Effect of water temperature on digestive enzyme activity and gut mass in sea cucumber Apostichopus japonicus （Selenka）, with special reference to aestivation

The effect of water temperature on gut mass and digestive enzyme activity in sea cucumber Apostichopus japonicus, including relative gut mass (RGM), amylase, lipase, pepsin and trypsin activities were studied at temperatures of 7, 14, 21, and 28°C over a period of 40 days. Results show that RGM significantly decreased after 40 days at 21°C and markedly decreased over the whole experiment period at 28°C; however, no significant effect of duration was observed at 7 or 14°C. At 14°C, trypsin activity significantly decreased over 10 and 20 days, then increased; amylase and trypsin activity significantly decreased after 40 days at 28°C. However, no significant effect of duration was found on amylase, pepsin or trypsin activities in the other temperature treatment groups. At 28°C, lipase activity peaked in 20 days and then markedly decreased to a minimum at the end of the experiment. On the other hand, pepsin activity at 28°C continuously increased over the whole experimental period. Principle component analysis showed that sea cucumbers on day 40 in the 21°C group and in the previous 20 days in the 28°C group were in the prophase of aestivation. At 28°C, sea cucumbers aestivated at 30–40 days after the start of the experiment. It is concluded that the effect of temperature on the digestion of A. japonicus is comparatively weak within a specific range of water temperatures and aestivation behavior is accompanied by significant changes in RGM and digestive enzyme activities.     

Establishment and characterization of a new cell line from the kidney of spotted halibut <Emphasis Type="Italic">Verasper variegates</Emphasis>

A cell line,SHK,was derived from the kidney of spotted halibut Verasper variegates.The cell line was subcultured more than 40 passages in minimum essential medium(MEM)supplemented with fetal bovine serum(FBS)and 10 ng ml-1 basic fibroblast growth factor(bFGF).Cell morphology from primary culture and subculture was observed continuously by microscopy.The SHK cell line consisted predominantly of fibroblast-like cells.The cell line was able to grow between 20℃ and 30℃ with the optimum growth at 24℃ and with a reduced growth between 12℃ and 20℃.The growth rate of the cells increased as the proportion of FBS increased from 10% to 20% at 28℃ with optimum growth at the concentration of 20%.The doubling time of the cells was determined to be 44.8 h.Chromosome analysis revealed that 52% of the SHK cells maintained a normal diploid chromosome number (2n=46).The cells were successfully transfected with green fluorescent protein(GFP)reporter plasmids and the expression of GFP gene in the cells indicated the possible utility of the cells in gene expression studies.The cells were infected by lymphosystis disease virus(LCDV)and found to be susceptible to the virus in cytopathic effect(CPE)observation.The infection was confirmed by PCR and electron microscopy experiments,which proved the existence of the viral particles in the cytoplasm of the virus-infected cells.     

Effect of temperature and photoperiod on maturation of Cephalothrix hongkongiensis （Nemertea： Palaeonemertea）

The limited knowledge about the relationships between environmental factors and the maturation and reproductive seasonality of nemerteans is mostly obtained from field studies, and in many cases is conjectural. Few species have been studied under well-controlled laboratory conditions. In this work, the effects of temperature and photoperiod on gonad development and spawning of Cephalothrix hongkongiensis were observed by rearing worms under different temperatures and photoperiods. Worms reared at 4℃ and 6℃ showed no growth or gonadal development, and all died within 109 days. At 8℃ and 12℃, gonads could be seen from 90 days to the termination of the 360-day experiment, but no spawning was observed. Worms maintained at 16, 24, and 28℃ showed both gonadal development and spawning, and matured faster at higher temperatures. Under a fixed temperature(16℃), photoperiod did not have any significant impact on maturation and spawning. It is likely that temperature plays a major role in regulating the maturation and reproductive cycle of C. hongkongiensis.     

Effects of temperature on oxygen consumption and ammonia—N excretion ofChlamys farreri

Effects of temperature on oxygen consumption rate (OCR) and ammonia—N excretion rate of scallopChlamys farreri (1.7–6.2 cm in shell height) were studied in laboratory from Dec. 30,1996 to Jan. 28,1997. Under the controlled conditions of ambient water temperature 10–31°C and salinity 32, the concentrations of dissolved oxygen and ammonia—N were determined by the Winkle method and the hypobromite method, respectively. Results showed that the OCR ranged from 1.20 mg/g (DW) · h to 5.76 mg/g (DW) · h. The OCR increased with temperature from 10°C to 23°C, but at 28°C the OCR of mature individuals decreased, and that of different size scallops reduced at 31°C. The ammonia—N excretion rate ranged from 113.03 μg NH₄-N/g (DW) · h to 486.63 μg NH₄-N/g (DW) · h, and increased with temperature from 10°C to 31°C. Contribution No.3180 from the Institute of Oceanology, Chinese Academy of Sciences. This work was supported by the National Commission of Science and Technology of China, Grant No.96-922-02-04.     

Physiological responses of Chinese longsnout catfish to water temperature

We evaluated the effect of water temperature on the growth and physiology of the Chinese longsnout catfish (Leiocassis longirostris Günther). The fish were reared at four temperatures (20, 25, 30, and 35°C) and sampled on days 7, 20, and 30. We measured plasma levels of insulin, free thyroxine (FT4), free 3,5,3′-triiodothyronine (FT3), lysozyme and leukocyte phagocytic activity. The optimum water temperature for growth was 27.7°C. The plasma levels of insulin and FT4 declined significantly (P<0.05) on day 30 at temperatures above 20°C. Lysozyme activity was significantly (P<0.05) lower at 25°C than at other temperatures. We conclude that final weight, insulin, FT4, and lysozyme were significantly affected by water temperature.     

Stability and changes in astaxanthin ester composition from Haematococcus pluvialis during storage

In this paper, we investigated the effects of temperature, oxygen, antioxidants, and corn germ oil on the stability of astaxanthin from Haematococcus pluvialis under different storage conditions, and changes in the composition of astaxanthin esters during storage using high performance liquid chromatography and spectrophotometry. Oxygen and high temperatures （22-25~C） significantly reduced the stability of astaxanthin esters. Corn germ oil and antioxidants （ascorbic acid and vitamin E） failed to protect astaxanthin from oxidation, and actually significantly increased the instability of astaxanthin. A change in the relative composition of astaxanthin esters was observed after 96 weeks of long-term storage. During storage, the relative amounts of free astaxanthin and astaxanthin monoesters declined, while the relative amount of astaxanthin diesters increased. Thus, the ratio of astaxanthin diester to monoester increased, and this ratio could be used to indicate if astaxanthin esters have been properly preserved. If the ratio is greater than 0.2, it suggests that the decrease in astaxanthin content could be higher than 20%. Our results show that storing algal powder from H. pluvialis or other natural astaxanthin products under vacuum and in the dark below 4~C is the most economical and applicable storage method for the large-scale production of astaxanthin from H. pluvialis. This storage method can produce an astaxanthin preservation rate of at least 80% after 96 weeks of storage.     

Pharmacokinetics and tissue behavior of enrofloxacin and its metabolite ciprofloxacin in turbot Scophthalmus maximus at two water temperatures

Turbot Scophthalmus maximus, an important aquaculture species in China, currently suffers from epizootic diseases because of high density aquaculture. Enrofloxacin has been used to treat various systemic bacterial fish infections. However, studies concerning the pharmacokinetics of enrofloxacin in turbot are limited. In this study, the pharmacokinetics of enrofloxacin and its metabolite ciprofloxacin, were investigated in the turbot following intravenous and oral administration at 10 mg enrofloxacin/kg body weight, at 16°C and 10°C water temperatures. The concentrations of enrofloxacin and ciprofloxacin in the main tissues (plasma, muscle, liver and kidney) were detected by HPLC. The results show that the plasma concentration-time data for enrofloxacin were best described as a two-compartment open model after intravenous and oral administration. Three pharmacokinetic equations were established between the concentrations and temperatures. The kinetic profile of enrofloxacin was temperature dependent. The absorption half-life of enrofloxacin was 1.99 h and 2.17 h after oral administration, whereas the elimination half-life of the drug was 98.63 h and 136.59 h at 16°C and 10°C, respectively. The peak concentration of enrofloxacin in plasma and tissues was higher at 16°C than that at 10°C, and the peak plasma concentration time in the liver was the shortest at both temperatures among those of other tissues. The plasma C max /MIC ratio varied between 11.08 and 5 540.00 at 16°C; and between 7.92 and 3 960.00 at 10°C. The AUC/MIC ratio was 467.82-280 690.00 at 16°C, and 359.48-215 690.00 at 10°C. These ratios indicate that it is possible to obtain therapeutic efficacy. Very low levels of ciprofloxacin were detected. The AUC ratios of ciprofloxacin and enrofloxacin in plasma suggest that plasma ciprofloxacin might play a minor role in enrofloxacin treatment for turbot.     

Purification and characterization of a new thermostable κ-carrageenase from the marine bacterium Pseudoalteromonas sp. QY203

A new extracellular κ-carrageenase, namely CgkP, 34.0 kDa in molecular weight, was purified from Pseudoalteromonas sp. QY203. CgkP showed relatively high activity at acidities ranging from pH6.0 to pH9.0 and temperatures ranging from 30℃ to 50℃ with the highest activity at 45℃ and pH7.2. Sodium chloride increased its activity markedly, and KCl increased its activity slightly. The divalent and trivalent metal ions including Cu2+ , Ni2+ , Zn2+ , Mn2+ , Al3+ and Fe3+ significantly inhibited its activity, while Mg2+ did not. CgkP remained 70% of original activity after being incubated at 40℃ for 48 h, and remained 80% of the activity after being incubated at 45℃ for 1 h. It exhibited endo-κ-carrageenase activity, mainly depolymerizing the κ-carrageenan into disaccharide and tetrasaccharide. CgkP was more thermostable than most of previously reported κ-carrageenases with a potential of being used in industry.     

Toxicity of five phenolic compounds to brine shrimp Artemia sinica (Crustacea: Artemiidae)

The acute toxicity of five phenolic compounds each to 15 d old Artemia sinica was determined in this study. The brine shrimp A. sinica was hatched from the encysted dry eggs(Bohai Bay Brand) produced by Dongying Ocean Artemia Co., Ltd., China at 27℃± 1℃ in pre-filtered(through pores of 0.45 μm in diameter) and autoclaved seawater(salinity 31, pH 7.5–8.0) in a cilindroconical glass beaker(2000 mL in volume) under continuous illumination(provided by a side set 20 W fluorescent lamp) with slight aeration. Ten Artemia individuals from the same batch of the hatched were cultured in 10 mL toxicant solution prepared with seawater(salinity 31, pH 7.5–8.0) at room temperature(about 20℃) to determine 24 h, 48 h and 72 h medium lethal concentration(LC50) of 5 phenolic compounds each. It was found that the toxicity of n-heptylphenol was the highest followed by nonylphenol, t-butylphenol, 2,4-dichlorophenol and bisphenol A in order. The LC50 values of the 5 compounds were calculated with regression analysis. The real concentration(in μg- L1) of 5 phenolic compounds each in toxicant solutions was measured with GC/MS analysis. Significant loss of phenolic compounds caused by either adsorption or desorption was not found. The significant difference of LC50 values was found among the five compounds 3 exposure times each. The range between the highest no-observed-effect concentration(NOEC) and 100% death causing concentration of five phenolic compounds each was determined. The toxicity in term of 24 h LC50 value of n-HP was 9.10 times higher than that of BPA, 1.71 times higher than t-BP, 1.53 times higher than 2,4-DCP and 1.36 times higher than NP, respectively.     

Effects of rearing temperature and density on growth, survival and development of sea cucumber larvae, Apostichopus japonicus (Selenka)

In laboratory conditions, effects of rearing temperature and stocking density were examined on hatching of fertilized egg and growth of auricularia larvae of Apostichopus japonicus respectively. Data series like larval length and density, metamorphic time, and survival rate of the larvae were recorded. Statistics showed that for A. japonicus, survival rate (from fertilized egg to late auricularia) decreased significantly with the increasing rearing temperature (P<0.05). At different temperatures SGR was statistically significant as well (P<0.05) from day 1, and maximal SGR was found on day 9 at 24°C (159.26±3.28). This study clearly indicated that at low temperature (<24°C), metamorphic rate was remarkably higher than at higher temperature (>26°C). Hatching rate was significantly different between 0.2–5 ind./ml groups and 20–50 ind./ml groups. Rearing larvae at the higher density had the smaller maximal-length, whereas needed longer time to complete metamorphosis. This study suggested that 21°C and 0.4 ind./ml can be used as the most suitable rearing temperature and stocking density for large -scale artificial breeding of A. japonicus’s larvae.     

Triploidy induction in Pacific oysterCrassostrea gigas by caffeine with thermal shock

Zygotes of Pacific oyster (Crassostrea gigas) were treated for triploid induction with caffeine (10 mmol/L, 15mmol/L and 20mmol/L) in combination with thermal shocks (at 40 minute post-fertilization) lasting for 5 and 10 minutes. The highest yield of triploids, 41.5%, was obtained from the treatment with 20 mmol/L caffeine at 34°C lasting 10 minutes. The triploid levels were less than 30.0% in other treatments. Triploid induction was more effective in treatment with 15–20 mmol/L caffeine at 34–38 °C than with lower concentrations of caffeine at temperatures below 32 °C. Our results suggest that triploid induction with caffeine in combination with thermal shocks is less efficient than some other methods reported previously.     

Low temperature stress on the hematological parameters and <Emphasis Type="Italic">HSP</Emphasis> gene expression in the turbot <Emphasis Type="Italic">Scophthalmus maximus</Emphasis>

To study the effect of low temperature stress on hematological parameters and HSP gene expression in the turbot (Scophthalmus maximus), water temperature was lowered rapidly from 18 to 1°C. During the cooling process, three individuals were removed from culture tanks at 18, 13, 8, 5, 3, and 1°C. Blood samples and tissues were taken from each individual, hematological indices and HSP gene expression in tissues were measured. The red blood cell count, white blood cell count, and hemoglobin concentration decreased significantly (P < 0.05) as temperature decreased. Enzyme activities of plasma alanine transaminase and creatine kinase increased as temperature decreased, whereas aspartic transaminase and γ-glutamyl transpeptidase activities displayed no obvious changes above 1°C and lactate dehydrogenase activity increased first and then decreased. Blood urea nitrogen and uric acid levels were highest at 8°C, and creatinine concentration was highest at 3°C. The concentrations of plasma cortisol, cholesterol, and triglyceride all increased significantly (P < 0.05) as temperature decreased. The serum glucose concentration increased first and then decreased to the initial level. The HSP70 mRNA expression showed various patterns in different tissues, whereas HSP90 mRNA expression showed the same tendency in all tissues. Overall, these results indicate that temperature decreases in the range of 8 to 5°C may induce a stress response in S. maximus and that temperature should be kept above 8°C in the aquaculture setting to avoid damage to the fish.     

Stress Responses to Rapid Temperature Changes of the Juvenile Sea Cucumber （Apostichopusjaponicus Selenka）

Activities of hexokinase （HK）, pyruvate kinase （PK）, superoxide dismutase （SOD） and catalase （CAT） and Hsp70 level were measured to evaluate the response of the commercially important sea cucumber （Apostichopus japonicus Selenka） to rapid temperature changes in laboratory. Animals were subjected to a higher temperature （from 10 to 20℃） （Tinc treatment） or to a lower temperature （from 20 to 10℃） （Tddec treatment） for 72 h. At 1, 3, 12, 24, 72 h of exposure, animals were removed and prepared for further analysis. Results showed that the effect of acute temperature changes on enzyme activities was significant. In Tinc treatment, activities of SOD and CAT increased immediately. The significant enhancement of SOD and CAT activities suggested that oxidative stress increases significantly when ambient temperature increasing from 10 to 20℃. The up-regulation of Flsp70 in Tinc and Tdec treatments indicated that Hsp70 was a bioindicator of thermal stress in the sea cucumber, and the expression pattern depended on the thermal treatment.     

Biofilm formation under high temperature causes the commensal bacteria Bacillus cereus WPySW2 to shift from friend to foe in Neoporphyra haitanensis in vitro model

Although biofilm formation may promote growth, biofilms are not always beneficial to their hosts. The biofilm formation characteristics of Bacillus cereus WPySW2 and its changes at different temperatures were studied. Results show that B. cereus WPySW2 promoted the growth of Neoporphyra haitanensis (an economically cultivated seaweed) at 20 °C but accelerated algal rot at 28 °C. Thicker B. cereus WPySW2 biofilms covered the surface of N. haitanensis thalli at 28 °C, which hindered material exchange between the algae and surrounding environment, inhibited algal photosynthesis and respiration, and accelerated algal decay. Compared with planktonic bacteria, mature biofilm cells had lower energy consumption and metabolic levels. The biofilm metabolic characteristics of B. cereus WPySW2 changed significantly with temperature. High temperature accelerated biofilm maturation, which made it thicker and more stable, allowing the bacteria to easily adapt to environmental changes and obtain greater benefits from their host. High temperature did not affect the production or increased the abundance of toxic metabolites, indicating that the negative effects of B. cereus WPySW2 on algae were not caused by toxins. This study shows that increased temperature can transform a harmless bacterium into a detrimental one, demonstrating that temperature may change the ecological function of phycospheric bacteria by affecting their morphology and metabolism.

     

Acclimation-dependent expression of heat shock protein 70 in Pacific abalone (Haliotis discus hannai Ino) and its acute response to thermal exposure

Heat shock protein 70 (Hsp70) is one important member of heat shock protein (Hsp) family that is responsible for various stresses, especially thermal stress. Here we examined the response of Hsp70 gene to both chronic and acute thermal exposure in Pacific abalone (Haliotis discus hannai Ino). For the chronic exposure, abalones were maintained at 8, 12, 20, and 30°C for four months and their mRNA levels were measured. The highest mRNA level of Hsp70 gene relative to actin gene was detected in the 30°C-acclimated group, followed by the 8°C-acclimated group and then the 12°C- and 20°C-acclimated groups. After the long-term acclimation, gills from each of the above acclimation groups were dissected and exposed to different temperatures between 8°C and 38°C for 30 min. Hsp70 expression in gills acclimated to different temperatures responded differentially to the same temperature exposure. The incubation temperature that induced maximum Hsp70 mRNA expression was higher in the higher temperature acclimation groups than lower temperature groups. Pacific abalones could alter the expression pattern of Hsp70 gene according to environmental thermal conditions, through which they deal with the stress of thermal variations.     

Effects of temperature and salinity on survival,growth and DNA methylation of juvenile Pacific abalone,Haliotis discus hannai Ino

Temperature and salinity are two of the most potent abiotic factors infl uencing marine mollusks.In this study,we investigated the individual and combined ef fects of temperature and salinity on the survival and growth of juvenile Pacifi c abalone,Haliotis discus hannai Ino,and also examined the DNA methylation alteration that may underpin the phenotypic variation of abalone exposed to dif ferent rearing conditions.The single-factor data showed that the suitable ranges of temperature and salinity were 16–28°C at a constant salinity of 32,and 24–40 at a constant temperature of 20°C,respectively.The two-factor data indicated that both survival and growth were signifi cantly af fected by temperature,salinity and their interaction.The optimal temperature-salinity combination for juveniles was 23–25°C and 30–36.To explore environment-induced DNA methylation alteration,the methylation-sensitive amplifi ed polymorphism(MSAP)technique was used to analyze the genomic methylation profi les of abalone reared in optimal and adverse conditions.Neither temperature nor salinity induced evident changes in the global methylation level,but 67 and 63 dif ferentially methylated loci were identifi ed in temperature and salinity treatments,respectively.The between-group eigen analysis also showed that both temperature and salinity could induce epigenetic dif ferentiation in H.discus hannai Ino.The results of our study provide optimal rearing conditions for juvenile H.discus hannai Ino,and represent the fi rst step toward revealing the epigenetic regulatory mechanism of abalone in response to thermal and salt stresses.     

Competition Among Dinoflagellate Alexandrium Tam—arense, Raphidophyte Heterosigma Carterae and Diatom Skeletonema Costatum under Combinations ofTwo Temperatures and Five Salinities

Competition among HAB (Harmful Algal Bloom) species Dinoflagellate Alexandrium tamarense, Raphidophyte Heterosigma carterae, and Diatom Skeletonema costatum was studied in the laboratory. Experiments with these three major HAB species under combinations of different salinities (10, 18, 25, 30, 35) and temperatures (19℃, 25℃) were carried out. The results showed that S. costatum successfully competed with the other two species at salinities of 18, 25,30, and 35 at temperatures of 19℃ and 25℃. However, H. carterae showed its advantage at low salinity of 10 and became the single dominant species at salinity 10 and 25℃. A. tamarense could not compete successfully with the other two species especially at low salinities. However, it could remain at low density in the presence of higher densities of other algae.     

Simulated thermal alteration of sterols and keto—Alcohols in marine sediments of the Okinawa Trough

Compositional data on the sterol and alcohol fractions isolated from deep-sea marine sediments from the Okinawa Trough were obtained to determine the relative contribution from marine and terrestrial inputs. Following extraction, the sterol plus alcohol fraction was isolated by layer chromatography, derivatized with BSTFA and then analysed by capillary GC and GC—MS. A suite of C₂₆−C₂₉ stenols and stanols and C₃₀−C₃₂ keto—alcohols were identified in the sediments. The thermal stability of the compounds in these sediments was studied by heating portions of the surface sediment in glass tubes for 16 hours at temperatures from 50°C to 200°C. The C₂₇ stanol/stenol ratio increased when temperatures went up to 175°C, but the distribution of C₃₀−C₃₂ Keto—alcohols remained unaffected. At 200°C most of the sterols and Keto—alcohols were destroyed. Supported by the National Natural Foundation of China.     

Embryology of Maldives clownfish, Amphiprion nigripes (Amphiprioninae)

This study investigated the embryonic development of Maldives clownfish Amphiprion nigripes under natural conditions (28 - 30℃) at a lagoon of Agatti Island, Lakshadweep, India. The newly deposited fish egg was capsule-shaped and orange-red, with a (0.73 ± 0.04) mm3 yolk containing 5 - 10 fat globules. The embryonic development of fertilized eggs was divided into 26 stages and the time elapsing for each stage was recorded. Results showed that the cleavage was rapid, with the first division observed 1 h 20 min after fertilization. Blastulation occurred 4 h later, followed by gastrulation 12 h after fertilization, with a yolk volume of (0.61 ± 0.06) mm3. The organogenesis process started 22 h after fertilization when the blastopores closed and notochord formation began. The embryonic stage was recorded 24 h later, with the appearance of forebrain, midbrain, hindbrain, melanophores on yolk-sac and 22 somites, and a decreased yolk volume of (0.54 ± 0.08) mm3. Other organs developed well 31 h after fertilization, whereas the heart started beating and blood circulation began 78 h later. Red pigmentation (erytrophores) appeared 96 h after fertilization, with a small yolk volume of (0.22 ± 0.02) mm3. Mouth developed well and eyes were noticeable 120 h later, with head, pectoral fin and tail frequently moving 144 h after fertilization. The embryo reached the pre-hatching stage 168 h later and started to hatch after 170 - 180 h incubation. This study first detailed the embryonic development and yolk absorption of A. nigripes under natural conditions.