Direct Profiling of Environmental Microbial Populations by Thermal Dissociation Analysis of Native rRNAs Hybridized to Oligonucleotide Microarrays

Oligonucleotide microarrays were used to profile directly extracted rRNA from environmental microbial populations without PCR amplification. In our initial inspection of two distinct estuarine study sites, the hybridization patterns were reproducible and varied between estuarine sediments of differing salinities. The determination of a thermal dissociation curve (i.e., melting profile) for each probe-target duplex provided information on hybridization specificity, which is essential for confirming adequate discrimination between target and nontarget sequences.     

Detection of hepatitis A virus in seeded estuarine samples by hybridization with cDNA probes.

The development and trials of a nucleic acid hybridization test for the detection of hepatitis A virus (HAV) in estuarine samples within 48 h are described. Approximately 10(4) physical particles of HAV per dot could be detected. Test sensitivity was optimized by the consideration of hybridization stringency, 32P energy level, probe concentration, and nucleic acid binding to filters. Test specificity was shown by a lack of cross-hybridization with other enteroviruses and unrelated nucleic acids. Potential false-positive reactions between bacterial DNA in samples and residual vector DNA contamination of purified nucleotide sequences in probes were eliminated by DNase treatment of samples. Humic acid at concentrations of up to 100 mg/liter caused only insignificant decreases in test sensitivity. Interference with hybridization by organic components of virus-containing eluates was removed by proteinase K digestion followed by phenol extraction and ethanol precipitation. The test is suitable for detecting naturally occurring HAV in samples from polluted estuarine environments.     

Detection of hepatitis A virus in seeded estuarine samples by hybridization with cDNA probes

The development and trials of a nucleic acid hybridization test for the detection of hepatitis A virus (HAV) in estuarine samples within 48 h are described. Approximately 10(4) physical particles of HAV per dot could be detected. Test sensitivity was optimized by the consideration of hybridization stringency, 32P energy level, probe concentration, and nucleic acid binding to filters. Test specificity was shown by a lack of cross-hybridization with other enteroviruses and unrelated nucleic acids. Potential false-positive reactions between bacterial DNA in samples and residual vector DNA contamination of purified nucleotide sequences in probes were eliminated by DNase treatment of samples. Humic acid at concentrations of up to 100 mg/liter caused only insignificant decreases in test sensitivity. Interference with hybridization by organic components of virus-containing eluates was removed by proteinase K digestion followed by phenol extraction and ethanol precipitation. The test is suitable for detecting naturally occurring HAV in samples from polluted estuarine environments.     

Evolutionary impacts of hybridization and interspecific gene flow on an obligately estuarine fish

For free-spawning estuarine taxa, gene flow among estuaries may occur via hybridization with mobile congeners. This phenomenon has rarely been investigated, but is probably susceptible to anthropogenic disturbance. In eastern Australia, the estuarine Black Bream Acanthopagrus butcheri and marine Yellowfin Bream Acanthopagrus australis have overlapping distributions and the potential to hybridize. We used surveys of microsatellite and mtDNA variation in 565 adults from 25 estuaries spanning their distributional range to characterize the species and their putative hybrids. Hybrids were widespread (68% of estuaries) and hybrid frequencies varied greatly among estuaries (0-58%). Most (88%) were classed as advanced generation backcrosses with A. butcheri and displayed A. butcheri mtDNA haplotypes. We found most hybrids in the three estuaries within the zone of sympatry (57%). Our study highlights the underemphasized importance of estuaries as sites of hybridization and suggests that hybridization is driven both by opportunity for contact and human activity.     

Marine genetic swamping: hybrids replace an obligately estuarine fish

Populations of obligately estuarine taxa are potentially small and isolated and may lack genetic variation and display regional differentiation as a result of drift and inbreeding. Hybridization with a wide‐ranging marine congener should introduce genetic variation and reduce the effects of inbreeding depression and genetic drift. However, high levels of hybridization can cause demographic and genetic swamping. In southeastern Australia hybridization occurs between obligately estuarine Black bream (Acanthopagrus butcheri) and migratory marine Yellowfin bream (Acanthopagrus australis). Here, we surveyed genetic variation at eight microsatellite loci and the mitochondrial control region of juvenile fish from five coastal lagoons (including temporal replication in two lagoons) (total n = 970) to determine the frequency and persistence of hybridization, and its likely consequence for the estuarine restricted A. butcheri. Of 688 juvenile fish genotyped 95% were either A. australis (347) or hybrids (309); only 5% (32) were A. butcheri. Most hybrids were later generation hybrids or A. butcheri backcrosses, which are likely multi‐generational residents within lagoons. Far greater proportions of hybrid juveniles were found within two lagoons that are generally closed to the ocean (>90% hybrid fish within generally closed lagoons vs. 12–27% in permanently or intermittently open lagoons). In both lagoons, this was consistent across multiple cohorts of fish [79–97% hybrid fish (n = 282)]. Hybridization and introgression represent a major threat to the persistence of A. butcheri and have yet to be investigated for large numbers of estuarine taxa.     

Ecology of Vibrio vulnificus in estuarine waters of eastern North Carolina

While several studies on the ecology of Vibrio vulnificus in Gulf Coast environments have been reported, there is little information on the distribution of this pathogen in East Coast waters. Thus, we conducted a multiyear study on the ecology of V. vulnificus in estuarine waters of the eastern United States, employing extensive multiple regression analyses to reveal the major environmental factors controlling the presence of this pathogen, and of Vibrio spp., in these environments. Monthly field samplings were conducted between July 2000 and April 2002 at six different estuarine sites along the eastern coast of North Carolina. At each site, water samples were taken and nine physicochemical parameters were measured. V. vulnificus isolates, along with estuarine bacteria, Vibrio spp., Escherichia coli organisms, and total coliforms, were enumerated in samples from each site by using selective media. During the last 6 months of the study, sediment samples were also analyzed for the presence of vibrios, including V. vulnificus. Isolates were confirmed as V. vulnificus by using hemolysin gene PCR or colony hybridization. V. vulnificus was isolated only when water temperatures were between 15 and 27 degrees C, and its presence correlated with water temperature and dissolved oxygen and vibrio levels. Levels of V. vulnificus in sediments were low, and no evidence for an overwintering in this environment was found. Multiple regression analysis indicated that vibrio levels were controlled primarily by temperature, turbidity, and levels of dissolved oxygen, estuarine bacteria, and coliforms. Water temperature accounted for most of the variability in the concentrations of both V. vulnificus (47%) and Vibrio spp. (48%).     

A highly permeable species boundary between two anadromous fishes

Meristic identification, mitochondrial DNA and a suite of microsatellite markers were employed to estimate the incidence of hybridization in wild populations of anadromous Allis shad Alosa alosa and twaite shad Alosa fallax in southern Irish riverine and estuarine waters. It was shown that 16% of the fishes examined were misclassified using meristic count of gill rakers. Next, a significant proportion of fishes that were robustly assigned to a species using nuclear markers were shown to possess the mtDNA of the other. The genomes of A. alosa and A. fallax in Ireland are extensively introgressed, which suggests a complex history of hybridization between these species, which can only partially be explained by recent man-made habitat changes.     

Specific detection of Arcobacter spp. in estuarine waters of Southern Italy by PCR and fluorescent in situ hybridization

Aim:  To evaluate the reliability of culture-independent methods in comparison with culture-dependent ones for the detection of Arcobacter spp. in estuarine waters of Southern Italy.
Methods and Results:  PCR and fluorescent in situ hybridization (FISH) procedures were used to detect arcobacters directly in water samples and after enrichment cultures. The samples totally were positive by molecular methods (PCR and FISH) but only 75% were culture positive, confirming the limitation of these latter to detect Arcobacter spp. in natural samples. Culturable arcobacters were retrieved in all times except in July, and isolated species were ascribed only to Arcobacter cryaerophilus .
Conclusions:  Culturable and nonculturable forms of Arcobacter in the estuarine environment were present. PCR assays were more sensitive than traditional culture in detecting Arcobacter butzleri and A. cryaerophilus . FISH comparatively to PCR technique may provide information about cell morphology and viability of single cells.
Significance and Impact of the Study:  Our investigation indicates the existence of an environmental reservoir of potential pathogenic arcobacters in an estuarine Italian area, which may survive under a viable but not culturable state.     

Ecology of Vibrio vulnificus in Estuarine Waters of Eastern North Carolina

While several studies on the ecology of Vibrio vulnificus in Gulf Coast environments have been reported, there is little information on the distribution of this pathogen in East Coast waters. Thus, we conducted a multiyear study on the ecology of V. vulnificus in estuarine waters of the eastern United States, employing extensive multiple regression analyses to reveal the major environmental factors controlling the presence of this pathogen, and of Vibrio spp., in these environments. Monthly field samplings were conducted between July 2000 and April 2002 at six different estuarine sites along the eastern coast of North Carolina. At each site, water samples were taken and nine physicochemical parameters were measured. V. vulnificus isolates, along with estuarine bacteria, Vibrio spp., Escherichia coli organisms, and total coliforms, were enumerated in samples from each site by using selective media. During the last 6 months of the study, sediment samples were also analyzed for the presence of vibrios, including V. vulnificus. Isolates were confirmed as V. vulnificus by using hemolysin gene PCR or colony hybridization. V. vulnificus was isolated only when water temperatures were between 15 and 27°C, and its presence correlated with water temperature and dissolved oxygen and vibrio levels. Levels of V. vulnificus in sediments were low, and no evidence for an overwintering in this environment was found. Multiple regression analysis indicated that vibrio levels were controlled primarily by temperature, turbidity, and levels of dissolved oxygen, estuarine bacteria, and coliforms. Water temperature accounted for most of the variability in the concentrations of both V. vulnificus (47%) and Vibrio spp. (48%).     

Molecular analysis of isolates of Streptococcus suis capsular type 2 by restriction-endonuclease-digested DNA separated on SDS-PAGE and by hybridization with an rDNA probe.

This study was undertaken to assess the discriminatory value of restriction endonuclease (RE) digestion patterns of Streptococcus suis chromosomal DNA using polyacrylamide gel electrophoresis (SDS-PAGE) and DNA-rDNA hybridization. For the RE digestion patterns, DNAs were digested separately with the enzymes BamHI and BglII and the resultant fragments were separated by SDS-PAGE. An Escherichia coli rDNA probe derived from pKK3535 was used for the hybridization. Twenty-three S. suis capsular type 2 isolates recovered from diseased and clinically healthy pigs, from a human case, and from a cow were compared in this study. The majority of isolates associated with septicaemia belonged to one restriction endonuclease analysis (REA) profile group. Isolates associated with pneumonia belonged either to the REA profile group of isolates associated with septicaemia or to a second REA profile group. The REA profiles of isolates from clinically healthy animals were more heterogeneous. The REA profile of the type 2 reference strain, S735, which was originally isolated from a pig, was very different from those of the porcine and bovine isolates but similar to the profile of the human isolate. The profiles obtained after rDNA hybridization were more homogeneous. Although different patterns were detected in the 23 isolates, there was no correlation between the source of the isolate and the patterns observed with this technique.     

Comparison of Vibrio parahaemolyticus grown in estuarine water and rich medium

Cell envelope composition and selected physiological traits of Vibrio parahaemolyticus were studied in regard to the Kanagawa phenomenon and growth conditions. Cell envelopes were prepared from cells cultured in Proteose Peptone-beef extract (Difco Laboratories, Detroit, Mich.) medium or filtered estuarine water. Protein, phospholipid, and lipopolysaccharide contents varied with culture conditions. The phospholipids present in the cell envelopes were identified as phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. Phosphatidylethanolamine decreased and phosphatidylglycerol increased in cells grown in estuarine water. Profiles of proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated numerous protein species, with four to six predominant proteins ranging from 26,000 to 120,000 in molecular weight. The profile of V. parahaemolyticus cell envelope proteins was unique and might be useful in the identification of the organism. Alkaline phosphatase activity was slightly higher in Kanagawa-negative strains and was higher in cells grown in estuarine water than in cells grown in rich laboratory medium. The DNA levels in estuarine water-grown cells increased, while RNA levels and cell volume decreased. Bacteriophage sensitivity typing demonstrated a close intraspecies relationship. Results indicated that Kanagawa-positive and -negative strains were closely related, but they could be grouped separately and may have undergone starvation-related physiological changes when cultured in estuarine water.     

Comparison of Vibrio parahaemolyticus grown in estuarine water and rich medium.

Cell envelope composition and selected physiological traits of Vibrio parahaemolyticus were studied in regard to the Kanagawa phenomenon and growth conditions. Cell envelopes were prepared from cells cultured in Proteose Peptone-beef extract (Difco Laboratories, Detroit, Mich.) medium or filtered estuarine water. Protein, phospholipid, and lipopolysaccharide contents varied with culture conditions. The phospholipids present in the cell envelopes were identified as phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. Phosphatidylethanolamine decreased and phosphatidylglycerol increased in cells grown in estuarine water. Profiles of proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated numerous protein species, with four to six predominant proteins ranging from 26,000 to 120,000 in molecular weight. The profile of V. parahaemolyticus cell envelope proteins was unique and might be useful in the identification of the organism. Alkaline phosphatase activity was slightly higher in Kanagawa-negative strains and was higher in cells grown in estuarine water than in cells grown in rich laboratory medium. The DNA levels in estuarine water-grown cells increased, while RNA levels and cell volume decreased. Bacteriophage sensitivity typing demonstrated a close intraspecies relationship. Results indicated that Kanagawa-positive and -negative strains were closely related, but they could be grouped separately and may have undergone starvation-related physiological changes when cultured in estuarine water.     

Geographical and typological changes in fish guilds of South African estuaries

A comparative analysis of fish estuary association guilds was undertaken on some 190 South African estuaries. This pioneering study spanned three zoogeographic regions and included three broad estuarine types. The guild compositions of the estuaries were compared based on an importance value, incorporating taxonomic composition, numerical abundance and relative biomass. Multivariate analyses included both inter‐regional (zoogeographic) and intra‐regional (estuarine typology) comparisons. The major estuary‐associated guilds (estuarine species and marine migrant species) were important in all estuary types within all biogeographic regions. Significant differences both between regions and between estuary types within regions, however, were recorded. Cool–temperate estuaries were generally dominated by migratory species (estuarine migrants and marine migrant opportunists) while the importance of species dependent on estuaries (estuarine residents and estuarine‐dependent marine migrants) was higher in warm–temperate and subtropical regions. The significance of estuarine nursery areas, particularly in regions where estuaries are few in number, is highlighted. In terms of typology, migratory species assumed a greater importance in predominantly open systems, while freshwater and estuarine‐resident species were more important in predominantly closed systems. Predominantly closed estuaries, however, were also important for marine migrant species, which further highlights the significance of these systems as nursery areas for fishes.     

Proportion of prokaryotes enumerated as viruses by epifluorescence microscopy

It is well known that there are prokaryotes small in size (e.g. ultra-microprokaryotes) that pass through a 0.2-μm filter. As bacterial and viral abundances are determined by epifluorescence microscopy and the differentiation between them is based on particle size, some bacteria can be erroneously enumerated as viruses, namely in marine waters where bacteria are small. However, there is no information on the proportion of prokaryotes that could be misidentified as viruses by epifluorescence microscopy. In this work, we assessed, in water samples collected in the estuarine system Ria de Aveiro (Portugal), the proportion of prokaryotes that could be counted as viruses by the current widespread epifluorescence microscopy and, for the first time, by fluorescence in situ hybridization (FISH). The total number of particles was determined on membranes of 0.2 and 0.02 μm after staining with 4′,6-diamidino-2-phenylindole (DAPI), and the number of prokaryotes (Bacteria and Archaea) was determined by FISH for both pore size membranes. The results show that, in the marine zone of the estuarine system, 28 % of particles enumerated as virus-like particles were prokaryotes, but, in the brackish water zone, only 13 % of the particles counted as viruses were actually prokaryotic cells. Epifluorescence microscopy overestimates viral abundance, and also the ratio viruses:prokaryotes, and this error must be taken into consideration because it can vary significantly within a system. In fact, in the marine zone of an estuarine system, the overestimation of viral abundance can be twice as high as in the brackish water zone.     

Hybridization of invasive <Emphasis Type="Italic">Phragmites australis</Emphasis> with a native subspecies in North America

Interspecific hybridization can lead to the extinction of native populations and increased aggressiveness in hybrid forms relative to their parental lineages. However, interbreeding among subspecies is less often recognized as a serious threat to native species. Phragmites australis offers an excellent opportunity to investigate intraspecific hybridization since both native and introduced lineages occur in North America. Introduced Phragmites is a highly successful estuarine plant invader throughout North America, but native Phragmites populations are declining in the eastern US. Despite range overlaps, hybridization has not yet been detected between the native and introduced lineages in the wild, suggesting that phenological or physiological barriers preclude cross-pollination. We demonstrate, for the first time, that native and introduced populations of Phragmites can hybridize. There is substantial overlap in flowering period between native and introduced populations from the same geographic locations. We manually cross-pollinated native individuals with pollen from introduced Phragmites and recovered viable offspring. We then used microsatellite markers to prove that alleles unique to the pollen parent were transferred to progeny. Our results imply a mechanism for the further decline of native Phragmites in North America and a potential for the formation of aggressive hybrid offspring.     

Biogeography,habitat transitions and hybridization in a radiation of South American silverside fishes revealed by mitochondrial and genomic RAD data

Rivers and lake systems in the southern cone of South America have been widely influenced by historical glaciations, carrying important implications for the evolution of aquatic organisms, including prompting transitions between marine and freshwater habitats and by triggering hybridization among incipient species via waterway connectivity and stream capture events. Silverside fishes (Odontesthes) in the region comprise a radiation of 19 marine and freshwater species that have been hypothesized on the basis of morphological or mitochondrial DNA data to have either transitioned repeatedly into continental waters from the sea or colonized marine habitats following freshwater diversification. New double digest restriction‐site associated DNA data presented here provide a robust framework to investigate the biogeographical history of and habitat transitions in Odontesthes. We show that Odontesthes silversides originally diversified in the Pacific but independently colonized the Atlantic three times, producing three independent marine‐to‐freshwater transitions. Our results also indicate recent introgression of marine mitochondrial haplotypes into two freshwater clades, with more recurring instances of hybridization among Atlantic‐ versus Pacific‐slope species. In Pacific freshwater drainages, hybridization with a marine species appears to be geographically isolated and may be related to glaciation events. Substantial structural differences of estuarine gradients between these two geographical areas may have influenced the frequency, intensity and evolutionary effects of hybridization events.     

The ecology of Vibrio vulnificus, Vibrio cholerae, and Vibrio parahaemolyticus in North Carolina Estuaries

While numerous studies have characterized the distribution and/or ecology of various pathogenic Vibrio spp., here we have simultaneously examined several estuarine sites for Vibrio vulnificus, V. cholerae, and V. parahaemolyticus. For a one year period, waters and sediment were monitored for the presence of these three pathogens at six different sites on the east coast of North Carolina in the United States. All three pathogens, identified using colony hybridization and PCR methods, occurred in these estuarine environments, although V. cholerae occurred only infrequently and at very low levels. Seventeen chemical, physical, and biological parameters were investigated, including salinity, water temperature, turbidity, dissolved oxygen, levels of various inorganic nutrients and dissolved organic carbon, as well as total vibrios, total coliforms, and E. coli. We found each of the Vibrio spp. in water and sediment to correlate to several of these environmental measurements, with water temperature and total Vibrio levels correlating highly (P<0.0001) with occurrence of the three pathogens. Thus, these two parameters may represent simple assays for characterizing the potential public health hazard of estuarine waters.     

Genetic Diversity of a Natural Population of Sinorhizobium melilotiRevealed in Analysis of Cryptic Plasmids and ISRm2011-2 Fingerprints

Fifty-six natural strains of alfalfa nodule bacteria were isolated from samples of the soil under wild legume and alfalfa in two different field sites of Irkutsk oblast. Based on the results of analysis of plasmid profile, 11 different types of strains were detected, and 43 types were identified based on the results of hybridization with the insertion sequence element ISRm2011-2. Significant differences were found in the plasmid profile and IS fingerprints between strains isolated from the soil under alfalfa and the soil under legume. In contrast, strains growing at some distance from each other differed only in the IS fingerprints. From a comparison of results obtained in the assessment of plasmid profile and in analysis of IS fingerprints with results of RFLP analysis in strains, the conclusion about the transference of cryptic plasmids between strains and genetic rearrangements in strains of this population was drawn.     

Estuarine recruitment of a marine goby reconstructed with an isotopic clock

Information on movement patterns of marine fishes between estuarine populations and stocks at sea is fundamental to understanding their population dynamics, life history tactics and behavior. Furthermore, understanding estuarine habitat use by marine fishes is crucial for their effective conservation and integrated estuarine management. Although large numbers of young marine fish make use of temperate estuaries in highly predictable abundance patterns, very little is known about how estuarine populations interact with the populations at sea. Recruitment of sand goby Pomatoschistus minutus (Pallas, 1770) into the low salinity zone of the Scheldt estuary (Belgium) was reconstructed over an entire year by means of an isotopic clock. These results were combined with a growth model to yield age and length at immigration. Sand gobies entered the upper Scheldt estuary almost continuously from May onwards, except in July when they appeared to avoid the estuary due to warm summer temperatures. About 70% of the fish caught in the upper estuary resided there for less than 1 month, which indicates a strong temporal overlap of immigration and emigration. This complex migration pattern suggests that estuarine residence is caused by trade-offs made at the individual level, whereby migration is probably triggered by temperature. The high turnover of individuals in the estuarine population leads us to question the functional role of the estuary for marine fishes. Sand gobies entering the upper estuary had a wide range of ages and body sizes, although they were at least 2 months old and had a minimum standard length of approximately 20 mm. This study shows that the use of an isotopic clock strongly complements catch data and is useful to describe the connectivity between populations.     

Genetic diversity of a natural population of Sinorhizobium meliloti, detected during analysis of a cryptic plasmid and ISRm2011-2 fingerprints]

Fifty-six natural strains of alfalfa nodule bacteria were isolated from samples of the soil under wild legume and alfalfa in two different field sites of Irkutsk oblast. Based on the results of analysis of plasmid profile, 11 different types of strains were detected, and 43 types were identified based on the results of hybridization with the insertion sequence element ISRm2011-2. Significant differences were found in the plasmid profile and IS fingerprints between strains isolated from the soil under alfalfa and the soil under legume. In contrast, strains growing at some distance from each other differed only in the IS fingerprints. From a comparison of results obtained in the assessment of plasmid profile and in analysis of IS fingerprints with results of RFLP analysis in strains, the conclusion about the transference of cryptic plasmids between strains and genetic rearrangements in strains of this population was drawn.