Ontogenic effects of early feeding of sea bass (Dicentrarchus labrax) larvae with a range of dietary n-3 HUFA levels on the functioning of PUFA desaturation pathways

Four replicated groups of sea bass (Dicentrarchus labrax) larvae were fed diets containing an extra-high level of highly unsaturated fatty acids (HUFA) (XH; 3·7% EPA + DHA), a high level of HUFA (HH; 1·7 %), a low level of HUFA (LH; 0·7%) or an extra-low level of HUFA (XLH; 0·5%) from day 6 to day 45 (experiment 1; XH1, HH1, LH1, XLH1). After a subsequent 1-month period feeding a commercial diet (2·7% EPA + DHA), the capacity of the four initial groups to adapt to an n-3 HUFA-restricted diet (0·3% EPA + DHA; R-groups: XH2R, HH2R, LH2R, XLH2R) was tested for 35 d. Larval dietary treatments had no effect on larval and juvenile survival rates. The wet weight of day 45 larvae was higher in XH1 and HH1 (P < 0·001), but the R-juvenile mass gains were similar in all treatments. D-6-desaturase (D6D) mRNA level was higher in LH1 and XLH1 at day 45 (

Coordinated gene expression during gilthead sea bream skeletogenesis and its disruption by nutritional hypervitaminosis A

Background: Vitamin A (VA) has a key role in vertebrate morphogenesis, determining body patterning and growth through the control of cell proliferation and differentiation processes. VA regulates primary molecular pathways of those processes by the binding of its active metabolite (retinoic acid) to two types of specific nuclear receptors: retinoic acid receptors (RARs) and retinoid X receptors (RXRs), which promote transcription of downstream target genes. This process is well known in most of higher vertebrates; however, scarce information is available regarding fishes. Therefore, in order to gain further knowledge of fish larval development and its disruption by nutritional VA imbalance, the relative expression of some RARs and RXRs, as well as several genes involved in morpho- and skeletogenesis such as peroxisome proliferator-activated receptors (PPARA, PPARB and PPARG); retinol-binding protein (RBP); insulin-like growth factors I and II (IGF1 and IGF2, respectively); bone morphogenetic protein 2 (Bmp2); transforming growth factor beta-1 (TGFB1); and genes encoding different extracellular matrix (ECM) proteins such as matrix Gla protein (mgp), osteocalcin (bglap), osteopontin (SPP1), secreted protein acidic and rich in cysteine (SPARC) and type I collagen alpha 1 chain (COL1A1) have been studied in gilthead sea bream. Results: During gilthead sea bream larval development, specific expression profiles for each gene were tightly regulated during fish morphogenesis and correlated with specific morphogenetic events and tissue development. Dietary hypervitaminosis A during early larval development disrupted the normal gene expression profile for genes involved in RA signalling (RARA), VA homeostasis (RBP) and several genes encoding ECM proteins that are linked to skeletogenesis, such as bglap and mgp. Conclusions: Present data reflects the specific gene expression patterns of several genes involved in larval fish RA signalling and skeletogenesis; and how specific gene disruption induced by a nutritional VA imbalance underlie the skeletal deformities. Our results are of basic interest for fish VA signalling and point out some of the potential molecular players involved in fish skeletogenesis. Increased incidences of skeletal deformities in gilthead sea bream fed with hypervitaminosis A were the likely ultimate consequence of specific gene expression disruption at critical development stages

Salinity reduction benefits European eel larvae: Insights at the morphological and molecular level

European eel (Anguilla anguilla) is a euryhaline species, that has adapted to cope with both, hyper- and hypo-osmotic environments. This study investigates the effect of salinity, from a morphological and molecular point of view on European eel larvae reared from 0 to 12 days post hatch (dph). Offspring reared in 36 practical salinity units (psu; control), were compared with larvae reared in six scenarios, where salinity was decreased on 0 or 3 dph and in rates of 1, 2 or 4 psu/day, towards iso-osmotic conditions. Results showed that several genes relating to osmoregulation (nkcc2α, nkcc2β, aqp1dup, aqpe), stress response (hsp70, hsp90), and thyroid metabolism (thrαA, thrαB, thrβB, dio1, dio2, dio3) were differentially expressed throughout larval development, while nkcc1α, nkcc2β, aqp3, aqp1dup, aqpe, hsp90, thrαA and dio3 showed lower expression in response to the salinity reduction. Moreover, larvae were able to keep energy metabolism related gene expression (atp6, cox1) at stable levels, irrespective of the salinity reduction. As such, when reducing salinity, an energy surplus associated to reduced osmoregulation demands and stress (lower nkcc, aqp and hsp expression), likely facilitated the observed increased survival, improved biometry and enhanced growth efficiency. Additionally, the salinity reduction decreased the amount of severe deformities such as spinal curvature and emaciation but also induced an edematous state of the larval heart, resulting in the most balanced mortality/deformity ratio when salinity was decreased on 3 dph and at 2 psu/day. However, the persistency of the pericardial edema and if or how it represents an obstacle in further larval development needs to be further clarified. In conclusion, this study clearly showed that salinity reduction regimes towards iso-osmotic conditions facilitated the European eel pre-leptocephalus development and revealed the existence of highly sensitive and regulated osmoregulation processes at such early life stage of this species

Effect of dietary vitamin A on Senegalese sole (Solea senegalensis) skeletogenesis and larval quality

16 páginas, 12 figuras, 4 tablas.The effects of different levels of vitamin A (VA) in Senegalese sole larval performance and development were evaluated by means of a dietary dose–response experiment using enriched Artemia metanauplii as a carrier of this micronutrient. Larvae were fed from 6 to 27 days post hatch (dph) with enriched Artemia containing graded levels of total VA (1.3, 2.1, 4.5 and 12.9 µg VA mg− 1 DW). The content of VA in live prey directly affected its accumulation in larvae and early juveniles. Retinyl palmitate accumulated during larval ontogeny, whereas retinol showed the opposite trend, decreasing from hatching until 41 dph and then remaining constant until the end of the study. In metamorphic larvae (10 and 15 dph), VA did not affect the number of thyroid follicles or the intensity of the immunoreactive staining of T3 and T4. However, at older stages of development (post-metamorphic larvae: 20, 30, 41 and 48 dph), VA decreased the number of thyroid follicles but increased their mean size and enhanced T3 and T4 immunoreactive staining. A dietary excess of VA did not affect either larval performance in terms of growth and survival or the maturation of the digestive system. However, the most remarkable impact of this morphogenetic nutrient was detected during skeletal morphogenesis. Dietary VA accelerated the intramembranous ossification of vertebral centrums, which led to the formation of a supranumerary haemal vertebra and a high incidence of fused and compressed vertebrae in fish fed 2.1, 4.5 and 12.9 mg VA mg− 1 DW. In addition, VA also affected those structures from vertebrae and caudal fin formed by chondral ossification, leading to defects in their shape and fusions with adjacent skeletal elements. In particular, the caudal fin was the region most affected by the dietary treatments. In order of importance, the bones with more developmental anomalies were the modified neural and haemal spines, epural, hypurals and parahypural. The impact of systemic factors such as thyroidal hormones in skeletogenesis should not be neglected since present results revealed that an excess of dietary VA affected the levels of T3 and T4, which might have affected bone formation and remodelling, leading to skeletal deformities.This work was funded by the Ministry of Education and Culture (MEC) of the Spanish Government (project AGL2005-02478).Peer reviewe

Effect of thermal and nutritional conditions on fatty acid metabolism and oxidative stress response in juvenile European sea bass (Dicentrarchus labrax)

Coastal nursery areas are subjected to a wide range of natural and anthropogenic stressors, including global warming, which indirectly influence trophic food webs. A global rarefaction of n-3 polyunsaturated fatty acids (PUFA) in trophic networks is in progress. The aim of this study was to assess the effect of a reduction in the dietary availability of n-3 PUFA on some molecular and biochemical parameters related to lipid metabolism and oxidative stress response in juvenile European sea bass (Dicentrarchus labrax) raised at two temperatures (15 °C and 20 °C). Fish were fed for five months with a reference diet (RD; 1.65% n-3 PUFA on a dry matter basis, DM), used as a proxy of trophic networks where n-3 PUFA is plentiful, and a lower n-3 PUFA diet (LD; 0.73% n-3 PUFA DM), designed to mimic a decrease in n-3 PUFA resulting from global changes (the n-3 PUFA levels tested remained above the nutritional minimum required for this species). Results showed that diet did not affect the hepatic expression of some mRNA coding for transcriptional factors involved in regulating the metabolic pathways related to fatty acid bioconversion. Although our molecular analysis was limited to transcript expression, these data suggest the presence of a threshold in the nutritional supply of PUFA above which the activation of these molecular pathways does not occur. However, the expression for most of the transcripts tested was up-regulated at 20 °C. Despite the high peroxidation index in fish fed RD, very few modifications of the oxidative stress response were associated with diet. At 20 °C, an increase of the enzymatic antioxidant response was observed, but there was no correlation with the peroxidation index or malondialdehyde products

Temperature induced variation in gene expression of thyroid hormone receptors and deiodinases of European eel (Anguilla anguilla) larvae

Thyroid hormones (THs) are key regulators of growth, development, and metabolism in vertebrates and influence early life development of fish. TH is produced in the thyroid gland (or thyroid follicles) mainly as T4 (thyroxine), which is metabolized to T3 (3, 5, 3’-triiodothyronine) and T2 (3, 5-diiodothyronine) by deiodinase (DIO) enzymes in peripheral tissues. The action of these hormones is mostly exerted by binding to a specific nuclear thyroid hormone receptor (THR). In this study, we i) cloned and characterized thr sequences, ii) investigated the expression pattern of the different subtypes of thrs and dios, and iii) studied how temperature affects the expression ofthose genes in artificially produced early life history stages of European eel (Anguilla anguilla), reared in different thermal regimes (16, 18, 20 and 22°C) from hatch until first-feeding. We identified 2 subtypes of thr (thrα and thrβ) with 2 isoforms each (thrαA, thrαB, thrβA, thrβB) and 3 subtypes of deiodinases (dio1, dio2, dio3). All thr genes identified showed high similarity to the closely related Japanese eel (Anguilla japonica). We found that all genes investigated in this study were affected by larval age (in real time or at specific developmental stages), temperature, and/or their interaction. More specifically, the warmer the temperature the earlier the expression response of a specific target gene. In real time, the expression profiles appeared very similar and only shifted with temperature. In developmental time, gene expression of all genes differed across selected developmental stages, such as at hatch, during teeth formation or at first-feeding. Thus, we demonstrate that the expression of thrs and dios show sensitivity to temperature and are involved in and during early life development of European eel