Miquel Planas

Control of Vibrio alginolyticus in Artemia culture by treatment with bacterial probiotics

Abstract In order to determine the influence of six lactic acid bacterial (LAB) strains, lactic acid and major extracellular products (ECPs) of LAB on the bacterial load of Artemia culture, two experiments were carried out. In both experiments, the dominant bacterial species were identified as Vibrio alginolyticus. The treatment with Lactobacillus brevis (1×108 bacteria/ml) and lactic acid (0.1 g/l) reduced the load of this bacterium in the Artemia culture water. Biochemical and morphological characteristics of the V. alginolyticus strains isolated from Artemia culture were determined. For comparative purposes, another V. alginolyticus strain isolated from diseased turbot larvae was also included. The in vitro antibacterial activity of extracellular products (ECPs) from LAB, as well as lactic acid (0.1 and 0.05 g/l), was assessed against both V. alginolyticus strains. ECPs from LAB were able to inhibit the growth of V. alginolyticus, especially those from L. brevis. In the case of lactic acid treatments, no significant inhibition of V. alginolyticus growth was observed.

Use of multivariate analysis to assess the nutritional condition of fish larvae from nucleic acids and protein content

The nutritional condition of turbot larvae (Scophthalmus maximus) was assessed by a multivariate analysis with DNA, RNA, and protein content as input variables. Special attention was given to the time that feeding began and to the timing and duration of starvation. The combination of the principal components analysis and the stepwise discriminant analysis, both techniques of multivariate analysis, made it possible to allocate the larvae into groups that were defined and identified based on similarities in developmental stage and nutritional condition. The developmental stage was mostly determined by the input variables DNA and protein content, while nutritional condition was determined by the RNA content. In the period studied, the more developed larvae were less resistant to starvation. Furthermore, when initial feeding was delayed as little as 6 h, the variables analyzed were markedly changed, and the effect on the deprived larvae was found to be equivalent to a 3-day delay in development—when compared with the larvae fed immediately after mouth opening. Through this technique, new samples of larvae with unknown history might be classified into groups, using their DNA, RNA, and protein content as input values in the defined classification functions. Results were compared to those obtained using RNA/DNA and RNA/dry weight indices, and the multivariate method was considered to be more sensitive and to provide extra information about larval nutritional history and development.

Bacillus galliciensis sp. nov., isolated from faeces of wild seahorses (Hippocampus guttulatus)

A Gram-positive-staining, motile, rod-shaped, endospore-forming bacterium (BFLP-1( T)) was isolated from faeces of wild long-snouted seahorses ( Hippocampus guttulatus) captured in north-west Spain (Toralla, Galicia). Strain BFLP-1(T) grew at 10-30 degrees C and pH 5.5-9 (optimally at 20 degrees C and pH 7.2) and with 0-7 % (w/v) NaCl (optimally with 2 % NaCl). The G+C content of the DNA was 48.1 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain BFLP-1(T) was a member of the genus Bacillus and was most closely related to Bacillus herbersteinensis D-1,5a(T) (96.6 %), B. shackletonii LMG 18435(T) (96.0 %) and B. isabeliae CVS-8(T) (95.9 %). Chemotaxonomic data (peptidoglycan type, meso-diaminopimelic acid; major menaquinone, MK-7; predominant fatty acids, anteiso-C(15 : 0 ), anteiso-C(17 : 0) and C(16 : 1 )omega11c; major polar lipids, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an unknown aminoglycophospholipid) supported the affiliation of strain BFLP-1(T) to the genus Bacillus . Comparative analysis of 16S rRNA gene sequences and chemotaxonomic and phenotypic features indicated that strain BFLP-1(T) represents a novel species within the genus Bacillus, for which the name Bacillus galliciensis sp. nov. is proposed. The type strain is BFLP-1( T) (=DSM 21539(T) =LMG 24668(T)).

Vibrio hippocampi sp. nov., a new species isolated from wild seahorses (Hippocampus guttulatus).

A Gram-negative, facultatively anaerobic, motile and slightly curved rod-shaped bacterium (BFLP-4(T)) was isolated from the faeces of wild seahorses (Hippocampus guttulatus) captured in northwest Spain (Toralla, Galicia). Strain BFLP-4(T) grew at 10-35 degrees C and pH 5-9 (optimally at 20 degrees C and pH 7.2) and at salt concentrations in the range 0-7% w/v NaCl. The G+C content of the DNA was 49.3 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain BFLP-4(T) was a member of the genus Vibrio, being most closely related to Vibrio ichthyoenteri (97.1%), Vibrio mediterranei (96.7%), Vibrio scophthalmi (96.7%) and Vibrio sinaloensis (96.6%). A phylogenetic analysis based on recA gene sequences also supported the affiliation of strain BFLP-4(T) to the genus Vibrio. Strain BFLP-4(T) could be readily differentiated from other closely related species by several phenotypic properties and fatty acid profiles. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain BFLP-4(T) represents a novel species within the genus Vibrio, for which the name Vibrio hippocampi sp. nov. is proposed. The type strain is BFLP-4(T) (=DSM 22717(T)=LMG 25354(T)).

Seahorse Aquaculture, Biology and Conservation: Knowledge Gaps and Research Opportunities

ABSTRACT Seahorses are currently experiencing an unprecedented level of anthropogenic pressure promoted by habitat destruction and increasing fishing effort to supply premium markets. This study provides an overview of the scientific literature on seahorses in the 21st century and critically discusses five major knowledge gaps and research opportunities to advance the state of the art on this research field. The average number of publications per year increased from 10 (2001–2002) to ∼40 (2001–2015), the majority addressing issues on seahorse ecology, biology, and aquaculture, with the most studied species being Hippocampus kuda, H. guttulatus, H. reidi, H. abdominalis, H. erectus, H. hippocampus, and H. trimaculatus. This study explores the opportunity of using seahorses as flagship species to foster mangrove conservation and decrease trawling fisheries. It also suggests that further scientific studies are needed to better understand and manage the populations of the most heavily traded seahorse species, as well as the need to monitor their vulnerability to emerging pollutants and climate change. Sustainable seahorse aquaculture can play an important role in seahorse conservation, as well as in the development of reliable traceability tools to fight the illegal trade of these highly priced organisms.

Simple techniques for labelling prey and gut content analysis in short-term feeding experiments with fish larvae

Techniques were developed for labelling prey, and for gut content analysis in fish larvae. Labelling consisted of a short feeding period of the prey (rotifer and Artemia metanauplii) on black-drawing ink before supplying them to the larvae. Prey labelling was rapid and remained stable for at least 3 h when transferred into seawater. In addition, a method was tested to facilitate the observation of larval gut contents. The method, based on Dawsons technique, involved fixation, removal of lipids and clearance of turbot (Psetta maxima) larvae, which become transparent and ready for gut content analysis. This treatment did not interfere with the black ink label of the ingested prey.

Conservation Genetics of Threatened Hippocampus guttulatus in Vulnerable Habitats in NW Spain: Temporal and Spatial Stability of Wild Populations with Flexible Polygamous Mating System in Captivity

This study was focused on conservation genetics of threatened Hippocampus guttulatus on the Atlantic coast of NW Iberian Peninsula. Information about spatial structure and temporal stability of wild populations was obtained based on microsatellite markers, and used for monitoring a captive breeding program firstly initiated in this zone at the facilities of the Institute of Marine Research (Vigo, Spain). No significant major genetic structure was observed regarding the biogeographical barrier of Cape Finisterre. However, two management units under continuous gene flow are proposed based on the allelic differentiation between South-Atlantic and Cantabrian subpopulations, with small to moderate contemporary effective size based on single-sample methods. Temporal stability was observed in South-Atlantic population samples of H. guttulatus for the six-year period studied, suggesting large enough effective population size to buffer the effects of genetic drift within the time frame of three generations. Genetic analysis of wild breeders and offspring in captivity since 2009 allowed us to monitor the breeding program founded in 2006 in NW Spain for this species. Similar genetic diversity in the renewed and founder broodstock, regarding the wild population of origin, supports suitable renewal and rearing processes to maintain genetic variation in captivity. Genetic parentage proved single-brood monogamy in the wild and in captivity, but flexible short- and long-term mating system under captive conditions, from strict monogamy to polygamy within and/or among breeding seasons. Family analysis showed high reproductive success in captivity under genetic management assisted by molecular relatedness estimates to avoid inbreeding. This study provides genetic information about H. guttulatus in the wild and captivity within an uncovered geographical range for this data deficient species, to be taken into account for management and conservation purposes.

Vibrio inhibens sp. nov., a novel bacterium with inhibitory activity against Vibrio species.

Strain BFLP-10T, isolated from faeces of wild long-snouted seahorses (Hippocampus guttulatus), is a Gram-negative, motile and facultatively anaerobic rod. This bacterium produces inhibitory activity against Vibrio species. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain BFLP-10T was a member of the genus Vibrio and was most closely related to Vibrio owensii (99%), Vibrio communis (98.9%), Vibrio sagamiensis (98.9%) and Vibrio rotiferianus (98.4%). However, multilocus sequence analysis using gyrB, pyrH, recA and topA genes revealed low levels of sequence similarity (<91.2%) with these closely related species. In addition, strain BFLP-10T could be readily differentiated from other closely related species by several phenotypic properties and fatty acid profiles. The G+C content of the DNA was 45.6 mol%. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain BFLP-10T represents a novel species within the genus Vibrio, for which the name Vibrio inhibens sp. nov. is proposed. The type strain is BFLP-10T (=CECT 7692T=DSM 23440T).

Dietary composition of endangered seahorses determined by stable isotope analysis

The lack of integrated measures for assessing the feeding ecology of seahorses may restrict the effectiveness of conservation actions on wild populations of worldwide threatened seahorse species. Identifying dietary sources will allow researchers to determine their degree of vulnerability to environmental changes, redefine their conservation status and apply appropriate management strategies. The resource use of the seahorse Hippocampus guttulatus inhabiting coastal waters of Galicia (north-western Iberian Peninsula) was assessed for three populations and 2 years using stable isotope mixing models. The Bayesian mixing model (MixSIAR) estimated the relative contributions of the dietary sources to the seahorse diet and revealed that Caprellidea were the primary source, followed by Gammaridea and Caridea. Mysidae and Annelida represented the less dominant prey. This prey preference can be explained by the foraging behaviour of seahorses. Different contributions of Gammaridea and Caridea to the diet were found among sites, indicating different habitat characteristics and hence different habitat use by seahorses within each site. In addition, differences were encountered among sexes. Caprellidea was the dominant prey for females, whereas Gammaridea was the dominant prey for males. The findings of the present study will contribute to the knowledge of feeding patterns of H. guttulatus, providing relevant data for conservation of this endangered species.

Histological development of the long-snouted seahorse Hippocampus guttulatus during ontogeny

The objective of the present study was to describe histological development of the European long-snouted seahorse Hippocampus guttulatus, to increase understanding of the biology and physiology of the species. Most vital organs were present in juveniles by the time of their release from the males pouch. Digestive tract specialization occurred at 89 effective day-degrees (D°eff ), corresponding to 15 days post partum (dpp), with development of the first intestinal loop and mucosal folding. At 118 D°eff (20 dpp), lipids were being mobilized from the liver and oocytes attained the perinuclear stage. The fovea emerged at 177 D°eff (30 dpp), contemporaneous with the shift from pelagic to benthic behaviour in juveniles. At this stage, the most interesting feature was the formation of the second intestinal loop. Male gonads were never observed during the study (from 0 to 354 D°eff ; 0-60 dpp), but the first oogonia were present at 30 D°eff (5 dpp). In 354 D°eff (60 dpp) juveniles, oocytes were observed in a cortical alveoli stage, indicating maturity. Low digestive efficiency was observed at early stages, which was due to a poorly developed gastrointestinal tract and an immature digestive tract prior to 89 D°eff . The present study demonstrates that approximately 89 and 177 D°eff represent two important transitional stages in the early development of H. guttulatus. At a temperature of approximately 19 ± 1°C and an age of 1 month (177 D°eff ), main organs were fully functional, suggesting that the adult phenotype was largely established by that age, with females becoming mature at the age of 2 months (354 D°eff ).