Characterization of digestive enzymes protease and alpha‐amylase activities in the thick‐lipped grey mullet (Chelon labrosus,Risso 1827)

Activities of pepsin, trypsin, chymotrypsin, alpha‐amylase and lipase, as well as their optima and stability to pH and temperature, were determined in digestive extracts of thick‐lipped grey mullet, Chelon labrosus, of three different sizes: Group 1 (45.2 ± 3.0 g), Group 2 (180.9 ± 4.2 g), and Group 3 (328.5 ± 43.3 g). SDS‐PAGE zymograms were also used to assess the role of serine proteases in the digestive tract of C. labrosus. On the other hand, possible changes in the digestive enzyme profile of C. labrosus during development were observed, with a comparatively lower pepsin activity, higher activities of alkaline proteases and alpha‐amylase and no lipase activity recorded in pre‐adult specimens. It is suggested that these variations are linked to the changes in diet composition with age, moving from a partly carnivorous to a more herbivorous feeding habit.     

Early development and allometric growth in hatchery‐reared characin Brycon orbignyanus

The characin piracanjuba, Brycon orbignyanus (Valenciennes, 1850), has been recognized as a candidate species for aquaculture. The early morphological development and allometric growth of hatchery‐reared piracanjuba were studied from hatching to the juvenile stage, at water temperature of 27.9 ± 0.6°C. Growth, in total length (TL), was linear during that period. At hatching (3.4 ± 0.2 mm TL), the non‐pigmented free embryo had most functional systems not fully differentiated. The primordial finfold was almost completely absorbed, except the preanal segment, in individuals measuring 9.1 ± 0.4 mm TL. Retinal pigmentation occurred as early as 24 hours posthatching (hph). The yolk sac was no longer observed after 60 hph. Body proportion and growth rates changed considerably during early morphological development. The head experienced positive allometric growth in length throughout the interval of study, and at the inflexion point of 6.6 mm TL, head growth had reduced significantly, but still remained allometrically positive. Trunk length showed negative allometric growth throughout the period of study. The growth of the postanal length was allometrically positive until the inflexion point at 7.1 mm TL, and thereafter decreased to near isometric. The allometric growth changes in the piracanjuba during initial life likely result from selective organogenesis directed towards survival priorities.     

Ontogeny of the digestive tract of thick lipped grey mullet (Chelon labrosus) larvae reared in “mesocosms”

This work describes the ontogeny of the digestive tract in thick lipped grey mullet (Chelon labrosus) larvae reared until day 36 post-hatching with the semi-extensive technology in mesocosms. Diet was constituted by live preys, rotifers, Artemia and wild zooplankton, then compound diet was added from day 20 (p. h.). Linear growth, weight growth and digestive enzymes specific activities were studied during larval ontogeny. Pancreatic enzymes (trypsin and amylase) and intestinal enzymes (leucine-alanine peptidase “Leu-ala”, aminopeptidase N “AN” and alkaline phosphatase “AP”) were assayed in larvae sampled throughout the rearing trial to evaluate gastrointestinal maturation along the development.The trypsin specific activities were very high during the first two weeks and then declined as observed in marine fish species. A following increase in trypsin specific activity from day 20 was attributed notably to ingestion of particle compound diet. In contrast to the pattern generally described in fish larvae, amylase specific activity showed a continuous increase. This could be attributed to the fact that C. labrosus is an omnivorous species and suggests that the fish might be able to use efficiently diets containing higher levels of starch or other carbohydrates since the end of larval development.Relative expression of intestinal brush border membrane enzymes (AP and AN) and cytosolic enzyme (Leu-ala), showed an abrupt increase of both AP/leu-ala and AN/leu-ala ratios at day 8 (p. h.), indicating that maturation of intestinal tract in C. labrosus larvae is particularly precocious. It is assumed that larvae of C. labrosus might support early co-feeding and weaning strategies, which could reasonably be initiated since mouth opening.     

Early development and allometric growth in hatchery‐reared Eurasian perch,Perca fluviatilis L.

The morphological development and allometric growth patterns of Eurasian perch, Perca fluviatilis L., a highly valued commercial species, were studied under intensive rearing conditions from hatching up to 50 DPH (Days Post Hatch). Based on the external morphology, four different phases during early development of Eurasian perch were identified: pre‐flexion larva 0–20 DPH (5.70–10.16 mm TL); flexion larva 22–30 DPH (11.09–15.14 mm TL) and post‐flexion larva/juvenile 32–50 DPH (18.00–24.75 mm TL). The results indicate that growth period when final replacement of all temporary (larval) structures and most important changes in the shape of P. fluviatilis occurred (between 13.95 and 24.06 mm TL, during flexion and post‐flexion phase) can be considered as a transitional period between the larva and juvenile. All body segments, except trunk length and tail length showed fast growth (positive allometry) throughout the entire studied period or up to the respective inflexion point with a common tendency to isometry. In addition, the specific behaviours (e.g. pelagic way of life) of Eurasian perch larvae resulted in some characteristic allometric growth patterns in the posterior region, different from the majority of other teleosts. The results are discussed with respect to the ontogeny of the functional morphology under both ecological and aquaculture considerations.     

The effect of temperature on the development of yolk‐sac larvae of European hake (Merluccius merluccius L.) under laboratory conditions

The effect of temperature (10, 13, 16, 19 and 22°C) on hatching, development and survival of yolk‐sac larvae of European hake, Merluccius merluccius, was studied. At 22°C the experiment was suspended because all eggs died a few hours after incubation. Five morphological indicators of larval development (standard length – SL, yolk‐sac volume – YSV, oil globule diameter – OD, body height – BH and body wet weight – BW) were analysed. SL, YSV, OD and BW of newly hatched larvae were biggest in size and weight at low temperatures (P < 0.05). Throughout the experiments, the rates of changes in SL, YSV and OD varied with temperature (P < 0.05). By contrast, BH and BW remained constant and did not show significant relationship with the effect of temperature (P > 0.05). The potential model showed that the SL growth rate varied from 0.05 to 0.08 mm day^?1 from 10.5 to 19.5°C respectively. However, at all temperatures two growth phases were identified: a rapid growth phase followed by a slow one. Rapid growth rate in length and depletion in yolk‐sac and oil globule occurred at high temperatures. After total YSV and OD consumption (death by starvation), larvae died between 8 and 14 days at 19.5 and 10.5°C respectively, reaching a maximum length of 4.3 mm at 10.5°C. Metabolic indices such as: lowest threshold temperature (T₀); effective larval development (); cumulative thermal units (TC) to complete larval development; the temperature coefficient on growth rate in length (Q₁₀) and the condition factor (K) were also studied.     

Survival and growth of geoduck clam larvae (Panopea generosa) in flow‐through culture tanks under laboratory conditions

The geoduck clam, Panopea generosa, is a species from the west coast of Baja California, Mexico, and the optimization of seed production systems is still a limiting factor for its aquaculture. In this study, a flow‐through culture system was designed and tested in P. generosa larvae. Survival and growth was compared in triplicate 45‐L fiberglass tanks using three larval densities (5, 10 and 15 larvae/ml). A head tank kept constant the water inflow, with a daily renewal rate of 1.8× tank volume. The food (Isochrysis spp.) was dosed according to the ingestion rate of larvae and the dilution rate. Survival decreased linearly during the first 10 days and reached asymptotic values of ca. 20% (15 larvae/ml) and 50% (densities of 5 and 10 larvae/ml) afterwards. Mean shell length at the end of the experiment (243 ± 1.8 to 270 ± 0.7 μm) was not statistically different among treatments, even though a trend towards higher gross growth rate was observed in the treatment with the lowest density (9.5 μm/day) relative to the rest of the treatments (8.5 μm/day). It is concluded that P. generosa larvae can be successfully grown in flow‐through systems at maximum densities of 10 larvae/ml without significantly affecting their survival and growth rates. The system design was reliable, kept a constant water flow with reduced maintenance, and may represent an important option in the laboratory for increasing the stocking density of Panopea species during the larval phase.     

Effects of dietary protein on growth performance,nutrient utilization,digestive enzymes and physiological status of grey mullet,Mugil cephalus L. fingerlings reared in inland saline water

A 60 days feeding trial was conducted to illustrate the effect of graded levels of protein on the growth and metabolic enzymes of grey mullet (Mugil cephalus L.) fingerlings reared in inland saline water (ISW). Six isoenergetic (16 MJ/kg) and isolipidic (60 g/kg) diets containing 240, 260, 280, 300, 320 and 340 g crude protein (CP)/kg diet were formulated and fed to triplicate. Weight gain %, specific growth rate, protein utilizing efficiency, feed efficiency and RNA:DNA ratio were significantly higher (p < .05) in 320 and 300 g CP/kg diets. Fish fed with 240 g CP/kg diet showed significantly higher (p < .05) feed intake, whole‐body lipid content, hepatosomatic index value and liver glycogen content. Transaminase enzymes and malate dehydrogenase activities were elevated in fish fed 340 g CP/kg diet. Protease activity increased with increasing dietary CP level, but amylase activities showed an inverse relationship. No significant (p > .05) variations were observed for lactate dehydrogenase, oxidative stress enzymes, blood parameters and serum osmolality among all the treatment groups, but red blood cell count increases with increasing dietary CP levels. Based on the results, feeding dietary protein level of 300 g CP/kg is economically viable for rearing of grey mullet in ISW.     

Dietary L‐tryptophan modulates growth and immuno‐metabolic status of Labeo rohita juveniles exposed to nitrite

Effect of nitrite exposure on growth and immuno‐metabolic responses of Labeo rohita fed L‐tryptophan (TRP) was studied. Fish previously fed normal and elevated levels of tryptophan for 60 days were exposed to nitrite (2.0 mg L^?1) for another 45 days with same feeding regime. There were four treatment groups, viz., TRP₀‐N (control), TRP₀+N, TRP_0.75+N (0.75% supplemental tryptophan in the diet) and TRP_1.5+N (1.5% supplemental tryptophan in the diet). Highest weight gain% and SGR were observed in control and lowest in TRP₀+N. Dietary supplementation of elevated levels of tryptophan augmented weight gain% and SGR. Nitrite exposed groups recorded higher catalase, SOD, LDH, AST and ALT activities compared with control. However, activities reduced with additional levels of tryptophan supplementation. Nitrite exposure reduced WBC count, total protein, albumin, globulin and lysozyme activity compared with unexposed group but groups which were fed additional amounts of tryptophan restored total protein, albumin and globulin similar to TRP₀‐N. In conclusion, nitrite exposure had adversely affected growth, increased activities of LDH, AST, ALT, catalase, but decreased WBC, serum protein, lysozyme and acetylcholine esterase activity of L. rohita. Normal requirement of tryptophan was unable to combat nitrite stress. However, dietary fortification with tryptophan (minimum 0.75% of diet + normal requirement) found effective in combating nitrite induced stress.     

Comparison of growth,digestive system maturation and skeletal development in sea bass larvae reared in an intensive or a mesocosm system

The quality of development in intensive or mesocosm hatchery‐reared Dicentrarchus labrax larvae was investigated using physiological indicators assessing ontogeny. Larvae were reared in intensive (120 L tanks) and in mesocosm systems (20 m³ enclosures) with the same feeding sequence, excluding the wild zooplankton naturally available in mesocosms. Faster growth was recorded since early development [16 day after hatching (DAH)] in the mesocosm. Maturation of the digestive system also occurred earlier as indicated by the higher amylase secretion ratios, the intestinal maturation index (alkaline phosphatase/leucine–alanine peptidase and aminopeptidase‐N/leucine–alanine peptidase ratios) and the more developed intestinal epithelium at 23 DAH. Nevertheless, the delay in digestive maturation in the intensive system seemed retrieved within few days. In both the groups, the number of vertebrae ranged between 24 and 26, with the dominant class being 25 vertebrae. However, the distributions differed between treatments for meristic characteristics, ossification stages and incidence of malformation types. Loss of a vertebra was more frequent in the intensive system, while the appearance of an additional vertebra was more frequent in the mesocosm. Ossification at 37 DAH was also more advanced in the mesocosm in addition to a lesser rate and severity of skeletal malformations. It is suggested that the early nutritional contribution of mesocosm wild zooplankton, yet at densities of 0.2–0.7 prey mL^?1, had key effects on larvae development since the early stages.     

The use of diatom Skeletonema costatum on aquaculture‐produced purple sea urchin (Paracentrotus lividus) larvae and post‐larvae diet

The quality of the microalgae provided on Paracentrotus lividus larvae rearing is a primordial factor having a direct (nutritional properties) and indirect (water quality) impact on growth, competence and survival. Skeletonema costatum is a diatom commonly used in the bivalve cultivation. However, the use of this diatom in P. lividus larval cultivations is poorly known. The Rhodomonas spp. is a microalgae commonly used in sea urchin larvae culture. Three different diets were tested on P. lividus larvae and post‐larvae cultivation (D1—Rhodomonas marina, D2—S. costatum, D3—mixture of both algae). Larvae fed with the D2 diet (55.8%) and D3 (39.9%) had a survival at 15 DAH higher than D1 (5.5%). The low survival in D1 could be due to the higher microbiological load on microalgae (Vibrio alginolyticus and V. pectenicide). Larvae fed with S. costatum (D2) showed a lower development than other diets. The competency index was lower for larvae fed with the D2. These results show that microalgae diversified diets contribute to a better development of P. lividus larvae. During the settlement and post‐settlement phase, there was also a lower growth of the sea urchin fed with the D2 and a higher survival for D3.     

Effects of tagging on migration behaviour,survival and growth of hatchery‐reared Atlantic salmon smolts

The effects of four different tagging methods (PIT, anchor T‐tag, Carlin tag and dummy radio transmitter) on survival, behaviour and growth of Atlantic salmon smolts during their downstream migration were examined in semi‐natural circular channels during a natural migration period in spring. Survival of smolts was high and tagging wounds healed well in all tagging groups. Tag loss rates were generally low, being the highest (2.5%) in the dummy radio transmitter group. Total length and body mass of the tagged and untagged smolts did not differ at the end of the experiment. Migration activity of smolts generally showed similar patterns among the treatments. However, Carlin‐tagged smolts started their migration slightly later than the PIT‐tagged fish, and smolts tagged with Carlin tag or dummy radio transmitter showed less overall migration activity than fish with PIT tag.     

Effects of starvation on survival,growth and development of Exopalaemon carinicauda larvae

This study aimed at evaluating the effects of two starvation modes (fasting‐feeding mode and feeding‐fasting mode) on survival, growth and development of mysis larvae of Exopalaemon carinicauda. It showed that food restriction significantly affected the survival, growth and development in both starvation modes. With the extension of initial starvation period, a significantly reduced survival rate during metamorphosis was observed. In the fasting‐feeding mode, the duration of metamorphosis was extended and the body lengths of the individuals, which experienced successful metamorphosis to the post‐larval stage 1, were much shorter. In the feeding‐fasting mode, the extension of initial feeding period led to dramatically increased survival rate and body lengths, while there was no significant difference in duration of metamorphosis of those groups that reached to post‐larval stage. The 50% point‐of‐no‐return and the 50% point‐of‐reserve‐saturation were 3.85 days and 4.81 days respectively. The results of this study indicate that E. carinicauda mysis have the ability to withstand or recover from relatively extended starvation, but to ensure the success of commercial seedling, timely and adequate feeding is necessary. The information obtained from this study could provide a basis to optimize the feeding schedule of artificial seedling of E. carinicauda.     

Effect of dietary protein levels on growth and nutrient utilization of fringe‐lipped carp,Labeo fimbriatus (Bloch) fingerlings

Effect of various dietary protein levels on growth and nutrient utilization were studied in fringe‐lipped carp, Labeo fimbriatus fingerlings for 60 days. Five practical diets containing graded protein levels of 200, 250, 300, 350 and 400 g kg^?1 with respective digestible protein (DP) contents of 192.4, 244.5, 291.6, 339.4 and 391.4 g kg^?1 were evaluated as five treatments, T1, T2, T3, T4 and T5, respectively, in triplicate. Each experimental tank (80‐L) contained eight fingerlings (4.9 ± 0.1 g) and was subjected to continuous aeration and 25% water replenishment daily. The fish were fed two times daily at 8:00 and 14:00 h to satiation. Significantly higher (P < 0.05) absolute growth and thermal growth coefficient, and lower feed conversion ratios (FCR) were observed in T2–T4 than T1 and T5. Protein efficiency ratio (PER) and protein productive values (PPV) were highest in T2 and lowest in T5. Significantly higher (P < 0.05) apparent protein digestibility was perceptible in T2–T4. While specific amylase activity declined linearly with increasing DP : DE, the protease, trypsin, aspartate aminotransferase and alanine aminotransferase established polynomial relationship. Based on live weight gain, PER and PPV fitted to quadratic model optimum DP levels were estimated as 301.4, 260.0 and 273.0 g kg^?1, respectively.     

Embryonic and larval development in the semelparous Nereid polychaete Hediste diversicolor (OF Müller, 1776) in Norway: Challenges and perspectives

Knowledge of broodstock manipulation, gametogenesis, artificial fertilization and larval nutrition is a prerequisite to reach a large‐scale production of the polychaete Hediste diversicolor. In this study, the characteristics of oogenesis and spermatogenesis and embryonic and larval development were observed. Moreover, the effect of temperature on embryonic development and effect of diets and sediment on earlier juvenile development were tested. Mature oocytes were characterized by a spherical shape with a diameter of about 200 μm. They showed radial symmetry with a large centric nucleus surrounded by four distinctive layers. Embryo cleavage started between 4 and 10 hr after fertilization, and developed to the swimming trochophore stage after 5–6 days after fertilization at 11°C. The rate of embryonic development increased with a temperature increase from 6.1 to 21.2°C. The trochophore larvae increased in length from day 9 and commenced differentiation into 3‐setiger stage larvae at day 12. The length growth of larvae until 7‐setiger stage was 0.43 mm/day when fed with fish feed, while those fed shellfish diet and smolt sludge both grew around 0.21–0.23 mm/day. The results suggest that fish feed is a superior diet compared to shellfish diets and smolt sludge for the early larval stage of H. diversicolor.     

Recombinant expression and comparative bioactivity of tongue sole insulin‐like growth factor (IGF)‐1 and IGF‐2 in Pichia pastoris

Insulin‐like growth factor (IGF) plays a key role in the complex system that regulates bony fish growth, differentiation, and reproduction. In the current study, recombinant tongue sole IGF‐1 and IGF‐2 were obtained using the Pichia pastoris expression system and their comparative bioactivities were investigated. Tricine–SDS–PAGE and western blot analysis showed that the recombinant tongue sole IGFs were secreted into the culture medium and had a molecular weight of 8.7 kDa. The optimal incubation time and pH for recombinant expression of IGFs were 36 hr and 5.0 respectively. Functional analysis demonstrated that both recombinant tongue sole IGF‐1 and IGF‐2 significantly promoted cell proliferation of MFC‐7 in vitro. In addition, the recombinant tongue sole IGF‐1 and IGF‐2 proteins could suppress hepatic mRNA levels of igf‐1 and igf‐2 in vitro, which showed that they have similar physiological functions. Taken together, the biologically active recombinant tongue sole IGF‐I and IGF‐II proteins will allow us to further investigate their physiological roles in growth regulation of this species. Furthermore, the present results also hinted at the potential application of these two recombinant IGF‐I and IGF‐II proteins into the tongue sole farming industry.     

Efficacy of calcein as a chemical marker of green‐lipped mussel (Perna canaliculus) larvae and its potential use for tracking larval dispersal

An optimal chemical shell marking protocol was developed for the New Zealand green‐lipped mussel, Perna canaliculus with a view to its future use in larval tracking experiments. Larval P. canaliculus aged either 10, 15 or 19 days post fertilization were immersed in treatments of 50, 100 and 200 mg L^?1 of calcein for a period of 24 h before measurements of shell mark brightness were taken. There was 100% marking success in all calcein treatments for all age classes, with 19‐day larvae immersed in 200 mg L^?1 calcein producing the brightest mark. Growth was not affected by calcein immersion; however, 10‐day larvae exhibited significantly higher levels of mortality compared with 15‐ and 19‐day larvae suggesting a reduced resilience to the marking protocols in younger larvae. In a mass staining experiment, a solution of 100 mg L^?1 calcein was used to successfully stain15.6 million hatchery reared P. canaliculus larvae. Calcein, therefore, offers a low impact method with which to stain the sensitive early life stages of this species thus providing a rapid method for identifying individuals of interest, i.e. individuals released in the wild or specific family lines within a hatchery environment.     

Combined effect of temperature and live feed period on growth and survival of Coregonus maraena (Bloch, 1779) larvae

Despite intensive stocking programmes, wild Maraena whitefish Coregonus maraena (Bloch, 1779)‐stocks are in danger of extinction in the Baltic sea region. Current aquaculture rearing methods in recirculating aquaculture systems (RAS) are lacking efficient protocols for transitioning larvae from endogenous to exogenous feeding. In this 34‐day‐experiment the offspring of wild fish was used. Maraena whitefish larvae were weaned at three different temperatures (15.9°C, 17.9°C, 19.7°C) and three feeding regimes, resulting in nine treatments. The first group received pure live feed (freshly hatched Artemia sp. nauplii) for 10 days and a 1:1‐mixture of live and dry feed for 3 days. The second group received the live and dry feed mixture for 13 days and the third group directly received a commercial dry feed. All nine treatments were stocked in triplicate with 500 larvae per tank (19 ind. L^?1). Feeding started 4 days post hatch. Survival was highest in the pure dry feed groups and lowest in the pure live feed groups. In contrast to growth, which was highest in the live feed groups and lowest in the dry feed group. Higher temperature increased growth in the live feed groups but had no effect on survival in all groups. These results will enhance the weaning of C. maraena in recirculation aquaculture.