Changes in RNA, DNA, protein contents and growth of turbot Scophthalmus maximus larvae and juveniles

The growth potential of turbot Scophthalmus maximus larvae and juveniles was studied using nucleic acid-based indices and protein variables. The experiment was carried out from 4 to 60 days post hatching (dph). A significant increase in instantaneous growth rate during metamorphosis and retarded growth rate during post-metamorphic phase were observed. Ontogenetic patterns of DNA, RNA and protein all showed developmental stage-specific traits. The RNA:DNA ratio decreased up to 12 dph, then increased rapidly till 19 dph and fluctuated until 35 dph followed by a decline to the end. The RNA:DNA ratio was positively correlated with growth rate of juveniles during the post-metamorphic phase, whereas this ratio was not a sensitive indicator of growth during the pre-metamorphic phase and metamorphosis. The protein:DNA ratio showed a similar tendency to the RNA:DNA ratio. Changes of DNA content and protein:DNA ratio revealed that growth of S. maximus performed mainly by hyperplasia from 4 to 12 dph and hypertrophy until 21 dph during the pre-metamorphic larval phase. Growth was dominantly hypertrophical from the early- to mid-metamorphosing phase and hyperplastic thereafter. The results show that the DNA content and protein:DNA ratio can evaluate growth rates of larval and juvenile S. maximus on a cellular level.

Ontogenetic development of digestive enzymes and effect of starvation in miiuy croaker Miichthys miiuy larvae

The ontogenetic development of the digestive enzymes amylase, lipase, trypsin, and alkaline phosphatase and the effect of starvation in miiuy croaker Miichthys miiuy larvae were studied. The activities of these enzymes were detected prior to exogenous feeding, but their developmental patterns differed remarkably. Trypsin activity continuously increased from 2 days after hatching (dah), peaked on 20 dah, and decreased to 25 dah at weaning. Alkaline phosphatase activity oscillated at low levels within a small range after the first feeding on 3 dah. In contrast, amylase and lipase activities followed the general developmental pattern that has been characterized in fish larvae, with a succession of increases or decreases. Amylase, lipase, and trypsin activities generally started to increase or decrease at transitions from endogenous to exogenous feeding or diet changes, suggesting that these enzymatic activities can be modulated by feeding modes. The activities of all the enzymes remained stable from 25 dah onwards, coinciding with the formation of gastric glands and pyloric caecum. These results imply that specific activities of these enzymes underwent changes due to morphological and physiological modifications or diet shift during larval development but that they became stable after the development of the digestive organs and associated glands was fully completed and the organs/glands functioned. Trypsin and alkaline phosphatase were more sensitive to starvation than amylase and lipase because delayed feeding up to 2 days after mouth opening was able to adversely affect their activities. Enzyme activities did not significantly differ among feeding groups during endogenous feeding; however, all activities were remarkably reduced when delayed feeding was within 3 days after mouth opening. Initiation of larvae feeding should occur within 2 days after mouth opening so that good growth and survival can be obtained in the culture.

Effect of stocking density on growth, settlement and survival of clam larvae, Meretrix meretrix

To determine the optimal larval density for hatchery culture of the clam Meretrix meretrix, experiments with stocking densities of 5, 10, 20, 40 and 60 larvae ml(-1) were designed, which included the developmental stages from D-veliger to 8 days postsettlement. Shell length, settlement time and survival rate of the larvae were recorded. Results showed that, at each sampling time, larvae reared at the highest density had the smallest mean size, whereas larvae reared at the lowest density had the largest mean size. Statistical differences in mean shell length at different stocking densities appeared from day 2, and greater differences occurred with increased culture time. Specific growth rate (SGR) in the rapid growing stage (day 0-3) was negatively correlated with density; however, no correlation was found between SGR and density in the slow growing stage (days 3-7). Settlement time was prolonged and shell length of settled larvae decreased as density increased. However, larval survival rate (74.8-79.1%) was independent of stocking density. Results showed that a high stocking density, in the designated range, is feasible for larval culture of the clam M. meretrix. However, for large-scale culture, in the interest of costs and safety, a stocking density of 10-20 larvae ml(-1) is recommended. (c) 2006 Elsevier B.V. All rights reserved.

Settling abalone veliger larvae in a free-swimming microalgal culture

In the current abalone hatchery in China, insufficient diatoms on vertically placed corrugated pvc plates at later stage often could not support the growth of postlarvae up to the stage that they can feed on live macroalgae. As a result, stripping the spats (35 mm) off by anaesthetization and switching the diet from live diatoms to artificial powdered diet in combination has to be performed in most of the abalone farms. This manipulation normally leads to more than 50% mortality. Here we report the direct use of the unicellular green alga Platymonas helgolandica Kylin var. tsingtaoensis as a potential alga to be used to settle the veliger larvae of the Pacific abalone Haliotis discus hannai and to feed the postlarvae. Settlement rate of 2-day-old veliger larvae in mono culture of P helgolandica could be as high as 92% ( +/- 4.2%) on day 10 in small scale trials, higher than that in the selected benthic diatom strain (53.6% +/- 12.7%) when settled in the water in which bacteria propagation was controlled by treatment of 2 ppm of benzylpenicillinum calcium and streptomycin sulfate. Postlarvae fed solely on P. helgolandica or the selected benthic diatom Navicula-2005-A grew at rates of 40.1 ( +/- 1.9) and 45.8 (+/- 13.4) mu m day(-1), respectively, when raised at 22 degrees C until day 50 postfertilization. P. helgolandica was shown to have distinct diurnal settling rhythm characterized with a peak of settled cells in the middle of the night for cell division and a peak of free-swimming cells in the middle of the day. High density of attached P. helgolandica cells on the inner surface of the culture facility in the night fits the nocturnal feeding behavior of the abalone spats. Judged by the promising larvae settling rate, growth and survival rates of the postlarvae fed with this alga, the free-swimming micro-green alga P. helgolandica constitutes a potential species for settling the veliger larvae and for supporting the growth of postlarvae as well. (c) 2006 Published by Elsevier B.V.

PRELIMINARY STUDIES ON CRYOPRESERVATION OF SYDNEY ROCK OYSTER (SACCOSTREA GLOMERATA) LARVAE

In this study several parameters critical to the success of cryopreserving Sydney rock oyster (Saccostrea glomerata) larvae were investigated. They were: (1) cryoprotectants (10% dimethyl sulfoxide and 10% propylene glycol). (2) freezing protocols (with or without the seeding step). (3) larval concentrations (1,000, 3,000, 5,000, 10,000, 30,000 individuals mL(-1)). and (4) larval ages (6, 12, 24, 48 and 96 h old). The survival rates were determined as percentages of postthaw larvae performing active movements for the 6 and 12 h larvae or active cilia movement for the 24, 48 and 96 h larvae. Analyses showed that the difference in survival rates between different age classses was significant in all the experiments conducted, with the maximum survival rate being achieved in the 24-h-old larvae the postthaw survival rates of larvae cryopreserved with 10% dimethyl sulfoxide (93.1 +/- 0.2%) were significantly higher (P < 0.001) that those with 10% propylene glycol (81.5 +/- 0.4%). Differences in postthaw survival rates between different concentrations (1,000 30,000 individuals mL(-1)) were not significant within each of the three larval age classes (6-, 12-, and 24-h-old ) used.

Effects of temperature and delayed initial feeding on the survival and growth of Japanese flounder larvae

The effects of the timing of initial feeding (0, 1, 2 3 and 4 days after yolk exhaustion) and temperature (15, 18 and 21degrees C) on the point-of-no-return (PNR), survival and growth of laboratory-reared Japanese flounder Paralichthys olivaceus larvae were studied under controlled conditions. The larvae reached PNR on 7(.)7, 5(.)2 and 4(.)2 days-post-hatching (dph) at 15, 18 and 2 V C, respectively. At each temperature, larval growth did not differ significantly among the delayed initial feedings 1 day before PNR but decreased significantly in larvae first fed after that. In the treatments where initial feeding was equally delayed, larvae grew significantly faster at 18 and 21degrees C than at 15degrees C. The larvae survived apparently better at 15 and 18degrees C than at 21degrees C when initial feeding was equally delayed. At each temperature, survival of the larvae first fed before PNR did not differ noticeably, while delayed initial feeding after that apparently reduced their survival. These results indicated that there existed a negatively temperature-dependent PNR in the Japanese flounder larvae. Survival and growth of the larvae strongly depended on temperature as well as the timing of initial feeding. High temperature accelerated the yolk exhaustion and growth of the larvae and thus reduced their starvation tolerance and survival. To avoid potential starvation mortality and obtain good growth, the Japanese flounder larvae must establish successful initial feeding within 2 days after yolk exhaustion at 15degrees C and within 1 day at both 18 and 21degrees C. (C) 2005 The Fisheries Society of the British Isles.

Feeding, morphological changes and allometric growth during starvation in miiuy croaker larvae

We investigated the effects of the timing of first feeding (larvae in F0, F1, F2, F3 and S were first fed on day 3, 4, 5, 6 days after hatching (DAH) and unfed, respectively) on feeding, morphological changes, survival and growth in miiuy croaker larvae at 24A degrees C. The fed larvae initiated feeding on 3 DAH and reached point of no return (PNR) on 6 DAH. Larvae in F0 and F1 groups survived apparently better than F2 group at the end of the experiment on 36 DAH. High larval mortality occurred from 3 to 7 DAH in all feeding groups, accounting for 40% (F0, F1 and F2 groups) to 90% (F3 and S groups) of the total mortality. Larvae in F0 and F1 groups grew better than F2 group throughout the experiment. Eye diameter, body height, head height and mouth gape of the first feeding larvae were more sensitive to starvation than other morphometrics and could be used as indicators for evaluating their nutritional status. Results indicated that delayed first feeding over 1 day after yolk exhaustion could lead to poor larval survival and growth. To avoid starvation and obtain good growth in culturing, larvae feeding should be initiated within 1 day after yolk exhaustion at 24A degrees C.

Toxicity of lead, cadmium and mercury on embryogenesis, survival, growth and metamorphosis of Meretrix meretrix larvae

In order to assess the toxicity of heavy metals on the early development of Meretrix meretrix, the effects of mercury (Hg), cadmium (Cd) and lead (Pb) on embryogenesis, survival, growth and metamorphosis of larvae were investigated. The EC50 for embryogenesis was 5.4 mu g l(-1) for Hg, 1014 mu g l(-1) for Cd and 297 mu g l(-1) for Pb, respectively. The 96 h LC50 for D-shaped larvae was 14.0 mu g l(-1) for Hg, 68 mu g l(-1) for Cd and 353 mu g l(-1) for Pb, respectively. Growth was significantly retarded at 18.5 mu g l(-1) (0.1 mu M) for Hg, 104 mu g l(-1) (1 mu M) for Cd and 197 mu g l(-1) (1 mu M) for Pb, respectively. The EC50 for metamorphosis, similar to 48 h LC50, was higher than 96 h LC50. Our results indicate that the early development of M. meretrix is highly sensitive to heavy metals and can be used as a test organism for ecotoxicology bioassays in temperate and subtropical regions.

Identification of factors affecting the growth and survival of the settling Japanese flounder larvae, Paralichthys olivaceus

A series of experiments were conducted to identify the factors that affected the growth and survival of the settling flounder larvae Paralichthys olivaceus. Settling larvae 24 days after hatching (DAH) were reared in 10-l experimental tanks up to 40 DAH, and two of the following factors were changed as controlled factors in each experiment: light regime (24L:0D or 12L:12D), prey density (1500, 3000, or 5000 Artemia l(-1)), shelter (sand or no sand) and stocking density (5, 10, or 15 fish l(-1)). Early settling larvae (24-35 DAH) experienced little mortality (less than 10% of the overall mortality) that was not significantly affected by above factors. In contrast, late settling larvae (36-40 DAH) suffered high cannibalistic mortality which was significantly influenced by each of the above factors. Larvae experienced significantly lower mortality at 10 fish l(-1) level than at other densities. Larvae at 15 fish l(-1) level had higher mortality than at 5 fish l(-1) when all other factors were identical. Larvae at 3000 and 5000 Artemia l(-1) treatments survived significantly better than at 1500 Artemia l(-1), but no significant differences in larval mortality were found between the two higher densities. Larvae suffered higher mortality at low prey density or at the absence of sand when they were exposed to longer photoperiod. Low stocking density significantly improved the growth of the settling larvae. The average daily instantaneous growth rate (G) at 5 and 15 fish l(-1) treatments were 0.050 and 0.034, with the coefficient of variation (CV) in final length at 16.4 and 23.5, respectively. Daily instantaneous growth rate increased significantly from 0.033 in the 1500 Artemia l(-1) to 0.041 and 0.045 in the 3000 and 5000 Artennia l(-1), respectively, but no significant difference in larval growth existed between the two higher prey densities. These findings suggested that the optimal prey density for growth and survival of the settling flounder larvae at a stocking density of 5 - 15 fish l(-1) was around 3000 Artemia l(-1) . Larvae that were exposed to 24L showed 20% increase in growth ( G = 0.046, CV = 18.7) than those exposed to 12L ( G = 0.037, CV = 20.5). Longer exposure to light significantly improved larval growth, provided sufficient food was available. Sand substrate did not show significant effects on larval growth, possibly because the larvae spent most of the time swimming or feeding in the water column during this stage. (C) 2003 Elsevier Science B.V. All rights reserved.

Feeding resumption, morphological changes and mortality during starvation in Japanese flounder larvae

Japanese flounder Paralichthys olivaceus larvae established first feeding 3 days after hatching (DAH) at c. 17degreesC. Non-fed fish reached irreversible starvation at age 5 DAH. Non-fed fish showed similar feeding rate and feeding intensity as the fed fish when they were provided with prey before 5 DAH, after which the starved larvae did not feed even when prey became available. None of the six morphological measurements examined (total length, body height, eye height, head height, gut height and myotome height) showed significant differences between the non-fed and fed larvae until 5 DAH. Normal development continued only in the fed group, and the non-fed larvae showed reverse growth or body collapse after 5 DAH. Owing to the shrinkage and collapse at the top of head due to starvation, head height could be a sensitive indicator of starvation in Japanese flounder larvae. In the fed treatments, high mortality occurred from first feeding (3 DAH) to irreversible starvation (5 DAH), accounting for about two-thirds to three-quarters of the overall mortality (46-52%) throughout the experiments. This mortality was not prey density or larval density dependent. Mortality during the same period in the non-fed larvae accounted for about a third of the overall mortality (100%). (C) 2002 The Fisheries Society of the British Isles. Published by Elsevier Science Ltd. All rights reserved.

Spatial distribution of ciliates, copepod nauplii and eggs, Engraulis japonicus post-larvae and microzooplankton herbivorous activity in the Yellow Sea, China

The abundance of anchovy Engraulis japonicus larvae, >20 mum ciliates, copepod eggs and nauplii, and microzooplankton herbivorous activity were studied in the Yellow Sea in June 2000. Anchovy juveniles and larvae were found in only 6 of the 19 stations sampled. The ciliate communities were dominated by 2 species: Laboea strobila and Strombidium compressum. In the surface waters, the abundance of L. strobila ranged between 0 and 560 ind. l(-1). S. compressum only appeared at Stns 15 to 18 (20 to 3300 ind. l(-1)). L. strobila was found mainly in the top 20 m. The abundance of L. strobila was less than 50 ind, l(-1) in waters deeper than 25 m. S, compressum showed subsurface abundance peaks at the salinity abnormality. Tintinnids occurred occasionally with abundance lower than 100 ind. l(-1), The total ciliate abundance fell in the range of 40 to 3420 ind. l(-1). The ciliate biomass in the surface water and the water column ranged between 0,15 and 6.76 mug C l(-1) and 0.4 and 134.4 mg C m(-2), respectively, In the surface waters, the abundance of copepod eggs and nauplii ranged from 0,3 to 3.1 and 1,1 to 15.6 ind, l(-1), respectively. The average abundance of copepod eggs and nauplii in 4 depth (0, 5, 10 and 20 m) fell in the range of 0.2 to 2.8 and 1.0 to 29.4 ind. l(-1), respectively. As a food item of the E. japonicus post-larvae, the abundance of copepod nauplii and eggs appeared to be low. The abundance peaks of ciliate and E, japonicus post-larvae coincided. Although not found in the gut of E, japonicus post-larvae, aloricate ciliates might be ingested by first-feeding anchovy larvae, preventing initial starvation and prolonging the time to irreversible starvation. On the basis of dilution experiments with positive microzooplankton grazing rates, microzooplankton grazed at rates of 0 to 0.61 d(-1). Grazing pressure of microzooplankton on chlorophyll a standing stock (P-i) and potential chlorophyll a primary production (P-p) were 17 to 46% and 35 to 109% d(-1), respectively.

Inhibition of egg hatching success and larvae survival of the scallop, Chlamys farreri, associated with exposure to cells and cell fragments of the dinoflagellate Alexandrium tamarense

We report an apparently novel toxic effect of the dinoflagellate Alexandrium tamarense, manifested by inhibition of the egg hatching success of the scallop, Chlamys farreri. The hatching rate of C. farreri approached only 30% of controls when its fertilised eggs were exposed for 36 h to A. tamarense cells or cellular fragments at a concentration of 100 cells/ml, and the hatching rate was just 5% after exposure to A. tamarense of 500 cells/ml. Similar exposures of the fertilised scallop eggs to two other algal species, the diatom Phaeodactylum tricornutum and the raphidophyte Heterosigma carterae, resulted in no such toxicity or inhibitory effects.. Likewise, exposure of eggs to standard STX toxin. as well as to A. tamarense cell contents (supernant of re-suspended algal cells following ultrasonication and centrifugation), did not elicit this inhibitory response. However, exposure of the scallop eggs to cell cultures, intact algal cells, or cell fragments of A. tamarense produced marked toxicity. The alga also influenced larvae at early D-shape stage of scallop. The survival rates began to decrease significantly after exposed for 6 days at concentration of 3000 cells/ml and above: no larvae could survive after 14-day exposure to A. tamarense at 10,000 cells/ml or 20-day at 5000 cells/ml. The results indicated the production of novel substances from A. tamarense which can cause adverse effects on egg hatching and survival of the scallop larvae, The experiment also found that the developmental stages before blastula was the developmental period most sensitive to the A. tamarense toxin(s) and the alga at early exponential stage had the strongest effect on egg hatching comparing with other growth phases. The adverse effect of A. tamarense on early development of scallops may cause decline of shellfish population and may have further impact on marine ecosystem. (C) 2001 Elsevier Science Ltd. All rights reserved.

Feeding behaviour of Japanese flounder larvae under laboratory conditions

Tank-reared Japanese flounder larvae, Paralichthys olivaceus, had a major feeding peak in the morning and a secondary peak in the afternoon throughout the larval development, with light being the primary factor regulating their feeding activity. The larvae consumed rotifers in preference to Artemia for up to 10 days, after which the food preference shifted to Artemia. Feeding rates of the larvae prior to 10 days post-batch depended on prey density, but in the old larvae, feeding rates were independent of prey density. Maximum feeding rate occurred at 19 degrees C. The occurrence of the attack posture, after its onset at first feeding (2 days post-hatch), increased up to 25 days, began to decrease when the larvae prepared to settle down, then disappeared after settlement. The occurrence frequency of the attack posture was positively related to fish density, but inversely related to starvation duration, and occurred most frequently at 19 degrees C. This posture depended on prey density in larvae prior to 10 days post-hatch, but became independent of prey density as the larvae developed. It was obvious that, for flounder larvae, attack posture was a behavioural character closely related to feeding and subject to larval development and environmental factors. (C) 2000 The Fisheries Society of the British Isles.

Effects of delayed first feeding on growth and survival of rock bream Oplegnathus fasciatus larvae

The effects of the timing of first feeding (0, 1 and 2 days after yolk exhaustion) and starvation on the point-of-no-return (PNR), survival and growth of laboratory-reared rock bream larvae were studied under controlled conditions. Larvae began to feed exogenously at 3 days after hatching (dah) and reached PNR on 54 h after yolk exhaustion at 22 +/- 1.5 degrees C. Larvae growth was significantly affected by the time of first exogenous feeding. The growth of 0 day delayed first feeding larvae was obviously faster than those of the other delayed first feeding larvae (P<0.05) whether at 7 dab (SL=3.40 mm, SGR=5.7, CV=4.0) or at 15 dah (SL=4.85 mm, SGR=6.1, CV=8.2) with a more uniform size distribution. Survival of 0 day delayed first feeding larvae and I day delayed first feeding larvae was 13% and 8% at the end of experiment, respectively, while no larvae survived up to 7 dah for 2 days delayed first feeding larvae and unfed larvae. Food resulted in a progressive deterioration of the larval digestive system and atrophy of skeletal muscle fibre. The ratios of head length to SL (standard length), body height to SL and eye diameter to SL were the most sensitive morphometric indices to detect the effects of fasting on larval condition. Present results showed that the combination of morphological and morphometric variables could be used to evaluate the nutritional condition of rock bream larvae. In order to avoid the potential mortality and gain better development, survival and growth in industrial production, the rock bream larvae must establish successful first feeding within 2 days after yolk exhaustion. (C) 2008 Elsevier B.V. All rights reserved.

Effects of photoperiod on growth, mortality and digestive enzymes in miiuy croaker larvae and juveniles

The growth, mortality and digestive enzymes (trypsin, amylase and lipase) in miiuy croaker Miichthys miiuy larvae and juveniles (2-53 dph) were investigated at four photoperiods: 24L:OD), 18L:6D, 12L:12D and OL:24D. Larvae could not feed at OL:24D and did not survive up to 7 dph. In the 24L:OD, 18L:6D, 12L:12D groups, photoperiod had not significant effects on the growth of the rniiuy croaker younger than 20 dph. However, their total length and specific growth rate (SGR) were significantly larger at 18L:6D and 24L:OD than 12L:12D after 20 dph. Photoperiod also affected the mortality of the first feeding larvae (5 dph). being apparently higher in 5 dph larvae at OL:24D (60%) than at other photopenods (20-27%), but no significant differences in mortality were found among other photoperiods. High mortality of the miiuy croaker in 12L:12D, 18L:6D and 24 L:OD groups mainly occurred from 5 (20-27%) to 11 dph (11-16%) and tended to decrease gradually from 15 dph onwards. Digestive enzymes activities in the rniiuy croaker larvae and juveniles had a similar change trend with age at all photoperiods. They underwent drastic changes with age. The specific activity of lipase was significantly higher at 18L:6D and 24L:0D than 12L:12D, but there were no significant differences in specific activities of either trypsin or amylase among photoperiods. With regard to the total length, SGR, survival and digestive enzyme activities, our findings suggested that the optimal light regime for the culture of miiuy croaker during the early life stage was 18L:6D. (C) 2008 Elsevier B.V. All rights reserved.