Dietary beauvericin and enniatin B exposure cause different adverse health effects in farmed Atlantic salmon.

The extensive use of plant ingredients in novel aquafeeds have introduced mycotoxins to the farming of seafood. The emerging enniatin B (ENNB) and beauvericin (BEA) mycotoxins have been found in the novel aquafeeds and farmed fish. Little is known about the potential toxicity of ENNs and BEA in farmed fish and their feed-to-organ transfer. Atlantic salmon (Salmo salar) pre-smolt (75.3 ± 8.10 g) were fed four graded levels of spiked chemical pure ENNB or BEA feeds for three months, in triplicate tanks. Organismal adverse health end-point assessment included intestinal function (protein digestibility), disturbed hematology (red blood cell formation), bone formation (spinal deformity), overall energy use (feed utilization), and lipid oxidative status (vitamin E). Both dietary BEA and ENNB had a low (<∼0.01%) transfer to organs (kidney > liver > brain > muscle), with a higher transfer for ENNB compared to BEA. BEA caused a growth reduction combined with a decreased protein digestion and feed conversion rate- ENNB caused a stunted growth, unrelated to feed utilization capacity. In addition, ENNB caused anemia while BEA gave an oxidative stress response. Lower bench-mark dose regression assessment showed that high background levels of ENNB in commercial salmon feed could pose a risk for animal health, but not in the case of BEA.

Micro-anatomical characterization of vertebral curvatures in Senegalese sole Solea senegalensis.

The micro-anatomical changes associated with lordotic and kyphotic vertebral curvatures (VC) in juvenile and adult Senegalese sole Solea senegalensis are described. In addition, it is demonstrated that the tissue and cellular structures of individual vertebrae can be severely affected. Two main conformations were found in deformed juvenile specimens: flattened vertebrae with dorso-ventral compression and trapezoidal vertebrae forming concave and convex sides under compressive and tensile stresses. Histological analyses revealed the occurrence of an ectopic cartilaginous tissue within the acellular bone, both in juveniles and adults, possibly to cope with altered mechanical stress in deformed vertebrae. The results suggest that the alteration in loading to which curved vertebral columns are subjected might trigger vertebral reshaping and differentiation of cells towards this ectopic tissue. In addition, mesenchymal cells appear to play an important role in its formation. It is here proposed that the acellular bone of S. senegalensis is capable of adaptively responding to altered loading regimes at the structural level by reshaping vertebrae and at the micro-anatomical level by recruiting chondrocyte-like cells to areas of altered mechanical stress.

Larval and juvenile development of dusky grouper Epinephelus marginatus reared in mesocosms.

The larval development of the dusky grouper Epinephelus marginatus up to the benthic juvenile stage is described in detail to establish a reference for their larval identification. Development is described in terms of ontogenetic changes in morphology, growth, pigmentation, fin structure and skeletal structure. Larvae were reared in mesocosms at a mean temperature of 24·3° C, salinity of 36·5, dissolved oxygen of 6·4 mg l(-1) and pH of 8·2. Newly hatched larvae had an estimated total length (LT ) of 2·3 mm. On the second day post hatching the yolk was almost fully absorbed with traces of the oil globule still present, the eyes were already pigmented and mouth and gut functional. At this stage the cranial skeletal elements for feeding and breathing (mouth and gills) and the pectoral-fin support were already present. About 50% of the observed larvae had food in their guts. Pigmentation was very characteristic, consisting of two large chromatophores visible on the edge of the primordial fin, close to the midpoint of the post-anal region of the body and over the midgut and hindgut and post-anal portion of the body. At 2·9 mm LT the emergence of the second dorsal-fin spine, characteristic of the Epinephilinae, was clearly visible. The pre-flexion stage started in larva of 3·2 mm LT . At 5·5 mm LT the larvae possessed posterior preopercular angle spines, and the dorsal and pelvic spines presented serrated edges and were pigmented. The water surface-tension-related death of the yolk sac and pre-flexion larvae described in the rearing of several other grouper species did not occur during E. marginatus culture. Notochord flexion, with initial ossification of the caudal-fin supporting elements, started at 6·6 mm LT . At this stage the major melanophores, preopercular, dorsal and pelvic spines and mandibular teeth were already present. Transformation of larvae into juveniles occurred when larvae averaged 13·8 mm LT . Juveniles with a mean LT of 20·1 mm started to settle and most of them were benthic with a mean LT of 26·8 mm.

Identification of a promoter element within the zebrafish colXalpha1 gene responsive to runx2 isoforms Osf2/Cbfa1 and til-1 but not to pebp2alphaA2.

Type X collagen is a short chain collagen specifically expressed by hypertrophic chondrocytes during endochondral ossification. We report here the functional analysis of the zebrafish (Danio rerio) collagen Xalpha1 gene (colXalpha1) promoter with the identification of a region responsive to two isoforms of the runt domain transcription factor runx2. Furthermore, we provide evidence for the presence of dual promoter usage in zebrafish, a finding that should be important to further understanding of the regulation of its restricted tissue distribution and spatial-temporal expression during early development. The zebrafish colXalpha1 gene structure is comparable to that recently identified by comparative genomics in takifugu and shows homology with corresponding mammalian genes, indicating that its general architecture has been maintained throughout vertebrate evolution. Our data suggest that, as in mammals, runx2 plays a role in the development of the osteogenic lineage, supporting zebrafish as a model for studies of bone and cartilage development.

Osteocalcin and matrix GLA protein in developing teleost teeth: identification of sites of mRNA and protein accumulation at single cell resolution.

In this study, the tissue distribution and accumulation of osteocalcin or bone Gla protein (BGP) and matrix Gla protein (MGP) were determined during tooth development in a teleost fish, Argyrosomus regius. In this species, the presence of BGP and MGP mRNA in teeth was revealed by in situ hybridization. mRNA for BGP was detected in the odontoblasts as well as in its cytoplasmic processes emerging through dentinal tubules, while mRNA for MGP was expressed in the enamel portion within the apical portion of the elongated cell bodies of enameloblasts, adjacent to the root of the teeth as well as in cells within the pulpal space. Immunolocalization of BGP and MGP demonstrated that these proteins accumulate mainly in the mineralized dentin or in enameloblastic processes, confirming in situ hybridization results. In this study, we examined for the first time the localization of both BGP and MGP gene expression and protein accumulation within the different regions of the vertebrate tooth. We clearly demonstrated that although the overall pattern of BGP and MGP gene expression and protein accumulation in A. regius teeth was in general agreement to what is known for other vertebrates such as rats or rodents, our study provided novel information and highlighted some species-differences between fish and higher vertebrates.

Characterization of osteocalcin (BGP) and matrix Gla protein (MGP) fish specific antibodies: validation for immunodetection studies in lower vertebrates.

In fish species the basic mechanisms of bone development and bone remodeling are not fully understood. The classification of bone tissue in teleosts as cellular or acellular and the presence of transitional states between bone and cartilage and the finding of different types of cartilage in teleosts not previously recognized in higher vertebrates emphasizes the need for a study on the accumulation of the Gla-containing proteins MGP and BGP at the cellular level. In the present study, polyclonal antibodies developed against BGP and MGP from A. regius (a local marine teleost fish) and against MGP from G. galeus (a Pacific Ocean shark), were tested by Western blot for their specificity against BGP and MGP from several other species of teleost fish and shark. For this purpose we extracted and purified both proteins from various marine and freshwater teleosts, identified them by N-terminal amino acid sequence analysis and confirmed the presence of gamma-carboxylation in the proteins with the use of a stain specific for Gla residues. Each antibody recognized either BGP or MGP with no cross-reaction between proteins detected. All purified fish BGPs and MGPs tested were shown to be specifically recognized, thus validating the use of these antibodies for further studies.

Matrix Gla protein gene expression and protein accumulation colocalize with cartilage distribution during development of the teleost fish Sparus aurata.

Matrix Gla protein (MGP) is a member of the family of extracellular mineral-binding Gla proteins, expressed in several tissues with high accumulation in bone and cartilage. Although the precise molecular mechanism of action of this protein remains unknown, all available evidence indicates that MGP plays a role as an inhibitor of mineralization. We investigated the sites of gene expression and protein accumulation of MGP throughout development of the bony fish Sparus aurata, by in situ hybridization, Northern and RT-PCR Southern hybridization, and immunohistochemistry. The results obtained were compared with the patterns of developmental appearance of cartilaginous and mineralized structures in this species, identified by histological techniques and by detection of mRNA presence and protein accumulation of osteocalcin (Bone Gla protein), a marker for osteoblasts known to accumulate in bone mineralized extracellular matrix. The expression of MGP mRNA was first detected at 2 days posthatching (dph) by Northern analysis, RT-PCR amplification, and in situ hybridization, and thereafter continuously detected at various levels of intensity, until 130 dph. In situ hybridization analysis performed in parallel with immunohistochemistry indicated that until ca. 45 dph, the MGP gene was highly expressed in a number of different tissues including skull, jaw, neural and hemal arches, and heart and the protein accumulated in cartilaginous tissues. At 85 dph, a stage when most skeletal structures are mineralized, MGP gene expression and protein accumulation were restricted to the remaining cartilaginous structures, whereas osteocalcin gene expression and protein accumulation were localized in most mineralized structures. MGP gene expression was also detected in heart and kidney, although in situ hybridization only detected MGP mRNA in heart, located in the arterial bulbus and not in the cardiac muscle. Our results are in agreement with those recently described for MGP localization in adult tissues of another teleost fish, as well as available data from higher vertebrates, strengthening the hypothesis of a conserved function for MGP from teleost fish to human, a period of more than 200 million years of evolution. In addition, Sparus aurata, a marine teleost fish routinely grown in captivity, appears to be a good model to further analyze MGP gene expression and regulation.

Detection of mineralized structures in early stages of development of marine Teleostei using a modified alcian blue-alizarin red double staining technique for bone and cartilage.

We have developed a procedure for staining cartilage and bone in fish larvae as small as 2 mm (notochord length), for which standard alcian blue/alizarin red procedures did not give positive and/or consistent results. Small calcified structures only 100-200 microns in length can be clearly visualized. The method is suitable for both ontogenic studies during early stages of skeletal development in most marine fishes (e.g., Sparus aurata L., Solea senegalensis Kaup), whose larvae at hatching are often only a few millimeters long and for detecting skeletal abnormalities in small larvae. This procedure can also be used for specimens that have been preserved in 100% ethanol for up to two years.