Microbiota of Atlantic cod (Gadus morhua L.) rearing systems at pre- and posthatch stages and the effect of different treatments.

AIMS: To study the effect of ova disinfection, antibiotic and microbial treatments on the dominant cultivable cod rearing microbiota at pre- and posthatch stages, determining some virulence-related phenotypic traits among bacterial isolates and their relation to larval survival. METHODS AND RESULTS: Sampling of rearing systems (rearing water, ova, larvae, feeds and supplement) for analysis of cultivable microbiota took place at early stages in 2004 and 2005. Cultivation, phenotypic and genotypic (16S rRNA gene) analyses were performed. The production of putative virulence factors (PVFs), including haemolysin, siderophores and quorum-sensing signals, by bacterial isolates was investigated and related to larval survival. The study was performed during two spawning seasons, evaluating current hatchery practices (ova disinfection and antibiotic treatment of unhealthy larvae) and specific putative probiotics applied to ova and larvae or rotifers. A diversified microbiota (75 operational taxonomic units, OTUs) was observed in cod rearing systems influenced by the feeds and treatments, with prevailing γ-Proteobacteria prior to hatching towards a multiphyla microbiota posthatch. Phenotypic tests demonstrated the heterogeneity within some OTUs. Multivariate analysis of survival data in larval silos and the corresponding larval microbiota was used to divide the genotypic groups into beneficial/harmless and detrimental/opportunistic clusters. PVFs were common among the proposed detrimental/opportunistic OTUs. CONCLUSIONS: The results clearly demonstrate the influence of exogeneous feeding and treatments on larval gastrointestinal microbiota and the role of bacteria in larval survival. SIGNIFICANCE AND IMPACT OF THE STUDY: Increased understanding of the microbiota in rearing systems may contribute to successful implementation of microbial management in cod aquaculture.

Effects of bacterial treatment at early stages of Atlantic cod (Gadus morhua L.) on larval survival and development.

AIMS: To assess the effects of bacterial treatment at the earliest stages of cod rearing on the microbial load, larval development and performance, testing three bacterial strains (Carnobacterium divergens V41, Arthrobacter sp. and Enterococcus sp.) in vivo that were previously shown to have inhibitory potential towards fish pathogens in vitro. METHODS AND RESULTS: A bacterial mixture was added eight times to the rearing water from the prehatch to the mid-larval stage (a 38-day period). Microbiological analysis of ova, larvae and rearing water was performed regularly. Larval performance and development were evaluated by survival rate, hypersalinity tolerance and physiological measurements. Different larval survival rates were observed within and between treatments, and possibly explained by variations in larval microflora and established probionts. Larvae from one silo, which had been bathed in the bacterial suspension, showed the highest survival rate (42.1%), lowest Vibrio levels, and were significantly heavier (19.3%) and more stress tolerant than control larvae (P < 0.01). This coincided with the intestinal establishment of two of the tested bacteria. CONCLUSIONS: Arthrobacter and Enterococcus strains added regularly to the rearing water from the postfertilized egg stage can become established in larval gastrointestinal tract. The Enterococcus strain was associated with increased larval growth, performance and microflora control, indicating its probiotic nature. SIGNIFICANCE AND IMPACT OF THE STUDY: Regular application of autochthonous probionts may promote larval welfare, development and stress tolerance at early stages, hence increasing production yield in intensive cod larviculture.

Discovery and description of giant submarine smectite cones on the seafloor in Eyjafjordur, northern Iceland, and a novel thermal microbial habitat.

With the submersible JAGO and by scuba diving we discovered three remarkable geothermal cones, rising 33, 25, and 45 m from the seafloor at a depth of 65 m in Eyjafjordur, northern Iceland. The greatest geothermal activity was on the highest cone, which discharged up to 50 liters of freshwater per s at 72 degrees C and pH 10.0. The cones were built up from precipitated smectite, formed by mixing of the hot SiO2-rich geothermal fluid with the cold Mg-rich seawater. By connecting a rubber hose to one outflow, about 240 liters of pure geothermal fluids was concentrated through a 0.2-microm-pore-size filter. Among 50 thermophilic isolates, we found members of Bacillus and Thermonema and a new unidentified low-G+C gram-positive member of the Bacteria as well as one member of the Archaea, Desulfurococcus mobilis. Analysis of small-subunit rRNA genes PCR amplified and cloned directly from environmental DNA showed that 41 out of 45 Bacteria sequences belonged to members of the Aquificales, whereas all of the 10 Archaea sequences belonged to the Korarchaeota. The physiological characteristics of isolates from different parts of the cones indicate a completely freshwater habitat, supporting the possibility of subterranean transmittance of terrestrial organisms.

Genetic diversity analysis of Rhodothermus reflects geographical origin of the isolates.

The genetic diversity and relationships of 81 Rhodothermus isolates from different geothermal environments in Iceland were examined by analysis of electrophoretically demonstrable allelic variation of 13 genes encoding enzymes. All the enzymes were polymorphic. A total of 71 distinctive multilocus genotypes (electrophoretic types, ETs) were identified. The mean genetic diversity per locus (H1) was 0.586. The relatively high genetic variance observed within Rhodothermus isolates from different locations is most likely the result of genetic changes occurring independently in the locations studied. A high Gst value (0.284) indicates that a considerable part of the variance observed is due to differences between locations. Cluster analysis revealed two major groups of ET clusters diverging at a genetic distance of 0.75, reflecting strongly the geographic origin of isolates. Estimation of the association index (I(A)) indicates that Rhodothermus marinus is a clonal species in which recombination events occur rarely. Partial or whole sequencing of the 16S rRNA genes of Rhodothermus isolates grouping at genetic distance of 0.40 confirmed that all the isolates belonged to the species Rhodothermus marinus. The results of this study confirm that, despite phylogenetic and phenotypic similarity, genetic diversity within Rhodothermus marinus is quite high.

Influence of sulfide and temperature on species composition and community structure of hot spring microbial mats.

In solfataric fields in southwestern Iceland, neutral and sulfide-rich hot springs are characterized by thick bacterial mats at 60 to 80 degrees C that are white or yellow from precipitated sulfur (sulfur mats). In low-sulfide hot springs in the same area, grey or pink streamers are formed at 80 to 90 degrees C, and a Chloroflexus mat is formed at 65 to 70 degrees C. We have studied the microbial diversity of one sulfur mat (high-sulfide) hot spring and one Chloroflexus mat (low-sulfide) hot spring by cloning and sequencing of small-subunit rRNA genes obtained by PCR amplification from mat DNA. Using 98% sequence identity as a cutoff value, a total of 14 bacterial operational taxonomic units (OTUs) and 5 archaeal OTUs were detected in the sulfur mat; 18 bacterial OTUs were detected in the Chloroflexus mat. Although representatives of novel divisions were found, the majority of the sequences were >95% related to currently known sequences. The molecular diversity analysis showed that Chloroflexus was the dominant mat organism in the low-sulfide spring (1 mg liter(-1)) below 70 degrees C, whereas Aquificales were dominant in the high-sulfide spring (12 mg liter(-1)) at the same temperature. Comparison of the present data to published data indicated that there is a relationship between mat type and composition of Aquificales on the one hand and temperature and sulfide concentration on the other hand.

Silicibacter lacuscaerulensis gen. nov., sp. nov., a mesophilic moderately halophilic bacterium characteristic of the Blue Lagoon geothermal lake in Iceland.

Mesophilic, moderately halophilic bacteria were isolated from a silica-rich geothermal lake, the Blue Lagoon in Iceland. The isolates are strictly aerobic, but reduce nitrate to nitrite, and are oxidase- and catalase-positive. The nonsporeforming and nonmotile Gram negative rods are 0.6-0.8 microm in diameter and variable in length (9-18 microm), and contain gas vacuoles. The GC content in their DNA is 66.15%. The minimum, optimum, and maximum temperatures for growth are 22 degrees C, 45 degrees C, and 50 degrees C, respectively. The isolates do not grow without added salt in the medium and can grow at up to 7% NaCl (w/v). The optimal salinity for growth is 3.5%-4% NaCl. The pH range for growth is 6.5-8.5, with the optimal pH at 7.0. At optimal conditions the bacterium has a doubling time of 80 min. The main cytochrome is a membrane-bound cytochrome c with an alpha-peak at 549nm. Sequencing of 16S rRNA from the type strain ITI-1157 revealed it to be a proteobacterium of the alpha-subclass with the closest relatives being Roseobacter litoralis and Paracoccuss kocuri. The new isolates do not contain bacteriochlorophyll a and are considered to represent a new genus and a new species, Silicibacter lacuscaerulensis.