Induced out-of-season spawning of the mud crab, Scylla paramamosain (Estampador) and effects of temperature on embryo development

Treated with combined bilateral eyestalk ablation and maintenance of water temperature at 22.5±1.5 °C, mud crab Scylla paramamosain females with mature ovaries were induced to produce eggs outside the natural spawning season in subtropical southern China. Newly extruded eggs from a crab were incubated in vitro at 10, 15, 20, 25, 27, 30, 35 °C, respectively, and the embryonic development was closely monitored. Abnormal cell division was observed at temperatures 10 and 35 °C. At 15 °C, development remained at the gastrula stage by day 32 post‐spawn, at which time the experiment was terminated. Hatching of in vitro incubated eggs occurred between 20 and 30 °C. An increase in incubation temperature from 20 to 25 °C reduced the incubation duration by 14 days, 2.6 times of that measured for a similar 5 °C increase from 25 to 30 °C. Embryonic development of S. paramamosain was divided into stage 0–10, and the duration of each stage was recorded for each incubation temperature. The information obtained allows accurate prediction of hatching time of female crabs incubated under variable temperatures. Larvae hatched from in vitro incubated eggs were reared to reach first juvenile crab stage and their dry weights were similar to those of larvae hatched naturally.     

The effect of ploidy and incubation temperature on survival and the prevalence of aplasia of the septum transversum in Atlantic salmon,Salmo salar L

Heart deformities are a concern in aquaculture and are linked to egg incubation temperature. Diploid and triploid Atlantic salmon, Salmo salar L., were incubated at 6, 8 and 10 °C and analysed for aplasia of the septum transversum (n = 150 ploidy^?1 incubation temperature^?1). Heart morphology (size and shape) was assessed in fish incubated at 6 °C and in fish with and without aplasia of the septum transversum (n = 9 group^?1) incubated at 10 °C. Egg mortality was significantly higher in triploids than in diploids at all incubation temperatures, and increased egg incubation temperatures increased mortality in both ploidy. Triploids grew quicker than diploids after egg incubation at 10 °C, but not at 6 °C. Aplasia of the septum transversum occurred only in triploid fish after incubation at 6 °C and 8 °C (0.7% and 3.3%, respectively) and was significantly greater (P ≤ 0.05) in triploids after incubation at 10 °C compared with diploids (30% and 18%, respectively). Aplasia of the septum transversum significantly increased heart mass and resulted in a long flat ventricle compared with fish displaying a septum transversum. The results suggest triploid salmon should be incubated below 8 °C.     

The effect of temperature and duration of incubation on the hatching of diapause eggs of Centropages hamatus (Lilljeborg) (Copepoda, Calanoida)

Diapause eggs of Centropages hamatus were used to investigate the effect of temperature and duration of incubation on egg hatching. Eggs were incubated for 10, 12, 14, 16, 20, 24, 28, 32, 36 and 40 h at 15°C and 14L–10D. After incubation for the designated period, eggs were transferred to 25°C and monitored periodically to determine egg hatching. Control eggs were incubated solely at 15°C and monitored for egg hatching. The greatest daily hatching success of eggs occurred within 1 or 2 days after transfer from 15°C to 25°C, while the controls required 3–4 days. The cumulative hatching success of eggs was significantly lower than the control, with the exception of eggs held for at least 36 h at 15°C before transfer to 25°C. These results indicate that overall time to hatching of diapause eggs of C. hamatus can be reduced by transferring the eggs to a higher temperature, for example, 25°C, following a minimum period of time (36 h) at reduced temperature, for example, 15°C. Exposure to 15°C for only 10 h does not appear to be sufficient to result in any subsequent hatching at higher temperature.     

虾夷扇贝胚胎及早期幼虫酸性磷酸酶和碱性磷酸酶的组织化学研究

采用组织化学方法对虾夷扇贝胚胎及早期幼虫内的酸性磷酸酶(ACP)和碱性磷酸酶(AKP)进行了定位研究。结果显示:从卵细胞到D型面盘幼虫各个时期ACP和AKP都呈阳性;卵细胞中ACP阳性颗粒比较均匀地分散在细胞内,受精卵中央着色较浅;卵裂期个体较大的分裂球内阳性颗粒较多,16细胞期到囊胚期的细胞近核区域着色深;面盘幼虫可见ACP集中在内脏团和外套膜区域。卵细胞中AKP阳性颗粒主要存在于细胞中央及细胞膜附近,卵裂期、囊胚、原肠胚和担轮幼虫各个发育时期都可观察到细胞膜附近着色较深,面盘幼虫AKP活性集中在外套膜边缘和内脏团。     

Effects of heat-shock selection during pelagic stages on thermal sensitivity of juvenile sea cucumber, <Emphasis Type="Italic">Apostichopus japonicus</Emphasis> Selenka

The effects of heat-shock during pelagic stages on growth and survival of juvenile sea cucumber Apostichopus japonicus were investigated to test the possibility to acquire high thermal tolerance individuals after the heat-shock selection. Larvae at the stages of blastula, gastrula, and auricularia were heat-shocked at selected temperatures (21.5, control; 26, 28, and 30°C) for 45 min and returned to 21.5°C for continuous rearing. There was a significant difference in thermal sensitivity among different developmental stages, and thermal tolerance of the larvae was correlated with the expression of heat shock protein 70 (Hsp70). Juveniles after the heat-shock selection at pelagic stages showed higher induced thermotolerance than those without heat-shock. Therefore, heat-shock application at early pelagic development stages is potentially a useful way to select high-thermotolerance variety of the sea cucumbers.     

Characterization of Herpes virus vitreum isolated from hyperplastic epidermal tissue of walleye,Stizostedion vitreum vitreum (Mitchill)*

Abstract. A new virus, provisionally named Herpesvirus vitreum, was isolated from hyperplastic epidermal tissue from a walleye, Stizostedion vitreum vitreum (Mitchill), taken in Saskatchewan, Canada. The virus, which was isolated in the walleye ovarian (WO) cell line, was identified as a herpesvirus on the basis of size (190–230 nm), morphology and apparent pattern of replication. The virus, which passes polycarbonate membranes of 200 nm mean pore diameter, was ether-labile. Virus replicated in WC-1 cells at 4 and 15°C, but not at 20°C. Although walleye cell lines (WO, WC-1, We-2) were susceptible to infection at 15°C, non-period cell lines were refractory. Syncytial formation and lysis occurred in susceptible cell lines. Virus was quantified by plaque assay at 13 to 15°C for two weeks. Replication was inhibited by 10^-3.0m phosphonoacetate and by 10^-5.0m 5-bromodeoxyuridine (BUDR), but addition of excess thymidine reversed the inhibition by BUDR. Viral replication in WO cells, but not in WC-1 cells, was inhibited by the antiherpetic drug acyclovir (10^-5.0m ). The relationship of the herpesvirus isolate and epithelial neoplasms was not determined.     

Effect of temperature and salinity on virulence of Edwardsiella tarda to Japanese flounder, Paralichthys olivaceus (Temminck et Schlegel)

Experiments were designed to determine the effects of temperature and salinity on the virulence of Edwardsiella tarda to Japanese flounder, Paralichthys olivaceus. In the temperature experiment, a two‐factor design was conducted to evaluate the effects of both pathogen incubation temperature and fish cultivation temperature on pathogen virulence. E. tarda was incubated at 15, 20, 25 and 30±1°C, and the fish (mean weight: 10 g) were reared at 15, 20 and 25±1°C respectively. The fish reared at different temperatures were infected with the E. tarda incubated at different temperatures. The results of a 4‐day LD₅₀ test showed that temperature significantly affected the virulence of E. tarda (P<0.01) and the interaction between the two factors was also significant (P<0.01). For fish reared at 15°C the virulence of E. tarda was the highest at 25°C of pathogen incubation, followed by 20, 15 and 30°C. When the fish rearing temperature was raised to 20 and 25°C, the virulence of E. tarda incubated at all temperatures increased. Isolation testing demonstrated results similar to those of LD₅₀. The higher rearing temperature increased the proliferation rate of the pathogen in fish. In the salinity experiment, the incubation salinity of E. tarda was at 0, 10, 20 and 30 g L^?1, respectively, and the fish with mean weight of 50 g were cultured in natural seawater of 30 g L^?1. The results of one‐way anova in 4‐day LD₅₀ test showed that incubation salinity significantly affected virulence. Virulence was lower when the salinity of the incubation medium was at 0 and 30 g L^?1, higher at 10 and 20 g L^?1. The results of isolation test were in accordance with those of LD₅₀. At 20 g L^?1E. tarda had a faster proliferation rate than that at 10 g L^?1.     

Stages of embryonic development and changes in enzyme activities in embryogenesis of turbot (Scophthalmus maximus L.)

The early development of turbot (Scophthalmus maximus) from fertilization to hatching was described. Hatching occurred at 108 h post-fertilization (hpf) in 14 °C. Yolk syncytial layer and blastocoel formed at morula stage and low stage, respectively. Neural rod derived from the ectoderm appeared and the first somite formed in the middle of the embryonic body at 90 % epiboly stage, and notochord primordium formed at complete epiboly stage. Kupffer’s vesicle appeared at 59 h 35 min hpf and degenerated at 89 hpf. At 72 hpf, the digestive tract formed in the ventral side of the embryonic body, and the posterior digestive tract of embryo was ciliated at 89 hpf. Enzymes play a key role in the catabolism of yolk during embryogenesis of fishes. In this study, the main enzymes alkaline phosphatase (AP), leucine aminopeptidase N (LAP), pepsin, trypsin and Leucine-alanine peptidase (Leu-ala) were all observed in unfertilized eggs and embryo of S. maximus, but amylase was not detected, speculating that amino acids appear to be the main energy substrate during embryonic development of S. maximus, while carbohydrates is less essential. AP reached the lowest value at the gastrula stage and then increased rapidly reaching the highest value at hatching. LAP showed the highest value in unfertilized eggs and kept on decreasing until the blastula stage with the lowest value and then increased at the gastrula stage, followed by a gradual decline thereafter. Trypsin reached the lowest value at the blastula stage and then fluctuated with the maximal value at hatching. Pepsin reached the highest and the lowest values at the unfertilized eggs and the cleavage stage, respectively, but disappeared at hatching. Leu-ala had the maximum activity at the blastula stage and then declined to the minimum at the gastrula stage followed by a gradual increase thereafter.     

Expression patterns of acidic and basic fibroblast growth factor in loach fish embryos

Heparin-binding fractions were taken from the heparin sepharose columns on which extracts of loach fish (Misgurnus anguillicaudatus) embryos from blastula, gastrula, 4–8 and 12–16 somites stages were applied. These heparin-binding fractions, except the fraction derived from 12–16 somite embryos, showed potent mitogenic activity on fibroblast-like cells derived from caudal fin blastema of goldfish. Western blot analysis of these heparin-binding fractions was carried out using monoclonal antibodies against human acidic and basic fibroblast growth factors (FGF-1 and-2). An immunoreactive FGF-1 band at 16 kD was detected in the heparin-binding fraction derived from embryos in each stage of blastula, gastrula and 4–8 somites. An immunoreactive FGF-2 band at 17 kDa was detected exclusively in the heparin-binding fraction derived from 4–8 somite embryos. In the heparin-binding fraction derived from 12–16 somite embryos neither immunoreactive FGF-1 nor-2 member band was detectable.     

Biodegradation potential of aquaculture chemotherapeutants in marine sediments

The commercial chemotherapeutant formulations SLICE^® and AlphaMax^® [active ingredients emamectin benzoate (EB) and deltamethrin respectively] are used in fin fish aquaculture to control parasitic sea lice. In some regions, the use of these substances has drawn concern from the commercial fishing industry regarding potential adverse effects on non‐target organisms. In the present work, biodegradation of EB and deltamethrin, and their commercial formulations, was investigated over 135 days at 4 and 10°C in fresh marine sediments collected from underneath an active open net‐pen salmon farm. EB incubated as either pure substance or commercial formulation was recalcitrant at both temperatures under abiotic and biotic conditions. Deltamethrin incubated alone or as its commercial formulation degraded slowly at 10°C (t_1/2 = 330 ± 107 and 201 ± 27.1 days respectively). At 4°C, deltamethrin degradation was only significant following incubation as commercial formulation (t_1/2 = 285 ±112 days). Degradation rates of EB and deltamethrin as pure substances versus their commercial formulations were not statistically different. Depletion of deltamethrin was observed in 10°C inactive sediments indicating that transformation occurred (at least in part) via an abiotic pathway. Overall, these data provide further insight into the fate and persistence of EB from the ongoing use of SLICE^® in British Columbia's salmon aquaculture industry. AlphaMax^® is not registered in Canada but is used in other salmon farming countries to control sea lice.     

Nile tilapia broodfish fed high‐protein diets: Digestive enzymes in eggs and larvae

The aim of this study was to assess the activity of gastric, pancreatic and intestinal digestive enzymes during the embryonic and larval development of Nile tilapia (Oreochromis niloticus) GIFT strain Aqua America^® 1 obtained from a broodstock fed two levels of crude protein (CP). A total of 72 females and 24 males, 10 hapas, two CP levels (32% and 38%) and six phases of embryonic (cleavage, blastula, gastrula) and larval (hatching, 7 and 10 days post hatch, dph) stages were used. The eggs were collected in cleavage, blastula and gastrula stages, 300 mg was collected, and kept in cryogenic tubes in liquid nitrogen. For the samples at larval stage, the remaining eggs were separated according to crude protein level and kept in hatcheries and samples were collected on 7 and 10 dph the same way as before. A total of 48 samples were collected: at each protein level (32% and 38% CP), four samples were collected in each phase of embryonic and larval development. Statistical differences were not observed during embryonic development for acid proteases, trypsin, amylase and lipase activity at both levels of crude protein (32 and 38% CP). Differences for acid proteases were noticed on 7 dph; trypsin and amylase on 7 dph and 10 dph. Significant differences on blastula and 7 dph for protease; as for aminopeptidase, there was significant difference on 7 dph. The data indicated early appearance of digestive enzymes in Nile tilapia broodfish receiving 32% CP taking into account the rapid growth and development of this species.     

Developmental rates,structural asymmetry,and metabolic fingerprints of steelhead trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>) eggs incubated at two temperatures

Temperature stress on developing steelhead (Oncorhynchus mykiss) was evaluated using asymmetry of skeletal characters, fish condition factor, and metabolic fingerprints. Eggs from three female hatchery steelhead were fertilized by a single male. The eggs from each female were divided into two groups and incubated at either 8°C or 18°C. Mortality, growth, and condition factor were measured at stage 6 (32 cells), stage 20 (eyed), and stage 21 (caudal flexing). In addition, ¹H-nuclear magnetic resonance (NMR) spectroscopy was used to establish metabolic fingerprints of developing eggs at the three stages. After hatching, all alevins were moved to tanks at 18°C and allowed to develop to 60 days post-emergence (DPE), at which point they were examined for structural asymmetry. Eggs incubated at 18°C experienced higher mortality, with all eggs from one hen dying at the higher temperature. Eggs incubated at the higher temperature that did survive hatched as larger larval fish than eggs incubated at the lower temperature. Fish incubated at the higher temperature exhibited greater structural asymmetry than fish incubated at the lower temperature. A principle components (PC) analysis of the metabolic fingerprints indicated that PC1 and PC2 accounted for 60% of the variance in the metabolites. Separation along PC1 corresponded to differences in developmental stage, and separation along PC2 corresponded to differences in hen. Eggs incubated at 18°C lagged behind eggs incubated at 8°C along PC1, indicating a potential problem with embryo staging. PC1 scores were highly correlated with the accumulated thermal units during development, indicating that scores along PC1 were a robust measure of developmental stage.     

Development of two cell culture systems from Asian seabass Lates calcarifer (Bloch)

Two new cell culture systems namely epitheloid cells of Lates (LCE) and fibroblastic cells of Lates (LCF) have been developed from fry and fingerling of the economically important brackishwater fish Lates calcarifer. Primary cultures were initiated by explant technique using caudal fin of fingerling and whole body tissue of the fry. The nutritional requirements and the growth pattern in response to different culture environment were similar for the two cell cultures. The culture medium used was Leibovitz‐15 supplemented with 20% fetal bovine serum (FBS) and 1% fish serum. The LCE comprised of epithelioid cells and LCF cells were fibroblastic. With a split ratio of 1:2, the confluency of cells was attained in 8–10 days at an incubation temperature of 28°C. The cells were found to grow well in a wide range of temperature (24–32°C) and stable at 20 and 36°C. The growth rate of LCF and LCE cells increased proportionately with the concentration of FBS from 5% to 20%. A decrease of serum level to 10% after eight subcultures produced no apparent change in cell morphology and growth rate. The viability of cells was found to be 70% when revived after a month of storage in liquid nitrogen (?196°C).     

Embryonic and larval developments of brackish water catfish,Mystus gulio (Hamilton and Buchanan, 1822) induced with human chorionic gonadotropin and consequent larval rearing

Mystus gulio, the long whiskers catfish, is a popular food fish and potential candidate species for aquaculture in Sundarban area of India and Bangladesh. Recently, catch of this species has declined due to overfishing and various ecological changes. In the present study, mature fish was induced to spawn in captivity through intramuscular injection of human chorionic gonadotropin at the doses of 10 IU/g to female and 5 IU/g to male. Photomicrographs of all developmental stages of live embryo and larvae were documented with the aid of a light microscope. Results demonstrated that morula, blastula, gastrula and neurula and organogenesis ended at 1:30‐, 3:00‐, 5:30‐, 7:30‐ and 17:15‐hour post‐spawning (hps) respectively. Heartbeat and muscular contraction of embryo commenced at 8:30 and 11.15 hps respectively. Hatching of embryo started after an incubation period of 17:30 hour at an ambient temperature of 29 ± 1°C. The newly hatched larvae measured 2.17 ± 0.29 mm in total length with a yolk volume of 0.165 ± 0.03 mm³ started feeding 36 hr after hatching. The present study, on induced breeding and chronological development of M. gulio embryo, will have significant implications on conservation and seed production for aquaculture.     

Physiological investigations of a neurotoxin-producing phytoflagellate, Chattonella marina (Raphidophyceae)

The effects of temperature, salinity, light intensity and pH on the growth and morphology of Chattonella marina (Subrahmanyan) Hara & Chihara were examined. Optimal growth was observed at temperatures of 20-25°C, salinities of 20-30%o, light intensities of 60-140 μE m^?2 s^_1 and pH 7.5-8.5. Growth did not occur at temperatures below 15°C or above 30°C, and at salinities below 10%o. The morphology (shape) of the cells was strongly affected by temperature. At 20°C and 25°C, the population occurred mostly in a spindle-like form, whereas at 10°C, 90% of the cells became spherical within 10 days of inoculation and stationary phase cultures consisted entirely of spherical cells. Morphology was also markedly affected at 30°C. The number of spindle-like cells was highest at 20-30%o and was less at lower salinities. Light intensity and pH did not influence morphology markedly under the range of light intensities (20-180 μE m ^?2 s-^?1) and pH (6.5-8.5) tested.     

Factors influencing in vitro respiratory burst assays with head kidney leucocytes from rainbow trout,Oncorhynchus mykiss (Walbaum)

Head kidney leucocytes are central elements in a number of in vivo and in vitro assays elucidating innate and adaptive immune mechanisms in teleosts following stimulation with various antigens. These systems are sensitive to several factors affecting the outcome of the assays. The present work describes the importance of temperature, cell concentration, exposure time and immune-modulatory molecules on the respiratory burst activity (RBA) of rainbow trout head kidney leucocytes in vitro. Some variation in RBA was observed among individual fish. However, use of cells pooled from four individuals produced satisfactory results following exposure to phorbol 12-myristate 13-acetate, zymosan and β-glucan. Temperature was shown to have a significant effect on production of reactive radicals as illustrated by a high activity in cells maintained at 15–20 °C and a reduced activity at temperature extremes (1, 4 and 30 °C). Highest activity was found at a cell concentration of 1 × 10⁷ cells mL⁻¹. Reactivity showed a clear decline when cells were exposed for more than 4 h. Moreover, incubation of cells with inhibitory substances viz., DiMePE2, cortisol and superoxide dismutase decreased the RBA. It is concluded that several biotic and abiotic factors should be taken into account when conducting RBA assays with head kidney leucocytes for elucidation of rainbow trout immune responses.     

The influence of rearing temperature on early development and growth of spotted wolffish Anarhichas minor (Olafsen)

Temperature influenced the developmental rate, survival and early growth of eggs and embryos of spotted wolffish, Anarhichas minor (Olafsen), an interesting candidate for cold water cultivation. The total incubation period decreased from 220 days at 4 °C (880 daydegrees), to 177 days at 6 °C (1062 daydegrees) and 150 days at 8 °C (1200 daydegrees) in these experiments. The proportion of normal embryos and survival of eggs until hatching were highest when the eggs were incubated at 6 °C. During the incubation period, the embryo and yolk sac size at 280 daydegrees was not significantly different but at 850 daydegrees the embryo size was inversely related to temperature and the remaining yolk sac size positively correlated with the incubation temperature. The transformation of yolk to body mass during incubation appeared to be most efficient at 4 °C, and the embryos hatched with a larger visible yolk sac at 6 and 8 °C. The largest larvae (wet‐weight) hatched from the largest eggs and the egg groups incubated at the lowest temperature (4 °C). There was no effect of temperature on meristic characters. During 6 weeks post‐hatching, all larvae from the three temperature groups were fed formulated dry feed in excess at 8 °C in low water‐level raceway systems. During startfeeding, the larvae from eggs incubated at the lowest temperature (4 °C) showed the highest growth rates (SGR). Best survival of larvae was noted among batches incubated at 6 °C.     

Low-temperature preservation (at 4 °C) of marine rotifer Brachionus

Mass production of rotifers is essential as live food during the larval rearing season, but a major problem of rotifer culture is unpredictable culture collapse. If mass‐produced rotifers could be kept alive at low temperature for an extended period of time, they could be supplied as live food to cultured marine fish larvae without interruption. Four experiments were performed to test this possibility in six strains of Brachionus plicatilis O. F. Müller and eight strains of Brachionus rotundiformis Tschugunoff. The results showed that: (1) B. rotundiformis strains were less tolerant to 4 °C than B. plicatilis strains. Among the B. rotundiformis strains, the strains known as SS type were the most susceptible and showed the lowest survival. (2) Exchange of culture media during the incubation at 4 °C in B. plicatilis and B. rotundiformis resulted in higher survival than not changing the culture media, but there were no differences in the regression slope with or without changing the culture media. (3) Acclimation at 15 °C for 96 h for B. plicatilis and B. rotundiformis before transfer to 4 °C resulted in higher survival rates than acclimation at 10 °C. (4) The combination of frequent exchange of culture media and acclimation significantly improved the survival of B. plicatilis and B. rotundiformis compared with controls that were maintained at 4 °C without exchange of the culture media. Large‐scale trials using B. plicatilis (Kamiura strain) cultured in 30‐L tanks were conducted in a hatchery at a density of 2000–20 000 individuals mL^?1. Rotifers were transferred directly from 25 °C to 4 °C. About 50% of the rotifers at 20 000 individuals mL^?1 survived after 14 days at 4 °C. These preserved rotifers could be cultured at 20 °C, recovering within 4 days.