Characterization and Expression Analysis of a Complement Component Gene in Sea Cucumber (Apostichopus japonicus)

The complement system plays a crucial role in the innate immune system of animals. It can be activated by distinct yet overlapping classical, alternative and lectin pathways. In the alternative pathway, complement factor B(Bf) serves as the catalytic subunit of complement component 3(C3) convertase, which plays the central role among three activation pathways. In this study, the Bf gene in sea cucumber(Apostichopus japonicus), termed Aj Bf, was obtained by rapid amplification of c DNA ends(RACE). The full-length c DNA of Aj Bf was 3231 bp in length barring the poly(A) tail. It contained an open reading frame(ORF) of 2742 bp encoding 913 amino acids, a 105 bp 5'-UTR(5'-terminal untranslated region) and a 384 bp 3'-UTR. Aj Bf was a mosaic protein with six CCP(complement control protein) domains, a VWA(von Willebrand factor A) domain, and a serine protease domain. The deduced molecular weight of Aj Bf protein was 101 k Da. Quantitative real time PCR(q RT-PCR) analysis indicated that the expression level of Aj Bf in A. japonicus was obviously higher at larval stage than that at embryonic stage. Expression detection in different tissues showed that Aj Bf expressed higher in coelomocytes than in other four tissues. In addation, Aj Bf expression in different tissues was induced significantly after LPS or Poly I:C challenge. These results indicated that Aj Bf plays an important role in immune responses to pathogen infection.     

Characterization and Phylogenetic Analysis of Lectin Gene cDNA Isolated from Sea Cucumber(Apostichopus japonicus) Body Wall

As a ‘living fossil' of species origin and ‘rich treasure' of food and nutrition development, sea cucumber has received a lot of attentions from researchers. The cDNA library construction and EST sequencing of blood had been conducted previously in our lab. The bioinformatic analysis provided a gene fragment which is highly homologous with the genes of lectin family, named AjL(Apostichopus japonicus lectin). To characterize and determine the phylogeny of AjL genes in early evolution, we isolated a fulllength cDNA of lectin gene from the body wall of A. japonicus. The open reading frame of this gene contained 489 bp and encoded a 163 amino acids secretory protein being homologous to lectins of mammals and aquatic organisms. The deduced protein included a lectin-like domain. SDS-PAGE analysis showed that AjL migrated as a specific band(about 36.09 kDa under reducing), and agglutinated against rabbit red blood cells. AjL was similar to chain A of CEL-IV in space structure. We predicted that AjL may play the same role of CEL-IV. Our results suggested that more than one lectin gene functioned in sea cucumber and most of other species, which was fused by uncertain sequences during the evolution and encoded different proteins with diverse functions. Our findings provided the insights into the function and characteristics of lectin genes invertebrates. The results will also be helpful for the identification and structural, functional, and evolutionary analyses of lectin genes.     

An Efficient Procedure for Isolating Microsatellite DNAs from Sea Cucumber (Apostichopus japonicus)

1 Introduction Microsatellite DNAs, also called simple sequence re-peats (SSRs), are stretches of DNA, consisting of tan-demly repeating mono-, di-, tri-, tetra- or penta-nucleotide units. They are evenly distributed throughout the ge-nomes of most eukaryotic species (Tohn et al., 2000). Their high information content about the divergence of genome, which is directly related to the number of alleles at each locus, and the ease of PCR assays make them ex-cellent molecular markers (Powell …     

Mechanical Stress Induces Neuroendocrine and Immune Responses of Sea Cucumber(Apostichopus japonicus)

Grading procedure in routine sea cucumber hatchery production is thought to affect juvenile sea cucumber immunological response. The present study investigated the impact of a 3-min mechanical perturbation mimicking the grading procedure on neuroendocrine and immune parameters of the sea cucumber Apostichopus japonicus. During the application of stress, concentrations of noradrenaline and dopamine in coelomic fluid increased significantly, indicating that the mechanical perturbation resulted in a transient state of stress in sea cucumbers. Coelomocytes concentration in coelomic fluid increased transiently after the beginning of stressing, and reached the maximum in 1 h. Whereas, coelomocytes phagocytosis at 3 min, superoxide anion production from 3 min to 0.5 h, acid phosphatase activity at 0.5 h, and phenoloxidase activity from 3 min to 0.5 h were all significantly down-regulated. All of the immune parameters recovered to baseline levels after the experiment was conducted for 8 h, and an immunostimulation occurred after the stress considering the phagocytosis and acid phosphatase activity. The results suggested that, as in other marine invertebrates, neuroendocrine/immune connections exist in sea cucumber A. japonicus. Mechanical stress can elicit a profound influence on sea cucumber neuroendocrine system. Neuroendocrine messengers act in turn to modulate the immunity functions. Therefore, these effects should be considered for developing better husbandry procedures.     

Effects of Different Microbes on Fermenting Feed for Sea Cucumber (Apostichopus japonicus)

The effects of different microbes on fermenting feed for sea cucumber(Apostichopus japonicus) were compared to select the optimal fermentation strain in this study. Saccharomgces cerevisae, Candida utilis, Bacillus subtilis and Geotrichum candidum were independently added into the experimental compound feed, while only saline was mixed with the control feed. The fermentation treatments were inoculated with 10% seed solution under the condition of 25℃ and 70% water content, which lasted for 5 days to elucidate the optimal microbe strain for fermenting effect. Physicochemical indexes and sensorial characteristics were measured per day during the fermentation. The indexes included dry matter recovery(DMR), crude protein(CP), the percentage of amino acid nitrogen to total nitrogen(AA-N/t N), the percentage of ammonia nitrogen to total nitrogen(NH3-N/t N), and the ratio of fermentation strains and vibrios to the total microbes, color, smell and viscosity. The results showed that DMR, CP and AA-N/t N of the S. cerevisae group reached the highest level on day 3, but the ratio of fermentation strain was second to C. utilis group. In addition, its NH3-N/t N and the ratio of vibrios were maintained at low levels, and the sensory evaluation score including smell, color and viscosity was the highest in S. cerevisae group on day 3. Therefore, S. cerevisae could be the optimal strain for the feed fermentation for sea cucumber. This research developed a new production method of fermentation feed for sea cucumber.     

Transcriptome Analysis Reveals New Insights into the Respiration Metabolism Mechanism of Different Feeding Rations of Sea Cucumber(Apostichopus japonicus)

Sea cucumber(Apostichopus japonicus) is an excellent model for investigating effects of bottom-dwellers on carbon migration and transformation. However, the molecular mechanism of respiratory metabolism process variation caused by feeding rations is poorly understood. In this study, treatment groups set as 1%(about 0.63 g), 3%, and 7% of total body weight(named F1, F3 and F7groups respectively). The potential molecular mechanisms behind the functions of respiratory tree and body wall were invest...     

Effect of Co-Culture of Chinese Shrimp(Fenneropenaeus chinensis) and Sea Cucumber(Apostichopus japonicus 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.     

Effect of Food on Specific Dynamic Action(SDA) of Green and Red Types of Sea Cucumber(Apostichopus japonicus Selenka)

Specific dynamic action(SDA), the energy expended on all physiological processes that is associated with meal digestion and absorption, is strongly affected by food type. Effects of formulated diet(FMD), macroalgae(ALG) and sea mud(SMD) diets on the postprandial metabolic response of the green type and the red type of sea cucumber(Apostichopus japonicus) were studied in order to understand their feeding physiology. Food offered to A. japonicus was different in protein, lipid content and energy but not in meal mass. SDA of A. japonicus resulted in a 1.3–2.7 folds of increase in oxygen consumption that can persist for up to 4.8–31.7 h after digesting three different diets. In a given type of sea cucumber, the magnitude of SDA was the highest when fed with FMD, medium with ALG, and the lowest with SMD, which is probably due to the differences in diet components and protein contents. The red type sea cucumber showed greater SDA magnitude than the green type with each diet treatment, which might result from the difference in factorial scope between the two types of sea cucumber. However, the smallest magnitude or even no difference was observed between the two types of A. japonicus in SMD group, perhaps owing to the poor nutrition and digestion of sea mud.     

Change of Digestive Physiology in Sea Cucumber Apostichopus japonicus(Selenka) Induced by Corn Kernels Meal and Soybean Meal in Diets

The present study was conducted to determine the change of digestive physiology in sea cucumber Apostichopus japonicus(Selenka) induced by corn kernels meal and soybean meal in diets. Four experimental diets were tested, in which Sargassum thunbergii was proportionally replaced by the mixture of corn kernels meal and soybean meal. The growth performance, body composition and intestinal digestive enzyme activities in A. japonicus fed these 4 diets were examined. Results showed that the sea cucumber exhibited the maximum growth rate when 20% of S. thunbergii in the diet was replaced by corn kernels meal and soybean meal, while 40% of S. thunbergii in the diet can be replaced by the mixture of corn kernels meal and soybean meal without adversely affecting growth performance of A. japonicus. The activities of intestinal trypsin and amylase in A. japonicus can be significantly altered by corn kernels meal and soybean meal in diets. Trypsin activity in the intestine of A. japonicus significantly increased in the treatment groups compared to the control, suggesting that the supplement of corn kernels meal and soybean meal in the diets might increase the intestinal trypsin activity of A. japonicus. However, amylase activity in the intestine of A. japonicus remarkably decreased with the increasing replacement level of S. thunbergii by the mixture of corn kernels meal and soybean meal, suggesting that supplement of corn kernels meal and soybean meal in the diets might decrease the intestinal amylase activity of A. japonicus.     

16S rRNA Gene Phylogenesis of Culturable Predominant Bacteria from Diseased Apostichopus japonicus(Holothuroidea,Echinodermata)

Cultured Apostichopusjaponicus in China suffers from a kind of skin ulceration disease that has caused severe economic loss in recent years. The disease, pathogens of which are supposed to be bacteria by most researchers, is highly infectious and can often cause all individuals in the same culture pool to die in a very short time. The 16S rRNA gene phylogenesis of the culturable bacteria from the lesions of diseased individuals was conducted to study the biodiversity of the bacterial communities in the lesions and to identify probable pathogen(s) associated with this kind of disease. S. japonica samples were selected from a hatchery located in the eastern part of Qingdao, China. Bacterial universal primers GM5F and DS907R were used to amplify the 16S rRNA gene of bacteria colonies, and touchdown PCR was performed to amplify the target sequences. The results suggest that γ- proteobacteria (Alteromonadales and Vibrionales) of CFB group, many strains of which have been also determined as pathogens in other marine species, are the predominant bacterial genera of the diseased Apostichopusjaponicus individuals.     

Effects of a Potential Autochthonous Probiotic Bacillus subtilis 2-1 on the Growth and Intestinal Microbiota of Juvenile Sea Cucumber,Apostichopus japonicus 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), 10~5, 10~7, and 10~9 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 10~9 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 10~9 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.     

Colonization and Probiotic Effect of Metschnikowia sp. C14 in the Intestine of Juvenile Sea Cucumber,Apostichopus japonicus

Viable cell count was used to determine whether Metschnikowia sp.C14 can colonize the intestine of juvenile sea cucumber Apostichopus japonicus.Sea cucumber individuals were divided into two groups,which were fed the control diet for 38 days or the C14-supplemented diet at 105 cells g−1 diet for 28 days,then the control diet from day 29 to day 38.The number of C14 cells in the intestine of sea cucumber fed the C14-supplemented diet significantly increased from day 7 to day 28,and decreased from day 29 to day 38.Sea cucumber fed with the diet containing C14 showed a significant increase in trypsin activity and lipase activity from day 21 to day 33 compared with the control.Feeding C14 significantly improved the phagocytic activity and respiratory burst in coelomocytes from day 21 to day 35 and from day 14 to day 38,respectively.In addition,there was an obvious enhancement in lysozyme activity(from day 21 to day 38 or day 33),phenoloxidase activity(from day 21 to day 28)and total nitric oxide synthase activity(from day 14 to day 38)in coelomic fluid supernatant and/or coelomocyte cell lysate supernatant compared with the control.There were significant positive correlations between the number of C14 cells colonizing the intestine and trypsin activity of the intestine,lysozyme activity of the coelomic fluid supernatant and coelomocyte lysate supernatant from sea cucumber.These data suggested that the number of C14 cells should be maintained at 105 cfu(colony-forming units)g−1 intestine material for the maximum benefit.     

Isolation,Expression and Characterization of rbcL Gene from Ulva prolifera J.Agardh (Ulvophyceae,Chlorophyta)

Ulva prolifera is a typical green alga in subtidal areas and can grow tremendously fast. A highly efficient Rubisco enzyme which is encoded by Up Rbc L gene may contribute to the rapid growth. In this study, the full-length Up Rbc L open reading frame(ORF) was identified, which encoded a protein of 474 amino acids. Phylogenetic analysis of Up Rbc L sequences revealed that Chlorophyta had a closer genetic relationship with higher plants than with Rhodophyta and Phaeophyta. The two distinct residues(aa11 and aa91) were presumed to be unique for Rubisco catalytic activity. The predicted three-dimensional structure showed that one α/β-barrel existed in the C-terminal region, and the sites for Mg~(2+)coordination and CO_2 fixation were also located in this region. Gene expression profile indicated that Up Rbc L was expressed at a higher level under light exposure than in darkness. When the culture temperature reached 35℃, the expression level of Up Rbc L was 2.5-fold lower than at 15℃, and the carboxylase activity exhibited 13.8-fold decrease. Up Rbc L was heterologously expressed in E. coli and was purified by Ni~(2+) affinity chromatography. The physiological and biochemical characterization of recombinant Rubisco will be explored in the future.     

Optimization of Stocking Density for the Sea Cucumber,Apostichopus japonicus Selenka, Under Feed-Supplement and Non-Feed-Supplement Regimes in Pond Culture

Optimal stocking densities were investigated for the sea cucumber Apostichopus japonicus Selenka under feed-supplement and non-feed-supplement regimes in net enclosures for 333 d. Substantial weight loss occurred during the aestivation phase (AE).Decreased growth rates were also observed during the winter phase (WT). In contrast, sea cucumbers showed rapid growth during the spring (SP) and autumn (AU) phases. Feeding regimes considerably influenced the growth performance, i.e., sea cucumbers grew faster under feed-supplement regime than under non-feed-supplement regime (P< 0.05). The average survival rates of sea cucumbers under feed-supplement regime were higher than those under non-feed-supplement regime for both the autumn phase and spring phase,but the differences were only significant for the latter phase (P<0.05). The fitted B-N curves showed that the optimal stocking densities, in terms of net production, were 22.3 ind. m-2 for feed-supplement regime and 14.1 ind. m-2 for non-feed-supplement regime.     

Characterization and identification of enzyme-producing microflora isolated from the gut of sea cucumber Apostichopus japonicus

Gut microorganisms play an important role in the digestion of their host animals. The purpose of this research was to isolate and assess the enzyme-producing microbes from the Apostichopus japonicus gut. Thirty-nine strains that can produce at least one of the three digestive enzymes(protease,amylase,and cellulase) were qualitatively screened based on their extracellular enzyme-producing abilities. The enzyme-producing strains clustered into eight groups at the genetic similarity level of 100% by analyzing the restriction patterns of 16 S rDNA amplified with Mbo I. Phylogenetic analysis revealed that 37 strains belonged to the genus Bacillus and two were members of the genus Virgibacillus. Enzyme-producing capability results indicate that the main enzyme-producing microflora in the A. japonicus gut was Bacillus,which can produce protease,amylase,and cellulase. Virgibacillus,however,can only produce protease. The high enzyme-producing capability of the isolates suggests that the gut microbiota play an important role in the sea cucumber digestive process.     

An Evaluation on the Ratio of Plant to Animal Protein in the Diet of Juvenile Sea Cucumber(Apostichopus japonicus): Growth,Nutrient Digestibility and Nonspecific Immunity

This study was conducted to evaluate the effects of plant/animal(P/A) protein ratios(viz.1:4, 1:3, 1:2, 1:1,2:1, 3:1, 4:1) on growth performance, body composition, apparent digestibility of diets, and nonspecific immunity of juvenile sea cucumber(Apostichopus japonicus). Sea cucumbers were divided into 21 plastic tanks, and each tank was stocked with 15 individuals(initial weight: about 23.73 g). Each feed was allocated to three replicates of sea cucumbers. The feeding experiment lasted for 50 days. Results indicated that weight gain rate(WGR) and body wall weight(BWW) significantly increased as dietary ratio of P/A increased from 1:4 to 3:1, and then decreased significantly with further increase of this ratio(P 0.05). The body wall coefficient(BWC) showed a similar tendency to WGR and BWW, but no significance was detected among dietary treatments(P 0.05). The apparent digestibility of dry matter, protein and lipid increased with ratio of P/A increasing from 1:4 to 2:1(P 0.05), and then decreased with further increase of this ratio. Correspondingly, activities of trypsin and amylase were significantly increased as P/A increased from 1:4 to 2:1(P 0.05). The activities of SOD and CAT showed a similar trend with WGR, with the highest value observed in the ratio of 1:2 and 1:1, respectively. Results above showed that moderate or relatively higher ratio of P/A protein(1:1-3:1) significantly increased the growth performance, apparent digestibility, and nonspecific immunity of sea cucumber. This will contribute to improving the feed formulation for juvenile cucumbers.     

Isolation of immune-relating 185/333-1 gene from Sea Urchin (Strongylocentrotus intermedius) and Its expression analysis

The 185/333 gene family involved in the immune response of sea urchin.One 185/333 c DNA was isolated from Strongylocentrotus intermedius,and named as Si185/333-1.Its full-length c DNA was 1246 bp in length with a 906 bp open reading frame encoding a protein of 301 aa.The molecular weight of the deduced protein was approximately 33.1 k D with an estimated PI of p H 6.26.Si185/333-1 had high identities(70%–86%) to most of Sp185/333.An extraordinary identity of 92% was found between Si185/333-1 and Sp185/333 C5 alpha(ABR22474).Moderate identities(63%–64%) were displayed between Si185/333-1 and He185/333.Si185/333-1 had similar structure to Sp185/333.A signal-peptide,a gly-rich region and a his-rich region were found in its secondary structure.RGD motif was found in gly-rich region at position 116–118aa.There was no transmembrane region in Si185/333-1.The element pattern of Si185/333-1 is different from any available pattern that identified in Sp185/333.Si185/333-1 clustered together with pattern C Sp185/333 in phylogenetic tree.The Si185/333-1 m RNA could be detected in tissues including peristomial membrane,coelomocytes,muscle of Aristotles lantern,gut and tube feet,with the highest expression level detected in peristomial membrane and a relatively low expression in ovary and testis.The temporal expression of Si185/333-1 in peristomial membrane and coelomocytes were up-regulated after bacterial,β-D-glucan and ds RNA challenges,reaching the maximum at 12 h post-stimulation.The up-regulation was more obvious in coelomocytes,and bacterial challenge triggered the highest response.These results proved that 185/333-1 gene was involved in the immune defense of S.intermedius,while more studies were necessary for its function in S.intermedius immunity.     

Characterization and application of monoclonal antibodies against Shewanella marisflavi, a novel pathogen of Apostichopus japonicus

Shewanella marisflavi strain AP629 was certified as a novel pathogen of the sea cucumber Apostichopus japonicus. In this study, four monoclonal antibodies (MAbs) (3C1, 3D9, 2F2, 2A8) against strain AP629 were developed by immunizing Balb/C mice. 3C1 and 3D9 recognized S. marisflavi only, showing no cross reactivity to other gram-negative bacteria. However, 2F2 and 2A8 showed cross reactivity to all tested bacteria. Indirect immunofluorescence, and immunogold electron microscopy, showed the binding antigens ...     

Characterization and Expression Analysis of Protein Kinase C Gene from Dunaliella salina

Protein kinase C(PKC) has a crucial role in signal transduction for a variety of biologically active substances which activate cellular functions and proliferation. We previously isolated the full-length PKC gene from Dunaliella salina(Ds PKC) using rapid amplification of cDNA ends(RACE) and RT-PCR methods. And we submitted the mRNA sequence of Ds PKC gene to NCBI(Genbank No. JN625213). In the present paper, the Ds PKC gene open reading frame obtained by PCR was cloned into pGS-21 a vector and transformed into Escherichia coli to generate the fusion protein. Bioinformatics analysis revealed that DsPKC gene was a member of serine/threonine kinase with two conserved domains and highly conserved motifs. The Ds PKC was highly expressed upon induction with isopropyl-β-d-thiogalactoside(IPTG) at a final concentration of 0.2 mmol L~(-1) at 37℃. Under salt stress, the fusion protein Green Fluorescent Protein(GFP)-DsPKC was transferred from the cytoplasm to the cell membrane. The expression pattern of Ds PKC gene was analyzed using real-time quantitative PCR, and indicated that Ds PKC gene was up-regulated by 3.0 mol L~(-1) NaCl at 12 h, which was significantly higher than in control values(P 0.05). These results suggest that the Ds PKC gene plays an important role in response to salt stress in D. salina.     

马氏珠母贝载脂蛋白-2基因的克隆与表达分析

以RACE技术克隆获得马氏珠母贝载脂蛋白-2(Pm-ApoL2)基因cDNA全长序列,以传统萃取法提取黄色与白色闭壳肌个体的类胡萝卜素,以荧光定量技术检测Pm-ApoL2基因在各个组织中的表达量及黄与白闭壳肌个体的Pm-ApoL2表达量。结果表明,Pm-ApoL2基因cDNA序列全长1 328 bp,开放阅读框(ORF)长705 bp,编码234个氨基酸,5′非翻译区(5′UTR)长342 bp,3′UTR长281 bp。Pm-ApoL2在肝胰腺中表达量最高,其后依次是外套膜、鳃、闭壳肌,其中肝胰脏表达量显著大于其它3个组织(P0.05)。黄、白色闭壳肌个体之间的类胡萝卜素含量和Pm-ApoL2的表达量都存在显著性差异(P0.05),且二者的类胡萝卜素含量与Pm-ApoL2表达量呈显著正相关。Pm-ApoL2基因为马氏珠母贝类胡萝卜素代谢候选基因。