Hyriopsis schlegelii is a fresh water mussel species farmed intensively and is chosen mainly to produce pearls in China.Cathepsin L is a member of the cysteine protease family and plays important roles in many physiological processes.In this paper,we report the full-length cDNA of cathepsin L(GenBank accession No.JN604558) from H.schlegelii based on cDNA library screening,homologous cloning strategies,and its immune response.cDNA of H.schlegelii(denoted as Hs-CtsL) was 1152 bp long and consisted of a 5′-untranslated region(UTR) of 1 bp,a 3′-UTR of 149 bp with one cytokine RNA instability motif(AATAAA) and one Poly(A) tail,and an open reading frame of 1002 bp,encoding a polypeptide of 333 amino acids,with an estimated molecular mass of 37.7 kD and a theoretical isoelectric point of 7.16.The protein sequence contained a typical signal peptide sequence with 20 amino acids,a propeptide inhibitor domain and a mature domain.The phylogenetic analysis showed that a clear clade division was observed between vertebrates and invertebrates cathepsin L proteins,and the Hs-CtsL of H.schlegelii clustered with the invertebrate cathepsin L cysteine proteases and was closely related to corresponding sequences in Hyriopsis cumingii and Cristaria plicata.Tissue expression detection with real-time fluorescence quantitative PCR demonstrated that the Hs-CtsL transcripts in healthy three-year adult H.schlegelii was expressed in intestine,gill,gonad,mantle,foot,adductor muscle,haemocytes,hepatopancrease,kidney and heart,with the highest expression level in haemocytes.After pathogenic Aeromonas hydrophila challenge,the Hs-CtsL mRNA expression was significantly up-regulated in haemocytes,gill,mantle and hepatopancrease,and the highest expression in hepatopancrease was observed at 6h after stimulation whereas its increased expression was detected at 4h,12h and 48h in haemocytes,gill and mantle,showing a fluctuant change during the early stage of bacterial challenge.These results suggested that the Hs-CtsL was not only involved in the immune defense of hemocytes but also involved in the immune response of hepatopancrease in H.schlegelii.

A 15-week feeding trial was conducted to study the effects of dietary potassium on growth and physiological responses of juvenile abalone,Haliotis discus hannai Ino.Six casein-gelatin based semi-purified diets containing graded levels of dietary potassium(KCl,0.10,2.12,4.39,9.79,20.08 and 27.26 g/kg dry diet,respectively) were fed to juvenile abalone [initial weight:(0.24 ± 0.00) g;initial shell length:(12.24 ± 0.04) mm] in triplicate groups in a flow-through system.During the experimental period,the water temperature ranged from 12 to 23℃ and potassium concentration was(472.94 ± 3.59) mg/L in the seawater.The results showed that there were no significant(P 0.05) differences in the weight gain ratio(WGR,%),daily increment in shell length(DISL,μm/d) and the survival of abalone among treatments.There were also no significant(P 0.05) differences in lipid,moisture contents in soft body and ash content in shell among the six dietary treatments.Compared with that in the treatment of 0.10 g/kg diet,the protein content in soft body increased significantly(P 0.05) when dietary potassium was more than 4.39 g/kg diet.The content of potassium in the soft body and potassium,sodium in the shell was maintained relatively constant regardless of dietary treatment.The soft body sodium content was significantly(P 0.05) lower in 20.08 and 27.26 g/kg of dietary treatment than any other treatments.The activity of gill Na+-K+ ATPase decreased steadily with the increasing of dietary potassium.Data from the growth parameters(WGR and DISL) showed that it was unnecessary to add potassium to the diets,and the potassium from diet sources and seawater was sufficient for the optimal growth of juvenile abalone.Based on the physical index(protein and sodium contents in soft body and Na+-K+ ATPase activity in gill),the optimal concentration of dietary potassium was 2.12 g/kg.

Spin protein family,with the conserved Spin/Ssty motif,play important role during gametogenesis and early embryo development.So far,no Spin genes have been identified in zebrafish,Danio rerio.In this study,to isolate the full-length cDNA of Spin gene from zebrafish,we designed two degenerate primers according to the conserved sequences of Spin protein family.Two 260 bp DNA fragments of DrSpin-1 and DrSpin-2 were isolated from SMART cDNA library of zebrafish mature eggs.The sequence homologous analysis indicated the identity of amino acid sequences between DrSpin-1 protein and CagSpin protein got up to 81%,and DrSpin-2 protein showed greater sequence identity with OlSpinB and OlSpinC.Further,two specific primers were designed according to the 260 bp DNA sequence of DrSpin-1 for RACE polymerase chain reaction,and the full-length cDNA sequences of DrSpin-1 was isolated from the SMART cDNA library referred above.Sequence analysis revealed DrSpin-1 cDNA contained 1082 base pairs with a 771 base pairs open reading frame encoding 257 amino acids and including three conserved Spin/Ssty motives and eight possible phosphorylation sites.These data confirmed the cloned DrSpin-1 gene was one of Spin gene family.Furthermore,the multiple alignments analysis of DrSpin-1 protein sequence and Spin protein sequences in other fishes by CLUSTRA W indicated that DrSpin-1 protein had the highest homology to CagSpin protein in gibel carp.Therefore,we suggested DrSpin-1 in zebrafish might have the similar expression pattern to CagSpin,and might play important roles in regulation during oogenesis and fertilization.Our studies provide the clue to further functional research of Spin in zefrafish.

Transferrin has a variety of biological functions such as iron transportation,anti-microbial,immunoregulation,etc.In this paper,the expression pattern and the antibacterial activity of purified recombinant protein of Mauremys mutica transferrin(MaTf) were analyzed.The ORF of MaTf were obtained by RT-PCR,then ligated into pET-32 a(+) vector to construct recombinant vector for prokaryotic expression,then transformed into E.coli BL21 competent cells.The putative protein was successfully expressed with the induction of IPTG(0.8 mmol/L) at 30℃ for 4 hours.The solubility of the expressed products was tested by PAGE electrophoresis of supernatant and precipitation separately resulted from smashed bacterial.The results indicated that the protein was in inclusion body and insoluable.The precipitation of smashed bacterial including the recombinant protein was washed with 1×binding buffer including 2M urea,resolved in 1×binding buffer including 6 M urea at 37℃ for 1h,purified with His bind column and then renatured by dialysis.The purity was tested by PAGE electrophoresis and western blot,which confirmed the high purity of the recombinant protein of MaTf obtained.The biological activity of anti-microbial was tested in vitro by assessing the effect on bacterial growth repression.According to the diameter of repressed bacteria circle on plate for equal dose of purified protein,the antibacterial effects to three kind of bacteria tested are different,which are Staphylococcus aureus Escherichia coli Serratia marcescens.The expression pattern in different tissues in M.mutica were quantified by western blot,which showed that the pattern at protein level is similar to the mRNA level,which is: liver spleen kidney heart.This research provides an important information for exploring the biological function of transferrin in non-specific immune response.

In the development of tilapia industry,excellent quality of tilapia fry is a key point to guarantee the rapid growth rate and good economical benefits of marketable fish.At present,the domestic fry market is increasing rapidly,but the tilapia fry are on various levels of quality.The hybrids of Oreochromis niloticus♀ × O.aureus♂ are one of the most popular commercial types for tilapia farmers,because of rapid growth rate,strong resistance to disease and low temperature for the hybrids.But due to complex origin of the fry parents,the hybrid effects and the cultured benefits are being reduced greatly.Therefore choosing superior hybrid combination of different parental strains is essential.In order to understand and utilize heterosis and select the perfect hybrid subgroups of combinations,differences of growth characters among numerous subgroups should be objectively evaluated.In our research,two strains of Oreochromis niloticus(XX,EE strain) and two strains of O.aureus(ZZ,AA strain) and their hybrid subgroups of Oreochromis niloticus♀ × O.aureus♂(XZ,EZ,ZA,XA) were produced and injected with RFID electronic tags to track and measure growth characteristics for comparative analysis.All the tilapia parents came from technology research center of national tilapia industrial system,which was one of the most important tilapia breeding bases in China.All the fry of combinations except ZZ strain had been hatched out,fifty individuals of each combination were picked out for our analysis.All the individuals for analysis were cultivated in the same pond and fed twice daily,the air pump had been installed in the pond.The results showed that the male ratios of XZ,XA,EZ,EA subgroups were 96.0%,94.0%,98.0%,95.7%,while the ratios of XX,EE,AA strains are 58.0%,66.0%,52.0%,respectively.After 380 days of cultivation,at least four morphological characteristics such as total length,standard length,head length and body depth of four hybrid subgroups were significantly more than those of their parental strains(P0.05),while there were more advantages of growth rate in hybrid tilapia subgroups than parental strains.Meanwhile,there was significant difference(P0.05) in growth characteristics among the four hybrid subgroups.Significant correlations were existed in body weight and other morphological characteristics,the correlation coefficients between body weight and caudal peduncle length(0.690-0.846) were obviously lower than that between body weight and total length,standard length,body depth and body width.Data on body weight and morphological characteristics were used for principal component analysis,the characteristics including body weight,total length,head length,body depth,caudal peduncle length,caudal peduncle depth and body width were divided by standard length.In order to eliminate the influence of differences in body size among the tilapia individuals,we obtained seven proportion characteristics including BS,TS,HS,BDS,CLS,CDS and BWS respectively.In the characteristic coefficients plots of principal components factor scores,those seven proportion characteristics were divided into two groups,the first group concluded BS,BDS,CDS and BWS,the other concluded TS,HS and CLS.We draw a scatter plot through the score coefficients of tilapia individuals in principal component factors PCR1 and PCR2.In the plot the individuals of AA strain could obviously differentiated from other subgroups.

Most of the decapod species of crustacean have comprehensive capacity of osmoregulation,and they can adapt to relative wide ambient salinity range.It is important to understand the underlying mechanisms of their comprehensive adaptability to salinity change because this knowledge is helpful for inland culturing of marine species.As a euryhaline crustacean,the Chinese mitten crab,Eriocheir sinensis,has been well studied in many aspects related to their physiological responses to salinity change,and many useful literature findings have been accumulated.Therefore,E.sinensis can be considered as an animal model for studying osmoregulation mechanism of the decapod species.However,the roles of nutrients in osmoregulation of E.sinensis are still limited.In this paper,male E.sinensis in freshwater(0.3‰) were transferred directly into waters at salinity values of 16‰ and 30‰,respectively,and the crabs kept at fresh water served as the control.Each treatment has three replicates.The soluble protein in haemolymph and hepatopancreas,hemocyanin,haemolymph glucose and hepatopancreas glycogen at 0h,6h,12h,24h,48h,72h and 96h of E.sinensis at different salinities were determined.Soluble protein of haemolymph and hepatopancreas were measured with a Folin reagent based on a standard curve using bovine serum albumin as the standard protein.Hemocyanin was assayed using an ultraviolet absorption methods at 335 nm and calculated according to Nickerson 1% 1cm E =2.83.Haemolymph glucose was assayed using glucose oxidase-peroxide enzyme(GOD-POD) method.Hepatopancreas glycogen was measured using the anthrone colorimetry method.The results showed that the crabs at salinity 16‰ and 30‰ had significantly lower soluble protein content of hepatopancreas than those at fresh water(P0.05) when crabs had encountered salt water for 12h—96h,and the soluble protein content of haemolymph decreased significantly from 6h to 48h,and then increased at 72h.The hemocyanin content showed a significantly decreasing trend from 0h to 24h,and then it increased after 48h.Hepatopancreas glycogen contents at salinity 16‰ and 30‰ were significantly lower than those at fresh water(P0.05) from 6h to 96h,while no significant differences were observed between crabs in waters with salinity 16‰ and 30‰(P0.05).Haemolymph glucose content at 16‰ reduced significantly(P0.05) from 24h to 48h,and gradually recovered up at 72h,but it showed a significantly decreasing trend from 6h to 12h at salinity 30‰,and then gradually increased after 24h.All these results indicated that E.sinensis could produce physiological and biochemical adaptation to maintain a stable osmotic pressure during acute salinity stress.Carbohydrate and protein play important roles in the osmoregulation of crustaceans.E.sinensis could use carbohydrate first to provide energy under acute salinity stress.The higher the salinity,the faster the haemolymph glucose was consumed,and it also recovered more quickly.This observation suggested that carbohydrate could be a direct source in osmoregulation.The crabs may use protein to maintain the balance of osmotic pressure under high salinity by metabolizing the protein into free amino acids.Beside providing free amino acids,hemocyanin also can carry oxygen to meet the needs for physiological activities.Therefore,more attention should be paid to carbohydrate and protein supplied under salinity stress.The ratio of free amino acids maintaining the balance of osmotic pressure and supplying energy by oxidation and the utilization of lipid in osmoregulation require further study.Since the lipid contents in haemolymph and hepatopancreas were not determined in this study,the role of body lipid in E.sinensis was not discussed here and should be further examined.

Tintinnid ciliates are an important component of plankton communities and widely distribute in the coastal waters. However, in morphological studies, the tintinnids belonged to the poorly known groups, especially in the water of the South China Sea. In the winter of 2008, the tintinnids sampled from the coastal waters in the South China Sea (Guangdong) were investigated. The water temperature was 17-20℃ and the salinity was about 30-31. Samples were collected by a horizontal tow net from 1.5 m under the water surface. Part of samples were immediately stored in 4℃ for in vivo observation and the others were fixed in formalin at a final concentration of 5% for future observations of lorica characteristics. The morphological characteristics including lorica features were observed and described by using light microscopical method. In this present study, 16 species were indentified and they belonged to Tintinnopsis, Codonella, Stenosemella and Favella. Three of them were believed to be the first records of China, Codonella apicata Kofoid Campbell, 1929, Tintinnopsis turbo Meunier, 1919 and Stenosemella expansa Wailes, 1925. In addition, another three species were the first records in the semi-tropical and tropical areas of China, Tintinnopsis tubulosoides Meunier, 1910; Tintinnopsis lohmanni (J?rgensen, 1927) Laackmann, 1906, and Codonella rapa Kofoid Campbell, 1929. The other 10 species included Tintinnopsis beroidea Stein, 1867, Tintinnopsis acuminata (Daday, 1887) Kofoid Campbell, 1929, Tintinnopsis tocantinensis Kofoid Campbell, 1929, Tintinnopsis tubulosoides Meunier, 1910, Tintinnopsis tublosa Levander, 1900, Tintinnopsis clongata Daday, 1887, Tintinnopsis lohmanni (J?rgensen, 1927) Laackmann, 1906, Stenosemella parvicollis (Marshall, 1934) Hada, 1935, Favella campanula (Schmidt, 1901)J?rgensen, 1924, and Codonella rapa Kofoid Campbell, 1929.  

The Three Gorges Dam was altogether finished in 2009 and water level will reach 175 meter during dry season (winter) and fall back to 145m during rainy season (summer). Impoundment of the Three Gorges Reservoir forms three significant different water types in the main stream of the Three Gorges Reservoir in Chongqing: Natural riverine zone (above 175 m) where the change of water environment fluctuate with seasons, Transition zone (145-175 m) where water environment is effected by seasons and impoundment and Analogous lacustrine zone where water level is below 145 m throughout the year. They are different in velocity of flow, nutritive level (such as the concentration of N and P) and so on. For investigating the response to these changes of algae, the physical and chemical characters of water in the main stream were tested and surveys on the phytoplankton communities in the Three Gorges Reservoir were carried out in September 2009 (time before impoundment of the Three Gorge Reservoir), January (storage period), March (drainage period) and September 2010 (time before the second impoundment). The result as follows: The mean of pH was 8.17 and Dissolved Oxygen (DO) changed from 6.54 mg/L to 11.44 mg/L during the investigation. Total nitrogen (TN) of all sample points was between 1.43 mg/L and 3.55 mg/L, and Total phosphorus (TP) was between 0.09 mg/L and 1.33 mg/L. The level of nitrogen and phosphorus nutrients were much higher than the international standard of eutrophication (0.2 mg/L and TP 0.02 mg/L) and were also a little higher than previous studies done by other researchers during initial water impoundment of reservoir. A total of 476 plankton species belonging to 7 phyla were identified, among which Bacillariphyta (63.9%) were dominant. Just like other subtropic riverine reservoirs, the Three Gorges Reservoir was full with phytoplankton in Chlorophyta and Cryptophyta during spring. algae in Cyanophyta began to increase in summer and autumn while Bacillariphyta dominated community of phytoplankton in winter. Yet different water types have changed with seasons differently: (1) with the decreasing of velocity of flow and the changes of water types from natural riverine zone to analogous lacustrine zone, the dominant species of water column changed from Bacillariphyta to Chlorophyta and the dominance degree index of Chlorophyta increased to 0.62, in the meanwhile the proportion also raised gradually and went up to 67.8% in analogous lacustrine zone in spring (March, 2010). In September, 2009, the proportion Cyanophyta increased to 52.6% and at the time of September, 2010, the proportion of Cyanophyta in phytoplankton even soar to 63.8% becoming main species. Such as Oscillatoria tenuis Ag whose dominance degree index was significant higher than any other species. Although Bacillariphyta were put one at an absolute predominance in all the water columns during winter, the abundance of algae in Chlorophyta in analogous lacustrine zone was larger than any other water types. (2) The mean Shannon-Wiener diversity index in the main stream was 2.43, and the variation in analogous lacustrine zone was in summer and autumnwinterspring. On the contrary, the index was the lowest in winter in natural riverine zone and transition zone. The evenness index changed between 0.52 and 0.85 and the evenness in spring was lower than any other seasons. Contrast previous studies, we can indicate that the water quality of he main stream of the Three Gorges Reservoir of Chongqing may become worse during these years and we should pay more attention to this trend and try to prevent it.