Characterization and identification of enzyme-producing microflora isolated from the gut of sea cucumber Apostichopus japonicus

Gut microorganisms play an important role in the digestion of their host animals. The purpose of this research was to isolate and assess the enzyme-producing microbes from the Apostichopus japonicus gut. Thirty-nine strains that can produce at least one of the three digestive enzymes(protease,amylase,and cellulase) were qualitatively screened based on their extracellular enzyme-producing abilities. The enzyme-producing strains clustered into eight groups at the genetic similarity level of 100% by analyzing the restriction patterns of 16 S rDNA amplified with Mbo I. Phylogenetic analysis revealed that 37 strains belonged to the genus Bacillus and two were members of the genus Virgibacillus. Enzyme-producing capability results indicate that the main enzyme-producing microflora in the A. japonicus gut was Bacillus,which can produce protease,amylase,and cellulase. Virgibacillus,however,can only produce protease. The high enzyme-producing capability of the isolates suggests that the gut microbiota play an important role in the sea cucumber digestive process.     

High-throughput sequencing of 16S r RNA amplicons characterizes gut microbiota shift of juvenile sea cucumber Apostichopus japonicus feeding with three antibiotics

Sea cucumber Apostichopus.japonicus is an important marine economic species in Asian countries due to its profound nutritional and medicinal value.So far,with the rapid development of intensified artificial aquaculture of sea cucumbers,the use of antibiotics is still an inexpensive and dispensable way to treat pathogenic infections,especially during the nursery phase.However,there is little information on the effects of antibiotics on the intestinal microbiota of sea cucumber.Therefore an Illumina based sequencing method was used to examine the intestinal bacterial composition of juvenile A.japonicas following diets with three typical antibiotics(tetracycline,erythromycin.,and norfloxacin)under 15,30,and 45 d.The findings reveal that different antibiotics have distinct effects on the growth performance of juvenile sea cucumbers.However,the richness and diversity of microbiota were barely affected by antibiotics but the community composition alterations indicated that the three antibiotics exhibited their respective patterns of reshaping the intestinal bacteria of juvenile sea cucumbers.In common,the abundance of some sensitive genera with helpful functions,such as Thalassotalea,Shewanella,Sulfitobacter,and Halomonas decreased significantly with exposure to antibiotics and the abundance of multiple potential pathogenic-and suspected antibiotic-resistant microorganisms like Arcobacter,Leucothrix,and Clostridium_sensu_stricto_1 was found increased significantly in the antibiotic groups.These results suggest that low doses of antibiotics could affect the composition of the intestinal microbiota of sea cucumbers and might increase the risk of infection of the hosts.This study could help us to explore how antibacterial compounds modify the gut microbiota of sea cucumbers and provide theoretical guidance in hatchery management by scientific antibiotic use in sea cucumber mariculture.     

A Comparative Study of Fish Assemblages Near Aquaculture,Artificial and Natural Habitats

Habitat plays a critical role in regulating fish community structure. Using the data collected from a monthly trammel net survey in Ma’an archipelago off the east coast of China, we evaluated impacts of five different habitats(artificial reefs, mussel farms, cage aquaculture, rocky reefs and soft bottom) on fish assemblages. This study suggests that artificial reefs(AR) have significantly higher species richness, abundance and diversity than mussel farms(MF) or soft bottom(SB) habitats during most seasons, and that fish taxa in the AR habitats are similar to those in the rocky reef(RR) habitats. Two different fish assemblage patterns were revealed in the study area using non-metric multidimensional scaling ordination: an assemblage dominated by reef fishes(especially by Scorpaenidae species) in AR, RR and cage aquaculture(CA) habitats and an assemblage dominated by Sciaenidae species in MF and SB habitats. We suggest that reef fishes play a key role in differentiating fish community structures in the study area. Although few differences in fish abundance and diversity were found between the CA and SB habitats, a more diverse age structure was observed in the CA habitats. A much more complex fish assemblage and enhanced population of local species were established as a result of the presence of both floating and fixed artificial structures, probably through improved survival rates.     

Single or combined effects of dietary arabinoxylan-oligosaccharide and inulin on growth performance,gut microbiota,and immune response in Pacific white shrimp Litopenaeus vannamei

The single and synergistic effects of dietary arabinoxylan-oligosaccharide(AXOS) and inulin at different doses(2,4,and 8 mg/g diet) on survival,growth performance,gut microbiota,and immune response in Pacific white shrimp Litopenaeus vannamei were assessed.Singular application of either inulin or AXOS at doses of 4 mg/g diet showed the most stimulatory effects on the growth rate and gene expression levels of chitinase,cathepsin L,chymotrypsin,and extracellular signal-regulated kinase(ERK) in each single prebiotic feeding trial.Compared with single prebiotic treatments,simultaneous application of AXOS and inulin at 4 mg/g diet enhanced remarkably the growth parameters of shrimp and the expression of related genes(chitinase,cathepsin L,chymotrypsin ERK,myeloid differentiation factor88,and phenoloxidase) after 8-week feeding(P0.05).Additionally,gut microbiota analysis indicated the dietary supplementation with combined prebiotics increased significantly the bacterial community richness and relative abundance of Bacillus,Pseudomonas,Bacteriovorax,and Lactobacillus,and reduced the abundance of Vibrio,Rhodococcus,and Photobacterium in the digestive tract of L.vannamei.Compared with the single prebiotic treatment and the control,combined prebiotics supplementation boosted notably the survival rate and expression levels of immune-related genes in shrimp infected with Vibrio alginolyticus or white spot syndrome virus.Therefore,simultaneous application of AXOS and inulin shall have a great potential of dietary supplement in the culture of L.vannamei.     

Characterization of dominant and cellulolytic bacterial communities along the gut of silver carp Hypophthalmichthys molitrix during cyanobacterial blooms

Silver carp is one of the most important planktivorous fish in Chinese aquaculture and plays a significant role controlling cyanobacterial blooms. A balanced gut microbiota is crucial for growth and health of the host because of its important roles in immune defense, digestion of complex carbohydrates, and production of enterocytes. In our study, the dominant bacterial and cellulolytic bacterial( Clostridium I, Clostridium III, Clostridium XIVab, and Fibrobacter) communities in the contents and mucus of the silver carp gut(foregut, midgut, and hindgut) were analyzed by denaturing gradient gel electrophoresis and quantitative polymerase chain reaction(q PCR) analyses. The results revealed that the dominant and cellulolytic bacterial communities were significantly different among gut regions as well as in contents and mucus. Bacterial diversity and richness in contents and mucus increased along the gut and were higher in contents than those in local mucus. A sequence analysis of gut samples exhibited the conservative phylotypes of Proteobacteria, Actinobacteria, and Firmicutes. The gut of silver carp harbored an abundance of cellulolytic bacteria, particularly Clostridium XIV ab. The foregut segment had the highest proportions of the four cellulolytic bacteria, followed by the midgut and hindgut. However, the proportions of cellulolytic species in the silver carp gut was much lower than those in the terrestrial vertebrate gastrointestinal tract. We conclude that gut bacteria could help silver carp obtain energy from cyanobacteria, which may be why silver carp can maintain high growth rates during cyanobacterial blooms.     

Characterization and identification of enzyme-producing microflora isolated from the gut of sea cucumber <Emphasis Type="Italic">Apostichopus japonicus</Emphasis>

Gut microorganisms play an important role in the digestion of their host animals. The purpose of this research was to isolate and assess the enzyme-producing microbes from the Apostichopus japonicus gut. Thirty-nine strains that can produce at least one of the three digestive enzymes (protease, amylase, and cellulase) were qualitatively screened based on their extracellular enzyme-producing abilities. The enzyme-producing strains clustered into eight groups at the genetic similarity level of 100% by analyzing the restriction patterns of 16S rDNA amplified with Mbo I. Phylogenetic analysis revealed that 37 strains belonged to the genus Bacillus and two were members of the genus Virgibacillus. Enzyme-producing capability results indicate that the main enzyme-producing microflora in the A. japonicus gut was Bacillus, which can produce protease, amylase, and cellulase. Virgibacillus, however, can only produce protease. The high enzyme-producing capability of the isolates suggests that the gut microbiota play an important role in the sea cucumber digestive process.     

Habitat values for artificial oyster (Crassostrea ariakensis) reefs compared with natural shallow-water habitats in Changjiang River estuary

studies have compared oyster reefs with adjacent natural shallow-water habitats.Here the resident benthic macroinvertebrate communities in an artificial oyster(Crassostrea ariakensis) reef and in adjacent natural estuarine shallow-water habitats(salt marsh,intertidal mudflat,and subtidal soft bottom) in the Changjiang(Yangtze) River estuary were described.The mean total densities and biomass,Margalef’s species richness,Pielou’s evenness and Shannon-Weaver biodiversity indices of the resident benthic macroinvertebrate communities differed significantly among the habitats.Significantly higher densities and biomass of benthic macroinvertebrates occurred in the oyster reef compared with the other three habitats.Ordination plots showed a clear separation in benthic macroinvertebrate communities among the four habitat types.The results demonstrated that the artificial oyster reef supported distinct and unique benthic communities,playing an important role in the complex estuarine habitat by supplying prey resources and contributing to biodiversity.In addition,the results suggested that the oyster reef had been restored successfully.     

Macroalgae blooms and their effects on seagrass ecosystems

Seagrass decline caused by the macroalgae blooms is becoming a common phenomenon throughout temperate and tropical regions. We summarized the incidence of macroalgae blooms throughout the world and their impact on seagrass beds by direct and indirect ways. The competition for living space and using resources is the most direct effect on seagrass beds when macroalgae are blooming in an aquatic ecosystem. The consequence of macroalgae blooms (e.g., light reduction, hypoxia, and decomposition) can produce significant indirect effects on seagrass beds. Light reduction by the macroalgae can decrease the growth and recruitment of seagrasses, and decomposition of macroalgae mats can increase the anoxic and eutrophic conditions, which can further constrict the seagrass growth. Meanwhile, the presence of seagrass shoots can provide substrate for the macroalgae blooms. Controlling nutrient sources from the land to coastal waters is a general efficient way for coastal management. Researching into the synergistical effect of climate change and anthropognic nutrient loads on the interaction between searsasses and macroalgae can provide valuable information to decrease the negative effects of macroalgae blooms on seagrasses in eutrophic areas.     

Bacterial diversity of gut content in sea cucumber (Apostichopus japonicus) and its habitat surface sediment

This study investigated the bacterial diversity of gut content of sea cucumber(Apostichopus japonicus) and its habitat surface sediment in a bottom enhancement area using PCR-based denaturing gradient gel electrophoresis(DGGE) technique. Bacterial diversity evaluation showed that the value of the Shannon-Wiener index of gut content in different intestinal segments of A. japonicus varied between 2.88 and 3.00, lower than that of the surrounding sediment(3.23). Phylogenetic analysis showed that bacterial phylotypes in gut content and the surrounding sediment of A. japonicus were closely related to Proteobacteria includingγ-, α-, δ-and ε-proteobacteria, Bacteroidetes, Firmicute, and Actinobacteria, of which γ-proteobacteria were predominant. These results suggested that the sea cucumber A. japonicus was capable of feeding selectively, and PCR-DGGE was applicable for characterizing the bacterial community composition in gut content and the surrounding sediment of sea cucumber. Further investigation targeting longer 16S rDNA gene fragments and/or functional genes was recommended for obtaining more information of the diversity and function of bacterial community in the gut content of sea cucumber.     

The application of stereo-video technology for the assessment on population change of black rockfish Sebastes schlegeli in a vessel reef area in Haizhou Bay,China

The assessment of population structure and abundance of fish assemblages associated with artificial reefs(ARs) is an important aspect of AR management.In the present study,we used a DiveOperated Stereo Video(stereo-DOV) technique to assess the population structure and abundance of Sebastes schlegeli associated with two metallic,and three wooden,vessel reefs in Haizhou Bay during 2012 and2013.The study used video systems to obtain length measurements and estimates of abundance.The size composition of S.schlegeli differed among reefs and individuals around vessel reefs were all adults,with total lengths(TL) of 20 cm.Juvenile fish were encountered by divers in a rocky area near the island away from the vessel reefs.The largest individual S.schlegeli(with the highest TL) among five reefs were found around a metallic vessel reef in both 2012 and 2013.TL of S.schlegeli from all reefs increased by an average of 3.2 cm(P0.05) from 2012 to 2013,with an estimated mean weight increase of 250.4 g(P0.05).The video survey also indicated a decrease in the biomass of schools near two metallic vessels between the years.Stereo-video technology was found to be suitable for rockfish surveys around the reefs.     

Change of Digestive Physiology in Sea Cucumber Apostichopus japonicus(Selenka) Induced by Corn Kernels Meal and Soybean Meal in Diets

The present study was conducted to determine the change of digestive physiology in sea cucumber Apostichopus japonicus(Selenka) induced by corn kernels meal and soybean meal in diets. Four experimental diets were tested, in which Sargassum thunbergii was proportionally replaced by the mixture of corn kernels meal and soybean meal. The growth performance, body composition and intestinal digestive enzyme activities in A. japonicus fed these 4 diets were examined. Results showed that the sea cucumber exhibited the maximum growth rate when 20% of S. thunbergii in the diet was replaced by corn kernels meal and soybean meal, while 40% of S. thunbergii in the diet can be replaced by the mixture of corn kernels meal and soybean meal without adversely affecting growth performance of A. japonicus. The activities of intestinal trypsin and amylase in A. japonicus can be significantly altered by corn kernels meal and soybean meal in diets. Trypsin activity in the intestine of A. japonicus significantly increased in the treatment groups compared to the control, suggesting that the supplement of corn kernels meal and soybean meal in the diets might increase the intestinal trypsin activity of A. japonicus. However, amylase activity in the intestine of A. japonicus remarkably decreased with the increasing replacement level of S. thunbergii by the mixture of corn kernels meal and soybean meal, suggesting that supplement of corn kernels meal and soybean meal in the diets might decrease the intestinal amylase activity of A. japonicus.     

Planaxidae (Mollusca,Gastropoda) from the South China Sea

Planaxidae is a family of tropical and subtropical marine gastropods that are adapted to an intertidal, rocky environment. The present study deals with three species in the family Planaxidae from the South China Sea: P lanaxis sulcatus(von Born, 1778), Angiola longispira(Smith, 1872), and S upplanaxis niger(Quoy and Gaimard, 1833), based on specimens deposited in the Marine Biodiversity Collections of the South China Sea, Chinese Academy of Sciences. The taxonomic status, main morphological characteristics of the shell and radula, distribution, and habitat of these three planaxid species are presented. We also briefly discuss their morphological dif ferences and the biogeographic distribution.     

Seasonal variation in species composition and abundance of demersal fish and invertebrates in a Seagrass Natural Reserve on the eastern coast of the Shandong Peninsula,China

Seagrass habitats are structurally complex ecosystems, which support high productivity and biodiversity. In temperate systems the density of seagrass may change seasonally, and this may influence the associated fish and invertebrate community. Little is known about the role of seagrass beds as possible nursery areas for fish and invertebrates in China. To study the functioning of a seagrass habitat in northern China, demersal fish and invertebrates were collected monthly using traps, from February 2009 to January 2010. The density, leaf length and biomass of the dominant seagrass Zostera marina and water temperature were also measured. The study was conducted in a Seagrass Natural Reserve(SNR) on the eastern coast of the Shandong Peninsula, China. A total of 22 fish species and five invertebrate species were recorded over the year. The dominant fish species were Synechogobius ommaturus, Sebastes schlegelii, Pholis fangi, Pagrus major and Hexagrammos otakii and these species accounted for 87% of the total number of fish. The dominant invertebrate species were Charybdis japonica and Octopus variabilis and these accounted for 98% of the total abundance of invertebrates. There was high temporal variation in species composition and abundance. The peak number of fish species occurred in August–October 2009, while the number of individual fish and biomass was highest during November 2009. Invertebrate numbers and biomass was highest in March, April, July and September 2009. Temporal changes in species abundance of fishes and invertebrates corresponded with changes in the shoot density and leaf length of the seagrass, Zostera marina.     

Two new marine benthic diatom species of Hippodonta (Bacillariophyceae) from the coast of China: H. nanjiensis sp. nov. and H. qingdaoensis sp. nov.

The diversity and distribution of Hippodonta species on the coast of China are largely unknown due to limited investigations. In this paper, we report the detailed morphology and ultrastructure of two new species: H. nanjiensis sp. nov., collected from intertidal sandy sediments of Nanji Islands, the East China Sea and H. qingdaoensis sp. nov., collected from intertidal sandy sediments of the No. 1 Bathing Beach in Qingdao City, the Yellow Sea. Both species occur mainly in the middle part of the intertidal zone. H. nanjiensis possesses a unique large round central area. H. qingdaoensis differs from congeners in having a unique stria pattern, wherein the areolae in the middle transapical striae are less developed or absent on the primary side of the valve, and two areolae per stria are apparent under light microscopy(LM). This study expands our knowledge of Hippodonta diversity and distribution on the coast of China.     

Succession and seasonal variation in epilithic biofilms on artificial reefs in culture waters of the sea cucumber Apostichopus japonicus

Periphytic biofilms in aquaculture waters are thought to improve water quality, provide an additional food source, and improve the survival and growth of some reared animals. In the AsiaPacific region, particularly in China, artificial reefs are commonly used in the commercial farming of sea cucumbers. However, few studies have examined the epilithic biofilms on the artificial reefs. To gain a better understanding of the succession of epilithic biofilms and their ecological processes in sea cucumber culture waters, two experiments were conducted in culture waters of the sea cucumber Apostichopus japonicus in Rongcheng, China, using artificial test panels. On the test panels of succession experiment, more than 67 species were identified in the biofilms. On the test panels of seasonal variation experiment, more than 46 species were recorded in the biofilms. In both experiments, communities of epilithic biofilms were dominated by diatoms, green algae and the annelid Spirorbis sp. In the initial colonization, the dominant diatoms were Cocconeis sp., Amphora spp. and Nitzschia closterium in June, which were succeeded by species of Navicula, Cocconeis and Nitzschia(July to September), and then by Licmophora abbreviata, Nitzschia closterium and Synedra spp. in the following months. A diatom bloom in the autumn and filamentous green algae burst in the summer were also observed. Ecological indices well annotated the succession and seasonal changes in epilithic communities. Multidimensional scaling(MDS) analysis found significant differences in diatom community composition among months and seasons. Fast growth of biofilms was observed in the summer and autumn, whereas the biomass of summer biofilms was largely made up of filamentous green algae. Present results show that the components of epilithic biofilms are mostly optimal foods of A. japonicus, suggesting that biofilms on artificial reefs may contribute important nutritional sources for sea cucumbers during their growth seasons. Future works should include quantitative determination of the contribution of epilithic biofilms to the diet of A. japonicus, potential roles of epilithic biofilms in regulating the water quality of sea cucumber ponds, and the regulation of epilithic biofilms in sea cucumber culture ponds.     

PCR-DGGE analysis of intestinal bacteria and effect of Bacillus spp.on intestinal microbial diversity in kuruma shrimp(Marsupenaeus japonicus)

In this study,the intestinal microbiota of kuruma shrimp(Marsupenaeus japonicus) was examined by molecular analysis of the 16S rDNA to identify the dominant intestinal bacteria and to investigate the effects of Bacillus spp.on intestinal microbial diversity.Samples of the intestines of kuruma shrimp fed normal feed and Bacillus spp.amended feed.PCR and denaturing gradient gel electrophoresis(DGGE) analyses were then performed on DNA extracted directly from the guts.Population fingerprints of the predominant organisms were generated by DGGE analysis of the universal V3 16S rDNA amplicons,and distinct bands in the gels were sequenced.The results suggested that the gut of kuruma shrimp was dominated by Vibrio sp.and uncultured gamma proteobacterium.Overall,the results of this study suggest that PCR-DGGE is a possible method of studying the intestinal microbial diversity of shrimp.     

Epi- and endobiont faunal communities on an Egyptian Mediterranean rocky shore: species composition and their competition for space

The exposed calcareous rocky area of the Abu Qir Headland,east of Alexandria,Egypt which range from coarse grains,through pebbles,cobbles,and boulders to rocky platforms,was significantly colonized by sclerobionts(epibionts and endobionts).The epibiont species diversity was comparatively higher than that of endobionts.Epibiont communities included bryozoans(the anascan-grade cheilostome Bifiustra savartii),serpulid(Hydroides elegans,Spirobranchus cf.tetraceros,Vermiliopsis striaticeps and Protula or Apomatus) and spirorbid(Spirorbis) wormtubes,and balanoid barnacles(Amphibalanus amphitrite,Balanus trigonus,and Perforatus perforatus).The cheilostome bryozoan colonies,which developed extensive sheets,proliferated on the study rocky shore either encrusting the bioeroded basement directly,or encrusting other epilithic taxa,particularly balanoid barnacles and serpulid worms.Encrusters displayed a remarkable marginal competitive interaction(overgrowth and stand-off) for space on the study rocky shore.On the other hand,endobionts were not well represented on the studied rocky shore exposure.It was possible to identify three ichnotaxa:Gastrochaenolites,Maeandropolydora,and Finichnus.The findings documented represent a significant contribution to our knowledge of sclerobionts composition,sequence of their colonization and/or bioero sion,and their mutual relationships on the intertidal rocky shore of the Abu Qir Headland.     

Comparison of intestinal bacterial communities in grass carp, Ctenopharyngodon idellus, from two different habitats

The intestinal bacteria of vertebrates form a close relationship with their host.External and internal conditions of the host,including its habitat,affect the intestinal bacterial community.Similarly,the intestinal bacterial community can,in turn,influence the host,particularly with respect to disease resistance.We compared the intestinal bacterial communities of grass carp that were collected from farm-ponds or a lake.We conducted denaturing gradient gel electrophoresis of amplified 16S rRNA genes,from which 66 different operational taxonomic units were identified.Using both the unweighted pair-group method with arithmetic means clustering and principal component analysis ordination,we found that the intestinal bacterial communities from the two groups of pond fish were clustered together and inset into the clusters of wild fish,except for DF-7,and there was no significant correlation between genetic diversity of grass carp and their intestinal bacterial communities(Mantel one-tailed test,R=0.157,P=0.175).Cetobacterium appeared more frequently in the intestine of grass carp collected from pond.A more thorough understanding of the role played by intestinal microbiota on fish health would be of considerable benefit to the aquaculture industry.     

Effects of a Potential Autochthonous Probiotic Bacillus subtilis 2-1 on the Growth and Intestinal Microbiota of Juvenile Sea Cucumber,Apostichopus japonicus Selenka

The effects of Bacillus subtilis 2-1 from the intestine of healthy sea cucumber on the growth, digestive enzyme activities and intestinal microbiota of juvenile sea cucumber(Apostichopus japonicus) were determined in the present study. Sea cucumber was fed with Sargassum thunbergii powder supplemented with B. subtilis 2-1 at different concentrations varying among 0(control), 10~5, 10~7, and 10~9 CFU g~(-1) for 8 weeks. Results showed that the growth performance and intestinal amylase and trypsin activities were significantly increased by dietary B. subtilis 2-1 at 10~9 CFU g~(-1)(P 0.05). However, dietary B. subtilis 2-1 had no significant influence on the lipase activity in sea cucumber(P 0.05). The polymerase chain reaction denaturing gradient gel electrophoresis and 16S rRNA gene sequencing analysis indicated that dietary B. subtilis 2-1 at 105 and 107 CFU g~(-1) inhibited most of the Proteobacteria including those in genus Vibrio. Dietary B. subtilis 2-1 at 10~9 CFU g~(-1) not only decreased the abundance and species of genus Vibrio, but also increased the intensity of genera Psychrobacter and Bacillus. A specific dosage of dietary B. subtilis 2-1 could increase the growth and modulate the intestinal microbiota of sea cucumber; thus it might be a novel probiotic for keeping the health of sea cucumber.     

A taxonomical and ecological survey of rotiffer communities in Krottensee (Austria)

The composition and distribution of benthic, periphytic and planktonic rotifers in different habitats in Krottensee were investigated. Sixty-five rotifer taxa were identified, their relative abundance estimated. Classification of the data set by cluster analysis allowed the recognition of distinct habitat groups confined to acid bog ponds, macrophytes, sediments and open water. Five groups of rotifer taxa were identified on the basis of their habitat preferences and the occurrence of the taxa. Highest diversities were found on macrophytes and in acid bog ponds.