Applying the ontogeny of digestive enzyme activity to guide early weaning of pigfish,Orthopristis chrysoptera (L.)

Weaning marine fish larvae from live prey to a dry microdiet is an important step towards optimizing the commercial production, but early weaning is constrained by the lack of sufficient digestive enzymes at first feeding. This study quantified the activity of five digestive enzymes throughout the larval period of pigfish (Orthopristis chrysoptera [L.]) to assess ontogenetic changes in digestive abilities, and then trials were conducted that determined the optimal time for weaning. The activity of all digestive enzymes was low or undetectable at first feeding (3 days post hatching, dph; 2.5 mm standard length, SL). A substantial increase in activity occurred at 5.7 mm SL (17 dph), 6.9 mm SL (21 dph), 7.7 mm SL (23 dph), 8.4 mm SL (25 dph) and 11.2 mm SL (30 dph) for bile salt‐dependent lipase, trypsin, chymotrypsin, amylase and acid protease respectively. During the weaning experiment, larvae were co‐fed live prey and microdiet beginning 15 dph (4.8 mm SL). Live prey was withdrawn from the diet at 24, 28, 32 or 36 dph, with the control receiving live prey and microdiet throughout (to 43 dph). There were no significant differences in mean final SL among treatments, but survival was significantly lower when larvae were weaned at 24 dph compared to 32–43 dph. Based on the digestive enzyme activity and survival, weaning larval pigfish at 32 dph (11.7 mm SL) when reared at 24°C is recommended.     

Muscle growth in full-cycle cultured Pacific bluefin tuna Thunnus orientalis from early larval to juvenile stage: histochemical,immunohistochemical and ultrastructural observation

With the development of successful aquaculture techniques, the Pacific bluefin tuna (PBT) has become the most important fish species in Japan. Early muscle growth and cellularity of muscle fibers have a profound effect on ultimate body size and meat quality of fish. Larvae and juveniles of full-cycle cultured PBT were sampled at 3, 15, 29, 41, 70, 128 and 218 days post hatch (dph). Transverse body sections until 41 dph and muscle sections from 41 dph thereafter from dorso-cranial or caudal areas were stained for muscle fiber morphological and morphometric analysis and muscle sections from the dorso-cranial region were used for ultra-structural observation. Red muscle fibers appeared by 15 dph at the horizontal septum both in epaxial and hypaxial regions. Other than a single layer of superficial red fibers and those at the horizontal septum, existence of red muscle fibers was apparent in the PBT myotome. The muscle fiber diameters varied in their size even in adjacent myotomes of PBT. Overall, growth throughout the various stages was a result of both hyperplasia and hypertrophy of muscle fibers. Muscle fiber diameters were greater in the dorso-cranial than in dorso-caudal region at 41, 70 and 128 dph. These results provide additional characterization of PBT growth, which may assist with managing fish production efficiency, harvest age and eating quality.     

Partial characterisation of digestive proteases of the Mayan cichlid Cichlasoma urophthalmus

The characterisation of digestive proteases in native freshwater fish such as the Mayan cichlid Cichlasoma urophthalmus provides scientific elements that may be used to design balanced feed that matches with the digestive capacity of the fish. The purpose of this study was to characterise the digestive proteases, including the effect of the pH and the temperature on enzyme activity and stability, as well as the effect of inhibitors using multienzymatic extracts of the stomach and intestine of C. urophthalmus juveniles. Results showed that the optimum activities of the acid and alkaline proteases occurred at pH values of 3 and 9, respectively, whereas their optimum temperatures were 55 and 65 °C, respectively. The acid proteases were most stable at pH values of 2–3 and at temperatures of 35–45 °C, whereas the alkaline proteases were most stable at pH values of 6–9 and at 25–55 °C. The inhibition assays recorded a residual activity of 4 % with pepstatin A for the acid proteases. The inhibition of the alkaline proteases was greater than 80 % with TPCK, TLCK, EDTA and ovalbumin, and of 60 and 43.8 % with PMSF and SBT1, respectively. The results obtained in this study make it possible to state that C. urophthalmus has a sufficiently complete digestive enzyme machinery to degrade food items characteristic of an omnivorous fish species, although specimens showed a tendency to carnivory.     

Horizontal and vertical movements of juvenile bluefin tuna (Thunnus orientalis) in relation to seasons and oceanographic conditions in the eastern Pacific Ocean

Electronically tagged juvenile Pacific bluefin, Thunnus orientalis, were released off Baja California in the summer of 2002. Time‐series data were analyzed for 18 fish that provided a record of 380 ± 120 days (mean ± SD) of ambient water and peritoneal cavity temperatures at 120 s intervals. Geolocations of tagged fish were estimated based on light‐based longitude and sea surface temperature‐based latitude algorithms. The horizontal and vertical movement patterns of Pacific bluefin were examined in relation to oceanographic conditions and the occurrence of feeding events inferred from thermal fluctuations in the peritoneal cavity. In summer, fish were located primarily in the Southern California Bight and over the continental shelf of Baja California, where juvenile Pacific bluefin use the top of the water column, undertaking occasional, brief forays to depths below the thermocline. In autumn, bluefin migrated north to the waters off the Central California coast when thermal fronts form as the result of weakened equatorward wind stress. An examination of ambient and peritoneal temperatures revealed that bluefin tuna fed during this period along the frontal boundaries. In mid‐winter, the bluefin returned to the Southern California Bight possibly because of strong downwelling and depletion of prey species off the Central California waters. The elevation of the mean peritoneal cavity temperature above the mean ambient water temperature increased as ambient water temperature decreased. The ability of juvenile bluefin tuna to maintain a thermal excess of 10°C occurred at ambient temperatures of 11–14°C when the fish were off the Central California coast. This suggests that the bluefin maintain peritoneal temperature by increasing heat conservation and possibly by increasing internal heat production when in cooler waters. For all of the Pacific bluefin tuna, there was a significant correlation between their mean nighttime depth and the visible disk area of the moon.     

Partial characterization and ontogenetic development of pancreatic digestive enzymes in Japanese eel Anguilla japonica larvae

The pancreatic digestive enzymes, trypsin, chymotrypsin, lipase and amylase were partially characterized, and changes in their activities were examined during the initial ontogeny of Japanese eel Anguilla japonica larvae from 5 to 34 days post-hatching (dph). The pH optima of the eel larval enzymes were narrower than those other fish species; trypsin activity was highest at pH 9, chymotrypsin and amylase activities were highest at pH 7 and 8, and lipase activity was highest at pH 8 and 9. In an analysis of thermal profiles, the larval pancreatic enzymes had a high optimal temperature and high thermal stability, which are typical of fish from the tropics. At 12 and 13 dph, lipase activity and gene expression levels of trypsin (-a and -b), lipase and amylase decreased markedly, suggesting a marked change in larval metabolism at that time. These data could be useful in the development of artificial larval diets in Japanese eel.     

Molecular characterization of southern bluefin tuna myoglobin (<Emphasis Type="Italic">Thunnus maccoyii</Emphasis>)

The primary structure of southern bluefin tuna Thunnus maccoyii Mb has been elucidated by molecular cloning techniques. The cDNA of this tuna encoding Mb contained 776 nucleotides, with an open reading frame of 444 nucleotides encoding 147 amino acids. The nucleotide sequence of the coding region was identical to those of other bluefin tunas (T. thynnus and T. orientalis), thus giving the same amino acid sequences. Based on the deduced amino acid sequence, bioinformatic analysis was performed including phylogenic tree, hydropathy plot and homology modeling. In order to investigate the autoxidation profiles, the isolation of Mb was performed from the dark muscle. The water soluble fraction was subjected to ammonium sulfate fractionation (60–90 % saturation) followed by preparative gel electrophoresis. Autoxidation profiles of Mb were delineated at pH 5.6, 6.5 and 7.4 at temperature 37 °C. The autoxidation rate of tuna Mb was slightly higher than that of horse Mb at all pH examined. These results revealed that tuna myoglobin was unstable than that of horse Mb mainly at acidic pH.     

Suppressive effect of ATP on autoxidation of tuna oxymyoglobin to metmyoglobin

The discoloration of tuna meat proceeds during frozen storage at around ?20 °C. On the other hand, the discoloration of highly fresh tuna meat could effectively be suppressed even if stored at ?20 °C. However, the suppressive mechanism of the discoloration is not well understood. Here the effects of ATP on the autoxidation rate and molecular structure of tuna myoglobin are reported. The autoxidation rate of southern bluefin tuna Thunnus maccoyii myoglobin at 25 °C was suppressed in the presence of ATP especially in acidic pH range. Mixing ATP with myoglobin induced a spectral perturbation in the soret region of myoglobin. This spectral perturbation was observed as a function of the ATP concentration. Quenching of myoglobin fluorescence was also caused by ATP, saturating at around 0.5 mM ATP. According to dynamic light-scattering measurements, the molecular weights of tuna Mb changed from 15.5 to 11.3 kDa with ATP and zeta-potential measurements gave also a negative surface charge without ATP and a positive one with ATP, respectively. The above results indicate that ATP-induces changes in the conformational structure of myoglobin. The effects of ATP on myoglobin could thus provide a possible mechanism to regulate the autoxidation of myoglobin.     

Partial characterization of the digestive enzymes of Pacific bluefin tuna <Emphasis Type="Italic">Thunnus orientalis</Emphasis> under culture conditions

The digestive enzyme activities of Pacific bluefin tuna Thunnus orientalis were evaluated for specific activity and characterized for pH and temperature optima in crude extracts of stomach, caecal mass, and proximal, middle and distal intestine. A higher level of alkaline proteolytic activity was detected in the caecal mass than in the proximal intestine. Total alkaline proteases, trypsin, chymotrypsin and leucine aminopeptidase (LAP) were tested. The temperature and pH analyses showed that proteolytic activity as well as lipase were maximal in the alkaline range, with a maximum at pH 9.0 and at temperatures between 35 and 60°C, except for the pepsin, which showed maximum activity at the same temperatures but in the acid range (pH 3.0). The α-amylase activity showed a broader range in activity, both for pH and temperature, with higher activity over the alkaline pH values and higher temperature. The lipase activity seems to be nondependent on bile salts under our assay conditions, resulting in a significant activity reduction in the presence of bile salts. This knowledge will allow the development of a gastrointestinal model (everted intestine) where food or feed will be hydrolysed with the fish’s own enzymes, a project that is being undertaken in our laboratory as a contribution to the development of novel diets for tuna fish.     

Ontogeny of digestive and immune system organs of larval and juvenile kelp grouper Epinephelus bruneus reared in the laboratory

ABSTRACT:   A histological examination was made of the ontogenetic development of the digestive and immune systems of the larval and juvenile kelp grouper Epinephelus bruneus reared in the laboratory. The liver, gall bladder, pancreas and the demarcating region between the intestines and rectum were formed within 3 days post-hatch (dph). During the preflexion phase (within 16 dph), revolution of the intestine concluded, and pharyngeal teeth and the mucous cells of the esophagus were differentiated. In the transitional period to the juvenile stage (25 dph), the blind sac of the stomach, gastric glands and pyloric caeca began to form. From the viewpoint of the differentiation phase of the adult-type digestive system, the kelp grouper is similar to Heterosomata, hitherto reported. The primordial thymus, kidney and spleen were present at 12, 1 and 6 dph, and the small lymphocytes in these lymphoid organs appeared at 21, 30 and 33 dph, respectively. The developmental sequence of the lymphoid organs and the appearance ages of the lymphoid organs and small lymphocytes in the lymphoid organs in the kelp grouper are similar to those of other marine fish previously reported, except for the Pacific bluefin tuna Thunnus orientalis .     

Influence of time,storage temperature and freeze/thaw cycles on the activity of digestive enzymes from gilthead sea bream (<Emphasis Type="Italic">Sparus aurata</Emphasis>)

In this study, we tested the effects of long-term storage (2 years) at ?20 °C and short-term storage (several hours) in ice and freeze/thaw cycles on the activities of pancreatic, gastric and intestinal (brush border and cytosolic) digestive enzymes in a teleost fish species. The results revealed a significant lose in activity of pancreatic (trypsin, chymotrypsin, total alkaline proteases and α-amylase) and intestinal cytosolic (leucine–alanine peptidase) enzymes between 140 and 270 days of storage at ?20 °C, whereas in contrast, the activity of all the assayed brush border enzymes remained constant during the first 2 years of storage at ?20 °C. During short-term storage conditions, the most stable enzymes assayed were those of the enterocytes of the brush border, which did not show any change in activity after being held for 5 h in ice. Five freezing and thawing cycles did not affect the activity of the intestinal brush border enzymes and the cytosolic ones, whereas the activity of trypsin, α-amylase and bile-salt-activated lipase was significantly affected by the number of freezing and thawing cycles. No changes in pepsin activity were found in samples exposed to 1 and 2 freezing and thawing cycles.     

Universal PCR primers for ribosomal protein gene introns of fish

Human ribosomal protein (RP) gene sequences with respect to intron/exon structures and corresponding cDNA or genomic data of fish species were obtained from the GenBank database. Based on conserved exon sequences, 128 primer pairs for 41 genes were designed for exon-primed intron-crossing (EPIC) polymerase chain reaction (PCR). In reference to the draft genome sequences of the Pacific bluefin tuna (Thunnus orientalis), 12 primer pairs expected to amplify introns of the bluefin tuna with lengths of 500–1000 bp were selected and applied to six distantly related fish species belonging to the Orders Clupeiformes, Tetraodontiformes, Pleuronectiformes, Perciformes, Scorpaeniformes, and Anguilliformes. PCR amplification was observed for at least four species in each primer pair, and all fragments were larger than those expected for intronless amplification. Single fragment amplification was observed for at least seven primer pairs per species. Fragment sizes of the bluefin tuna for nine primer pairs corresponded to those expected from the genomic data. Thus, our primer pairs are potentially applicable to a wide variety of fish species and serve as an initial step for isolating single-copy nuclear DNA sequences.     

Changes in the scotopic vision of juvenile Pacific bluefin tuna (Thunnus orientalis) with growth

In cultured juvenile Pacific bluefin tuna (Thunnus orientalis), reducing the mass deaths caused by collision or contact with tank or net walls at night is a priority for seedling production. Pacific bluefin tuna is a visually dependant species, although its scotopic vision is poor. We recorded electroretinograms to investigate the visual function with growth in the dark-adapted eyes of juvenile Pacific bluefin tuna. Peak wavelengths of spectral sensitivity [38–62 days posthatch (dph), 77–167 mm standard length (SL)] were observed between 474 and 494 nm. Visual light sensitivity has a tendency to increase slightly with growth at 28–64 dph in individuals that measured 29–175 mm SL. However, visual temporal resolution did not significantly increase with growth at 38–62 days dph in individuals that measured 77–167 mm SL. These results suggest that the mass death continues between 28 and 64 dph because of low visual function and increasing swimming speed with growth.     

Effect of water temperature on the feeding activity and the resultant mercury levels in the muscle of cultured bluefin tuna Thunnus orientalis (Temminck and Schlegel)

Tuna muscle often contains high levels of mercury, and fish samples with mercury concentrations ten times higher than the specified safety standards have been reported. Here, we report on the relationship between water temperature and the concentration of mercury in the tail muscle tissue of cultured bluefin tuna Thunnus orientalis. The fish used in this study were cultured at Fisheries Laboratory of Kinki University (Amami Experimental Station, Kagoshima, Japan). One hundred fish weighing 26.2–89.4 kg were selected for analysis between February 2007 and January 2008. Water temperature during rearing ranged from 21 to 29 °C. The total mercury levels were measured using the reduction vaporizing atomic absorption method after acid digestion. Body weight increased approximately 1.5 times that observed in a previous study, despite feeding activity either being the same or less than that observed previously. The average mercury concentration in white muscle was 0.353 mg kg^?1, remaining almost constant and independent of body growth. Unlike previous studies, seasonality was not observed in this study. Based on these findings, water temperatures within a certain range were considered to stabilize feeding activity and increase feeding efficiency. Consequently, water temperature is considered to have a moderating effect on seasonal fluctuations in muscle mercury concentrations in cultured bluefin tuna.     

Vertical behavior of juvenile yellowfin tuna Thunnus albacares in the southwestern part of Japan based on archival tagging

The behavior of juvenile yellowfin tuna Thunnus albacares in southwestern Japan was investigated using archival tag data from five fish (fork length 52.5–92 cm, days at liberty 26–280 days) released near the Nansei Islands (24–29°N, 122–130°E). Vertical behavior was classified into three patterns: “shallow” (≥50 % of daytime hours at depth of <50 m), “deep” (≥50 % of daytime hours at ≥100 m), and “intermediate” (other than “shallow” or “deep”). The pooled proportion of the number of days of each behavior was 29, 25 and 46 %, respectively. The proportion of “shallow” behavior increased with fish size. The proportion of time spent near the surface at nighttime increased in the colder season, when the thermal gradient was relatively small. Surface-oriented behavior (fish remained at a depth of <10 m for more than 10 min) occurred mainly during nighttime and between November and January. Dives exceeding 500 m were occasionally observed (0.02 day^?1), and one fish dived to 1230 m. The results of our study show that yellowfin tuna were typically distributed in the mixed layer or upper thermocline where the water temperature was close to the sea surface temperature and that the vertical behavior was variable.     

Histomorphological changes in digestive tract of golden mahseer (<Emphasis Type="Italic">Tor putitora</Emphasis>) during different developmental stages

Histomorphological changes in digestive tract of golden mahseer (Tor putitora) were examined in larvae [starting from hatching to 45 days post-hatching (dph)], fry, fingerling, and adult. Digestive tract appeared during hatching, on the dorsal side of yolk sac, as a straight tube with a narrow lumen. Mouth opening and appearance of liver and pancreas were observed at 2 dph, and subsequently anal opening, appearance of goblet cells in esophagus, and posterior intestine were evident at 3 dph. The remodeling of oral cavity in terms of epithelial stratification, appearance of taste buds, and goblet cells were observed in a window of 4–5 dph. Intestinal folding was found to be initiated at 8 dph. From 12 to 45 dph, thickening of oral and esophageal mucosal/extramucosal layers, increase in intestinal folding, increases in the density of goblet cells in entire gut were observed. Within the same time window, other histological changes such as disappearance of vacuoles in liver, and abundance of zymogen granules in pancreas were also observed. Supranuclear vesicles in mid-to-posterior intestine were found to be prominent from first feeding to 45 dph; however, this phenomenon was no longer evident in fry and fingerling. Overall, the increase in intestinal folding and complexity of extramucosal layer were found to be continuous from the first appearance to adult, and this inferred the fact that the nutritional physiology, in terms of digestion and assimilation, progressively changes throughout the life stages of golden mahseer. Findings of this study will, therefore, help in preparing diets for different life stages of this fish, and in addition, the present information widens the understanding of digestive physiology of golden mahseer.     

Food ingestion,prey selectivity,feeding incidence,and performance of yellowtail kingfish Seriola lalandi larvae under constant and varying temperatures

This study tested the effects of constant and varying temperatures on newly hatched yellowtail kingfish Seriola lalandi larvae in two experiments. In Experiment I, four constant temperatures (21, 23, 25, and 27 °C) were tested under fed or unfed conditions with the fish age from the day of hatch to 24 days post hatch (DPH). Temperatures at 25 and 27 °C reduced the time of fish to reach irreversible starvation, but did not affect the percentage of fish that were able to ingest food. Fish survivals at 21 and 23 °C were significantly higher than those at 25 °C by 24 DPH, but all fish died at 27 °C by 24 DPH in the treatment with food. In Experiment II, three constant temperatures (21, 23, and 25 °C) and two varying temperatures (21–23 and 21–25 °C) were compared using fish from hatch to 28 DPH. On 4 DPH, fish ingested more rotifers, but from 6 to 9 DPH, fish ingested fewer rotifers at 25 °C than at other temperatures. On 19 and 23 DPH, fish ingested more Artemia at 25 °C than at other temperatures. At 25 °C, fish selected for Artemia nauplii earlier than at other temperatures. Fish length and survival between constant temperatures (21 and 23 °C) were not significantly different, but fish survival at the constant 21 °C or at the 21–25 °C varying temperature was significantly higher than that at the constant 23 °C or at the 21–23 °C varying temperature. This study indicates that within the range of temperature tested, the optimal temperature for the first feeding larvae is 21–23 °C after hatch and mortality is likely to occur at ≥25 °C in the first 10 DPH, but fish grew faster at 25 °C after they adapted to the increasing temperature.     

Environmental influences on Atlantic bluefin tuna (Thunnus thynnus) catch per unit effort in the southern Gulf of St. Lawrence

The Atlantic bluefin tuna (Thunnus thynnus) population in the western Atlantic supports substantial commercial and recreational fisheries. Despite quota establishment and management under the auspices of the International Commission for the Conservation of Atlantic Tunas, only small increases in population growth have been estimated. In contrast to other western bluefin tuna fisheries indices, contemporary estimates of catch per unit effort (CPUE) in the southern Gulf of St. Lawrence have increased rapidly and are at record highs. This area is characterized by the Cold Intermediate Layer (CIL) that is defined by waters <3°C and located at depths of 30–40 m in September. We investigated the influence of several in situ environmental variables on the bluefin tuna fishery CPUE using delta‐lognormal modelling and relatively extensive and consistent oceanographic survey data, as well as dockside monitoring and mandatory logbook data associated with the fishery. Although there is considerable spatial and temporal variation of water mass characteristics, the amount of available habitat in the southern Gulf of St. Lawrence (assuming a > 3°C thermal ambit) for bluefin tuna has been increasing. The percentage of the water column occupied by the CIL was a significant environmental variable in the standardization of CPUE estimates. There was also a negative relationship between the spatial extents of the CIL and the fishery. Properties of the CIL account for variation in the bluefin tuna CPUE and may be a factor in determining the amount of available feeding habitat for bluefin tuna in the southern Gulf of St. Lawrence.     

Effect of the early temperature on the growth of larvae and postlarvae turbot, <Emphasis Type="Italic">Scophthalmus maximus</Emphasis> L.: muscle structural and ultrastructural study

Turbot specimens were kept at three temperatures (T _s ): warm (W) (21–22 °C), ambient (A) (17–18 °C) and cold (C) (13–14 °C) during the larval and early postlarval stages. At 90 days posthatching (dph), all of them were transferred to ambient T until 190 dph. At 2–3 dph, the specimens showed a monolayer of red muscle and immature white fibres; external or dermomyotome cells (presumptive myogenic cells) were observed on the surface of the red muscle. In the following stages, many myogenic cells and presumptive myogenic precursors were observed within the myotome, presumably derived of the dermomyotome. When comparing the growth at the same age (2, 10, 25, 37 dph), the body length and the muscle growth were positively influenced by the warm T, being the hyperplasia the muscle parameter more significantly influenced. The development rate was also positively correlated with the high T: the beginning of the metamorphosis took place at 15, 23 and 25 dph at W, A and C temperatures, respectively, with the highest body length values at ambient temperature. The metamorphosis finished at 25, 30 and 37 dph at W, A and C temperatures, respectively, with the highest body length values at warm temperature. However, the muscle cellularity was similar in all the groups at the end of the metamorphosis. At 90 and 190 dph, the largest body length was observed at W temperature. However, the muscle cellularity was similar between A and W; the number of fibres was similar in all the groups at 190 dph, which shows the beginning of a compensatory muscle growth in A and C, mainly in A.