Post-metamorphic Larvae Research Articles

Searching integrated strategies for the evaluation of the physiological status in fish fed functional diets: The example of SDPP in gilthead sea bream (Sparus aurata)

In this study we evaluated the effects of high dietary arachidonic acid (ARA) levels on prostaglandin E2 (PGE2) and E3 (PGE3) production and their effect on two morphogenetic processes occurring during metamorphosis: the establishment of the juvenile pigmentation pattern and eye migration and remodeling of cranial bones. In this sense, Senegalese sole larvae were fed from 2 to 50 days post-hatch (dph) with live prey enriched with an experimental emulsion containing high levels of ARA (ARA-H; 10.2 and 7.1% TFA in enriched rotifer and Artemia, respectively) versus a reference commercial enriching product (Algamac 3050®, AGM; 1.0 and 1.4% TFA in enriched rotifer and Artemia, respectively). High dietary ARA levels did not affect larval growth performance at 50 dph, but significantly induced malpigmentation (81.4 ± 7.5%, versus 0.9 ± 0.3% in larvae fed the AGM diet). This malpigmentation was linked to the higher prostaglandin E2 (PGE2) levels observed in pseudo-albino fish as compared to normally pigmented individuals. The PGE2 levels were higher in normally pigmented specimens fed the ARA-H diet than in those fed the AGM diet. The effects of ARA on normally pigmented fish fed the AGM diet and pseudo-albino specimens fed the ARA-H diet were evaluated by means of the density of melanophores and the texture and image segmentation analyses in the dorsal skin of post-metamorphic fish. The skin of pseudo-albino specimens had a more uniform and homogeneous melanophore pattern than normally pigmented fish. Melanophores in pseudo-albino specimens were less abundant and not so aggregated in patches as they were in normally pigmented ones, whereas their shape differed (round vs. dendritic) suggesting their inability to disperse melanin. In addition, fish fed the ARA-H diet presented a higher percentage of cranial deformities (95.1 ± 1.5%) than those fed the control diet (1.9 ± 1.9%) that was significantly and negatively correlated with the incidence of normally-pigmented animals (R2 = − 0.88, P < 0.001). Cranial deformities in pseudo-albino fish were associated with an impaired migration of the eye from the ocular side (the right eye), whereas the left eye migrated from the blind side into the ocular side almost normally. The effects of high dietary ARA levels in the eye migration and cranial bone remodeling processes in post-metamorphic larvae were evaluated by means of the staining of cranial skeletal elements. Pseudo-albino fish showed higher interocular distance and head height than normally pigmented individuals, a different disposition of the eyes with regard to the vertebral column and mouth axes, and a distinct osteological development of some skeletal structures from the neuro- and splanchnocranium, in relation to high dietary ARA contents and high PGE2 production. These results brought new information about possible nutritional forcing factors and physiological mechanisms of pigmentary disorders and impaired eye migration, which are current major bottlenecks in flatfish aquaculture.

Transcriptional regulation of genes involved in retinoic acid metabolism in Senegalese sole larvae.

The aim of this study was the characterization of transcriptional regulatory pathways mediated by retinoic acid (RA) in Senegalese sole larvae. For this purpose, pre-metamorphic larvae were treated with a low concentration of DEAB, an inhibitor of RALDH enzyme, until the end of metamorphosis. No differences in growth, eye migration or survival were observed. Nevertheless, gene expression analysis revealed a total of 20 transcripts differentially expressed during larval development and only six related with DEAB treatments directly involved in RA metabolism and actions (rdh10a, aldh1a2, crbp1, igf2r, rarg and cyp26a1) to adapt to a low-RA environment. In a second experiment, post-metamorphic larvae were exposed to the all-trans RA (atRA) observing an opposite regulation for those genes involved in RA synthesis and degradation (rdh10a, aldh1a2, crbp1 and cyp26a1) as well as other related with thyroid- (dio2) and IGF-axes (igfbp1, igf2r and igfbp5) to balance RA levels. In a third experiment, DEAB-pretreated post-metamorphic larvae were exposed to atRA and TTNPB (a specific RAR agonist). Both drugs down-regulated rdh10a and aldh1a2 and up-regulated cyp26a1 expression demonstrating their important role in RA homeostasis. Moreover, five retinoic receptors that mediate RA actions, the thyroid receptor thrb, and five IGF binding proteins changed differentially their expression. Overall, this study demonstrates that exogenous RA modulates the expression of some genes involved in the RA synthesis, degradation and cellular transport through RAR-mediated regulatory pathways establishing a negative feedback regulatory mechanism necessary to balance endogenous RA levels and gradients.

Otolith chemistry chimes in: migratory environmental histories of Atlantic tarpon (Megalops atlanticus) caught from offshore waters of French Guiana

The migratory life histories of 37 Atlantic tarpon (Megalops atlanticus) caught from offshore waters of French Guiana were quantified using otolith Sr:Ca and Ba:Ca ratios. In general Sr:Ca ratio was more informative marker for inferring movements along salinity gradients, but Ba:Ca ratio was used to infer movements to estuarine mixing zones with high Ba availability. All sampled individuals had made use of marine and brackish water but only 8 % had experienced fresh water and 24 % hypersaline water. The newly-hatched post-metamorphic larvae recruited to brackish (49 %), sea (43 %), fresh (5 %) and hypersaline (3 %) water nursery areas. Following the settlement to nursery areas Atlantic tarpon likely displayed facultative habitat selection and complex set of inter-habitat shifting among waters of various salinity. The combined use of both markers allowed us to conclude that brackish (estuarine) areas are likely the main habitat for Atlantic tarpon in French Guiana, and fresh water use is surprisingly rare. As no general migratory life-history emerged, and because Atlantic tarpon seemed rather facultative in its habitat use, it should be regarded as ‘euryhaline marine migrantʼ.

Oxidative Stress and Digestive Enzyme Activity of Flatfish Larvae in a Changing Ocean.

Until now, it is not known how the antioxidant and digestive enzymatic machinery of fish early life stages will change with the combined effects of future ocean acidification and warming. Here we show that high pCO2 (~1600 μatm) significantly decreased metabolic rates (up to 27.4 %) of flatfish larvae, Solea senegalensis, at both present (18 °C) and warmer temperatures (+4 °C). Moreover, both warming and hypercapnia increased the heat shock response and the activity of antioxidant enzymes, namely catalase (CAT) and glutathione S-transferase (GST), mainly in post-metamorphic larvae (30 dph). The lack of changes in the activity of CAT and GST of pre-metamorphic larvae (10 dph) seems to indicate that earlier stages lack a fully-developed antioxidant defense system. Nevertheless, the heat shock and antioxidant responses of post-metamorphic larvae were not enough to avoid the peroxidative damage, which was greatly increased under future environmental conditions. Digestive enzymatic activity of S. senegalensis larvae was also affected by future predictions. Hypercapnic conditions led to a decrease in the activity of digestive enzymes, both pancreatic (up to 26.1 % for trypsin and 74.5 % for amylase) and intestinal enzymes (up to 36.1 % for alkaline phosphatase) in post-metamorphic larvae. Moreover, the impact of ocean acidification and warming on some of these physiological and biochemical variables (namely, lower OCR and higher HSP and MDA levels) were translated into larvae performance, being significantly correlated with decreased larval growth and survival or increased incidence of skeletal deformities. The increased vulnerability of flatfish early life stages under future ocean conditions is expected to potentially determine recruitment and population dynamics in marine ecosystems.

Diel food intake and digestive enzyme production patterns in Solea senegalensis larvae

Abstract The aim of this study was to examine patterns of food intake and production of the main digestive enzymes of Senegalese sole larvae under a fixed 12 h light:12 h dark cycle. Daily gut content, soluble protein content and digestive enzyme activities were studied at different times of day in pelagic sole larvae (onset of feeding and 6 days post-hatch; dph) and in benthonic sole post-larvae (20 and 30 dph). The larvae displayed temporal changes in food intake throughout development. Pre-metamorphic larvae ceased feeding after lights were turned off. In contrast, post-metamorphic larvae continued to feed during the day, with a higher food intake occurring during dark phase. It was demonstrated that larval digestive enzymes, mainly lipases, were active before mouth opening. Tryptic activity varied with food intake at pre-metamorphic larvae, whereas its activity levels were kept along the day in post-metamorphic larvae.

Senegalese sole (Solea senegalensis) metamorphic larvae are more sensitive to pseudo-albinism induced by high dietary arachidonic acid levels than post-metamorphic larvae

Abstract High dietary levels of arachidonic acid (ARA) and its relative proportions with eicosapentaenoic acid (EPA), fed during early larval stages, have been associated with malpigmentation in various flatfish species. This study investigated whether the nutritional induction of pigmentary disorders at larval stages was related to a specific larval period of increased sensitivity to ARA in Senegalese sole (Solea senegalensis Kaup, 1858). Senegalese sole larvae were fed high dietary ARA levels during pre- and pro-metamorphosis (2–15 dph) and/or post-metamorphosis (15–50 dph). Larval tissues reflected the dietary fatty acid composition. Malpigmentations were significantly related to elevated dietary and larval ARA contents and ARA/EPA ratio. This study reports evidence for a “pigmentation window”, with a higher larval sensitivity to dietary ARA during pre- and pro-metamorphosis than post-metamorphosis. High dietary ARA fed to larvae during pre-metamorphosis enhanced survival, but did not affect growth nor eye migration. The aspect and density of melanophores in the skin of the ocular side of ARA-induced pseudo-albinos were significantly reduced in comparison to normally pigmented individuals, even more in the pseudo-albino fish fed high dietary ARA levels during the pre-metamorphic stage. Pseudo-albino fish fed high dietary ARA levels during post-metamorphosis showed higher concentrations of 2- and 3-series prostaglandins (PGE2 and PGE3) than normally pigmented specimens fed the same diets. An increased sensitivity to ARA-induced malpigmentations has been identified at pre-metamorphosis and early metamorphosis in Senegalese sole. Supplying high dietary ARA amounts imbalanced the dietary ARA/EPA ratio and disrupted the relative concentrations of derived PGE2 and PGE3, resulting in 20 to 81.7% pseudo-albino individuals, depending on the dietary treatment. The administration of high levels of dietary ARA at different developmental stages did not only affect the incidence of animals with pigmentary disorders, but it also affect the melanophore density and skin aspect in normally pigmented and pseudoalbino fish as image segmentation and texture analyses indicated.

The effects of dietary arachidonic acid on Senegalese sole morphogenesis: A synthesis of recent findings

In this study we evaluated the effects of high dietary arachidonic acid (ARA) levels on prostaglandin E 2 (PGE 2 ) and E 3 (PGE 3 ) production and their effect on two morphogenetic processes occurring during metamorphosis: the establishment of the juvenile pigmentation pattern and eye migration and remodeling of cranial bones. In this sense, Senegalese sole larvae were fed from 2 to 50 days post-hatch (dph) with live prey enriched with an experimental emulsion containing high levels of ARA (ARA-H; 10.2 and 7.1% TFA in enriched rotifer and Artemia , respectively) versus a reference commercial enriching product (Algamac 3050®, AGM; 1.0 and 1.4% TFA in enriched rotifer and Artemia , respectively). High dietary ARA levels did not affect larval growth performance at 50 dph, but significantly induced malpigmentation (81.4 ± 7.5%, versus 0.9 ± 0.3% in larvae fed the AGM diet). This malpigmentation was linked to the higher prostaglandin E2 (PGE 2 ) levels observed in pseudo-albino fish as compared to normally pigmented individuals. The PGE 2 levels were higher in normally pigmented specimens fed the ARA-H diet than in those fed the AGM diet. The effects of ARA on normally pigmented fish fed the AGM diet and pseudo-albino specimens fed the ARA-H diet were evaluated by means of the density of melanophores and the texture and image segmentation analyses in the dorsal skin of post-metamorphic fish. The skin of pseudo-albino specimens had a more uniform and homogeneous melanophore pattern than normally pigmented fish. Melanophores in pseudo-albino specimens were less abundant and not so aggregated in patches as they were in normally pigmented ones, whereas their shape differed (round vs. dendritic) suggesting their inability to disperse melanin. In addition, fish fed the ARA-H diet presented a higher percentage of cranial deformities (95.1 ± 1.5%) than those fed the control diet (1.9 ± 1.9%) that was significantly and negatively correlated with the incidence of normally-pigmented animals (R 2 = − 0.88, P < 0.001). Cranial deformities in pseudo-albino fish were associated with an impaired migration of the eye from the ocular side (the right eye), whereas the left eye migrated from the blind side into the ocular side almost normally. The effects of high dietary ARA levels in the eye migration and cranial bone remodeling processes in post-metamorphic larvae were evaluated by means of the staining of cranial skeletal elements. Pseudo-albino fish showed higher interocular distance and head height than normally pigmented individuals, a different disposition of the eyes with regard to the vertebral column and mouth axes, and a distinct osteological development of some skeletal structures from the neuro- and splanchnocranium, in relation to high dietary ARA contents and high PGE 2 production. These results brought new information about possible nutritional forcing factors and physiological mechanisms of pigmentary disorders and impaired eye migration, which are current major bottlenecks in flatfish aquaculture. • High dietary arachidonic acid levels induced pseudo-albinism in Senegalese sole. • Pseudo-albinism was significantly correlated with impaired eye migration. • The process of cranial bone remodeling was affected in pseudo-albino fish. • High ARA levels increased PGE 2 production in pseudo-albino deformed fish.

Nanoparticles as a novel delivery system for vitamin C administration in aquaculture

Abstract The potential of chitosan-based nanoencapsulation as a tool for delivering ascorbic acid (AA) to marine and freshwater organisms was investigated. Polymeric non-loaded and loaded vitamin C nanoparticles (NPs) were made by ionic gelation and the particles were characterized. In vitro performance of nanoparticles was evaluated in a zebrafish liver cell-line (ZFL) and in vivo studies were carried out in fish (post-metamorphic larvae of Solea senegalensis) and rotifers (Brachionus plicatilis) to assess the potential use of these NPs to be used as a tool in nutritional aquaculture studies. The results showed that NPs are suitable to trap hydrosoluble compounds such as AA by forming positively charged complexes (30–35 mV), in a nanosize range ( 90% of loaded AA remained within nanoparticles after 2 h in seawater). The potential cytotoxicity of the NPs was evaluated in ZFL cells and no decrease in cell viability was noted up to 2.5 mg/ml of nanoparticle concentration. The NP uptake was analyzed in ZFL cells by FACS cytometry and confocal laser scanning microscopy (CLSM). Time course and dose–response experiments were performed using fluorescein isothiocyanate labeled NPs (FITC-NPs). The in vitro endocytosis assays with ZFL cells showed a maximum uptake after 6 h of incubation and a dose-dependent increase of fluorescence intensity directly proportional to the FITC-NP concentration. The antioxidant properties of vitamin C nanoparticles (AA-NPs) were also analyzed in ZFL cell extracts. Lipopolysaccharide (LPS) was added to ZFL cells to induce oxidative stress. The total antioxidant capacity of the AA-NP-treated cells showed a statistically significant increase with respect to the control with non-loaded nanoparticles (71.00 ± 9.6 and 25.36 ± 3.96 μM Trolox equivalent; p

Thermal conditions during larval pelagic phase influence subsequent somatic growth of Senegalese sole by modulating gene expression and muscle growth dynamics

In the present study, Senegalese sole eggs incubated at 20°C were reared at three different temperatures (15°C, 18°C or 21°C) during the pelagic phase, and then transferred to a common temperature (20°C) from benthic stage until 100days post-hatch (dph). Somatic growth, fast muscle cellularity and expression of 15 growth-related genes were compared at the same developmental stage for each temperature (pre-metamorphic larvae, metamorphic larvae, post-metamorphic larvae) and at 83dph and 100dph early juveniles. During pre-metamorphosis and metamorphosis, larvae from 21°C weighed significantly more than those reared at 18°C or 15°C (P<0.001). Relative growth rate (RGR) of pelagic larvae and survival of newly-settled larvae were also higher at 21°C (P<0.05). Furthermore, an increase in muscle growth towards the highest temperatures was observed concomitantly with an increase in gene expression, namely myogenic regulatory factors (MRFs), myosins, igf-I and fgf6. After transfer to a common temperature (20°C), the 15°C group initiated a process of compensatory growth, inverting relative growth rate values that became the highest, particularly between 83dph and 100dph (P<0.05). This increased growth effort in the 15°C group was accompanied by an up-regulation of gene expression in fast muscle, particularly in the 83dph juveniles. Mrf4 and myHC were up-regulated at 15°C and positive correlations with growth were also found for myog or pax7 (P<0.05). In contrast, mstn1 was down-regulated at 15°C (P<0.05), suggesting a boost in muscle growth. By 100dph, the 15°C group had already reached the same weight as the 18°C and the same length as the 21°C ones. At this age, the 18°C juveniles had the largest mean muscle fibre diameter (P<0.001), and thus fibre hypertrophy seems to be a major growth mechanism relatively to the other groups. Our work shows that temperature during pelagic phase greatly influences the growth trajectory of Senegalese sole juveniles.

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

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 T 3 and T 4. 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 T 3 and T 4 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 T 3 and T 4, which might have affected bone formation and remodelling, leading to skeletal deformities.

Digestive enzymes activity during ontogenetic development and effect of starvation in Japanese flounder, Paralichthys olivaceus

Japanese flounder larvae and early juveniles were reared for 39 days after hatching (DAH) in order to determine pancreatic enzymes activities (trypsin and neutral lipase) using fluorogenic substrates during ontogeny and to compare enzymes activities of starved fish at different developmental stages. Larvae were fed L-type rotifers Brachionus plicatilis from 3 to 28 DAH, Artemia nauplii from 14 to 39 DAH and then gradually switched to pelleted feed from 25 DAH. Temperature was kept at 18 °C and photoperiod was 12L : 12D. Three starvation trials were made: from 2 to 8 DAH (premetamorphic larvae), 23–29 DAH (metamorphic) and 31–39 DAH (metamorphic and postmetamorphic). Fluorometric determinations on individual larvae were made using specific substrates for trypsin and lipase activity. Trypsin and neutral lipase activities were found in 2 DAH larvae (0.18 ± 0.09 U μg protein − 1 and 25.12 ± 7.36 nmoles 10 − 3 4MU μg protein − 1 min − 1 , mean ± SD, respectively), just at the time of first feeding. The evolution of both enzymes activities showed a profile marked by decreases during settlement (27–36 DAH). There is a significant increment ( P < 0.05) on specific trypsin activity at 15 DAH, corresponding with the beginning of Artemia feeding and a significant decrease of lipase activity at 6 DAH. In the first starvation experiment, larval mortality was 100% after 6 days. In the second trial a starvation-feeding experiment was done with two and four days starved larvae. Survival at day 6 was 50% and 25%, respectively. On the third trial post-metamorphic larvae were starved 5 days, survival at day 6 was 81%. As a general trend, enzymes activities get significantly lower ( P < 0.05) in starved fish. On two day starved–fed fish, lipase and trypsin activities levels took two days to reach control levels; on the other hand four days starved–fed fish showed significant differences two days after feeding again. During the last trial (31–39 DAH) lipase and trypsin activities went down, coinciding with juvenile settlement. These results show that trypsin and also neutral lipase activity measurements could be used for determining the critical periods during ontogeny and the nutritional status of larvae.

Growth and Temperature-Induced Mortality of Young-of-the-Year Summer Flounder (Paralichthys dentatus) in Southern New Jersey

From Nov. 1988 through Oct. 1989, we examined the spatial and temporal distribution, growth, and temperature-induced mortality of young-of-the-year summer flounder (Paralichthys dentatus) from Great Bay and Little Egg Harbor, New Jersey. We collected P. dentatus undergoing metamorphosis (11-15 mm TL) with plankton nets from Nov. 1988 through May 1989, as they entered the study area from adjacent continental shelf waters. Temperature-induced mortality of early postmetamorphic individuals in the laboratory was largely limited to the one instance that ambient winter temperatures dropped below 2 C (58% mortality). Mortality of a control group held in heated water (approx. 15 C) was low (3%). More important, we observed recruitment of postmetamorphic larvae well after this low temperature period in Feb. In mid-June we collected by trawl, benthic young-of-the-year fish almost exclusively (98%) at the mouth of two mesohaline subtidal marsh creeks. After June, young-of-the-year fish became more widespread, and we collected P. dentatus in a variety of habitats throughout the study area. In Aug. and Sept. P. dentatus were apparently able to avoid the trawl, but we collected large numbers by block nets in polyhaline subtidal marsh creeks. Young-of-the-year P. dentatus ranged 200-326 mm TL by Sept. In early Oct. 1989, we collected only two individuals by any method, and presumably most fish had migrated offshore by this time. We estimated growth rate by linear regression of TL on collection date at 1.9 mm/d from May through Sept. (n = 579, r2 = 0.89). Examination of otoliths for annuli formation (n = 313, range = 126-418 mm TL) showed that the first annulus formed over a range of 275-326 mm TL. The growth rate of young-of-the-year P. dentatus in New Jersey was among the fastest for juvenile estuarine fishes. This fast growth rate and the size at first annulus formation indicate that the first mark on otoliths is indeed the first annulus, a conclusion contrary to some earlier interpretations. Thus, P. dentatus can survive New Jersey winter temperatures, grow very quickly, and attain a large size at the end of the first summer. These characteristics imply that southern New Jersey estuaries act as important nurseries for this species.