Influence of diet on pepsin and some pancreatic enzymes in sea bass (Dicentrarchus labrax) larvae

Sea bass (Dicentrarchus labrax) larvae were weaned on day 25 with a basal compound diet as microparticles, enriched with a mixture of amino acid (SLAA) or protein (SLP). The control group was fed live prey (LP). Larval growth was significantly lower in the weaned groups. Trypsin activity was enhanced by the amino acid mixture. Pepsin was detected from day 24. Pepsin specific activity was significantly higher in the weaned groups, suggesting that the presence of pepsin is not such a determinant factor in the digestive process of sea bass larvae, since a poor growth was observed in these groups.     

Growth of Perca fluviatilis larvae fed with Artemia spp. nauplii and the effects of initial starvation

Experiments were performed to test the growth and survival of perch larvae fed with Artemia spp. nauplii and to determine the most advantageous time of first feeding. During a first experiment, perch larvae were divided into groups according to their hatching dates. Daily ingestion of Artemia spp. nauplii began significantly 2 days after mass larvae hatching. By day 7, the daily prey acceptance was over 75% among the separate larvae groups. During a second 14-day feeding experiment, larvae fed after a 2- or a 3-day delay showed the best growths: 12.5 mm and 13.7 mg. There was no significant difference (P > 0.05) among survival rates (85.3%) of perch larvae fed after a 1-, 2- or 3-day delay.     

Growth and survival of tench larvae fed under different feeding strategies

Two feeding experiments were conducted to evaluate the growth and survival rates of tench Tinca tinca L. larvae when initially fed with a combination of three different brands of Artemia nauplii under two conditions: (A) in the laboratory and (B) on a commercial farm. At the same time, a protozoan culture of the freshwater ciliate Colpoda cucullus was additionally tested in one of the experiments to possibly enhance the initial hunting behaviour of the larvae. The larvae were fed every 4 h from the onset of exogenous feeding up to 14 days of age. Three types of commercial Artemia products, mainly differing in size and high unsaturated fatty acid (HUFA) content, were used. One group was fed with C. cucullus as a starter. The different combinations of Artemia nauplii were used to evaluate possible effects on larval growth. The final growth at 26.3 °C, expressed in length and weight, did not show significant differences, suggesting the use of the most economically feasible Artemia strain studied. The experiments confirmed that using smaller prey during the first 2 days of feeding increases their survival rate, although the mean final survival rate was high (89%). In the experiment carried out at the commercial fish farm facilities, experimental groups were also fed with Artemia nauplii, using the EG type either enriched or not enriched with HUFA. Again, one of the groups was offered the ciliate C. cucullus as initial feed. Final growth showed significant differences when using Colpoda culture as a starter feed, although this test resulted in the lowest survival rate (69%), indicating that further studies on the management of its culture should be undertaken to improve the applicability of the technique. The mean final survival rate was 83%.     

Growth response of African catfish,Clarias gariepinus (B.), larvae and fingerlings fed protease‐incorporated diets

African catfish, Clarias gariepinus (B.), is one of the promising freshwater fish species in African aquaculture but the expansion of its farming needs more production of its larvae. The use of live food organisms at first feeding for larvae is still obligatory. That increases the cost of larvae production. Hence, the incorporating of exogenous enzymes especially protease in artificial microdiets may provide affordable alternatives for enhancing the larvae performance. The present study was carried out to evaluate the growth and survival of larvae or fingerlings of African catfish fed artificial diets incorporated with different protease levels. Four artificial diets were formulated and enriched with protease enzyme at levels of 0.0, 750, 1,000, and 1,250 unit/kg diet; after that diets were made into crumbles (100–200 µm diameter). After absorption of the yolk sac, diets were offered to fish larvae (3.6 ± 0.2 mg) in triplicates as a starter feed up to apparent satiation every two hours for 30 days. In another treatment, fish larvae were fed on newly hatched Artemia nauplii (2,500 Artemia/L) as a starter food. In another experiment, African catfish fingerlings (10.1 ± 1.6 g) were fed on the same diets up to satiation twice a day for 2 months. It was noticed that the dietary protease improved larval growth and survival but not as Artemia nauplii did where fish larvae fed on Artemia nauplii showed highest growth and survival followed by those fed a diet enriched with 1,250 unit/kg diet of protease. The mortality of larvae fed protease‐enriched diets as well as the control diet was occurred mostly at the first week reaching its maximum at the third week. The poor growth was observed with fish larvae fed the control diet. Meanwhile, catfish fingerlings fed protease‐enriched diets showed higher growth over those fed the control diet. The larvae survival (11.0%–41.7%) was enhanced by increasing protease levels and it was lower than that of fingerlings (95.6%–100.0%). Furthermore, protein retention and digestibility were significantly improved with protease supplementation over the control diet especially at a level of 1,000 unit/kg diet. As compared with the previous studies, live food should be used in larvae rearing for the first week after that a starter diet enriched with protease at levels of 1,250 unit/kg diet should be used. In case of fish fingerlings, the dry diets should be enriched with 1,100 unit/kg diet to improve diet digestibility and subsequently enhance their growth.     

Diet composition,feed preferences and mouth morphology of early stage silver therapon (Leiopotherapon plumbeus,Kner 1864) larvae reared in outdoor tanks

This study examined the diet composition, feeding preferences, and mouth morphology of the silver therapon (Leiopotherapon plumbeus, Kner 1864) larvae under captive conditions. Larvae were reared in outdoor tanks (4 m³) with natural food grown 2 weeks prior to start of larval rearing. Food preference was measured by the Chesson's electivity index (α_i). Gut content analysis of larvae sampled between 5 and 25 days after hatching (DAH) showed the dominance in the diet by zooplankton, mainly copepod nauplii, cladocerans and insect larvae. Small fish larvae (5–9 DAH; 3.32–6.29 mm standard length) preferred cladocerans, ciliates and copepod nauplii; whereas older larvae (12–25 DAH; 5.45–19.26 mm standard length) preferred insect larvae over cladocerans and adult insects. The mouth gape size at 5 DAH was 359 μm and increased to 3.75 mm at 40 DAH when body size grew at an average rate of 0.59 mm d^?1. The standard length (SL) of L. plumbeus larvae was strongly associated with mouth size (r² = 0.98, P < 0.05), indicating a progressive increase of ingested prey size of the fish larvae. These results clarified the early life feeding ecology of this species, which is essential in developing effective hatchery techniques.     

Malnutrition may affect common sole (Solea solea L.) growth, pigmentation and stress response: molecular, biochemical and histological implications

In the last decades there have been several evidences that traditionally used live preys like rotifers and Artemia salina have nutritional deficiencies that result in a general decrease of fish health, causing anomalies in the development, in growth and in pigmentation. In this study a partial of total replacement of traditional live preys with preserved copepods that represent the natural food of the larvae was evaluated during Solea solea culture. In this study a positive effect of co-feeding preserved copepods in sole larviculture was observed since larvae fed this diet growth and survived better, showed a better tolerance to captive conditions and had a better response to the final thermal/density stress-test with respect to larvae fed a traditional diet. Morphometric data were fully supported by molecular and biochemical ones. Moreover, liver histological investigations, revealed that the inclusion of preserved copepods in the larval diet was able to improve lipid assimilation. In conclusion, preserved copepods may be considered a suitable food for sole when used as a supplement to the traditional diet based on rotifers and Artemia nauplii.     

Evaluation of the probiotics Bacillus subtilis and Lactobacillus plantarum bioencapsulated in Artemia nauplii against vibriosis in European sea bass larvae (Dicentrarchus labrax, L.)

Two potential probiotics Bacillus subtilis and Lactobacillus plantarum were evaluated for use in aquaculture as preventive measures against vibriosis. In vitro evaluation of the probiotics using co-culture assays with the pathogen Vibrio anguillarum and testing for the production of antibacterial substances showed the presence of antagonism and confirmed the production of antibacterial substances. Both potential probiotics were administered to the live fish feed Artemia franciscana nauplii, offering protection against a subsequent challenge of the nauplii with the fish pathogen V. anguillarum, with best survival rates of the nauplii and the most efficient protection offered by B. subtilis. Nauplii enriched with B. subtilis were further used to evaluate the protection of sea bass larvae against vibriosis. The untreated group of fish challenged with V. anguillarum presented low survival of 36.7 %, while the fish treated with nauplii enriched with the probiotic B. subtilis showed significantly increased survival rates of 86.7 % after challenge with the pathogen. The survival of healthy unchallenged fish treated with the probiotic was not significantly different from control unchallenged fish (90-94 %). Our results indicate that B. subtilis is a probiotic suitable to be used for the prevention of vibriosis in fish larvae and can be safely administered through their live feed Artemia nauplii.     

The use of live and frozen Artemia salina nauplii enriched with fluorochromes for mass‐marking vendace Coregonus albula (L.) larvae

The aim of this study was to investigate the use of Artemia salina nauplii enriched with chosen fluorochromes for mass marking vendace Coregonus albula (L.) larvae. In the experiment, vendace larvae (6 DPH) were fed with live or frozen A. salina nauplii immersed in tetracycline hydrochloride (TC), calcein (Cal) or alizarin red S (ARS) for four subsequent days. More successful effects (marking otoliths) were obtained by feeding the fish with live nauplii enriched with fluorochromes, regardless of the dye type. The highest percentage of marked otoliths (100%) was recorded in the group fed with live nauplii immersed in TC. In the groups fed live or frozen nauplii enriched with Cal and ARS, a lower percentage of marked individuals (63.3%–73.7% and 56.7%–63.3%, respectively) were recorded. The survival rate of vendace larvae in particular groups oscillated between 93.7% and 95.7%. There were no significant differences in the total body length and body weight of the reared vendace larvae among different groups. In conclusion, for mass marking of vendace larvae using a feeding method, fish fed A. salinalive nauplii enriched with TC at a dose of 600 ppm is recommended for fishery practice.     

Consumption and gut evacuation rate of laboratory‐reared spotted seatrout (Sciaenidae) larvae and juveniles

The temperature and mass dependence of maximum consumption rate was measured for larval and early juvenile spotted seatrout Cynoscion nebulosus . Maximum consumption ( C _MAX) estimates were obtained from feeding and gut evacuation experiments on larvae (3·8–19 mm standard length, L _S) at three temperatures (24, 28 and 32° C), and maximum consumption experiments on juveniles at three temperatures (20, 26 and 32° C). Feeding levels were determined for larvae fed live prey ( Brachionus plicatilis and Artemia salina ) ad libitum . The midgut and total evacuation times were estimated for fish feeding continuously and discontinuously using alternate meals of tagged and untagged live prey. Temperature and fish size had significant effects on gut evacuation and consumption. The gut evacuation time increased with increasing fish size, and decreased with increasing temperatures. Mass‐specific midgut contents increased for small larvae <0·156 mg dry mass ( M _D)( c . 4 mm L _S), and decreased for larger larvae and juveniles. Maximum consumption was modelled by fitting a polynomial function to a reduced dataset of individuals feeding at high levels. The C _MAX model predicted an initial increase in specific feeding rate from 70 to 155% M _D day⁻¹ for small larvae, before declining for larger larvae and juveniles.     

Releasing of the European sea bass Dicentrarchus labrax (Linnaeus) in the Adriatic Sea: Large-volume versus intensively cultured juveniles

The main goals of this project were to evaluate if artificial reefs are suitable sites for releasing hatchery-reared sea bass and if intensively and extensive large-volume cultured sea bass are suitable to be released into the wild for stock enhancement purposes. Large-volume cultured bass were reared in lower densities compared to intensively cultured ones and, when fry were about 90 days old, were transferred into external ponds connected to the channels of the surrounding marsh, where that they could integrate pellet food sources with live prey. Intensively cultured bass were fed for 55 days with Artemia salina (Linnaeus, 1758) and then with pellets. Underwater visual census (UVC) and fishing sampling were carried out to verify the presence of the tagged specimens at the artificial reef. A low mortality rate after tagging was observed and good tag retention was recorded. Individuals dead after tagging were statistically smaller than survivors. A total of 45 tagged bass (42 large-volume and 3 intensively cultured) were returned by fishers and 16 specimens were observed during UVC (15 large-volume and 1 intensively cultured). The majority of recaptured bass concentrated in the surroundings of river mouths and harbours suggesting that, after release, sea bass migrated towards shallower and brackish waters. Subsequently, as they grew, they came back towards deeper waters and tended to aggregate around artificial structures. Analysis of stomach contents of returned individuals confirmed their dependence on hard-substrate food items. The study provides evidence to support the suitability of large-volume juveniles for restocking purposes, due to their ability to prey on wild food and their endurance to the stress caused by release operations.     

Food selection and growth of young snakehead Channa striatus

Food selection and growth of young snakehead Channa striatus were studied in the laboratory and in a field trial. In the laboratory, first-feeding snakehead larvae of 6–7 mm total length (TL) with a mouth opening of 0.55 mm selected for Artemia nauplii, and against formulated feed. Fish began feeding on formulated feed at 12mm TL when their mouth width reached 1.0mm. In both laboratory and field trials, snakehead diets changed as fish size increased. For fish 15–20 mm TL, cladocerans and copepods were 96.5% of their diet. With fish 30–40 mm TL, zooplankton consumption was greatly reduced while benthic organism consumption increased. Fish 45–50 mm TL fed exclusively on benthic invertebrates. Diet shift from zoo-plankton to benthic invertebrates was not due to reduced zoo-plankton availability, but was instead related to changes in gill raker structure. Low density of benthic invertebrates in the field trial caused reduced fish growth rates when fish switched diets from zooplankton to benthos. Our results indicate that snake-head can take Artemia nauplii as a larval starter food, then accept formulated feed at ≥12 mm TL. Zooplankton can serve as food for snakehead < 40 mm, but formulated feed should be provided for larger fish which are unable to catch zoo-plankton.     

The occurrence of juvenile pigmentation abnormalities in plaice (Pleuronectes platessa) larvae fed on enriched and unenriched Artemia salina nauplii

The effect of diet on the frequency of malpigmentation was investigated in laboratory-reared plaice ( Pleuronectes platessa ) fed on either enriched or unenriched Artemia salina nauplii . The juveniles fed continuously on unenriched Artemia during their larval period showed 29.0% normal pigmentation whereas those fed solely on enriched Artemia , showed 63% normal pigmentation. The possibility of a critical period for pigment development was tested by changing the diet of larvae at different ages. Results suggested that a critical period is less important than the duration of time fed on a nutritionally complete diet.     

Drug assimilation in the tissue of European sea bass (Dicentrarchus labrax) fry delivered orally through bioencapsulation

Assimilation levels of the antibacterials trimethoprim (TMP) and sulfamethoxazole (SMX) in sea bass ( Dicentrarchus labrax ) fry tissue administered orally were investigated. A 1:5 TMP and SMX combination incorporated in an oil emulsion (Selco) at 20 % and 40 % concentrations (w/w) were bioencapsulated in Artemia (Instar II) nauplii. Chemotherapeutics-loaded ('medicated') nauplii were fed to the sea bass fry and drug concentrations in the tissue were analysed by high-performance liquid chromatrography (HPLC). Fish fed 40 %'medicated' Artemia assimilated significantly higher levels of chemotherapeutics compared with those fed 20 %'medicated' Artemia. Chemotherapeutics given at 40 % reached peak levels (19.3 μg TMP/g DW and 23.31 μg SMX/g DW) within 2 h while those at 20 % peaked (8.74 μg TMP/g DW and 6.73 fig SMX/g DW) after 5 h. TMP persisted in the tissues longer (up to 72 h) than SMX (12–16 h), suggesting a more efficient uptake and retention of the former and/or faster metabolism and elimination of the latter.     

Manipulation of dietary lipids, fatty acids and vitamins in zooplankton cultures

1. A wide range of species that are cultivated in commercial mariculture are planktonic during at least part of their life cycle; for example, the larval stages of shellfish (shrimp and molluscs) and the live feeds (rotifers, brine shrimp, copepods) used in the larviculture of marine fish and shellfish. Over the last decades various techniques have been developed to deliver nutrients to these zooplanktonic organisms either through artificial diets or by manipulating the composition of the live prey fed to the carnivorous stages. This paper reviews the methodology that has allowed aquaculturists to gain knowledge of nutritional requirements and may offer interesting opportunities for ecologists to verify the importance of key nutrients in the natural food chain of marine as well as freshwater ecosystems.
2. Live micro-algae can be replaced partially or completely in the diet of filter-feeders such as rotifers, Artemia , shrimp larvae and bivalves, by various types of preserved algae, micro-encapsulated diets and yeast-based diets, whereas lipid emulsions and liposomes may be utilized to supplement specific lipid-and water-soluble nutrients, respectively. Microbound and micro-encapsulated diets have been designed to supplement live feed in the culture of micro-predators such as fish and shrimp larvae.
3. Live prey organisms, in particular rotifers and Artemia , can be 'bio-encapsulated' with a variety of enrichment diets to manipulate their content in certain nutrients, including ω3 highly unsaturated fatty acids (FA) and the vitamins C, A and E. Nevertheless, the enrichment techniques are not applicable for all nutrients and prey organisms. Phospholipid composition is difficult to manipulate through the diet of live feed and the enrichment of the essential FA docosahexaenoic acid (DHA) is hampered in most Artemia species due to the catabolism of this FA following enrichment.     

Live food mediated vitamin C transfer to Dicentrarchus labrax and Clarias gariepinus

Live food enrichment techniques, using formulated diets and emulsions for improving the nutritional quality of Brachionus and Artemia , were studied as a tool for transferring ascorbic acid (AA) to fish larvae.
Artemia nauplii enriched for 24 h with an experimental emulsion containing 20% HUFA and 0%, 10% and 20% ascorbyl palmitate (AP) were administered to catfish larvae in a 20-day feeding trial. Survival was not affected by the dietary AA, but from day 7 onwards a significantly positive effect of supplemental AA on growth was demonstrated. At the end of the experiment the 20% AP group weighed 30% more than the control (0% AP), i.e. 9.5 and 6.3 mg DW, respectively. Evaluation of the physiological condition was demonstrated by salinity tests. In all three treatments larval growth was relatively low, and it still has to be verified if extra vitamin C in the diet really promotes growth. Seabass larvae fed on AP-enriched rotifers (days 4–12) and Artemia nauplii (days 13–46) showed no significant differences in production characteristics nor in stress resistance, however, for all salinity stress tests the 20% AP group performed better. AA was well incorporated into the predator larvae from the Brachionus feeding onwards.     

Allometric growth and functional development of the gut in developing cod Gadus morhua L. larvae

The development of the gut epithelium in cod Gadus morhua was studied during the larval period in intensive rearing systems. Cod larvae were fed enriched rotifers from mouth opening. On 17 days post‐hatch (dph) one group of larvae were fed Artemia sp. nauplii while another group were fed both rotifers and a formulated diet (co‐fed). At the end of the experiment (30 dph) larvae receiving live feed were almost three times larger than the co‐fed larvae, although no clear signs of pathological effects due to feeding regime were found in any larvae sampled for morphological studies. The midgut volume in larvae fed live feed increased by a factor of 38 during the experiment, and in particular volume increased rapidly between 24 and 30 dph. The enterocyte size increased between 12 and 24 dph from 652 ± 64 to 1479 ± 144 μm³ (mean ±s .e .). When enterocytes reached their maximum size, several morphological changes in the gut epithelium were initiated, such as increased number of mitochondria per enterocyte, increased size of the nuclei and a considerable increase in microvilli surface area. The mitochondrial membrane structures changed during the experiment, suggesting a maturation process of the mitochondria. The midgut development was strongly related to larval size rather than age. On 30 dph co‐fed larvae were equal in size to Artemia sp. fed larvae on 24 dph. This was reflected by equal values of estimated midgut volume, midgut length and total number of enterocytes and the number of mitochondria per enterocyte. The microvilli surface area, however, was significantly larger in co‐fed larvae on 24 dph compared to live‐feed larvae on 30 dph. This increase in absorptive surface was probably a response to suboptimal feeding conditions. The strong correlation between gut development and larval size and the lack of clear pathological effects, suggested that the gut tissue is flexible and can withstand periods of suboptimal nutrition at this stage.     

Development of a bacterial challenge test for gnotobiotic sea bass (Dicentrarchus labrax) larvae

The use of probiotic microorganisms in aquaculture is gaining a lot of interest. Gnotobiotic model systems are required in order to fully understand the effects and modes-of-action of these microorganisms, as the native microbial communities present in non-sterile animals can lead to false conclusions. In this study, a gnotobiotic sea bass larvae ( Dicentrarchus labrax ) test system was developed. In order to obtain bacteria-free animals, the eggs were disinfected with glutaraldehyde and subsequently incubated in a solution of rifampicin and ampicillin. Axenity was confirmed using culture-dependent and -independent techniques. The gnotobiotic larvae were fed axenic Artemia sp. from 7 days after hatching onwards. In the challenge test, one of the three opportunistic pathogens, Aeromonas hydrophila , Listonella anguillarum serovar O1 and O2a, was added to the model system via the water and encapsulated in Artemia sp. Only serovar O2a led to increased mortality in the sea bass larvae. The presented gnotobiotic model can be used for research on, among others, reciprocal metabolic effects between microorganisms and the host (e.g. as measured by gene expression), immunostimulants, pharmacological research and the histological development of the gastrointestinal tract and growth of larvae.     

Seasonal differences in muscle fibre recruitment of pilchard larvae in the north‐western Mediterranean

The recruitment of slow and fast myotomal muscle fibres with respect to growth in body length in European pilchard larvae Sardina pilchardus [(3·5–13·5 mm standard length ( L _S)] was significantly higher in November 1998 than February 1999. This resulted in a significant seasonal difference in the relationship between fibre number and L _S, particularly for the fast muscle. Mean sea surface temperature was c . 6° C higher in November than February, whereas the mean abundance of potential prey items (copepod nauplii) was comparable between cruises. Laboratory and field data obtained from other clupeid species have indicated the importance of early thermal experience on muscle fibre recruitment patterns. Differences in average sea temperature therefore provide a plausible mechanism for the observed seasonal differences in muscle growth characteristics.     

Uptake and processing of a Vibrio anguillarum bacterin in Artemia franciscana measured by ELISA and immunohistochemistry

Nauplii of Artemia franciscana were incubated in two different concentrations (undiluted and 1:9 in autoclaved sea water) of a divalent bacterin composed of two different serovars of Vibrio anguillarum. In order to investigate uptake and further processing of a bacterin in the live feed organism A. franciscana, immunohistochemistry was applied, visualising the presence of whole bacterial cells and antigens from the bacterin in individual nauplii. By using ELISA, it was shown that approximately 1.5-2-5 x 10(5) cells were incorporated into each Artemia under the conditions used. Maximum incorporation of cells was measured after 30 min, whereas after 60 min there was a decline to levels of 0.9-1.6 x 10(5) cells per Artemia. Immediately after incubation in the bacterin solution, the nauplii were transferred to a culture of the alga Isochrysis galbana, in order to simulate transfer of the nauplii to rearing tanks for fish larvae. From the ELISA, it could be concluded that the incorporated bacterial cells were excreted from the Artemia nauplii rapidly, however a large variation among different nauplii could be visualised by immunohistochemistry.