Toxic effects of several phthalate esters on the embryos and larvae of abalone Haliotis diversicolor supertexta

As the most widely used plasticizers in the world, phthalate esters (PAEs) are potential endocrine disruption compounds (EDCs). In the present study, the toxicity of dimethyl phthalate (DMP),diethyl phthalate (DEP), dibutyl phthalate (DBP), di (2-ethylhexyl) phthalate (DEHP) on embryogenesis and larvae development of the marine univalve Haliotis diversicolor supertexta was examined in laboratory. The results show that the malformation of embryos appeared during the experiment, such as embryos died or lysed, small transparent flocculent rings studded on the periphery of the embryo, and the larvae could failed to hatch. In embryo toxic test, embryos incubated at the highest concentration of DMP,DEP and DBP solutions showed significantly high abnormal rate compared with the control, while DEHP solutions displayed no significant difference. In larval toxic test, in all concentrations of DMP, DEP and DBP solutions, larval settlement rates were low significantly than that of the control. Similarly, DEHP solutions show nearly no effect on the larval settlement. The order of toxicity on embryos and larvae is DBP>DEP>DMP>DEHP. Being a simple and easy stimulation to indoor spawn, sensitive to environmental factors, and short culture time, the embryos of 1-1. diversicolor supertexta can be used to indicate toxicity of the PAEs.     

The hydrogen peroxide impact on larval settlement and metamorphosis of abalone <Emphasis Type="Italic">Haliotis diversicolor supertexta</Emphasis>

Abalone Haliotis diversicolor supertexta is an important economic mollusk. The settlement and metamorphosis are two critical stages during its development period, which has direct influence on abalone survival and production. The influence of reactive oxygen species （hydrogen peroxide） on abalone embryo and juvenile development were examined in this study. Larvae of Haliotis diversicolor supertexta were induced to settlement and metamorphose by exposure to seawater supplemented with hydrogen peroxide. They had the best performance at 800 μmol/L. The concentration of 1 000 μmol/L or higher was toxic to the larvae, as the larvae could settle down only at benthic diatom plates without complete metamorphosis. In addition, H2O2 adding time was critical to the larval performance. 24h after two-day post-fertilization was proved to be the optimal adding time. In this paper, two action mechanisms of hydrogen peroxide are discussed： （1） hydrogen peroxide has direct toxicity to ciliated cells, thus cause apoptosis; （2） hydrogen peroxide, as a product from catecholamines＇ autoxidation process in vivo, can reverse this process to produce neuro-transmitters to induce abalone metamorphosis.     

The hydrogen peroxide impact on larval settlement and metamorphosis of abalone Haliotis diversicolor supertexta

Abalone Haliotis diversicolor supertexta is an important economic mollusk.The settlement and metamorphosis are two critical stages during its development period,which has direct influence on abalone survival and production.The influence of reactive oxygen species(hydrogen peroxide) on abalone embryo and juvenile development were examined in this study.Larvae of Haliotis diversicolor supertexta were induced to settlement and metamorphose by exposure to seawater supplemented with hydrogen peroxide.They had the best performance at 800 μmol/L.The concentration of 1 000 μmol/L or higher was toxic to the larvae,as the larvae could settle down only at benthic diatom plates without complete metamorphosis.In addition,H2O2 adding time was critical to the larval performance.24h after two-day post-fertilization was proved to be the optimal adding time.In this paper,two action mechanisms of hydrogen peroxide are discussed:(1) hydrogen peroxide has direct toxicity to ciliated cells,thus cause apoptosis;(2) hydrogen peroxide,as a product from catecholamines' autoxidation process in vivo,can reverse this process to produce neuro-transmitters to induce abalone metamorphosis.     

The effects of temperature and substrate on ontogenetic behavior of bastard halibut, Paralichthys olivaceus （T.et S.）

The effects of temperature and substrate on ontogenetic patterns and settlement behavior of bastard halibut, Paralichthys olivaceus （T. et S.） were studied in a series of laboratory experiments. Analysis on stage-based data revealed that except for settlement, and compared to the substrate, the ambient temperature dominantly controlled the hatch success and subsequent development of the species. The oosperm optimum survival rate of 60% during hatching occurred at 20℃, and survival rate of 29%, at 18℃ for larval rearing. The survival rate of larvae reared under variable temperature of 3.5-4.5℃ was higher than that of those reared under stable temperature. The maximum survival rate of 29%, corresponded to variation at temperature of 4℃. With increase in age newly settled juveniles preferred to lie on the fine substrate and gradually moved on the coarse substrate. GLM （general linear model） analysis showed that the combination of temperature and substrate had no significant impact on the survival of settling larvae, but definitely affected the duration of settlement.     

Induction of larval settlement and metamorphosis ofHaliotis discus hannai Ino (Gastropoda,Mollusca)

Conspecific foot mucus, excessive [K⁺] and gamma-aminobutyric acid (GABA) showed different metamorphosis-inductive effect on the veliger ofHaliotis discus hannai. The inductive effect of excessive [K⁺] and GABA was developmental stage-dependent and dose-dependent, while that of conspecific foot mucus was only developmental stage-dependent. At 20°C the veliger larvae became competent within 4 days after fertilization.H. discus hannai larvae showed gregarious settlement pattern on the conspecific foot mucus under the conditions of either presence or absence of KCl or GABA. The present studies showed that the effect of conspecific foot mucus on abalone larvae metamorphosis could be dose-independent.     

EFFECTS OF TEMPERATURE AND Ca^2＋ ON THE LARVAL DEVELOPMENT OF THE DECAPODA CRUSTACEAN ： ERIOCHEIR SINENSIS

With increasing demand in China for the mitten crab larvae, understanding its survival mechanism gets more important. This research focused on the effects of temperature and Ca^2 on the larval growth and development. Eriocheir sinensis larvae were reared in laboratory under 21 different combinations of temperature (15, 20, 25℃) and Ca^2 content (120, 130, 140, 150, 160, 170, 180mg/l) and constant salinity (20) and pH (8), The results suggested that the survival rate increases with temperature and Ca^2 content. These combinations of temperature and Ca2 content maximized survival rate inour study and it may be the optimum water environmental conditions for culturing the larvae. To predict surviving larvae number under different water environmental conditions, 21 dynamic mathematical models were developed. This for the first time observation of the zoeal Ⅵ larvae of the Changjiang River E.sinensis population showed that they occurred under stressed water environmental conditions: temperature of 15℃ and Ca^2 content of 120,130mg/l.     

Ontogenic study of isozymes in giant tiger prawn,Penaeus monodon (Crustacea: Decapoda)

The patterns of isozyme variation throughout the life cycle of the giant tiger prawn,Penaeus monodon were described for ten enzyme systems (G3PD, MDH, LGG, GPI, MPI, FBP, PGM, LDH, PGD, and PROT). The zymograms showed little variation in the number of isozymic loci expressed during larval development compared with results reported for other penaeid prawns. There were specific ontogenic patterns at some loci. As larvae developed beyond the embryonic stage, there was a tendency for intensity of staining and complexity of banding patterns to increase.     

Effects of base analogues 5-bromouracil and 6-aminopurine on development of zebrafish Danio Rerio

Zebrafish (Danio rerio) genetic screens allow isolation of a wide array of problems in vertebrate biology. The effects of base analogues 5-bromouracil and 6-aminopurine on the development of zebrafish embryos are reported for the first time in this study. The early development of the zebrafish embryos was little affected by 5-bromouracil and 6-aminopurine, while the late development (organogenesis) was significantly impaired. Embryos exposed to 5-bromouracil mainly showed curled tail, wavy body, golden pigmentation and the mouth with protruding lower jaw. 6-aminopurine-treated embryos had defective anterior structures, curled tails and wavy body. RAPD analysis showed that the majority of 5-bromouracil- and 6-aminopurine-treated larvae and fish shared banding patterns in common with the control, suggesting that most mutagenesis induced by these agents are point mutations. However, some fish derived from 5-bromouracil-treated embryos had golden (gol) pigmentation; and RAPD analysis revealed that their band patterns differed from those of the control. Possibly, 5-bromouracil can occasionally cause relatively extensive changes in the fish genome. The results of this study may provide valuable help for detailed studies of mutagenesis. This work was supported by Ministry of Science and Technology (MOST) of China (G 1999012005).     

Cryopreservation of Veliger Larvae of Trumpet Shell, Charonia sauliae： an Essential Preparation to Artificial Propagation

Trumpet shell, Charonia sauliae, is an endangered and valuable species, but its artificial propagation protocol has not been successfully established. To estimate the possibility of cryopreservation for larvae of C. sauliae, which is a potential preparation for its artificial reproduction and further research, in this study a protocol for the cryopreservation of veliger larvae of trumpet shell was optimized. Through a two-step cryopreservation procedure, four kinds of cryoprotectants (ethylene glycol, 1, 2-propanediol, dimethyl sulfoxide and glycerol) were employed at three concentrations (1.0, 1.5 and 2.0 molL⁻¹) respectively and survival rates of larvae were determined after a storage of 1h. The larvae frozen with these four cryoprotectants after 1 h storage were cultured, and then survival rates were determined at 24, 72 and 120 h after thawing. Dimethyl sulfoxide at a concentration of 1.5 molL⁻¹ showed the best protective effect in all experiments (p<0.05). And survival rates of larvae frozen with dimethyl sulfoxide were determined after 1, 7 and 15 d of storage. The survival rates of larvae frozen with 1.5 molL⁻¹ dimethyl sulfoxide after 1 h, 1 d, 7 d and 15 d of storage were 80.77% ±7.51%, 80.34% ±11.28%, 83.10% ±9.14% and 77.23% ±6.22% respectively. No significant differences in survival rates of larvae frozen with dimethyl sulfoxide were observed after various storage periods (p>0.05).     

Production of bioplastics and hydrogen gas by photosynthetic microorganisms

Experiments on salimity tolerance of embryos and larvae of the southern bay scallopArgopecten irradians concentricus Say under controlled temperature of 28°C showed that although the eggs were fertilized and cleaved at salinity 18.1–37.4, they could further develop into normal D-shaped larvae only at salinity 23.4–36.1, while the trochophore larvae could develop into normal D—shaped larvae at salinity 22.1–39.9. For 72 hours, the larvae could tolerate salinity of 18.4–43.9, the salinity favoring growth was 19.8–42.4; the suitable range was 21–36, and the optimum at about 28. They metamorphosed at salinity 18.9–43.5, optimally at 24–32. Contribution No. 3301 from the Institute of Oceanology, Chinese Academy of Sciences. The research was supported by the National Climbing Project B(PDB6-3-2).     

Effects of environmental oxygen deficiency on embryos and larvae of bay scallop,Argopecten irradians irradians

The experimental results showed that: 1) The embryonic development of bay scallop is inhibited at a dissolved oxygen range of 1.38–3.64×10⁻³ at 23°C, and completely blocked below the lower limit. 2) The tolerance of larvae to anoxia increased with larval sizes and was related to their oxygen debt. 3) The scallop larvae exhibited specific behavioral responses to oxygen deficiency, which finally led to velum disintegration and larval death. The possible relationship between environmental oxygen deficiency and the disease of disintegration of the larval velum is also discussed. In this study, considerable oxygen debt was found in bay scallop larvae, which was greater in small animals. Based on the works of previous authors, a new concept is proposed for the estimation of oxygen debt, namely, the compensatory rate of oxygen debt (CROD). This can be used in intra—or interspecific comparison of oxygen debt. The results can be helpful in the management of water quality and for the prevention of larval diseases encountered in scallop culture. Contribution No. 2167 from the Institute of Oceanology, Chinese Academy of Sciences.     

Effect of n?3 HUFA levels in rotifers and Artemia on growth and survival of larval black sea bream ( Sparus macrocephalus )

Requirement for dietary n−3 HUFA (n−3 highly unsaturated fatty acid) for growth and survival of black sea bream (Sparus macrocephalus) larvae was studied using rotifers andArtemia at various levels of n−3 HUFA. Five treatments with rotifers andArtemia differing in n−3 HUFA were prepared by enriching them with various oil emulsions. Results indicated that dietary n−3 HUFA significantly influence fish n−3 HUFA levels and are essential for growth and survival of black sea bream larvae. The results also indicated that the incorporation of n−3HUFA TG (triacylglycerols) into tissues of larval black sea bream was more effective from natural fish oil in comparison with n−3 HUFA fatty acid ethyl esters from ethyl-esterified oil. Project No. 39770588 supported by the NSFC.     

Comparison of growth and survival of larvae among different shell color stocks of bay scallop Argopecten irradians irradians (Lamarck 1819)

In order to improve production and breed new broods of bay scallopArgopecten irradians irradians, different-colored orange, purple and white lines were established by two mating methods of self-fertilization and mass spawning at a commercial scallop hatchery in spring, 2002. And then larval growth and survival of different lines was compared to test whether there is a relationship between shell color and growth and survival at early developmental stage. Both growth and survival have no significant differences among different experimental larvae of self-fertilization or mass spawning. Results are as following in the order of orange, purple and white shell stock. For the self-fertilization, growth rates of larvae were 6.174, 6.412, and 6.599 μm/d, respectively. Survival rates of larvae at Day 3 were 74.41%, 76.86%, and 82.05%; Day 6 were 49.14%, 65.63%, and 52.79%; and Day 9 were 25.06%, 20.80%, and 26.47%, respectively. For the mass spawning, the growth rates were 7.836, 7.941, and 7.878 μm/d, respectively. Survival rates at Day 3 were 93.05%, 91.95%, and 92.50%; Day 6 were 79.17%, 78.05%, and 82.50%; and Day 9 were 34.72%, 36.67%, and 38.33%, respectively. The absence of any relationship between shell color and growth and survival at the larval stage may be resulted from their common genetic basis. This work was financially supported by Project of Scientific Innovation, Chinese Academy of Sciences (ZKCX2-211).     

Toxic effects of Zn2+, Cu2+, Cd2+ and NH3 on Chinese prawn

This study deals with the toxic effects of Zn²⁺, Cu²⁺, Cd²⁺ and NH₃ on Chinese prawn (Penaeus orientalis). The median lethal concentrations of the toxic matters on Chinese prawn were determined. Cd²⁺>Cu²⁺>Zn²⁺>NH₃-Nm is the toxic order of these chemicals to Chinese prawn. The toxic order of concentrated Zn²⁺ and Cd²⁺ in tissues and organs of Chinese prawn is in the order. gill>shell+appendage>viscera>muscle.     

Histological study on the skin of Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

Histological development of Japanese flounder Paralichthys olivaceus larval skin and ultrastructural difference of skin between reared normal and malpigmented Japanese flounder were studied with light microscopy (LM) and transmission electron microscopy (TEM). The results show that the skin develops slowly before the metamorphosis, while at the onset of metamorphosis, the skin develops quickly and becomes complete in structure till about 50d after being hatched. Ultrastructural observation on the normal and malpigmented skins shows that the iridophore and melanophore are adjacent to each other. Profile and structure of the two kinds of pigmcnt cells are more complete in the skin of normal ocular side than in the skin of pigmented blind side. The ultrastructure of typical chloride cell was observed in the skin of Japanese flounder larvae for the first time.     

化学物质诱导对马氏珠母贝眼点幼虫附着的影响

【目的】研究血清素(5-HT)、蜕皮激素、γ-氨基丁酸(GABA)、腺嘌呤核苷、次黄嘌呤核苷和次黄嘌呤对马氏珠母贝眼点幼虫附着的影响。【方法】以不同浓度的5-HT、蜕皮激素、GABA、腺嘌呤核苷、次黄嘌呤核苷和次黄嘌呤溶液处理马氏珠母贝眼点幼虫,不同时间后,分析眼点幼虫附着率的变化。【结果】在24~96 h作用时间里,5-HT、蜕皮激素、GABA对眼点幼虫附着的诱导效果整体上均呈上升趋势;在24~120 h作用时间里,腺嘌呤核苷、次黄嘌呤核苷和次黄嘌呤对眼点幼虫附着的诱导效果整体上呈上升趋势。眼点幼虫在10^-5 mol/L的5-HT和GABA处理24、48、72和96 h后,附着率均显著高于对照组(P<0.01);10^-5 mol/L的蜕皮激素处理24 h和72 h,10^-6 mol/L的蜕皮激素处理24 h和96 h,10^-7 mol/L的蜕皮激素处理24 h,附着率均显著高于对照组(P<0.01);10μmol/L腺嘌呤核苷处理24、48、72和120 h,1μmol/L腺嘌呤核苷处理24、48、72、96和120 h,附着率均显著高于对照组(P<0.01);10μmol/L次黄嘌呤核苷处理48和72 h,1μmol/L次黄嘌呤核苷处理48、72、96和120 h,附着率均显著高于对照组(P<0.01);10μmol/L的次黄嘌呤处理72、96和120 h,附着率均显著高于对照组(P<0.01)。【结论】5-HT、蜕皮激素、GABA、腺嘌呤核苷、次黄嘌呤核苷和次黄嘌呤溶液在适宜的浓度和处理时间均可诱导马氏珠母贝眼点幼虫附着,其中10^-5 mol/L的5-HT处理24 h对附着率的诱导效果最佳,可达对照组的6.3倍。     

Immunotoxicity effect of benzo[α]pyrene on scallop Chlamys farreri

The toxic effects of benzo[α]pyrene (B[α]P) at different concentrations (0.1, 0.5, 1, 2.5 and 7.5 μgL⁻¹) on scallop (Chlamy farreri) immune system were studied. The results showed that B[α]P had significant toxic effects on the haemocyte counts, neutral red uptake, phagocytosis, bacteriolytic and antibacterial activity (P<0.05), while the seawater control and acetone control had no significant differences. The haemocyte counts, neutral red uptake, phagocytosis and bacteriolytic activity in all B[α]P treatment groups as well as antibacterial activity in groups of 0.5, 1, 2.5 and 7.5 μgL⁻¹ B[α]P decreased significantly (P<0.05). Some of these indices tended to be stable on the sixth day and others on the ninth day, and the indices showed clear time- and concentration-response to B[α]P. Bacteriolytic activity in 0.1μgL⁻¹ B[α]P treatment group and antibacterial activity in 0.1 μgL⁻¹ and 0.5 μgL- B[α]P treatment groups increased at the beginning of exposure and reached their peaks on day 1 and day 6, respectively. Following that, both activities decreased gradually and became stable after day 9. When all the indices reached stability, they were significantly lower than those in control group (P<0.05), except for antibacterial activity in 0.1 μgL⁻¹ B[α]P treatment group (P>0.05). Thus, B[α] has evident toxic effects on scallop immune system, which supports the view that a relationship exists between pollution and immunomodulation in aquatic organisms.     

Morphological and toxicity characteristics of Pseudo-nitzschia pungens strain PP0201-01 isolated from the East China Sea

The morphology and toxicity ofPseudo-nitzschia strain PP0201-01, isolated near the estuary of Changjiang (Yangtze) River in Year 2002 and tentatively identified asPseudo-nitzschia pungens, were studied. The strain was identified morphologically asP. pungens using scanning electronic microscope (SEM). The analytical results of LC-PDA (liquid chromatography with photodiode array detector), LC-MS (liquid chromatography with mass detector), and the toxicity data from mouse bioassay, showed that no domoic acid was produced by the strain. However, the mechanism of domoic acid production byPseudo-nitzschia species is complicated. More works are needed to assess the toxic species and to evaluate the potential risk of amnesic shellfish poisoning (ASP) in China. Supported by the projects of Ministry of Science and Technology (No. 2001CB409704 and No. 2001BA804A20), and the innovation project of the Institute of Oceanology, CAS (No. 200223107).     

The effect of chemical cues on the settlement of sea cucumber (Apostichopus japonicus) larvae

The effects of four ions and eight neuroactive compounds on inducing larval settlement of A. japonicus were assessed in the present study. All bioassays were conducted in 60 × 9 mm Petri dishes, each contained 10 mL of the test solution and 10 doliolaria larvae. There were significant inductive effects of K+(10- mmol L-1), NH+4(0.1 mmol L1), GABA(10-3 mol L-1), acetylcholine(10-5 mol L-1), L-DOPA(10-5 mol L-1), norepinephrine(10-5 mol L-1) and dopamine(10-7 mol L-1 and 10-5 mol L-1) on the settlement of sea cucumber larvae. L-DOPA and dopamine are the most efficient chemical cues to induce A. japonicus larvae to settle. The highest percentage of larval settlement was induced by 10-5 mol L-1 L-DOPA and dopamine(33% and 40%) compared to the control(7%). However, Ca2+, Mg2+, choline, serotonin, and epinephrine were less effective on larval settlement at all tested concentrations. This study evaluated the stability and feasibility of chemical cues for larval settlement in different culture systems, which can be applied to improve the hatchery production of this valuable species.     

Tetraploid induction by inhibiting mitosis I in scallopChlamys farreri

Tetraploid induction was carried out by inhibiting mitosis I in fertilized eggs ofChlamys farreri. Mitosis I was blocked with cold shock (5–7°C), Cytochalasin B (0.75 mg/L) and 6-dimethylaminopurine (6-DMAP) (60–75 mg/L) when 60% fertilized eggs released polar body II at 20°C. At 4-cells embryo stage, the ploidy was determined by counting chromosome number. In control groups, most embryos were diploids (72.22%) and aneuploids (24.78%). In Cytochalasin B, cold shock and 6-DMAP treated groups, tetraploids were respectively 10.51%, 4.08%, and 13.34%; aneuploids were 43.10%, 35.93% and 29.16%, and triploids were 7.84%, 8.52% and 18.33%. At D-larva stage, ploidy was determined by flow cytometry (FCM). The ploidy analysis of day 2 larvae showed diploids in control group and also in three treated groups. Juvenile scallops (0.2–0.3cm) which were harvested in two control groups and two CB treated groups were all diploids through checking ploidy individually by FCM. Contribution No. 3648 from the Institute of Oceanology, Chinese Academy of Sciences. Contribution No. 238 from the Experimental Marine Biology Laboratory, Chinese Academy of Sciences. Project funded by grant (No. 819-01-07) from the Chinese Science and Technology Ministry and also by “Hundred Talents Plan” grant from the Chinese Academy of Sciences.