Effects of dietary arachidonic and eicosapentaenoic acids on common dentex (Dentex dentex Linnaeus 1758) larval performance

Oily emulsions containing constant levels of total fatty acids (FAs) and varying eicosapentaenoic acid (EPA) and arachidonic acid (ARA) levels were used to enrich rotifers. Common dentex larval survival and growth were compared between groups fed different enriched live prey. Growth, survival rate, and lipid composition of larvae suggest that feeding common dentex in the first 15 days posthatching with 2.5–3% EPA, 6–8% docosahexaenoic acid (DHA), and DHA/EPA ratio of 2.0–2.5 is sufficient to fulfill their EPA requirements. Higher amounts of dietary EPA did not result in any significant improvement in growth or survival. EPA requirement during this period of larval development does not seem to be as critical as other FAs during the first 15 days of common dentex larval development, but it does not exclude its essentiality later in development. In the case of ARA, nutritional requirements are low compared to other marine finfish species, with the upper limit of this essential FA being around 2% of total FAs provided in the live prey composition.     

Development of the common dentex (Dentex dentex) eggs in relation to temperature

Embryonic development of common dentex (Dentex dentex) was investigated at nine different constant water temperatures (8°C, 10°C, 12°C, 14°C, 16°C, 18°C, 20°C, 22°C and 24°C). The observed effects were compared using of regression analysis. Constant water temperatures between 12°C and 18°C were found to support successful embryonic development. A negative relationship between the rate of embryonic development and incubation temperature was observed. While embryonic development was completed within this range (12°C–18°C), there was no cell division at water temperatures of 8°C and 24°C. Total mortalities were observed at the 128 cleavage stage at a temperature of 10°C, and after the beginning of gastrulation at 20°C and 22°C.     

The effect of light intensity on early life development of common dentex Dentex dentex (L. 1758) larvae

The effects of different levels of illumination on common dentex Dentex dentex (L.) larvae have been examined. Illumination affected the relationships between total length and digestive tube length, total length and oil globule volume, and total length and yolk sac. The relationship between total length development and yolk sac utilization in dark and light conditions was found to be significant (P<0.05). However, it was determined that the relationship between digestive tube development and oil globule absorption was not significant (P>0.05). Besides, there is no effect of different light intensities on absorption of yolk sac and oil globule and digestive tube development (P>0.05). In addition, total length development was not significant (P>0.05). Survival rate was found to be not significantly different in dark conditions (P>0.05).     

Assessing the spawning season in common dentex (Dentex dentex) using microsatellites

A set of five variable microsatellite markers was used for the genetic characterization of two common dentex ( Dentex dentex ) broodstocks from an experimental hatchery and for the screening of the egg batches spawned during the complete spawn season in 2006. After parentage assignment, simulations were performed for the hatchery 1 broodstocks; the microsatellite set correctly assigned over 93% of descendants to parents when one parent was known and 80% when neither parent was known. Of the 261 eggs that the DNA was correctly amplified from, 254 were successfully assigned to a parental couple. More than one female or male took part (at the same point in time) in the spawning season. However, we observed 'dominant' individuals that produced high proportions of the descendants (unequal reproductive success) in all the sampling periods, although the contributions of these dominant individuals could change over time. This phenomenon reduces the effective breeding numbers, and could lead to inbreeding if this factor is not taken into account when the next parental generation is obtained. This factor should also be considered in aquaculture selection programmes because the dominant breeders may not be the most interesting individuals for commercial or productive schemes.     

Characterization and functional properties of digestive proteases in two sparids; gilthead seabream (Sparus aurata) and common dentex (Dentex dentex)

Digestive proteases present in two sparids, seabream (Sparus aurata) and common dentex (Dentex dentex), have been characterized using both biochemical and electrophoretic techniques. Although optimum pH and temperature for maximum activity of both acid and alkaline proteases were similar in the two species, important differences in total activity, as well as in thermal and pH stability were found. Specific inhibitors and SDS-PAGE zymograms were used to clarify such differences. Evidences support the existence of a more active and complex protease set in dentex. Results are discussed from the perspective of their application to the formulation of feeds for each species.     

Effects of two culturing techniques on the growth, survival and larval quality of Dentex dentex Linnaeus, 1758

Common dentex larvae were reared using two culturing techniques, mesocosms and intensive rearing, to determine the principal culture parameters involved in the differences observed in growth, skeletal deformations and survival between the two rearing techniques. In growth, only dry weight of larvae of 40 days post‐hatching (dph) from mesocosms was significantly higher than larvae from intensive rearing. Significant differences were observed in survival at 40 dph (6.58% in mesocosms and 1.58% in intensive rearing) and in the incidence of skeletal deformations, both for percentage of deformed larvae and for some deformation types such as those related to vertebral column and to the caudal complex. Initial larval density and initial prey density and quality are the factors suspected to affect growth and survival performance, while skeletal deformities might also be affected by tank hydrodynamics.     

Variations in Bacterial Community of Rearing Water and Gut of Common Dentex,Dentex dentex (Linnaeus 1758), Larvae Using Three Microalgae Management Approaches

Microorganisms present in the rearing water colonize the gut of first feeding larvae and represent the first barrier against opportunistic pathogens. The aim of the experiments presented herein was to standardize a protocol for the management of rearing water and microalgae suitable for the larval rearing of common dentex. In Experiment 1, bacteria–algae interactions were tested using a monospecific microalgal community, Tetraselmis chuii, suitable for nutritional experiments and with known antibacterial activity. In Experiment 2, the evolution of the bacterial community and larval performance (growth and survival) were monitored daily, in three conditions: (1) mature water: T. chuii was added 5 d before the rearing of common dentex larvae, (2) green water: T. chuii was added 1 d before, and (3) clear water: no T. chuii addition. The results show the influence of the presence of T. chuii on the evolution of the bacterial communities, both in terms of bacterial density and morphology, and indicate green water is the most suitable water treatment for management of larval rearing for common dentex.     

Changes in lipid content, fatty acid composition and lipid class composition of eggs and developing larvae (0–40 days old) of cultured common dentex (Dentex dentex Linnaeus 1758)

Total lipid content, fatty acid (FA) composition and lipid class composition of common dentex eggs spawned at different times and larvae reared under different culture conditions until 40 days posthatch (dph) were analysed to get a general pattern of lipid composition during larval development. Two groups of larvae were kept under starvation to compare their FA composition with that obtained from normally fed larvae. To compare FA use or accumulation during larval development, results were grouped according to the developmental stage of the larvae instead of age in days posthatch. Saturated and monounsaturated FAs decreased along larval development, while polyunsaturated fatty acid (PUFA) content increased. The ratio of docosahexaenoic acid (DHA)/eicosapentaenoic acid shifted from 4 to 5 in early developmental stages to lower than 1 after metamorphosis. Arachidonic acid levels remained constant along larval development. Larvae kept 6 days under starvation consumed most of their n-3 PUFA while conserving the DHA to values at day 0. The results presented here are useful for the design of nutritional experiments, because there were differences detected in terms of lipid and FA composition between developmental stages with higher differences mainly found in first-feeding larvae and early developmental stages.     

Egg quality determination based on the shape of the lipid vesicle in common dentex, Dentex dentex

The shape of the lipid vesicle (the ratio between the maximal and the minimal diameter and the shape coefficient) of the eggs of Diplodus puntazzo and Sparus aurata is correlated with larval survival rate (%) and can be used as an egg quality marker as reported previously by Lahnsteiner and Patarnello (2005) . In the present study, this method for egg quality determination has also been validated for common dentex, Dentex dentex, after introducing several modifications in the calculation and evaluation procedure in order to obtain higher sensitivity. In the present study, it could be demonstrated that the shape of the lipid vesicle is not only correlated with the hatching rate but also with the survival rate of 1, 2 and 3 days post hatch larvae and therefore it can also be used as an indicator of larval viability at early stages of development.     

Reproductive performance of common dentex, Dentex dentex, broodstock held under different photoperiod and constant temperature conditions

The effect of photoperiod and temperature on the timing and the quality of spawning, and on associated endocrine changes in circulating 17β-oestradiol, 11-ketotestoserone (11-KT) and vitellogenin (Vtg) were investigated in common dentex, Dentex dentex, undergoing their second reproductive cycle. The possibility was also explored of using the measurement of steroids in the culture water of broodstock tanks, rather than in individual blood plasma samples, as a potentially useful tool for assessing the physiological state of a fish without disturbing them. One group of fish was exposed to a simulated natural seasonal cycle and ambient temperature (CONTROL). The other two groups were exposed to simulated seasonal photoperiod cycles (12 month-long) but which were phase-shifted either three months before (ADVANCED) or after (DELAYED) the natural cycle. Temperature was kept at 19.4±0.9 °C all-year-round. In the CONTROL, spawning started in mid-April and lasted until mid-June, while in the ADVANCED group, spawning started 4 months earlier and in the DELAYED group 2 months later than the CONTROL. The total egg production, egg quality, hatching rate, relative fecundity, and spawning index of the experimental groups were similar to the controls. The differences in spawning time induced by photothermal manipulation were associated with a difference in the timing of peak concentrations of plasma E₂, 11-KT and Vtg. In all three groups, the amounts of conjugated 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) and free and sulfated 11-KT which could be extracted from the water during the spawning period were significantly higher than those found in the preovulatory period. However, the differences were mostly less than 2-fold suggesting that, at least under the conditions employed in this study, the method was of limited use for non-intrusive detection of gonadogenesis and spawning (as had been hoped). The observed differences in spawning time and in the seasonal changes of sex steroids and Vtg confirm and extend the findings on marine fish. This revised version was published online in August 2006 with corrections to the Cover Date.     

Growth performance,feed utilization and body composition of Dentex dentex fed on different macronutrient combinations

Determining an adequate macronutrient balance is essential to guarantee the production success. As protein is the limiting component for fish food, the utilization of lipids or carbohydrates as partial substitutes of this nutrient is a challenge to improve its use. In order to get an approximation of the maximum levels of utilization for carbohydrates and/or lipids and determine the most adequate macronutrient to partly replace protein as the main energy source of diets for dentex (91.7 ± 1.4 g mean weight), four experimental diets with different protein:lipid:carbohydrate percentages (43/16/28, 43/24/4, 38/19/28 and 38/24/13) were tested for 13 weeks. The results indicated the possibility of using 38% of dietary protein without affecting growth performance, under the experimental conditions. There were no differences among the four diets either in most of the nutritive utilization indicators or in the body composition and haematological parameters. The influence of dietary composition was only observed in the feed intake, being higher with more dietary carbohydrates, and the hepatosomatic index and protein efficiency ratio, showing more elevated values in diets with a higher lipid level. The dentex capacity of using both carbohydrates and lipids efficiently to obtain the necessary energy for its correct growth, as well as to compensate the energetic ‘vacuum’ caused by the dietary protein reduction, under the assayed conditions, was confirmed.     

Egg quality criteria in common dentex (Dentex dentex)

The spawning quality, in terms of hatching rate, larval mortality at 3 and 5 days post-hatching (dph) and day of total mortality of two broodstock groups of common dentex was evaluated for 1 month in 2005. Several biochemical parameters including total lipid content, lipid class and fatty acid composition, carbohydrate content and metabolic enzyme activities were analysed in all the egg batches collected. Comparison was carried out between low- (mortality at 3 dph higher than 35%) and high-quality (mortality at 3 dph lower than 10%) batches. No differences were observed in lipid content and/or lipid class and fatty acid composition although a slightly higher content of neutral lipids was detected in high-quality batches. However, significant differences were obtained regarding carbohydrate composition and the activity of enzymes such as alkaline phosphatase and pyruvate kinase being higher in low-quality egg batches.     

Plasma proteins,hepatic enzymes,thyroid hormones and liver histopathology of Cyprinus carpio (Linnaeus, 1758) exposed to an oxadiazin pesticide,indoxacarb

Indoxacarb is a relatively new pesticide from oxadiazin class, which is used near carp ponds for agricultural purposes. Thus, the aim of this study was to determine indoxacarb effects on common carp (Cyprinus carpio). The fish were exposed to 0 (control), 0.75, 1.5 and 3 ppm of indoxacarb over 21 days and plasma biochemical characteristics and liver histopathology were examined. Exposure to indoxacarb induced fall in total protein after 21 days. Globulin increased after 7 days and then decreased after 14‐ and 21‐day exposure. Plasma alanine transaminase and aspartate transaminase activities increased in 1.5 and 3 ppm treatments after 7‐day exposure. Indoxacarb exposure significantly decreased plasma alkaline phosphatase after 7 days with no change at the second and third samplings. After 7 days, plasma T₃ levels had no significant change; however, it decreased after 14 days in the 1.5 and 3 ppm treatments and also reduced after 21‐day exposure to the 3 ppm treatment compared to the control group. Plasma T₄ level only decreased significantly in the 3 ppm treatment compared to the control group after 21 days. Different histopathological symptoms such as necrosis, hyperaemia, sinusoidal space extension, pyknotic nuclei, leucocyte infiltration and melanomacrophage aggregates were observed after 21‐day exposure to indoxacarb. The symptoms intensity was dependent on indoxacarb concentration. In conclusion, the present results show that indoxacarb exposure adversely affects common carp health and welfare, which consequently may induce serious problems in this species aquaculture.     

Utilization of lipids by Dentex dentex L. (Osteichthyes, Sparidae) larvae during lecitotrophia and subsequent starvation

Total lipids, lipid classes and their associated fatty acids were measured in developing eggs, yolksac larvae and starving larvae (from fertilized egg to day 9 after hatch) of the common dentex Dentex dentex (L., 1758). The larvae of common dentex during lecitotrophia and subsequent starvation consumed 1.6 g of total lipid per larvae per day. The overall decrease was mainly due to utilization of the major neutral lipids, TAG and SE (0.5 and 0.6 g larvae^–1 day^–1, respectively) which was 3.4-fold greater than that of the the major phosphoglycerides (primarily PC by 0.2 g larvae^–1 day^–1). There was net synthesis/conservation of PE during the first half of the study period before it decreased rapidly (0.2 g larvae^–1 day^–1) during the second half. PUFAs were principally catabolized (468.6 ng larvae^–1 day^–1), primarily 22:6(n-3), 20:5(n-3) and 20:4(n-6) (221.8, 58.5 and 12.1 ng larvae^–1 day^–1, respectively). Saturated and monounsaturated fatty acids were also utilized (227.2 and 256.7 ng larvae^–1 day^–1, respectively), principally 16:0 and 18:1(n-9) that were both consumed at 149.8 and 156.7 ng larvae^–1 day^–1, respectively. The rank order of utilization of fatty acids (ng larvae^–1 day^–1) by D. dentex larvae from total lipids, PC and TAG coincided with the order of abundance of the different fatty acids in the respective lipid fractions. However, in PE, the most abundant fatty acid, DHA, was relatively conserved and 16:0, the second most abundant fatty acid, was catabolized to the greatest extent. D. dentex showed a pattern of lipid metabolism during early development similar to that of marine larval fish from temperate waters whose eggs contain high levels of total lipids, including an oil globule, and which preferentially utilize neutral lipids as the primary energy source.     

Bacteria associated with winter mortalities in laboratory-reared common dentex (Dentex dentex L.)

A pathological study was conducted on laboratory‐reared juvenile common dentex (Dentex dentex) suffering trickling and continuous mortalities. During a 3‐month period (October–December), water temperature, clinical signs and mortalities were recorded. Moribund or dead fish were examined for bacteria and parasites. Bacteria were isolated from head kidney and external ulcers, and samples from the gills, intestine, stomach, trunk kidney, gall bladder and liver were taken for histology. Cumulative mortality reached 73%, and 80% of fish examined were positive for bacteria (102 isolates). Vibrio splendidus was the most prevalent in head kidney (59.7%) and ulcers (88.9%), and it was frequently isolated as pure culture (74.7% and 100% respectively). This is the first report of this bacterium in association with mortality in common dentex. Vibrio scophthalmi was the second most prevalent bacterium (29.2%) and accounted for 63.6% of the mixed infections with V. splendidus, V. harveyi, Pseudoalteromonas spp. and other species were rarely isolated. No parasites were found in histological sections. Bacteria were frequently observed in the lumen of the intestine and stomach, destroying the epithelium. A cellular reaction was suggested by the high numbers of rodlet cells (RC) in the intestinal epithelium, and the abundance of eosinophilic granular cells (EGC) in the intestinal lamina propria. In the stomach, vacuolized cells containing unidentified debris, sometimes of crystalloid appearance, were very common. The high prevalence of V. splendidus in pure cultures and the absence of other aetiological agents suggest that V. splendidus is substantially involved in the registered mortalities, although the implication of V. scophthalmi and even of some nutritional factors cannot be discounted.     

Effect of larval and prey density, prey dose and light conditions on first feeding common dentex (Dentex dentex L.) larvae

Initial larval stocking density, prey density, daily prey ration and light conditions (light intensity and photoperiod) were tested for common dentex larval rearing under experimental conditions. Experiments continued until the first peak of larval mortality. The best results in larval survival were obtained with an initial stocking density of between 10 and 40 larvae L^?1, fed with at least 10 rotifers mL^?1, maintaining ratios of 500–1000 rotifers larva^?1, with one or two adjustments of prey density per day. The use of more than 2000 rotifers larva^?1 or three daily adjustments of live prey density had negative effects on larval survival. The best light conditions for common dentex larval rearing were found using a photoperiod of 24 h L:0 h D and an intensity of at least 3.4 μmol m^?2 s^?1.     

Protein sparing effect of dietary lipids in common dentex (Dentex dentex): A comparative study with sea bream (Sparus aurata) and sea bass (Dicentrarchus labrax)

The common dentex (Dentex dentex) is a sparid fish which is considered a suitable candidate for Mediterranean aqua-culture. A seven-weeks feeding trial was conducted over the summer period with common dentex, sea bream (Sparus aurata) and sea bass (Dicentrarchus labrax). All three species were fed to visual satiety with two practical diets with varying crude protein and crude fat levels (55 % protein, 9 % lipids; 46 % protein, 17 % lipids). The initial body weights were 8.2, 11.6 and 17.3 g for common dentex, sea bass and sea bream, respectively. In all cases, specific growth rates were not significantly affected by dietary treatment, but they varied among species (sea bass 1.7–1.8 %; sea bream 2.1–2.3 %; common dentex 3.1–3.2 %). When comparisons between fish species were made, we observed that the increase in growth rates was linked to a decrease of the whole body fat content, as a consequence of a greater utilisation of dietary lipids as energy substrates. Furthermore, in common dentex, the 17 % lipid diet was able to improve protein retention, but this diet effect was less significant in sea bream and sea bass. In contrast to sea bream, the 17 % lipid diet did not up-regulate plasma GH levels in common dentex, which provides additional evidence for a more efficient utilisation of dietary lipids.     

黄带拟鲹qRT-PCR内参基因筛选及验证

实时荧光定量PCR (qRT-PCR)是研究基因表达的一种广泛使用且有效的方法,选用黄带拟鲹(Pseudocaranx dentex)合适的内参基因,是qRT-PCR技术获得该物种基因表达可靠结果的关键。本研究首先测定了9个常用内参基因(β-actin、RPL13、EF-1α、GAPDH、HPRT、PPIA、β2M、TUB和PP2A)在黄带拟鲹成鱼组织中的表达丰度;同时,利用BestKeeper、NormFinder、geNorm和RefFinder 4种模型预测了内参基因的表达稳定性,发现其表达稳定性排序为RPL13>EF-1α> PP2A>HPRT>PPIA>TUB>β2M>β-actin>GAPDH。进一步通过定量检测目标基因myod1在不同组织的表达情况,验证上述预测结果的准确性。研究发现,RPL13和EF-1α单独或联合作为黄带拟鲹qRT-PCR内参基因,可显著提高目标基因表达量检测结果的稳定性与可靠性。结果表明,RPL13和EF-1α可作为黄带拟鲹不同组织q RT-PCR分析的内参基因。同时,也证实了并不是管家基因的表达在所有...     

Physiological responses of reared sea bream (Sparus aurata Linnaeus, 1758) to an Amyloodinium ocellatum outbreak

Amyloodiniosis represents a major bottleneck for semi‐intensive aquaculture production in Southern Europe, causing extremely high mortalities. Amyloodinium ocellatum is a parasitic dinoflagellate that can infest almost all fish, crustacean and bivalves that live within its ecological range. Fish mortalities are usually attributed to anoxia, associated with serious gill hyperplasia, inflammation, haemorrhage and necrosis in heavy infestations; or with osmoregulatory impairment and secondary microbial infections due to severe epithelial damage in mild infestation. However, physiological information about the host responses to A. ocellatum infestation is scarce. In this work, we analysed the proteome of gilthead sea bream (Sparus aurata) plasma and relate it with haematological and immunological indicators, in order to enlighten the different physiological responses when exposed to an A. ocellatum outbreak. Using 2D‐DIGE, immunological and haematological analysis and in response to the A. ocellatum contamination we have identified several proteins associated with acute‐phase response, inflammation, lipid transport, homoeostasis, and osmoregulation, wound healing, neoplasia and iron transport. Overall, this preliminary study revealed that amyloodiniosis affects some fish functional pathways as revealed by the changes in the plasma proteome of S. aurata, and that the innate immunological system is not activated in the presence of the parasite.