C.M Hernandez-Cruz

Recent advances in lipid nutrition in fish larvae

Due to the importance of dietary lipid utilization for larval rearing success, increasing attention has been paid during the last years to different aspects of larval lipid nutrition such as digestion, absorption, transport and metabolism, which are frequently studied by different research groups. The present study reviews the published information on these aspects, including some recent results obtained in our laboratory, that contribute to a better understanding of larval lipid nutrition.Neutral lipase activity was found in the digesta of larval gilthead seabream as early as first feeding, followed by a significant increase which reached up 8 times the initial levels at day 15 and was clearly influenced by the fatty acid composition of dietary lipids. Accordingly, the capacity for lipid absorption by the intestinal epithelium has been also observed at the onset of exogenous feeding, although the specific location in the different digestive tract segments differ with species. Whereas the capacity to absorb lipid increases with development in live prey-fed larvae, this improvemment is delayed in larvae fed formulated diet. Increasing dietary phosphatidyl cholines levels enhanced lipid absorption regardless of whether it is of soybean or marine origin, but the latter improved hepatic lipid utilization. Enzymatic, histological and biochemical evidences suggest that marine fish larvae are able to effectively digest and absorb n-3 HUFA-rich triacylglycerols, but feeding with phosphoacylglycerols, particularly if they are rich in n-3 HUFA, would enhance phosphoacylglycerols digestion and specially lipid transport alowing a better n-3 HUFA incorporation into larval membrane lipids and promoting fish growth. Although the essentiality of n-3 HUFA for larval marine fish has been studied extensively, only recently has the importance of dietary arachidonic acid in the larvae of few species been recognised. Evidences for competitive interactions among these essential fatty acids suggest that besides a minimum dietary requirement for each essential fatty acid, their relative ratios must also be considered.

Effect of dietary arachidonic acid levels on growth and survival of gilthead sea bream /Sparus aurata L. larvae

In order to determine the effect of different levels of arachidonic acid (AA) in microdiets on growth and survival of gilthead seabream larvae, two experiments were carried out. In the first experiment, 17-day old larvae were fed microdiets for 14 days. In this trial, we tested four diets with a similar docosahexaenoic acid (DHA)/eicosapentaenoic acid (EPA) ratio and AA in the range of 0.1%–1.8% dry weight (d.w.) and a fifth diet with 1.0% d.w. of AA and a different DHA/EPA ratio. In the second experiment, 16-day old larvae were fed for 21 days on two microdiets with levels of 0.1% and 1.0% d.w. AA. All diets in the two experiments had the same total n−3 HUFA level. Growth was measured by total length and dry body weight of larvae. Larval lipids were extracted and fatty acid compositions of total neutral lipid (NL) and total polar lipid (PL) fractions were determined by gas chromatography. In the first experiment, the diet containing 1.8% d.w. of AA showed the best survival (P<0.05) but did not improve growth rate. In the second experiment, an increase in the AA dietary level from 0.1% to 1.0% d.w. significantly improved (P<0.05) larval growth. EPA accumulation in the PLs of the larvae was negatively affected by the inclusion of dietary AA. When working with a fixed dietary n−3 HUFA level (2.2% d.w.), the effect of dietary AA on larval growth was masked by that of the dietary DHA/EPA ratio. However, when DHA/EPA ratio in diet was unchanged (∼1.8), it was possible to improve larval growth by supplementing the diet with 1.0 d.w. of AA. The negative effect produced by the increment of dietary AA on the EPA incorporation into the larval PLs may be related to a competition interaction.

Effect of different dietary polar lipid levels and different n−3 HUFA content in polar lipids on gut and liver histological structure of gilthead seabream (Sparus aurata) larvae

Abstract In a previous study feeding larval gilthead seabream on microdiets, an improved growth rate was observed when dietary n−3 HUFA (n−3 highly unsaturated fatty acids) were contained in the polar lipid fraction. A beneficial effect of dietary lecithin on n−3 HUFA incorporation to the larval polar lipids was also observed. To confirm those results and in order to obtain additional information on larval gut and liver histological structure, 11-day-old larvae were fed 4 microdiets differing in lipid composition. In diets N′ and NL′, the main sources of n−3 HUFA were neutral lipids, and polar lipids in diets P′ and PL′. Three lecithin levels were tested, being the highest in diet NL′ and the lowest in diet P′. The total n−3 HUFA level in diets N′ and NL′ was enough to meet the essential fatty acid requirement of larval gilthead seabream obtained in a previous study with a triacylglyceride based diet, while diets P′ and PL′ had a total n−3 HUFA level lower than the minimum determined. A reduction in the larval essential fatty acid requirement to 1.5% d.w. was obtained by feeding dietary n−3 HUFA present in the polar lipid fraction. Feeding larvae with microdiets with low polar lipid levels produced an accumulation of lipid droplets in the enterocytes and a high number of lipid vacuoles in the hepatocytes producing nuclear migration and suggesting a need of dietary polar lipid for lipid transport.

Development of red porgy Pagrus pagrus visual system in relation with changes in the digestive tract and larval feeding habits

Abstract Red porgy larvae, like other sparids such as red seabream are visual feeders. The normal development of the visual system is essential for successful prey capture and predator avoidance, leading to increased larval growth and survival. The aim of this work is to characterise the development of visual organs in relation to changes in the digestive system and feeding habits. Twenty-five larvae from hatching to day 29 were daily collected from the rearing tank, fixed in formalin, embedded in paraffin, 5 μm sectioned and stained with haematoxylin and eosin (H&E) and Periodic Acid Shift Reactive-Haematoxiline (PAS-Hx). Light microscopy was used to study changes in ocular morphology with respect to digestive system development. At hatching, eye and digestive systems of Pagrus pagrus larvae have no function. However, at day 3 post-hatch, when the mouth opens, the larvae must be ready for prey capture and digestion. Despite this, few day 3 larvae had food in the digestive tract. At day 4 photoreceptors were well developed in the eye, pigmentation pattern was complete and thus the visual system was completely ready for prey capture. This development coincided with detection of digestive activity in the midgut and most of larvae starting to take food. The results of this study suggest that adequate development of the visual system is important to establish the start of exogenous feeding. Besides, the appearance of rod cells increases larval photosensitivity and suggests that changes in lighting regimes could be necessary throughout the larval phase.

Effect of lipid and n−3 HUFA levels in microdiets on growth, survival and fatty acid composition of larval gilthead seabream (Sparus aurata)

Abstract The results of studies on the essential fatty acid requirement of larval gilthead seabream differ in the recommended n −3 HUFA levels in rotifers ( Brachionus plicatilis ) necessary to obtain optimal larval growth and survival. Some authors have suggested that the total lipid content of the rotifers may influence the fatty acid requirement of the larvae. In order to study this hypothesis, an experiment was conducted by feeding gilthead seabream larvae microdiets containing combinations of two lipid and two essential fatty acid levels. Fifteen-day-old gilthead seabream larvae were fed with 4 microparticulated diets containing the following lipid and n −3 HUFA levels: (A) 12.44% and 0.82%; (B) 12.72% and 2.16%; (C) 19.45% and 0.74%; and (D) 20.03% and 2.05%. Larvae were fed the experimental diets together with a small amount of rotifers cultured on bakers yeast for 12 days. Larvae fed Diet A had the lowest growth rate ( P n −3 HUFA (B and D) supported the best survival rates regardless of their lipid content. These results showed that for the lipid levels tested, the larval n −3 HUFA requirement did not decrease as the lipid level in the diet decreased.

Rearing techniques for red porgy Pagrus pagrus during larval development

Red porgy can be considered as a potential candidate to diversify aquaculture production of sparids, due both to the importance of red porgy fisheries and market within Europe, and to its phylogenetic closeness to gilthead seabream. Spontaneously spawned eggs were obtained from a broodstock of wild individuals captured in Canarian waters. Different rearing trials were conducted to study the success of: (a) different egg stocking densities (100, 150 and 200 eggs/l); (b) first feeding with rotifers enriched with different products (Powersh-fish oil, freeze-dried shark eggs and Selco supplemented with Nannochloropsis sp); and (c) supply of rotifers to larval cultures at different concentrations (5, 7 and 10 rotifers/ml). In all cases, larval feeding lasted for 14 days. Larval length was measured at the time of mouth opening, at day 10 of life, and at the end of the experiments. Larval survival was estimated at the end of each experiment by counting all the remaining live larvae. Total lipid and fatty acid composition of rotifers and larvae were analyzed and discussed in relation to larval growth and survival. In this study, egg densities between 100 and 150 eggs/l and the use of rotifers enriched with Powersh-W, containing a high level of 22:6n−3, at concentrations of 5–7 rotifers/ml produced the best larval growth and survival.

Regulation of growth, fatty acid composition and delta 6 desaturase expression by dietary lipids in gilthead seabream larvae (Sparus aurata)

The Δ6 and Δ5 desaturases and elongases show only very limited activity in marine fish, and little is known of the possibility of enhancing Δ6 desaturase gene expression in these fish. The use of plant oils in marine fish diets is limited by their lack of n−3 highly unsaturated fatty acids (HUFA) despite an abundant content of the 18C fatty acid precursor linoleic and α-linolenic acids. The objective of the present study was to determine the ability of larval gilthead seabream to utilize vegetable oils and assess the nutritional regulation of Δ6 desaturase gene expression. Seventeen-day-old gilthead seabream larvae were fed during a 17-day period with one of four different microdiets formulated with either sardine fish oil (FO), soybean, rapeseed or linseed oils, respectively, or a fifth diet containing defatted squid meal and linseed oil. Good larval survival and growth, both in terms of total length and body weight, were obtained by feeding the larvae either rapeseed, soybean or linseed oils. The presence of vegetable oils in the diet increased the levels of 20:2n−9 and 20:2n−6, 18:2n−9, 18:3n−6, 20:3n−6 and 20:4n−6, in larvae fed rapeseed and soybean oils in comparison to those fed FO. In addition, a sixfold increase in the relative expression of Δ6 desaturase-like gene was found in larvae fed rapeseed and soybean oils, denoting the nutritional regulation of desaturase activity through its gene expression in this fish species. However, feeding linseed oil did not increase the expression of the Δ6 desaturase gene to such a high extent.

Improvements in the culture of Sparus aurata L. larvae in relation to the use of antibiotics, phytoplankton and rearing system

Rotifers were enriched for 24 h with a lipid emulsion and the algae Nannochloropsis sp. Some of the rotifers were treated with antibiotics (chloramphenicol and oxolinic acid). Larval sizes were recorded initially, at day 7 and at the end of the experiment. Larval survival rates were estimated at the end of the experiment. Rotifers and larvae were analysed for lipid and moisture as well as the fatty acid profile of total lipids. The number of bacterial colonies from rotifer and larvae culture was also recorded. The best growth rate was found in larvae reared in a recirculating system with addition of phytoplankton and antibiotics, although no significant differences (P < 0.05) were apparent with the same treatment without antibiotics. Larvae reared in a flow-through system with phytoplankton showed the best survival rates, but larvae receiving rotifers treated with antibiotics showed the lowest survival rates. The n−3 HUFA levels in rotifers and larvae treated with antibiotics were significantly lower than those in treatments without these chemicals. The results of this study suggest that antibiotics should not be used indiscriminately in hatcheries as they may exert an adverse effect on larval cultures.

Occurrence of skeletal deformities and osteological development in red porgy Pagrus pagrus larvae cultured under different rearing techniques

The present study describes the osteological development and the occurrence of skeletal deformities in red porgy Pagrus pagrus larvae in relation to the intensification of the rearing system. Eggs obtained from natural spawning were cultured under two different rearing systems: intensive (100 eggs l⁻¹) in 2000 l and semi-intensive (mesocosm) system (5 eggs l⁻¹) in 40,000 l conico-cylindrical tanks. Fish samples were periodically collected along the development from hatching to juveniles at 95 days post hatching (dph). Osteological development, meristic counts and the presence of skeletal deformities were evaluated. Despite the external appearance of the juveniles being similar to wild standards, X-ray studies revealed a high number of fish (semi-intensive: 37·8%; intensive: 45·5%) with skeletal deformities. Regardless of the rearing system, no significant interaction was found between the per cent of the most common deformities, axial deviations (lordosis and presence of fused vertebrae). Cranial deformities and kyphosis incidences, however, were significantly higher in intensively cultured P. pagrus. Also, the fused vertebrae in these fish were located mainly in the caudal area instead of pre-haemal area for semi-intensively reared P. pagrus. Moreover, a significant interaction was found between the total number of vertebrae and the type of rearing system used; fish from the intensive system showing a higher number of fish with an extra vertebrae (10 abdominal + 15 caudal). Present results suggest a relationship among feeding sequence, osteological development and deformity incidence and location in P. pagrus larvae.

Control of Shell-Boring Polychaetes in Haliotis Tuberculata Coccinea (Reeve 1846) Aquaculture: Species Identification and Effectiveness of Mebendazole

ABSTRACT The experimental culture of Haliotis tuberculata coccinea is carried out at the Institute of Marine Science of Canary Islands (Gran Canaria, Spain) based on specimens captured in their natural habitat and conditioned as broodstock. In 2008, an outbreak of shell-boring polychaetes affected the culture. This study aims to identify some of the species of shell-boring polychaetes that affect H. tuberculata coccinea and, second, to assess the effectiveness of multiple mebendazole applications for the control of these pests. Mebendazole was applied as baths of Lomper (Esteve Laboratory, Barcelona, Spain) using the concentrations 6 mL/L, 8 mL/L, and 10 mL/L, with 0 mL/L as a control measure. Abalone were exposed to 3 mebendazole baths over a 3-day period (1× 3-h bath applied for 3 consecutive days), as recommended in the drug directions for human use. After each bath, the abalone were returned to their original culture tanks until the following day. After these 3 days, the abalone were kept in their original culture tanks for a term of 1 mo. Each month, as a result of the effect of mebendazole, moribund and/or dead polychaetes became detached from the burrows and were found at the bottom of the tank during the first 15 days after the baths of mebendazole were applied. The polychaetes expelled from the shells were counted, collected, and fixed in 10% buffered formaldehyde for subsequent identification. This process was repeated month after month until no moribund and/or dead shell-boring polychaetes were found after the application of the baths of mebendazole. The shell-boring polychaete species was identified as Polydora hoplura (Claparède 1870). The efficacy rate of Lomper was around 99% in all the concentrations tested when it was applied during a 7-mo term (each month, 1 bath was applied for 3 consecutive days), and abalone mortality was limited to highly infested animals only. Monitoring of abalone weight gain suggested that the mebendazole treatments did not affect growth significantly during the study period.