PCR-amplified 16S rDNA Research Articles

One nutritional symbiosis begat another: Phylogenetic evidence that the ant tribe Camponotini acquired Blochmannia by tending sap-feeding insects

BackgroundBacterial endosymbiosis has a recurring significance in the evolution of insects. An estimated 10-20% of insect species depend on bacterial associates for their nutrition and reproductive viability. Members of the ant tribe Camponotini, the focus of this study, possess a stable, intracellular bacterial mutualist. The bacterium, Blochmannia, was first discovered in Camponotus and has since been documented in a distinct subgenus of Camponotus, Colobopsis, and in the related genus Polyrhachis. However, the distribution of Blochmannia throughout the Camponotini remains in question. Documenting the true host range of this bacterial mutualist is an important first step toward understanding the various ecological contexts in which it has evolved, and toward identifying its closest bacterial relatives. In this study, we performed a molecular screen, based on PCR amplification of 16S rDNA, to identify bacterial associates of diverse Camponotini species.ResultsPhylogenetic analyses of 16S rDNA gave four important insights: (i) Blochmannia occurs in a broad range of Camponotini genera including Calomyrmex, Echinopla, and Opisthopsis, and did not occur in outgroups related to this tribe (e.g., Notostigma). This suggests that the mutualism originated in the ancestor of the tribe Camponotini. (ii) The known bacteriocyte-associated symbionts of ants, in Formica, Plagiolepis, and the Camponotini, arose independently. (iii) Blochmannia is nestled within a diverse clade of endosymbionts of sap-feeding hemipteran insects, such as mealybugs, aphids, and psyllids. In our analyses, a group of secondary symbionts of mealybugs are the closest relatives of Blochmannia. (iv) Blochmannia has cospeciated with its known hosts, although deep divergences at the genus level remain uncertain.ConclusionsThe Blochmannia mutualism occurs in Calomyrmex, Echinopla, and Opisthopsis, in addition to Camponotus, and probably originated in the ancestral lineage leading to the Camponotini. This significant expansion of its known host range implies that the mutualism is more ancient and ecologically diverse than previously documented. Blochmannia is most closely related to endosymbionts of sap-feeding hemipterans, which ants tend for their carbohydrate-rich honeydew. Based on phylogenetic results, we propose Camponotini might have originally acquired this bacterial mutualist through a nutritional symbiosis with other insects.

Comparative study of three analysis methods (TTGE, flow cytometry and HPLC) for xenobiotic impact assessment on phytoplankton communities

The impacts of the fungicide Opus (epoxiconazole) on marine phytoplankton communities were assessed in a 12-day field experiment using in situ microcosms maintained underwater at 6 m depth. Three community analysis methods were compared for their sensitivity threshold in fungicide impact detection. When phytoplankton communities were exposed to 1 microg l(-1) of epoxiconazole, no effects could be demonstrated using TTGE (Temporal Temperature Gradient gel Electrophoresis), flow cytometry or HPLC. When exposed to 10 microg l(-1), TTGE fingerprints from PCR amplified 18S rDNA of communities exhibited significant differences compared with controls (ANOSIM, P = 0.028). Neither flow cytometry counts, nor HPLC pigment profiles allowed to show significant differences in microcosms exposed to 10 microg l(-1) of epoxiconazole. When exposed to 100 microg l(-1), all three methods allowed to detect significant differences in treated microcosms, as compared to controls. The TTGE analysis appears in this study as the most sensitive method for fungicide impact assessment on eukaryote microbial communities.

Comparisons of different hypervariable regions of rrs genes for fingerprinting of microbial communities in paddy soils

The use of molecular approaches based on 16S rDNA-PCR in microbial ecology has revealed a tremendous prokaryotic diversity in environmental samples. However, there is little or no systematic evaluation of the impacts of hypervariable (V) regions of rrs genes choice on microbial community analysis in soil samples, especially the detailed information about the dominant groups preferentially amplified by different primer pairs. In the present study, eight primer pairs were detected to compare the different V regions for fingerprinting microbial communities in a paddy soil irrigated with petroleum-wastewater, using denaturing gradient gel electrophoresis (DGGE) and amplified ribosomal DNA restriction analysis (ARDRA) techniques. Results reveal the obvious PCR bias produced by different V regions. Both ARDRA analysis of 16S rDNA clone library and DGGE suggest that V 1–V 3 region amplified with primer pair 8f–519r produced the most informative fingerprinting profiles. Additionally, V 3–V 5 region amplified with 341f–907r was another preferable choice for microbial diversity in petroleum-contaminated soil. The V 4–V 5 region and single V region (V 1, V 3, and V 8) were not recommended for the future study of microbial diversity in soil samples. Phylogenetic analysis of 123 sequences from libraries constructed by amplicons generated from six different V regions suggests that different dominant groups were amplified with distinct primer sets. In detail, V 1–V 3 library (amplified with 8f–519r) and V 3–V 5 library were dominated by Actinobacteria (20.4%) (particularly in genus Arthrobacter), V 1–V 3 library (amplified with 63f–518r) was dominated by γ-Proteobacteria (25.0%) and α-Proteobacteria (22.0%) (particularly in genus Brevundimonas), V 3 library was dominated by β-Proteobacteria (22.3%) (particularly in genus Gallionella) and α-Proteobacteria (20.0%), V 6–V 8 library was dominated by Chlamydiae (20.4%) and β-Proteobacteria (20.4%), V 8 library was dominated by γ-Proteobacteria (27.2%) (particularly in genus Acinetobacter) and β-Proteobacteria (14.0%). The present work strongly recommends that primer pairs should be chosen cautiously in community diversity analysis based on PCR amplification of 16S rDNA, and involving at least two different 16S rDNA universal primer pairs would perform better.

Rapid discrimination of the common species of the stored product pest Liposcelis (Psocoptera: Liposcelididae) from China and the Czech Republic, based on PCR-RFLP analysis

Psocids of the genus Liposcelis (Psocoptera: Liposcelididae) are stored product pests that are difficult to identify morphologically. A molecular method based on Restriction Fragment Length Polymorphism (RFLP) of the PCR-amplified 16S rDNA gene was developed for the rapid discrimination of four common species (L. bostrychophila, L. entomophila, L. decolor, and L. paeta). Different developmental stages and populations (P.R. China and Czech Republic) were tested. One DNA fragment of about 500 bp in length was amplified from genomic DNA and the fragment was then digested using the restriction endonuclease DraI. Identification of the relevant banding pattern allowed all the developmental stages and both sexes to be discriminated in the species tested. The banding patterns of L. entomophila from all populations were identical, while the relevant restriction digests and sequence analysis confirmed that the Chinese and Czech populations of L. bostrychophila, L. decolor, and L. paeta differed. In conclusion, PCR-RFLP with one pair of primers (16Sar and 16Sbr) and one restrictive endonuclease, DraI, proved a reliable method for rapidly discriminating the Liposcelis species tested.

Comparison of bacterial communities in fish farm sediments along an organic enrichment gradient

Aquaculture is of major industrial importance world wide and this has led to concerns over the impact of fish farming on previously pristine marine environments. Fish farm sediments receive a large amount of organic matter due to uneaten food and fecal materials. This organic enrichment of sediment influences the biogeochemical process and benthic microbial communities. This study was performed to investigate the effect of pollution from marine fish farms on the bacterial community structure in the underlying sediments. Sediment samples were collected at four stations along an organic enrichment gradient on the coast of Wakasa Bay in southern Fukui Prefecture, Japan between April 2004 and December 2006. Sagami Bay sediments were also used as a non-fish farming reference site. The bacterial community composition was assessed using PCR amplification, cloning, sequencing, and phylogenetic analysis of 16S rDNA using directly extracted sediment DNA. Most of the clones sequenced fell into six major lineages of the domain Bacteria: γ-, δ-, ε-Proteobacteria, Acidobacteria/ Holophaga, Bacteroidetes, and Planctomycetes. Although there were no significant differences in the bacterial community structures among the sediment samples except for Sagami Bay, a minor change in the composition of 16S rDNA operational taxonomy units (OTUs) between sites was recognized. 16S rDNA sequences related to the γ-Proteobacteria were recovered with relatively higher frequencies from unpolluted sediments than from polluted sediments. In contrast, the most abundant clone sequences from polluted sediment samples were grouped with δ-Proteobacteria. Our data suggests organic enrichment of sediment may influence the composition of bacterial communities; and the δ-Proteobacteria were found and selected for within polluted fish farm sediments.

Diversity of bacteria that nodulate Prosopis juliflora in the eastern area of Morocco

A total of 274 bacterial strains were isolated from the root nodules of Prosopis juliflora, growing in two arid soils of the eastern area of Morocco. A physiological plate screening allowed the selection of 15 strains that could tolerate NaCl concentrations between 175 and 500 mM. These were compared with 15 strains chosen from among the ones which did not tolerate high salinity. The diversity of strains was first assessed by rep-PCR amplification fingerprinting using BOXA1R and ERIC primers. An analysis of the PCR-amplified 16S rDNA gene digestion profiles using five endonucleases indicated the presence of different lineages among the taxa associated with P. juliflora nodules in the soils studied. Nucleotide sequencing of the small subunit rRNA gene and BLAST analysis showed that P. juliflora could host at least six bacterial species in this region and that the identity of those associated with high salt tolerance was clearly distinct from that of the salt-sensitive ones. Among the former, the first type displayed 99% similarity with different members of the genus Sinorhizobium, the second 97% similarity with species within the genus Rhizobium, while the third ribosomal type had 100% homology to Achromobacter xylosoxidans. Within the salt-sensitive isolates the prevailing type observed showed 98% similarity with Rhizobium multihospitium and R. tropici, a second type had 98% similarity to R. giardinii, and a further case displayed 97% colinearity with the Ensifer group including E. maghrebium and E. xericitae. All of the thirty strains encompassing these types re-nodulated P. juliflora in microbiologically controlled conditions and all of them were shown to possess a copy of the nodC gene. This is the first report detecting the betaproteobacterial genus Achromobacter as nodule-forming species for legumes. The observed variability in symbiont species and the abundance of nodulation-proficient strains is in line with the observation that the plant always appears to be nodulated and efficiently fixing nitrogen in spite of a wide range of soil and environmental conditions.

Survival and quality of halibut larvae ( Hippoglossus hippoglossus L.) in intensive farming: Possible impact of the intestinal bacterial community

The high mortality commonly observed during the early life stages of intensively reared halibut ( Hippoglossus hippoglossus L) is believed to be caused by e.g. opportunistic bacteria. However, the impact of particular bacterial species is poorly defined and still remains disputable. The study describes the bacterial diversity in the gastrointestinal tract of halibut larvae in a large number of incubators at a commercial production site. The overall success of larvae was found to be highly variable and analysis of the gut microbiota revealed high variation of the cultivable part as well as the bacterial community of surface sterilised larvae analysed by denaturing gradient gel electrophoresis (DGGE) of PCR amplified 16s rDNA products. Analysis of the bacterial community of unfed yolk sac larvae revealed higher diversity than previously reported, with Marinomonas, Marinobacter, Aeromonas and Shewanella dominating the community structure. There are indications that Marinomonas is found only in the overall most successful first feeding larvae of the period where the Vibrio group dominated the bacterial community together with Shewanella. Vibrio wodanis was identified as a part of the bacterial community of feeding larvae that yielded the poorest overall success of the period. α -Proteobacteria, not previously reported in halibut, were also found as a part of the bacterial community of first feeding larvae. The diverse bacterial community was only partly reflected in the cultivable part which, however, may reflect the dominating bacterial groups of the highly heterogeneous bacterial community of larvae in the production system as a whole. The bacterial community of the Artemia was found to be highly variable in different samples collected through the period. Only a small part of the different groups observed in the bacterial community of surface sterilised larvae was reflected in the cultivable part which was dominated by highly variable groups in different samples of Artemia. Also, the numbers of cultivable bacteria were found to positively correlate with jaw deformation of unfed yolk sac larvae as well as incomplete metamorphosis of feeding larvae.

Application of 16S rDNA-DGGE and Plate Culture to Characterization of Bacterial Communities Associated with the Sawfly, Acantholyda erythrocephala (Hymenoptera, Pamphiliidae)

Culture-based analysis was employed in parallel with PCR amplification of 16S rDNA, coupled with denaturing gradient gel electrophoresis (DGGE), to profile bacterial species associated with different developmental stages of the pine false webworm (PFW), Acantholyda erythrocephala, a sawfly pest responsible for incidents of severe defoliation in commercially important tree plantations in North America. Culture-based analysis revealed that Pseudomonas spp. along with Bacillus sphaericus and Arthrobacter sp. were the predominant components of the microflora of the internal organs and identified life-stage-specific associations including Photorhabdus temperata with egg and larval samples and a Janthinobacterium sp. with eonymphs. PCR-DGGE confirmed the predominance of Pseudomonas spp. and B. sphaericus in the majority of samples but did not detect Arthrobacter sp., P. temperate, or Janthinobacterium sp. In contrast, DGGE revealed the presence of a Chryseobacterium sp. as the predominant component of the PFW micoflora at all life stages, with the exception of adults. This species had been infrequently cultured, at low levels, from a limited number of samples and the existence of a possible relationship between this bacterium and the PFW had gone unnoticed using the culture-based approach. Our findings highlight the advantages of applying a dual approach to the study of microbe-insect associations and demonstrate that the benefits of one system can be used to overcome some of the limitations of the other.

Physical, chemical and microbiological properties of an Andisol as related to land use and tillage practice

The effects of land use and management practice on soil physical, chemical and microbiological properties may provide essential information for assessing sustainability and environmental impact. This study compared the effects of 41 years of no-tillage (NT) with continuous apple orchard, with those of conventional tillage (CT) with wheat–soybean rotation and another of puddling (PD) with continuous rice on the characteristics of a pumice Andisol in a temperate region of northern Japan. Higher values for bulk density, penetration resistance, pH, C/N ratio, exchangeable Na (X-Na), Fe, and Mn were observed for PD than NT and CT. On the other hand, organic matter, EC, N, exchangeable K (X-K), exchangeable Ca (X-Ca) and Cu were significantly higher for NT than CT and PD. Highest content of Zn was found in CT compared to other practices. The three-phase composition at pF 2.0 was significantly affected by land use and tillage practices. The solid phase and liquid phase were greater under PD than under NT and CT, while air phase was greater under CT than under NT and PD. Significantly higher values for saturated hydraulic conductivity was found in CT than NT and PD. Total phospholipid fatty acid (PLFA) and PLFA for bacteria, aerobes and cyanobacteria were remarkably higher in NT than CT and PD, regardless of depth. On the other hand, PLFA for methane-oxidizing bacteria, sulfate-reducing bacteria and mycorrhizae were significantly higher in CT than NT and PD. PLFA for fungi was significantly higher in surface (0–10 cm) soils than subsurface (10–20 cm) soils regardless of treatments. Highest bacterial and fungal diversity evaluated by DNA band number in DGGE analysis based on PCR amplification of 16S rDNA and 18S rDNA fragments, respectively, were observed in surface soil of PD. The result suggests a linkage between microbial community and tillage practices in temperate Andisol. This study also justifies the need of measuring soil characteristics based on soil microbial communities.

Biodiversity and phylogenetic analysis of culturable bacteria indigenous to Khewra salt mine of Pakistan and their industrial importance

Culturable bacterial biodiversity and industrial importance of the isolates indigenous to Khewra salt mine, Pakistan was assessed. PCR Amplification of 16S rDNA of isolates was carried out by using universal primers FD1 and rP1and products were sequenced commercially. These gene sequences were compared with other gene sequences in the GenBank databases to find the closely related sequences. The alignment of these sequences with sequences available from GenBank database was carried out to construct a phylogenetic tree for these bacteria. These genes were deposited to GenBank and accession numbers were obtained. Most of the isolates belonged to different species of genus Bacillus, sharing 92-99% 16S rDNA identity with the respective type strain. Other isolates had close similarities with Escherichia coli, Staphylococcus arlettae and Staphylococcus gallinarum with 97%, 98% and 99% 16S rDNA similarity respectively. The abilities of isolates to produce industrial enzymes (amylase, carboxymethylcellulase, xylanase, cellulase and protease) were checked. All isolates were tested against starch, carboxymethylcellulose (CMC), xylane, cellulose, and casein degradation in plate assays. BPT-5, 11,18,19 and 25 indicated the production of copious amounts of carbohydrates and protein degrading enzymes. Based on this study it can be concluded that Khewra salt mine is populated with diverse bacterial groups, which are potential source of industrial enzymes for commercial applications.

DNA Microarrays for Identifying Fishes

In many cases marine organisms and especially their diverse developmental stages are difficult to identify by morphological characters. DNA-based identification methods offer an analytically powerful addition or even an alternative. In this study, a DNA microarray has been developed to be able to investigate its potential as a tool for the identification of fish species from European seas based on mitochondrial 16S rDNA sequences. Eleven commercially important fish species were selected for a first prototype. Oligonucleotide probes were designed based on the 16S rDNA sequences obtained from 230 individuals of 27 fish species. In addition, more than 1200 sequences of 380 species served as sequence background against which the specificity of the probes was tested in silico. Single target hybridisations with Cy5-labelled, PCR-amplified 16S rDNA fragments from each of the 11 species on microarrays containing the complete set of probes confirmed their suitability. True-positive, fluorescence signals obtained were at least one order of magnitude stronger than false-positive cross-hybridisations. Single nontarget hybridisations resulted in cross-hybridisation signals at approximately 27% of the cases tested, but all of them were at least one order of magnitude lower than true-positive signals. This study demonstrates that the 16S rDNA gene is suitable for designing oligonucleotide probes, which can be used to differentiate 11 fish species. These data are a solid basis for the second step to create a “Fish Chip” for approximately 50 fish species relevant in marine environmental and fisheries research, as well as control of fisheries products.

Monitoring the opportunistic bacteria Pseudoalteromonas sp. LT-13 in a great scallop, Pecten maximus hatchery

PCR with specific primers against Pseudoalteromonas sp. LT-13, and Denaturing Gradient Gel Electrophoresis (DGGE) of PCR-amplified 16SrDNA, was used to monitor the bacterial community in samples of early stages of scallop larvae suffering high mortality. Samples were taken of larvae from different tanks, water in the tanks, eggs/sperm, inlet water and algal cultures. The DGGE profiles revealed one dominating band that was always present in samples of scallop larvae suffering high mortality. The band was sequenced and demonstrated high sequence homology to Pseudoalteromonas sp. LT-13, a bacterium previously isolated from the hatchery. Samples from different locations in the production unit were collected four times in an 11-month period and tested for the presence of LT13 by PCR. Seventy-four percent of the samples collected in April 2003 contained LT-13. In this period the mortality of the scallop larvae was no less than 96.8%. In two of the other samples the mortality was moderate (83.3–78.0%), and LT-13 was detected in 30 and 57% of these samples. High mortality (98.3%) also occurred in the February 2003 sampling period; however, LT-13 was detected in just 39% of the samples. These results indicate that the bacterium is not an obligate pathogen, but rather an opportunistic bacterium that may be used as an early indicator of the conditions in the hatchery.

Comparison of the bacterial communities established on the mycorrhizae formed on Pinus thunbergii root tips by eight species of fungi

The ectomycorrhizal (ECM) fungi associated with Pinus thunbergii seedlings grown on sand dune were identified by molecular method, and the diversity of bacteria associated with ECM and Extraradical mycelium were examined by Denaturing Gradient Gel Electrophoresis (DGGE) of PCR-amplified 16S rDNA. The mycorrhizal formation rate of 1-year old P. thunbergii seedlings was more than 95%. Cenococcum geophilum was the most dominant ECM fungus, followed by T01, RFLP-8, Russula spp., and Suillus sp. Bacterial community was most diverse with C. geophilum- and RFLP-8-mycorrhiza. Sequencing analysis showed that Burkholderia spp. and Bradyrhizobium spp. were on the surface of ECM short root of seven ECM. The fungi detected as extraradical mycelium using DGGE of 18S rDNA were Suillus bovinus and RFLP-8-mycorrhiza. Bacterial community on the extraradical mycelium was more diverse than those on ECM root tip. Burkholderia spp. and Bradyrhizobium spp. were found also on extraradical mycelium.

The characterization of lactic acid producing bacteria from the rumen of dairy cattle grazing on improved pasture supplemented with wheat and barley grain

To identify and characterize the major lactic acid bacteria in the rumen of dairy cattle grazing improved pasture of rye grass and white clover and receiving a maize silage and grain supplement with and without virginiamycin. Eighty-five bacterial isolates were obtained from the rumen of 16 Holstein-Friesian dairy cows. The isolates were initially grouped on the basis of their Gram morphology and by restriction fragment length polymorphism analysis of the PCR amplified 16S rDNA. A more definitive analysis was undertaken by comparing the 16S rDNA sequences. Many of the isolates were closely related to other previously characterized rumen bacteria, including Streptococcus bovis, Lactobacillus vitulinus, Butyrivibrio fibrisolvens, Prevotella bryantii and Selenomonas ruminantium. The in vitro production of L- and/or D-lactate was seen with all but five of the isolates examined, many of which were also resistant to virginiamycin. Supplementation of grain with virginiamycin may reduce the risk of acidosis but does not prevent its occurrence in dairy cattle grazing improved pasture. This study shows that lactic acid production is caused, not only by various thoroughly researched types of bacteria, but also by others previously identified in the rumen but not further characterized.

Biodiversity and phylogenetic analysis of culturable bacteria indigenous to Khewra salt mine of pakistan and their industrial importance

Culturable bacterial biodiversity and industrial importance of the isolates indigenous to Khewra salt mine, Pakistan was assessed. PCR Amplification of 16S rDNA of isolates was carried out by using universal primers FD1 and rP1and products were sequenced commercially. These gene sequences were compared with other gene sequences in the GenBank databases to find the closely related sequences. The alignment of these sequences with sequences available from GenBank database was carried out to construct a phylogenetic tree for these bacteria. These genes were deposited to GenBank and accession numbers were obtained. Most of the isolates belonged to different species of genus Bacillus, sharing 92-99% 16S rDNA identity with the respective type strain. Other isolates had close similarities with Escherichia coli, Staphylococcus arlettae and Staphylococcus gallinarum with 97%, 98% and 99% 16S rDNA similarity respectively. The abilities of isolates to produce industrial enzymes (amylase, carboxymethylcellulase, xylanase, cellulase and protease) were checked. All isolates were tested against starch, carboxymethylcellulose (CMC), xylane, cellulose, and casein degradation in plate assays. BPT-5, 11,18,19 and 25 indicated the production of copious amounts of carbohydrates and protein degrading enzymes. Based on this study it can be concluded that Khewra salt mine is populated with diverse bacterial groups, which are potential source of industrial enzymes for commercial applications.

PAHs contamination and bacterial communities in mangrove surface sediments of the Jiulong River Estuary, China

Sixteen sediment samples collected from eight transects in a mangrove swamp of the Jiulong River Estuary, Fujian, China were investigated for their content of polycyclic aromatic hydrocarbons (PAHs) and the biodegradation potential of the indigenous microorganisms. The bacterial community structures in the mangrove sediments and in enrichment cultures were also investigated. The results showed that the total PAHs concentration of mangrove sediments ranged from 280 to 1074 ng g −1 dry weight, that the PAHs composition pattern in the mangrove sediments was dominated by high molecular weight PAH components (4–6 rings), and that Benzo[ghi]perylene and Indeno[1,2,3-cd]pyrene were the most dominant at different stations. Abundant PAH-degrading bacteria were found in all the stations, the values of phenanthrene-degrading bacteria ranged from 5.85 × 10 4 to 7.80 × 10 5 CFU g −1 dry weight, fluoranthene-degrading bacteria ranged from 5.25 × 10 4 to 5.79 × 10 5 CFU g −1 dry weight, pyrene-degrading bacteria ranged from 3.10 × 10 4 to 6.97 × 10 5 CFU g −1 dry weight and the benzo(a)pyrene-degrading bacteria ranged from 5.25 × 10 4 to 7.26 × 10 5 CFU g −1 dry weight. DGGE analysis of PCR-amplified 16S rDNA gene fragments confirmed that there was a remarkable shift in the composition of the bacterial community due to the addition of the different model PAH compound phenanthrene (three ring PAH), fluoranthene(four ring PAH), pyrene(four ring PAH) and benzo(a)pyrene(five ring PAH) during enrichment batch culture. Eleven strains were obtained with different morphology and different degradation ability. The presence of common bands for microbial species in the cultures and in the native mangrove sediment DNA indicated that these strains could be potential in situ PAH-degraders.

Culture-independent characterization of the bacterial populations associated with cod (Gadus morhua L.) and live feed at an experimental hatchery facility using denaturing gradient gel electrophoresis

Atlantic Cod (Gadus morhua L.) represents an attractive species for cold water aquaculture. However, like many so called ‘alternative species’ early larval culture remains a challenge to commercial production. Indeed in comparison to other cultured finfish species, there exists limited information concerning the bacterial species associated with cod larviculture. Herein, we have employed PCR amplification of 16S rDNA coupled to Denaturing Gradient Gel Electrophoresis (DGGE) and sequencing of isolated amplicons, to profile the bacterial species associated with cod (both eggs and larvae) and the live feed (rotifer and Artemia) provided to the larvae during the first 50 days of development. A stable core microflora, comprising a total of six bacterial species with a putative Arcobacter sp. as the dominant component, was recorded in the majority of the rotifer samples. In contrast, Artemia showed a highly variable microflora composed of Vibrio spp, Alteromonas spp and Pseudoalteromonas spp. An association between Colwellia spp and cod eggs was demonstrated, although these species rapidly disappeared from the larval microflora which subsequently showed a reproducible (in 6 tanks over 2 years), and well-defined bacterial succession. Vibrio logei, V.fischeri and Listonella anguillarum predominated during the first 3 weeks of feeding but were gradually replaced by Flexibacter aurantiacus, a species of Sulfitobacter, and a putative Mycoplamsa sp. At 50 days post feeding the putative Mycoplasma sp. emerged as the dominant component of the microflora in all tanks. The culture-independent approach employed in this study generated a dynamic description of bacterial colonization of cod larvae and live feed and revealed the existence of previously undescribed bacterial associations with eggs and rotifers which would almost certainly have gone undetected using conventional culture based methods.

Microflora Assessments Using PCR–Denaturing Gradient Gel Electrophoresis of Ozone-Treated and Modified-Atmosphere-Packaged Farmed Cod Fillets

Denaturing gradient gel electrophoresis (DGGE) of a PCR-amplified 16S rDNA sequence was used to characterize changes in the microbial flora caused by ozone (O3) treatment of farmed cod (Gadus morhua). Portions of cod were produced under controlled conditions, bathed in fresh water supplemented with 2 ppm of O3 for 30 min, and packaged in modified atmosphere (MA: 60% CO2 and 40% N2) before 4°C storage. Control samples were packaged in MA or air, without prior O3 treatment. Samples were analyzed by PCR-DGGE to determine the predominant bacterial flora and to examine possible differences in the microbial community due to O3 treatment. The DGGE analysis during the storage period showed that the O3 treatment produced no significant difference in the microbial flora compared with the controls. Sequencing of 16S rDNA detected the specific spoilage bacteria Photobacterium phosphoreum, Pseudomonas spp., Shewanella baltica, and Shewanella putrefaciens as the predominant bacteria in all samples. PCR-DGGE results were supported by culture and sensory analyses used in predicting product shelf life. Aerobic plate count, H2S-producing bacteria, and psychrotrophic bacterial counts demonstrated no significant extension of the shelf life of MA-packaged, O3-treated cod fillets.

Characterisation of bacterial communities associated with early stages of intensively reared cod (Gadus morhua) using Denaturing Gradient Gel Electrophoresis (DGGE)

High mortality is often observed during the early life stages of intensively reared cod, and believed to be at least partly caused by opportunistic bacteria. The aim of the present study was to use Denaturing Gradient Gel Electrophoresis (DGGE) of PCR-amplified 16S rDNA to characterise the bacterial populations associated with early life stages of cod larvae in intensive hatcheries. At one hatchery the analysis was carried out during a period of approximately 4 weeks post hatch, and confirmed that cod larvae are associated with bacteria before and after the onset of exogenous feeding. A change in the number of bands and banding positions indicate that new bacteria or bacterial community were introduced between the samples taken at day 5 and 13, probably as a result of the onset of exogenous feeding. The post-feeding analyses were dominated by α-proteobacteria. An additional study from two other hatcheries of moribund fry, were dominated by Vibrio spp., including V. xuii and V. logei. It is concluded that DGGE is a suitable method for characterising bacterial communities in hatcheries. However, other genes than 16S rDNA might be more suitable for the discrimination of closely related taxa, particularly different Vibrio spp.

Wolbachia identified in a new crustacean host: an explanation of the prevalence of asexual reproduction in non-marine ostracods?

Wolbachia are cytoplasmic proteobacteria which induce severe modifications in the reproductive behaviour of their arthropod hosts. Although common in insects and terrestrial isopods, Wolbachia have seldom been found in other arthropod groups. Based on PCR amplification and DNA sequencing of Wolbachia 16S rDNA we report the first record of these endosymbiont bacteria in ostracods (Ostracoda), an exclusively aquatic crustacean group. The implications of this finding and the likely links between Wolbachia and ostracod reproductive life-history traits (parthenogenesis and sex-ratio distortion) are discussed.