Recovery of bdellovibrios from submerged surfaces and other aquatic habitats

The distribution of bdellovibrios was investigated over a wide geographical area of the Chesapeake Bay including some tributaries and subestuaries. Bdellovibrios were recovered from five aquatic habitats; water, sediment, oyster shell surface biofilm, zooplankton, and plants over a wide range of temperature and salinity measurements. Consistently, the greatest number of the predators was recovered from samples of biofilm irrespective of temperature and salinity. A decrease in the numbers and frequency of predators recovered from all habitats was observed at temperatures below 10°C. Only the shell surface biofilm samples yielded bdellovibrios 100% of the time. The organisms were recovered from 79% of water samples and 44% of sediment samples. The results reveal that bdellovibrios are surface-associated organisms and that this association appears to provide some protection for the predators at low temperatures.     

A study of the occurrence and distribution of bdellovibrios in estuarine sediment over an annual cycle

The recovery of bdellovibrios from estuarine sediments over an annual cycle was studied. Greater numbers of the predators were recovered in sediment than in the water column. Increases in the number of bdellovibrios recovered from sediment over various periods of time suggest that multiplication of the predators occurred. Sediment was observed to be an important ecosystem for the survival of bdellovibrios in the winter months. As has been observed in water, the number of bdellovibrios in sediment fluctuated, with seasonal and temperature changes declining to very low numbers during the winter months. In the colder months, low numbers of the predators appeared to winter-over in sediment, with greater numbers of the organisms being recovered from deeper sediment. As the water temperature warmed in the spring, increases in the number of bdellovibrios occurred first in sediment and subsequently in water. This increase of bdellovibrios in sediment may have resulted in the shedding of the organisms into the water column where their numbers subsequently increased. Population fluctuations of bdellovibrios were similar in both water and sediment. Although the temperature may account for much of the observed fluctuation in the number of bdellovibrios, other factors, including salinity and the number of host bacteria, may also play a major role. The number of bdellovibrios recovered from sediment correlated positively with the water temperature, and negatively with the water salinity and the number of bacterial colony-forming units from sediment. The results of this study revealed the significance of sediment to the seasonal cycle, survival, and growth of the bdellovibrios in an estuarine environment.     

Distribution of bdellovibrios in the water column of an estuary

The distribution of bdellovibrios in the water column of the Miles River has been studied. Water samples were collected every 4 h over a 24-h period from five depths in the water column. The samples were cultured for the recovery of bdellovibrios lytic against Vibrio parahaemolyticus. Environmental parameters, i.e., salinity, temperature, turbidity, and dissolved oxygen (DO) were measured for each sample. Bdellovibrios were observed to be uniformly distributed at all depths measured in the water columns. There were no significant differences between the number of bdellovibrios recovered at the various depths. There were significant differences between the number of bdellovibrios recovered at various sampling times. However, no basis for these significant differences could be established. No association was found between the number of bdellovibrios recovered and the environmental parameters measured. Of interest was the observation that the distribution of the aerobic bdellovibrios did not correlate with DO measurements. The results suggest that neither depth nor DO content influenced the recovery of bdellovibrios from the Miles River.     

Dissolution of calcite in the twilight zone: bacterial control of dissolution of sinking planktonic carbonates is unlikely

We investigated the ability of bacterial communities to colonize and dissolve two biogenic carbonates (Foraminifera and oyster shells). Bacterial carbonate dissolution in the upper water column is postulated to be driven by metabolic activity of bacteria directly colonising carbonate surfaces and the subsequent development of acidic microenvironments. We employed a combination of microsensor measurements, scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM) and image analysis and molecular documentation of colonising bacteria to monitor microbial processes and document changes in shell surface topography. Bacterial communities rapidly colonised shell surfaces, forming dense biofilms with extracellular polymeric substance (EPS) deposits. Despite this, we found no evidence of bacterially mediated carbonate dissolution. Dissolution was not indicated by Ca²⁺ microprofiles, nor was changes in shell surface structure related to the presence of colonizing bacteria. Given the short time (days) settling carbonate material is actually in the twilight zone (500–1000 m), it is highly unlikely that microbial metabolic activity on directly colonised shells plays a significant role in dissolving settling carbonates in the shallow ocean.     

Bdellovibrios in Callinectus sapidus, the Blue Crab

Bdellovibrios were recovered from the gill tissue of all of 31 crabs sampled and from all samples of epibiota obtained from the ventral shell surface of 15 crabs. The results suggest that the blue crab is a reservoir for bdellovibrios. The association with crabs may be an important factor in the ecology of the bdellovibrios.     

Activity of antioxidant enzymes and physiological responses in ark shell,Scapharca broughtonii,exposed to thermal and osmotic stress: Effects on hemolymph and biochemical parameters

Changes in water temperature and salinity are responsible for a variety of physiological stress responses in aquatic organisms. Stress induced by these factors was recently associated with enhanced reactive oxygen species (ROS) generation, which caused oxidative damage. In the present study, we investigated the time-related effects of changes in water temperature and salinity on mRNA expression and the activities of antioxidant enzymes (SOD and CAT) and lipid peroxidation (LPO) in the gills and digestive glands of the ark shell, Scapharca broughtonii. To investigate physiological responses, hydrogen peroxide (H₂O₂), lysozyme activity, aspartate aminotransferase (AspAT), and alanine aminotransferase (AlaAT) were measured in the hemolymph. Water temperature and salinity changes significantly increased antioxidant enzyme mRNA expression and activity in the digestive glands and gills in a time-dependent manner. H₂O₂ concentrations increased significantly in the high-temperature and hyposalinity treatments. LPO, AspAT and AlaAT levels also increased significantly in a time-dependent manner, while lysozyme activity decreased. These results suggest that antioxidant enzymes play important roles in reducing oxidative stress in ark shells exposed to changes in water temperature and salinity.     

The effects of natural biofilms on the reattachment of young adult zebra mussels to artificial substrata

This laboratory study examined the effects of natural biofilms on the reattachment of young adult zebra mussels, Dreissena polymorpha, in Petri dishes. Natural biofilms were developed in glass and polystyrene Petri dishes using water samples collected at various times of the year. Biofilms were developed over 1, 3, 8, and 14 d. Controls were clean glass and polystyrene Petri dishes. Zebra mussels collected from the field (< or = 10 mm, ventral length) were placed in the dishes and their reattachment by byssal threads was recorded after 1 d. Zebra mussels reattached to the dish surface or the shells of other mussels in the dish, or remained unattached. The data indicate that reattachment to clean glass was greater than to clean polystyrene (p < or = 0.05, ANOVA), but there were no consistent differences between reattachment to filmed polystyrene and filmed glass dish surfaces. Zebra mussels in control and filmed glass dishes reattached in higher percentages to the dish surface compared to the shells of other mussels (p < or = 0.05, ANOVA). There was no difference in mussel of reattachment between the dish surface and the shells of other mussels in most control polystyrene dishes (p > 0.05, ANOVA), whereas in filmed polystyrene the percentage of reattachment to the dish surface was greater than to the shells of other mussels (p < or = 0.05, ANOVA). These results indicate that substratum wettability and the presence of biofilms on some types of substrata can be factors in the reattachment of young adult zebra mussels.     

Combined effects of temperature and salinity on larval development and attachment of the subtidal barnacle Balanus trigonus Darwin

The combined effects of temperature and salinity on larval development and attachment of Balanus trigonus Darwin (Cirripedia, Balanidae) was examined under controlled laboratory conditions. Whilst larval survivorship was not affected (>70%), the duration of larval development was significantly affected by temperature and salinity. The effect of temperature was comparatively stronger than that of salinity. The majority of nauplius II larvae metamorphosed into cypris stage after 4-5 and 10-11 days at 28 °C (22-34‰) and 18 °C (22-34‰), respectively. Temperature, salinity and the duration of assay had a significant effect on cypris attachment with significant interaction among these main effects. Maximum (>80% in 6 days) and minimum percent attachment (0% in 6 days) on polystyrene surfaces were observed at 24 °C (34‰) and 18 °C (22‰), respectively. At high temperature (28 °C) and low salinity (22-26‰), larvae rapidly (4 days) developed into cyprids, but less than 33% attached. These results suggest that low larval attachment rates may lead to the low recruitment of B. trigonus in Hong Kong waters during summer when the water temperature is high (about 28 °C) and salinity is low (<26‰).     

Epiphytic bacteria: development of a method for determining respiring bacteria on leaves

A method was developed for studying the total numbers and the proportion of active bacteria on leaf surfaces. It involves staining gelatin impressions of leaves treated with an electron transport system indicator, 2-( p -iodophenyl)-3-( p -nitrophenyl)-5-phenyl tetrazolium chloride (INT). The method is rapid, inexpensive and allows simultaneous observation of numbers, ecology and respiratory activity of epiphytic bacteria. The total numbers of epiphytic bacteria for four species of aquatic plants varied between 0.6 to 10.2 times 10⁶/cm². The proportion of active bacteria on leaves of aquatic plants ranged from 2.2 to 42.9%. The method was also applied to a comparison of surface fouling of glass slides and aquatic leaf surfaces, indicating significant differences in numbers of bacteria but little difference in the proportion active on the two surfaces.     

Germination of Phyllosticta ampelicidaPycnidiospores: Prerequisite of Adhesion to the Substratum and the Relationship of Substratum Wettability

Pycnidiospores of Phyllosticta ampelicida, the causal agent of black rot of grape, were found to germinate only on substrata on which they were firmly attached. Such surfaces were poorly wettable and had advancing contact angles (straight thetaa) formed by a water drop of >80°, e.g., grape leaf, polystyrene, Teflon, polycarbonate, collodion, and glass treated with the silanes n-octadecyltrichlorosilane, dimethyldichlorosilane, or diphenyldichlorosilane. When pycnidiospores were deposited on more wettable surfaces they did not attach firmly and did not germinate. Such highly wettable surfaces had straight thetaa 相似文献   

Reproductive and recruitment dynamics of clionaid sponges on oyster reefs in North Carolina

Boring sponges belonging to the family Clionaidae have become a destructive nuisance to eastern oyster (Crassostrea virginica) aquaculture and restoration efforts in the southeastern USA. Clionaid sponges colonize the inner layers of oyster shells and remove carbonate material, compromising the quality and marketability of the oyster; however, relatively little is known about reproduction and recruitment of these sponges. Using histological techniques, reproductive activity of clionaid sponges was monitored at two sites (Cedar Island and Masonboro Sound) in coastal North Carolina. Sponge recruitment to limestone tiles (5×5 cm), oyster shells, and clam shells was monitored in 2013 and 2014; recruitment to the limestone tiles was statistically higher than recruitment to clam or oyster shells. Overall, seasonal patterns in reproduction and recruitment of clionaid sponges were generally similar at the two sites. Three species of clionaid sponge were found during field sampling (Cliona celata, C. lobata, and C. truitti), and reproductive activity (eggs and spermatocysts) of these species was observed from April to November, with peak reproduction occurring from June to September for C. lobata and from August to September for C. celata. Recruitment peaked in late summer/early fall. Additionally, the relationship between environmental conditions (temperature, salinity, dissolved oxygen, pH, and chlorophyll a) and clionaid recruitment was explored using a regression model. At Cedar Island, the best‐fit model included salinity and dissolved oxygen, while the best‐fit model at Masonboro Sound included temperature, pH, and salinity. The data from this study show that the primary reproduction and recruitment pulses occur in the fall for local clionaids, and thus mitigation strategies should be applied in the late fall or winter to minimize infestations.     

Metagenomic characterization of oyster shell dump reveals predominance of Firmicutes bacteria

Metagenomic analyses were conducted to evaluate the biodiversity of oyster shell bacteria, under storage conditions, on the basis of 16s rDNA sequences. Temperature was recorded during a one year storage period, and the highest temperature (about 60 degrees C) was observed after five months ofstorage. Bacterial diversity was greatest in the initial stage sample, with 33 different phylotypes classified under seven phyla (Proteobacteria, Bacteroidetes, Firmicutes, Actinobacteria, Planctomycetes, Verrucomicrobia and unclassified bacteria), with 42.22% ofphylotypes belonging to Proteobacteria. The lowest diversity was found in the high temperature (fermentation) stage sample, with 10 different phylotypes belonging to Firmicutes (78.57%) and Bacteroidetes. In the final stage sample, bacteria were found belonging to Proteobacteria, Bacteroidetes, and Firmicutes, and some were unclassified bacteria. Of the bacteria constituting the final stage metagenome, 69.70% belonged to Firmicutes. Our results show that bacteria belonging to phylum Firmicutes were predominant during fermentation, and during the final stages of oyster shell storage, which suggests that these bacteria supposed to be the key players for oyster shell biodegradation.     

A latitudinal gradient of seasonal temperature variation recorded in oyster shells from the coastal waters of France and The Netherlands

Cathodoluminescence (CL) microscopy of the foliated calcite shell hinge sections of live-collected oyster Crassostrea gigas collected at seven locations along a latitudinal gradient from the Netherlands in the North Sea to the Atlantic coast of France, revealed variations in luminescence that were attributable to seasonal variations in calcification of the hinge. Photomicrographs of hinge sections and luminescence profiles were analyzed to define a micro-sampling strategy that was adopted to drill the hinge samples to determine their isotopic composition. Reconstructed seasonal seawater temperatures determined from the stable oxygen isotope (δ¹⁸O) composition along growth profiles from 32 oyster shell hinges showed distinct seasonal isotopic cycles that were compared with in situ measured seawater temperatures and salinities at each location. Comparison of the amplitude of the (δ¹⁸O) signal and the annual maximum and minimum seawater temperatures demonstrated that C. gigas shells from several locations provided a reliable record of seasonal seawater temperature variation. The exception to this was oysters from the Netherlands and northern France where winter growth rates at low temperatures were slow so that insufficient shell was deposited to allow adequate spatial resolution of sampling and this resulted in time-averaging of the reconstructed seawater temperatures and an overestimation of winter seawater temperature. A potential variability in δ¹⁸O_w–salinity relationship at low salinities could also explain the high difference between measured and predicted seawater temperatures in Dutch areas. The finding that latitudinal differences in oyster hinge growth rates and/or changes in the δ¹⁸O_w–salinity relationship can result in bias of the seawater temperature deduced from the stable isotopic composition of the hinge should be taken into account when reconstructing latitudinal gradients in seawater temperature.     

Assessing cockle shells (Anadara granosa) for reconstruction subdaily environmental parameters: Implication for paleoclimate studies

This study aims to investigate the potential of cockle shells as an environmental recorder, examining the environmental factors controlling the shell growth of the intertidal Anadara granosa from west coast of Malaysia. Subdaily environmental factors were recorded from December 2011 to November 2012. A total of 600 individuals were collected on a monthly basis and the shells sectioned from umbo to ventral margin, polished, etched and photographed under a light microscope to observe microgrowth bands and increments. Comparison of correlation matrix between mean increment width and each environmental factor indicated that shell growth had the highest positive correlation with seawater temperature (+0.72) and weak positive correlation with salinity (+0.53). Multiple regression analysis was used to assess independent associations between shell mean increment width and environmental parameters. Study model showed that 60.8% of the variation in shell growth could be explained by temperature, salinity, rainfall and tidal change. Individually, temperature and salinity made the greatest unique contribution to explain shell growth, respectively (p < 0.01). Laboratory results showed shell growth was in a linear trend to optimum temperature and salinity. These findings provide a basis for the interpretation of the temporal changes in shell microgrowth patterns in terms of environmental conditions of cockle shells.     

Use of Hoechst Dyes 33258 and 33342 for Enumeration of Attached and Planktonic Bacteria

The DNA-specific fluorochromes Hoechst 33258 and 33342 were used to enumerate aquatic bacteria by epifluorescent direct counts. Cultures of estuarine bacteria gave identical counts when stained with Hoechst 33258 or acridine orange, whereas natural populations of aquatic bacteria gave 92 to 98.5% of the acridine orange counts. The technique had distinct advantages over acridine orange when enumerating bacteria on surfaces which bind acridine orange, such as polystyrene.     

Effect of reduced pH on shells of brooded veligers in the estuarine bivalve Ostrea chilensis Philippi 1845

The oyster Ostrea chilensis develops in estuaries and incubates its embryos inside the pallial cavity. Reduced external salinity triggers valve closure and isolation of the brood chamber, which in turn alters conditions in the pallial fluid, including pH reductions ranging from 7.65 (SD = 0.05) to 6.97 (SD = 0.02) in non-brooding females and 6.96 (SD = 0.10) in brooding females after 12 h of isolation. Exposing veliger larvae to acidic pH's (5 and 3) decreased the thickness of the embryonic shell valves. Calcium, sodium, chlorine, tin, sulphur, and magnesium were the most important (> 99%) elements constituting the veliger shells. Calcium content in the pallial cavity fluid increased with continued isolation in both brooding and non-brooding females, but calcium remained as the primary component (about 94%) of embryonic shells and maintained its normal proportion with respect to the rest of the elements at all tested pH's. The increased calcium in the pallial fluid was identified mainly as coming from the shells of brooding females. If any neutralizing elements were coming from embryonic shell valves, all elements in those shells must have been solubilising to the same degree. Veligers ceased all further shell growth while isolated within the female's brood chamber. However, veliger shell growth resumed once females were returned to high-salinity seawater. Our study shows that female isolation of the mantle cavity, often considered an adaptive response to the periodically reduced salinities of estuarine waters, can in fact generate adverse effects on brooded embryos.     

香港巨牡蛎和长牡蛎幼虫及稚贝的表型性状

为了评估香港巨牡蛎和长牡蛎在北方沿海的早期表型性状,于2010年7月,以2009年6月在青岛繁育的两种牡蛎为材料,在大连研究了温度(Mt:(22±1.0)℃及Ht:(28±1.0)℃)、盐度(S₂₀:20±1.0及S₃₀:30±1.0)及中间育成环境(ID:室内及OD:室外)对两种牡蛎幼虫及稚贝表型性状的影响。结果表明:香港巨牡蛎壳宽显著大于长牡蛎(P<0.05),壳高及怀卵量显著小于长牡蛎(P<0.05),壳长、鲜重及壳重两者间无显著差异(P>0.05)。在温度和盐度相同情况下,长牡蛎卵径、受精率、孵化率及D形幼虫均大于香港巨牡蛎;香港巨牡蛎幼虫浮游前期生长较慢,而后快于长牡蛎。两种牡蛎幼虫存活能力在15日龄时高温组>中温组;相同温度下,香港巨牡蛎中盐组>高盐组,长牡蛎高盐组>中盐组。幼虫变态期间,较低的温度延迟了变态时间,降低了变态率,使得两种幼虫变态规格大型化。温度是影响幼虫生长、存活、变态的最主要因素,其次为盐度,交互作用几乎尚未起到作用。中间育成阶段,室外比室内培育效果更好,且香港巨牡蛎稚贝壳高在60日龄以后显著大于长牡蛎(P<0.05),环境是影响稚贝生长的最主要因素;无论室内还是室外两种牡蛎稚贝的存活率均在90%以上,且各实验组间无显著差异(P>0.05)。     

Localization of the bacterial agent of juvenile oyster disease (Roseovarius crassostreae) within affected eastern oysters (Crassostrea virginica)

The bacterium Roseovarius crassostreae causes seasonal mortalities among commercially produced eastern oysters (Crassostrea virginica) grown in the Northeastern United States. Phylogenetically, the species belongs to a major lineage of marine bacteria (the Roseobacter clade), within which Roseovarius crassostreae is the only known pathogen to be isolated in laboratory culture. The objective of the current study was to determine the location and nature of R. crassostreae interactions with oysters affected by juvenile oyster disease (JOD). Scanning electron microscopy of diseased individuals revealed abundant colonization of the inner shell surfaces by bacteria which were morphologically similar to R. crassostreae. The same types of cells were also observed on and within layers of host-derived conchiolin on the inner valves. Most bacterial cells were alive as determined by the use of a fluorescent viability stain. Further, most were clearly attached at the cell poles, which is consistent with the ability of R. crassostreae to express polar fimbriae. When material from the pallial fluid, soft tissue and inner valve surfaces was cultured, the highest numbers of R. crassostreae were recovered from the inner valves. These samples also contained the greatest abundance of R. crassostreae as a percentage of total colonies. Cloning and sequencing of 16S rRNA genes provided culture-independent evidence of the numerical dominance of R. crassostreae among the bacterial consortia associated with the inner shell surfaces of JOD-affected animals. The ability of R. crassostreae to colonize shell and conchiolin is consistent with the described JOD-pathology and may aid the bacteria in avoiding hemocyte-mediated killing.     

Aragonite shells are more ancient than calcite ones in bivalves: new evidence based on omics

Two calcium carbonate crystal polymorphs, aragonite and calcite, are the main inorganic components of mollusk shells. Some fossil evidences suggest that aragonite shell is more ancient than calcite shell for the Bivalvia. But, the molecular biology evidence for the above deduction is absent. In this study, we searched for homologs of bivalve aragonite-related and calcite-related shell proteins in the oyster genome, and found that no homologs of calcite-related shell protein but some homologs of aragonite-related shell proteins in the oyster genome. We explained the results as the new evidence to support that aragonite shells are more ancient than calcite shells in bivalves combined the published biogeological and seawater chemistry data.     

Rapid Immunocapture of Pseudomonas putida Cells from Lake Water by Using Bacterial Flagella

Monoclonal antibodies to Pseudomonas putida Paw340 cells were produced. In an enzyme-linked immunosorbent assay (ELISA) against whole bacterial cells, a hybridoma cell line termed MLV1 produced a monoclonal antibody that reacted with P. putida Paw340 but showed no cross-reaction with 100 medical isolates and 150 aquatic isolates. By ELISA, immunogold electron microscopy, and Western blot (immunoblot) analysis, MLV1 antibody was found to react with purified bacterial flagella. The surfaces of magnetic polystyrene beads were coated with MLV1 antibody. By mixing MLV1 antibody-coated beads with lake water samples containing the target P. putida host, bead-cell complexes which could be recovered by attraction towards a magnet were formed. Prevention of nonspecific attachment of cells to the beads required the incorporation of detergents in the isolation protocol. These detergents affected colony-forming ability; however, the cells remained intact for direct detection. When reisolated by standard cultural methods, approximately 20% of the initial target population was recovered. Since the beads and bead-cell complexes were recovered in a magnetic field, target bacteria were separated from other lake water organisms and from particulate material which was not attracted towards the magnet and were thereby enriched. This method may now provide a useful system for recovering recombinant bacteria selectively from environmental samples.