Cattle Fed on Diets with Different Doses of Gelatinized Starch-Urea

Gelatinized starch-urea (Starea, SU) is an effective and economical source of urea for ruminants. Here we assessed the influence of dietary supplementation with gelatinized starch-urea on the diversity of intestinal bacteria in finishing cattle. Fifty steers were randomly allotted to five treatments with diets supplemented with different doses of Starea [0 % (SU0), 8 % (SU8), 16 % (SU16), 24 % (SU24), and 32 % (SU32) of urea-N in total nitrogen]. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes was used to examine the effect of dietary supplementation of Starea on intestinal bacterial flora. Shannon–Weaver and Simpson diversity indices consistently showed the lowest bacterial diversity in the SU0 treatment. Increasing doses of Starea increased the diversity up to SU24 after which, diversity decreased. Cluster analysis of 16S rRNA gene DGGE profiles indicates that the intestinal bacterial communities associated with cattle that were not supplemented with Starea in feed differed in composition and structure from those supplemented with Starea. The amount of Starea supplemented in cattle diets influenced the abundance of several key species affiliated with Lachnospiraceae, Ruminococcaceae, Peptostreptococcaceae, Comamonadaceae and Moraxellaceae. These results suggest that Starea influences the composition and structure of intestinal bacteria which may play a role in promoting ruminant health and production performance.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-015-0526-8) contains supplementary material, which is available to authorized users.     

三倍体毛白杨不同组织内生细菌多样性分析

【背景】三倍体毛白杨非常适合黄河区域生态经济发展,是我国林业推广项目的重要树种。内生细菌在三倍体毛白杨不同组织中广泛存在,对三倍体毛白杨具有防病、促生、固氮和生物修复等生物学作用。【目的】通过分析三倍体毛白杨不同组织内生细菌多样性,可以充分挖掘其蕴含的丰富微生物资源。【方法】以北京林业大学山东冠县毛白杨基地的三倍体毛白杨为材料,应用16S rRNA基因高通量测序技术对其根、茎、叶中内生细菌多样性进行了分析,阐述三倍体毛白杨不同组织内生细菌多样性的变化趋势和规律,为其内生细菌的进一步应用奠定理论基础。【结果】三倍体毛白杨根部内生细菌群落丰富度及多样性最高,叶片中最低。高通量测序结果显示,在全部样本中,假单胞菌门和放线菌门为优势门,伯克氏菌属(Burkholderia)、假诺卡氏菌属(Pseudonocardia)和食酸菌属(Acidovorax)为优势属,不同组织的内生细菌群落结构组成差异显著;16S rRNA基因功能预测结果显示,三倍体毛白杨内生细菌的功能主要涉及氨基酸代谢、维生素代谢、芳香族化合物降解和糖酵解等。通过分离培养共获得217株内生细菌,分属于23个属44个种,其中有4株1...     

Genotypic characterization of soil bacteria in the Umm Al-Namil Island,Kuwait

Microflora is an integral part of soil ecosystem, in which bacteria are the largest group of soil microbes. This is a pioneer study for establishing baseline data on the diversity of soil bacteria among different regions in Kuwait. The aim is to understand biodiversity in different settings, how bacteria adapt to different niches in the environment as well as in different hosts. The identification of bacterial 16S rRNA molecules from environmental soil samples was investigated. Genomic Deoxyribonucleic acid DNA was extracted from 25 soil samples derived from five different test regions in the Umm Al-Namil Island, Kuwait. After amplification of bacterial 16S rRNA molecules by the Polymerase chain reaction PCR, the products were characterized and complex band patterns were obtained, indicating high bacterial diversity. A sample of the 16 s rRNA amplicons were sequenced in order to identify the species. The spatial distribution of bacterial taxa in the different soil samples was homogeneous, suggesting a stable and widespread community. Forty-nine isolates from Umm Al-Namil island were identified by comparative analysis of partial 16S rRNA gene sequences. Phylogenetic analysis was carried out in order to study the connection between the isolates to identify species. A large proportion of these isolates represent correspond to known or novel species within the Pseudomonus and Bacillus genera, which are common soil bacteria. Our results provided a reference for future studies to facilitate bacterial identification and ecological research in Kuwait.     

Microbial and pigment profile of the reddish patch occurring within Tuber magnatum ascomata

Tuber magnatum Pico, the delectable white truffle, is the most prized truffle species. In this study, we examined the reddish pigmentation that frequently occurs in T. magnatum ascomata for the presence of pigment-producing bacteria.The inner part of the reddish-pigmented region of three T. magnatum ascomata collected in North–Central Italy was analysed. This reddish part was used to establish a bacterial culture collection and to extract the total genomic DNA in order to obtain a library of 16S rRNA genes representative of the bacterial community. The molecular approach revealed limited microbial diversity within the reddish-pigmented regions compared to the wider range of bacterial species commonly found at the same maturation stage and season in T. magnatum ascomata. The pigmented regions showed a prevalence of specific bacterial species belonging to α-, β- and γ- Proteobacteria, Actinobacteria and Firmicutes. From the tandem mass spectrometry analysis of the extracted pigment, four compounds were identified: i) bixin, ii) β-carotene, iii) cis-1-glycosyl-apo-8′- lycopene and iv) the fucoxanthin. Carotenoid producing species such as Microbacterium and Chryseobacterium emerged as the most likely cause of the peculiar reddish pigment production. Indeed, our findings suggest that the peculiar reddish pigment might be produced by these bacterial species.     

Characteristics and Diversity of Endophytic Bacteria in Endangered Chinese Herb Glehnia littoralis Based on Illumina Sequencing

Glehnia littoralis is an endangered medicinal plant growing in the coastal ecological environment and plays an important role in coastal ecosystems. The endophytes in the plant have a significant role in promoting plant growth and enhancing plant stress resistance. However, the endophytic bacterial structure associated with halophyte G. littoralis is still not revealed. In this project, the construction and diversity of endophytic bacterial consortium associated with different tissues of G. littoralis were illustrated with high throughput sequencing of the V3-V4 region of the bacterial 16S rRNA. The results resolved that the diversity and richness of endophytic bacteria were significantly higher in root than in leaf and stem. The operational taxonomic units (OTU) analysis demonstrated that the Actinobacteria and Proteobacteria were dominant in all the samples at the phylum level, and Pseudomonas, Bacillus, Rhizobium were the dominant genera. Our results unraveled that the bacterial communities differed among different tissues of G. littoralis. Endophytic bacterial communities in leaf and stem shared more similarity than that in the root. Furthermore, the difference of bacteria community and structure among different tissues were also detected by principal coordinate analysis. Taken altogether, we can conclude that the bacterial communities of different tissues are unique, which could facilitate understanding the diversity of endophytic bacteria in G. littoralis.Key words: Glehnia littoralis, halophyte, endophytic bacteria, diversity, Illumina sequencing     

Gut bacteria associated with different diets in reared Nephrops norvegicus

The impact of different diets on the gut microbiota of reared Nephrops norvegicus was investigated based on bacterial 16S rRNA gene diversity. Specimens were collected from Pagasitikos Gulf (Greece) and kept in experimental rearing tanks, under in situ conditions, for 6 months. Treatments included three diets: frozen natural (mussel) food (M), dry formulated pellet (P) and starvation (S). Gut samples were collected at the initiation of the experiment, and after 3 and 6 months. Tank water and diet samples were also analyzed for bacterial 16S rRNA gene diversity. Statistical analysis separated the two groups fed or starved (M and P vs. S samples). Most gut bacteria were not related to the water or diet bacteria, while bacterial diversity was higher in the starvation samples. M and P samples were dominated by Gammaproteobacteria, Epsilonproteobacteria and Tenericutes. Phylotypes clustering in Photobacterium leiognathi, Shewanella sp. and Entomoplasmatales had high frequencies in the M and P samples but low sequence frequencies in S samples. The study showed that feeding resulted in the selection of specific species, which also occurs in the natural population, and might be associated with the animal's nutrition.