Impact of <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis> and white spot syndrome virus (WSSV) co-infection on survival of penaeid shrimp <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>

White spot syndrome virus (WSSV) is an important viral pathogen that infects farmed penaeid shrimp, and the threat of Vibrio parahaemolyticus infection to shrimp farming has become increasingly severe. Viral and bacterial cross or superimposed infections may induce higher shrimp mortality. We used a feeding method to infect Litopenaeus vannamei with WSSV and then injected a low dose of V. parahaemolyticus (WSSV+Vp), or we first infected L. vannamei with a low-dose injection of V. parahaemolyticus and then fed the shrimp WSSV to achieve viral infection (Vp+WSSV). The eff ect of V. parahaemolyticus and WSSV co-infection on survival of L. vannamei was evaluated by comparing cumulative mortality rates between experimental and control groups. We also spread L. vannamei hemolymph on thiosulfate citrate bile salt sucrose agar plates to determine the number of Vibrio, and the WSSV copy number in L. vannamei gills was determined using an absolute quantitative polymerase chain reaction (PCR) method. LvMyD88 and Lvakt gene expression levels were detected in gills of L. vannamei by real-time PCR to determine the cause of the diff erent mortality rates. Our results show that (1) the cumulative mortality rate of L. vannamei in the WSSV+Vp group reached 100% on day 10 after WSSV infection, whereas the cumulative mortality rate of L. vannamei in the Vp+WSSV group and the WSSV-alone control group approached 100% on days 11 and 13 of infection; (2) the number of Vibrio in the L. vannamei group infected with V. parahaemolyticus alone declined gradually, whereas the other groups showed significant increases in the numbers of Vibrio (P<0.05); (3) the WSSV copy numbers in the gills of the WSSV+Vp, Vp+WSSV, and the WSSV-alone groups increased from 10⁵ to 10⁷ /mg tissue 72, 96, and 144 h after infection, respectively. These results suggest that V. parahaemolyticus infection accelerated proliferation of WSSV in L. vannamei and vice versa. The combined accelerated proliferation of both V. parahaemolyticus and WSSV led to massive death of L. vannamei.     

Spatial variation of stable isotopes in different waters during melt season in the Laohugou Glacial Catchment,Shule River basin

To evaluate isotopic tracers at natural abundances by providing basic isotope data of the hydrological investigations and assessing the impacts of different factors on the water cycle, a total of 197 water samples were collected from the Laohugou Glacial catchment in the Shule River basin northwestern China during the 2013 ablation seasons and analyzed their H- and O-isotope composition. The results showed that the isotopic composition of precipitation in the Qilianshan Station in the Laohugou Glacial catchment was remarkable variability. Correspondingly, a higher slope of δ~(18)O-δD diagram, with an average of 8.74, is obtained based on the precipitation samples collected on the Glacier No.12, mainly attributed to the lower temperature on the glacier surface. Because of percolation and elution, the isotopic composition at the bottom of the firn is nearly steady. The δ~(18)O /altitude gradients for precipitation and melt water were -0.37‰/100 m and -0.34‰/100 m, respectively. Exposed to the air and influenced by strong ablation and evaporation, the isotopic values and the δ~(18)O vs δD diagram of the glacial surface ice show no altitudinal effect, indicating that glacier ice has the similar origins with the firn. The variation of isotopic composition in the melt water, varying from -10.7‰ to -16.9‰(δ~(18)O) and from -61.1‰ to -122.1‰(δD) indicates the recharging of snowmelt and glacial ice melt water produced at different altitudes. With a mean value of -13.3‰ for δ~(18)O and -89.7‰ for δD, the isotopic composition of the stream water is much closer to the melt water, indicating that stream water is mainly recharged by the ablation water. Our results of the stable isotopic compositions in natural water in the Laohugou Glacial catchment indicate the fractionations and the smoothing fluctuations of the stable isotopes during evaporation, infiltration and mixture.     

Effect of co-culture of Chinese shrimp (<Emphasis Type="Italic">Fenneropenaeus chinensis</Emphasis>) and sea cucumber (<Emphasis Type="Italic">Apostichopus japonicus</Emphasis> Selenka) on pond environment

Monoculture of sea cucumber (pond S) and polyculture of shrimp with sea cucumber (pond SS) were established to evaluate the effect of shrimp on the environmental conditions of sea cucumber farming pond. Contributions of sediment organic matter (SOM2) resuspended from benthic sediment and the suspended particulate organic matter (SPOM) deposited from the water column to the precipitated organic matter (SOM1) collected with sediment traps were estimated with carbon stable isotope analysis. The results showed that the levels of SPOM and SOM2 in pond SS significantly decreased in comparison with those in pond S at the end of experiment (P < 0.05), indicating that co-culturing shrimp in sea cucumber farming pond could purify the farming water. Carbon stable isotope analysis showed that the proportion of SOM2 in SOM1 in pond SS (84.97% ± 0.38%) was significantly lower than that in pond S (95.20% ± 0.30%) (P < 0.05), suggesting that the resuspension of organic matter from benthic sediment into overlying water was reduced in polyculture pond. In contrast, the proportion of SPOM in SOM1 in pond SS (15.03% ± 0.38%) was significantly higher than that in pond S (4.80% ± 0.30%) (P < 0.05), indicating that the sedimentation of SPOM from water column was enhanced in pond SS owing to the biodeposition effect of shrimp.     

Nitrogen Budget in Recirculating Aquaculture and Water Exchange Systems for Culturing <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>

In order to investigate the culture characteristics of two indoor intensive Litopenaeus vannamei farming modes, recirculating aquaculture system (RAS) and water exchange system (WES), this study was carried out to analyze the water quality and nitrogen budget including various forms of nitrogen, microorganism and chlorophyll-a. Nitrogen budget was calculated based on feed input, shrimp harvest, water quality and renewal rate, and collection of bottom mud. Input nitrogen retained in shrimp was 23.58% and 19.10% respectively for WES and RAS, and most of nitrogen waste retained in water and bottom mud. In addition, most of nitrogen in the water of WES was TAN (21.32%) and nitrite (15.30%), while in RAS was nitrate (25.97%), which means that more than 76% of ammonia and nitrite were removed. The effect of microalgae in RAS and WES was negligible. However, bacteria played a great role in the culture system considering the highest cultivable cultivable bacterial populations in RAS and WES were 1.03×10¹⁰ cfu mL^?1 and 2.92×10⁹ cfu mL^?1, respectively. Meanwhile the proportion of bacteria in nitrogen budget was 29.61% and 24.61% in RAS and WES, respectively. RAS and WES could realize shrimp high stocking culture with water consuming rate of 1.25 m³ per kg shrimp and 3.89 m³ per kg shrimp, and power consuming rates of 3.60 kw h per kg shrimp and 2.51 kw h per kg shrimp, respectively. This study revealed the aquatic environment and nitrogen budget of intensive shrimp farming in detail, which provided the scientific basis for improving the industrial shrimp farming.     

Temporal and spatial variations of abundance of phycocyanin- and phycoerythrin-rich <Emphasis Type="Italic">Synechococcus</Emphasis> in Pearl River Estuary and adjacent coastal area

Three surveys were carried out in Pearl River Estuary and adjacent coastal area in May, August, and November, 2013, to investigate the temporal and spatial variations of abundance of phycoerythrin-rich Synechococcus (PE-rich SYN) and phycocyanin-rich Synechococcus (PC-rich SYN). The effects of environmental factors on the alternation of the different Synechococcus groups were also elucidated. PE-rich SYN was detected in three surveys, whereas PC-rich SYN was detected in May and August, but not in November. The highest abundances of PE-rich SYN and PC-rich SYN were recorded in August and May, with mean values of 74.17×10³ and 189.92×10³ cells mL^?1, respectively. From May to November, the relative abundance of PE-rich SYN increased, whereas that of PC-rich SYN declined. PE-rich and PC-rich SYN presented similar horizontal distributions with high abundance in the southern estuary in May, and in the western estuary in August. The abundances of PE-rich and PC-rich SYN were high at 27–32°C and salinity of 10–20. PC-rich SYN was not detected at < 24°C, and PC:PE-rich SYN decreased in abundance with salinity increase. When less than 20 mg L^?1, suspended particulate matter (SPM) was helpful for Synechococcus growth. PE-rich SYN decreased in abundance when the concentration of dissolved inorganic nitrogen increased in May and November, and the concentration of phosphate increased in November. However, PC-rich SYN abundance and nutrients showed no correlation. Principal component analysis and regression analysis indicated that PE-rich SYN significantly correlated with the principal components that were affected by environmental factors.     

Antioxidant response of ridgetail white prawn <Emphasis Type="Italic">Exopalaemon carinicauda</Emphasis> to harmful dinoflagellate <Emphasis Type="Italic">Prorocentrum minimum</Emphasis> exposure and its histological change

The dinoflagellate Prorocentrum minimum, one of the most widespread red tide causing species, affects marine aquaculture and ecosystems worldwide. In this study, ridgetail white prawn Exopalaemon carinicauda were exposed to P. minimum cells (5 × 10⁴ cells mL^?1) to investigate its harmful effects on the shrimp. Antioxidant activities and histological changes were used as indicators of health status of the shrimp. In 72 hours, the mortality of E. carinicauda was not affected, but its antioxidant response and histology were statistically different from those of control. Elevated superoxide dismutase (SOD) and glutathione peroxidase (GPX) activities and depressed catalase (CAT) activity were observed in gill; while increased SOD, glutathione S-transferase (GST), CAT activities and modulated GPX activity were observed in hepatopancreas. Thus, antioxidant activities in gill and hepatopancreas seem to respond differentially to harmful alga exposure. Increased malondialdehyde (MDA) content in early a few hours indicates the damage of the antioxidant defense system. Although MDA content recovered to a low level thereafter, a series of histological abnormalities including accumulation or infiltration of hemocytes, tissue lesions and necrosis were discovered in gill and hepatopancreas. Exposure to P. minimum induced sublethal effects on E. carinicauda, including temporary oxidative damage and histological injury.     

Effects of dietary genistein on GH/IGF-I axis of Nile tilapia <Emphasis Type="Italic">Oreochromis niloticus</Emphasis>

There is considerable concern that isoflavones, such as genistein in fish feed composed of soybean protein, aff ects somatic growth in fish. Our previous works demonstrated that 30 and 300 μg/g dietary genistein had no significant eff ect on growth performance in Nile tilapia (Oreochromis niloticus), but the higher level of genistein (3 000 μg/g) significantly depressed growth. This study was conducted to further examine the eff ects of dietary genistein on the endocrine disruption on growth hormone/insulin-like growth factor-I (GH/IGF-I) axis in Nile tilapia (O. niloticus). Juvenile fish were fed by hand twice daily to satiation with one of four isonitrogenous and isoenergetic diets, each containing either 0, 30, 300 or 3 000 μg/g genistein. Following an 8-week feeding period, plasma GH and IGF-I levels were investigated by radioimmunoassay and gene expression levels of gh, ghrelin, gnrhs, ghr, npy, npyrs, pacap, ghrs, igf-I, igf-Ir, and igfbp3 were examined by real-time PCR. The results show that no significant change in plasma GH and IGF-I levels in fish fed with diets containing 30 μg/g and 300 μg/g genistein. mRNA expression of genes along the GH/IGF-I axis remained unaff ected, except for igf-Ir, which was stimulated by the 300 μg/g genistein diet. While in fish fed the 3 000 μg/g genistein diet, the plasma GH and IGF-I levels decreased, and mRNA expression of gh, ghr2, npyr1, igf-I, and igf-Ir were also significantly depressed. In contrast, npy and igfbp3 mRNA expression were enhanced. This study provides convincing evidence for growth impediment by genistein by disturbing the GH/IGF-I axis in Nile tilapia O. niloticus.     

Food sources of the Manila clam Ruditapes philippinarum in intertidal areas: evidence from stable isotope analysis

Based on stable isotope analysis, we characterized the dietary regime of the Manila clam Ruditapes philippinarum inhabiting intertidal areas along the Liaodong Peninsula, Northern China. Samples, including particulate organic matter (POM; n =30), benthic microalgae (BMI; n =30) and R. philippinarum (n =60), were collected from six sampling sites displaying the same ecological conditions. Of the two primary food sources, POM was more depleted in δ ¹³C (?20.61‰ to ?22.89‰) than BMI was (?13.90‰ to ?16.66‰). With respect to ¹⁵N, BMI was more enriched (2.90‰ to 4.07‰) than POM was (4.13‰ to 5.12‰). The δ ¹³C values of R. philippinarum ranged from ?18.78‰ to ?19.35‰ and the δ ¹⁵N values from 7.96‰ to 8.63‰, which were intermediate between the POM and BMI values. In a two-source isotope mixing model, we estimated the relative contributions of POM and BMI to the diet of R. philippinarum to be 74.2% and 25.8%, respectively. We conclude that R. philippinarum feeds mainly on POM, and BMI is also an important supplemental food source in intertidal areas.     

Biodeposition,respiration, and excretion rates of an introduced clam <Emphasis Type="Italic">Mercenaria mercenaria</Emphasis> in ponds with implications for potential competition with the native clam <Emphasis Type="Italic">Meretrix meretrix</Emphasis> in Shuangtaizi estuary,China

This study aimed to evaluate the potential impacts of an introduced clam Mercenaria mercenaria on estuarine ecosystem, and implications for the niche competition with a native clam Meretrix meretrix. The biodeposition, respiration, and excretion rates of M. mercenaria were determined seasonally using a sediment trap and a closed respirator in field. The biodeposition rates of M. mercenaria were 0.06–0.37 g/ (ind.·d), and the respiration rates were 0.31–14.66 mg/(ind.·d). The ammonia and phosphate excretion rates were 0.18–36.70 and 1.44–14.87 μg/(ind.·d), respectively. The hard clam M. mercenaria may discharge dry deposits up to 2.1×10⁵ t, contribute 18.3 t ammonia and 9.0 t phosphate to culture ponds, and consume 7.9×10³ t O₂ from ponds annually. It suggested that the hard clam M. mercenaria might play an important role in pelagic-benthic coupling in pond ecosystem through biodeposition and excretion. A comparison of the key physiological parameters of the introduced clam M. mercenaria and the native clam Meretrix meretrix suggested that M. mercenaria had a niche similar to that of Meretrix meretrix in Shuangtaizi estuary and might have a potential competition with Meretrix meretrix for habitat and food if M. mercenaria species escaped from the culture pond or artificially released in estuarine ecosystem.     

Effect of bioactive peptides (BPs) on the development of Pacific white shrimp (<Emphasis Type="Italic">Litopenaeus vannamei</Emphasis> Boone, 1931)

The present study was conducted to evaluate the feasibility of replacing fish meal (FM) with bioactive peptides (BPs) in diet of white shrimp (Litopenaeus vannamei). The changes in growth performance, body composition, non-specific immunity, and water quality were examined after the shrimp were fed four diets, in which 0% (control), 33.3%, 66.7% and 100% of FM was replaced by BPs, respectively. The groups were designated as Con, 1/3BPs, 2/3BPs, and 3/3BPs. A total of 720 shrimp with an initial body weight of 1.46 ± 0.78 g were fed the experimental diets for 56 days. The results revealed that: 1) the weight gain rate (WGR) in 1/3BPs, 2/3BPs, and 3/3BPs was significantly higher than that in Con (P < 0.05), while no significant difference was found on survival rate and feed conversion ratio (FCR); 2) the whole-body crude protein (CP) and crude lipids (CL) were significantly different among groups, while there was no significant difference between crude ash and phosphorus contents; 3) the levels of acid phosphatase (ACP), lysozyme (LZM), superoxide dismutase (SOD), phenol oxidase (PO) and bactericidal activity increased significantly with the inclusion of BPs; 4) in terms of water quality, no significant difference was found in pH and dissolved oxygen among diets during the whole experimental period. Moreover, even though nitrite and ammonium levels tended to increase with time, there was no significant difference among groups. The results indicated that BPs is an applicable alternative of protein source, which can substitute FM in the diets of L. vannamei; it is able to effectively promote growth performance and improve immunity. Moreover, BPs in the diets had no negative impact on water quality.     

Use of antioxidant enzymes of clam <Emphasis Type="Italic">Ruditapes philippinarum</Emphasis> as biomarker to polycyclic aromatic hydrocarbon pollution

The typical organic pollutant polycyclic aromatic hydrocarbon (PAH) anthracene was selected as a contaminant to investigate its effects on the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) in the clam Ruditapes philippinarum. The results show that SOD, CAT and GSH-Px had diff erent induction and inhibition reactions to anthracene stress, and that three diff erent organs in R. philippinarum (visceral mass, muscle tissue and mantle) had diff erent sensitivities to anthracene stress. This study suggest that SOD activities of the visceral mass, CAT activitities of the mantle and the visceral mass, and GSH-Px activity of the muscle tissue could be used as sensitive indicators of anthracene stress in R. philippinarum.     

Effects of nitrogen and phosphorus on the growth of <Emphasis Type="Italic">Levanderina fissa</Emphasis>: How it blooms in Pearl River Estuary

Effects of nitrogen (N) and phosphorus (P) from different sources and at different concentrations on the growth of Levanderina fissa (= Gyrodinium instriatum) were studied in laboratory conditions. The findings might explain the recurrent blooms of this species in Pearl River Estuary, China. Results showed that nutrient limitation significantly inhibited the growth of L. fissa. The values of specific growth rate (μ _max) and half-saturation nutrient concentration (K _S) were 0.37 divisions/d and 8.49 μmol L^?1 for N, and 0.39 divisions/d and 1.99 μmol L^?1 for P, respectively. Based on K _S values, dissolved inorganic N level in PRE was sufficient to support the high proliferation of L. fissa, while dissolved inorganic P concentration was far lower than the minimum requirement for its effective growth. L. fissa was not able to utilize dissolved organic N (DON) compounds such as urea, amino acids, and uric acid. However, it grew well by using a wide variety of dissolved organic P (DOP) sources like nucleotides, glycerophosphate, and 4-nitrophenylphosphate. The results from this study suggested that the ability in DOP utilization of L. fissa offers this species a competitive advantage in phytoplankton communities. The high level and continuous supply of DIN, enrichment of DOP, together with warm climate and low salinity in the Pearl River Estuary provided a suitable nutrient niche for the growth of L. fissa, and resulted in the recurrent blooms in the estuary.     

Effects of a Potential Autochthonous Probiotic <Emphasis Type="Italic">Bacillus subtilis</Emphasis> 2-1 on the Growth and Intestinal Microbiota of Juvenile Sea Cucumber, <Emphasis Type="Italic">Apostichopus japonicus</Emphasis> Selenka

The effects of Bacillus subtilis 2-1 from the intestine of healthy sea cucumber on the growth, digestive enzyme activities and intestinal microbiota of juvenile sea cucumber (Apostichopus japonicus) were determined in the present study. Sea cucumber was fed with Sargassum thunbergii powder supplemented with B. subtilis 2-1 at different concentrations varying among 0 (control), 10⁵, 10⁷, and 10⁹ CFU g^?1 for 8 weeks. Results showed that the growth performance and intestinal amylase and trypsin activities were significantly increased by dietary B. subtilis 2-1 at 10⁹ CFU g^?1 (P < 0.05). However, dietary B. subtilis 2-1 had no significant influence on the lipase activity in sea cucumber (P > 0.05). The polymerase chain reaction denaturing gradient gel electrophoresis and 16S rRNA gene sequencing analysis indicated that dietary B. subtilis 2-1 at 10⁵ and 10⁷ CFU g^?1 inhibited most of the Proteobacteria including those in genus Vibrio. Dietary B. subtilis 2-1 at 10⁹ CFU g^?1 not only decreased the abundance and species of genus Vibrio, but also increased the intensity of genera Psychrobacter and Bacillus. A specific dosage of dietary B. subtilis 2-1 could increase the growth and modulate the intestinal microbiota of sea cucumber; thus it might be a novel probiotic for keeping the health of sea cucumber.     

Carbon budget of bastard halibut<Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> in relation to body weight and temperature

The effects of body weight and temperature on the carbon budget of the juvenile bastard halibut ,Paralichthys olivaceus ,were studied at temperature 13.5,18,21.5 and 24℃,respectively.The carbon intake,faecal and growth carbon were measured ,and the carbon respiration was calculated using the carbon budget equation (Cc=Gc Fc Rc),The combined relationship between different components of the carbon budgent,body weight and temperature could be described by regression equations:Cc=1.0206 W^0.8126E^0.1483T;Gc=0.0042w^1.4096(-5.11 T^3 285.90T^2-5173.72T 30314.03);Fc=0.0485W^0.7711e^0.1624T;Uc=1.4333W^0.6715e^0.1487t,Body weight had no significant effect on the carbon absorption efficiency and the conversion efficiency.     

Biodegradation of <Emphasis Type="Italic">Enteromorpha</Emphasis> polysaccharides by intestinal micro-community from <Emphasis Type="Italic">Siganus oramin</Emphasis>

Micro-communities are supposed to have more potential functions of biodegradation of polysaccharides than single strain; however, the intestinal micro-communities involved in the biodegradation of Enteromorpha polysaccharides (EP) were seldom reported. In order to obtain the EP-degrading micro-community, the intestines of Siganus oramin was obtained to isolate the micro-communities, which were enriched by 0.3% of EP as the sole carbon source. A stable micro-community with EP degradative capability was achieved after seven generations of subculture, named H1. Results showed that H1 was able to degrade 75% of EP within 24 hours, and the activity of EP lyases reached 500 U mL^?1 in 32 hours. With denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone library analysis, ten bacteria closely related to Marinomonas pontica, Microbacterium sp., Leucobacter chironomi, Cyclobacterium sp., Algoriphagus winogradskyi, Pseudoalteromonas sp. and Vibrio sp. were determined. Furthermore, compared with the DGGE bands sequence and the clone library analysis, the dominant bacteria of the EP-biodegrading micro- community were Pseudoalteromonas sp. and Vibrio sp., with the respective proportion of 38% and 46%, and they should play an important role in EP degradation together with other degrading bacteria in the micro-community H1.     

The community structure and seasonal dynamics of plankton in Bange Lake,northern Tibet,China

The seasonal variations in biomass, abundance, and species composition of plankton in relation to hydrography were studied in the saline Bange Lake, northern Tibet, China. Sampling was carried out between one to three times per month from May 2001 to July 2002. Salinity ranged from 14 to 146. The air and water temperature exhibited a clear seasonal pattern, and mean annual temperatures were approximately 4.8°C and 7.3°C, respectively. The lowest water temperature occurred in winter from December to March at-2°C and the highest in June and July at 17.7°C. Forty-one phytoplankton taxa, 21 zooplankton, and 5 benthic or facultative zooplankton were identifi ed. The predominant phytoplankton species were Gloeothece linearis, Oscillatoria tenuis, Gloeocapsa punctata, Ctenocladus circinnatus, Dunaliella salina, and Spirulina major. The predominant zooplankton species included H olophrya actra, Brachionus plicatilis, Daphniopsis tibetana, Cletocamptus dertersi, and A rctodiaptomus salinus. The mean annual total phytoplankton density and biomass for the entire lake were 4.52×10~7 cells/L and 1.60 mg/L, respectively. The annual mean zooplankton abundance was 52, 162, 322, and 57, 144 ind./L, in the three sublakes. The annual mean total zooplankton biomass in Lakes 1–3 was 1.23, 9.98, and 2.13 mg/L, respectively. The annual mean tychoplankton abundances in Bg1, 2, and 3 were 47, 67, and 654 ind./L. The annual mean tychoplankton biomass was 2.36, 0.16, and 2.03 mg/L, respectively. The zooplankton biomass(including tychoplankton) in the lake was 9.11 mg/L. The total number of plankton species in the salt lake was signifi cantly negatively correlated with salinity.     

Diel variation in metabolism and ammonia excretion of <Emphasis Type="Italic">Marphysa sanguinea</Emphasis> (Polychaeta: Eunicidae)

Polychaetes provide an excellent food resource for fish and represent the dominant zoobenthos in marine ecosystems. Diel variation in the rates of metabolism and ammonia-N excretion of Marphysa sanguinea were studied. The worms were grouped according to their wet body weight into small (S; 1.24±0.06 g), medium (M; 4.00±0.30 g), and large (L; 8.54±1.08 g) categories. Their weight-specific metabolic rates, based on aerobic respiration (R), were measured at 16°C (±0.2°C) and classed as either routine (R _R) or standard (R _S) rates. Both respiration types decreased with increasing body weight. Respiration was described by R = a W ^b, where b was -0.400 9 and -0.532 0 for R _R and R _S, respectively. Diurnal changes in R _S for each group was relatively flat, with a slightly increasing trend with time, but was relatively stable as a whole. R _R of the diurnal variation of worms was higher than R S, but both had similar overall trends. The peak values of specific dynamic action (SDA) (R _SDA) in the S, M, and L groups were 2.704, 1.149, and 0.682 mg/(g?h), respectively. The durations of SDA were 13, 6, and 6 h, respectively and the energy expenditures of SDA were 377.98, 117.34, and 74.94 J/g, respectively. These data indicate that the metabolic rates were higher in smaller individuals, which is advantageous for their rapid growth.     

Inbreeding depression on growth and survival of full-sib family of Manila clam (<Emphasis Type="Italic">Ruditapes philippinarum</Emphasis>)

In present study, the inbreeding depression (ID) of growth and survival of Manila clam (Ruditapes philippinarum) was investigated at larval and juvenile stages. Nine inbred families (A ₂, B ₂, C ₂, D ₂, E ₂, F ₂, G ₂, H ₂ and I ₂) were established by mating within nine full-sib families with expected inbreeding coefficient of 0.25. Inbred families showed significant differences in shell length and hatching rate of D-larvae (straight-hinged larvae). The larvae of the nine inbred families grew slower than those of control group (CG), and their ID value ranged from 0.81% ± 6.09% to 16.10% ± 1.49%. The ID value of larval survival rate varied between 27.47% ± 9.36% and 70.50% ± 13.66%. The ID was also detected for juvenile growth in A ₂, B ₂, C ₂, and D ₂, which ranged from 4.60 ± 2.21 to 17.71 ± 7.73. The A ₂ family maintained the highest juvenile survival rate, whereas the other inbred families exhibited ID values varying between 62.79% ± 4.54% and 96.14% ± 0.87%. The linear relationship of estimated ID between growth and survival was negatively correlated (R = ?0.434, P < 0.05). The results of this study suggested that the ID of growth was common at the larval stage but was less prevalent at juvenile stage. In contrast, the ID of survival increased from larval to juvenile stage. A better understanding of the effect of inbreeding may aid to selective breeding of Manila clam.     

Genetic transformation of <Emphasis Type="Italic">Nannochloropsis oculata</Emphasis> with a bacterial phleomycin resistance gene as dominant selective marker

The gene ble from Streptoalloteichus hindustanus is widely used as a selective antibiotic marker. It can control the phleomycin resistance, and significantly increase the tolerance of hosts to zeocin. The unicellular marine microalga Nannochloropsis oculata is extremely sensitive to zeocin. We selected ble as the selective marker for the genetic transformation of N. oculata. After the algal cells at a density of 2×10⁷ cells mL^?1 was digested with 4% hemicellulase and 2% driselase for 1 h, the protoplasts accounted for 90% of the total. The ble was placed at the downstream of promoter HSP70A-RUBS2 isolated from Chlamydomonas reinhardtii, yielding a recombinant expression construct pMS188. The construct was transferred into the protoplasts through electroporation (1 kV, 15 μS). The transformed protoplasts were cultured in fresh f/2 liquid medium, and selected on solid f/2 medium supplemented with 500 ng mL^?1 zeocin. The PCR result proved that ble existed in the transformants. Three transformants had been cultured for at least 5 generations without losing ble. Southern blotting analysis showed that the ble has been integrated into the genome of N. oculata. The ble will serve as a new dominant selective marker in genetic engineering N. oculata.