Comparative transcriptomic analysis of surf clams (Paphia undulate) infected with two strains of Vibrio spp. reveals the identity of key immune genes involved in host defense.

BACKGROUND: Vibrio spp. is the major infection-producing marine bacteria in commercially important bivalve Paphia undulata. The host resistance is the major determining factor for the development of pathogenesis. To explore defense mechanisms, researchers have focused primarily on the study of differential expression of individual or specific groups of host immune genes during pathogen-challenge. RESULTS: We compared the expression profile in the surf clams infected with avirulent V. alginolyticus and virulent V. parahaemolyticus to mark the possible molecular mechanisms of pathogenesis. Comparison of the differentially expressed genes between the two groups of Vibrio-infected clams revealed that the number of down-regulate genes in V. parahaemolyticus injected clams (1433) were significantly higher than the other group (169). Based on Gene Ontology classification, a large proportion of these down-regulate genes were found to be associated with cellular and molecular mechanisms for pathogen recognition, and immunity development thereby explaining the low survival rate for the V. parahaemolyticus-treated clams and suggesting a higher virulence of this bacterium towards the surf clams. Quantitative real-time PCR of 24 candidate genes related to immunity involving the JAK-STAT signaling pathway, complementary cascade, cytokine signaling pathway, oxidative stress, phagocytosis and apoptosis down regulated under V. parahaemolyticus infection, indicating compromised host defense. Furthermore, we could demonstrate a central role of JAK-STAT pathway in bacterial clearance. dsRNA mediated depletion of a clam STAT homolog gene results in dramatic increase in the infection by V. alginolyticus, a mildly pathogenic strain under control conditions. CONCLUSIONS: The difference in gene expression profiles in surf clams treated with two Vibrio species with a differential pathogenicity to P. undulate and downstream molecular analysis could enlighten on the probable molecular mechanisms of the Vibrio pathogenesis and the virulence of V. parahaemolyticus in surf clams, which also benefits to develop new strategies for disease control in surf calm aquaculture.

Molecular cloning and expression analysis of a cytosolic heat shock protein 70 gene from mud crab Scylla serrata.

Heat shock protein 70s (Hsp70s) play important roles in resisting environmental stresses and stimulating innate immune system. To understand the immune defense mechanisms of Scylla serrata, a full-length cytosolic Hsp70 cDNA of S. serrata (designated as SSHsp70) was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) coupled with rapid amplification of cDNA ends (RACE). The full-length of SSHsp70 cDNA was 2235 bp, with a 5' untranslated region of 105 bp, a 3' untranslated region of 174 bp, and an open reading frame of 1956 bp encoding a polypeptide of 651 amino acids with an estimated molecular mass of 71.3 kDa and an estimated isoelectric point of 5.55. The cloned SSHsp70 belonged to a cytosolic Hsp70 family. Three typical Hsp70 signature motifs were detected in SSHsp70 by InterPro analysis. Quantitative PCR (qPCR) was used to detect tissue distribution and mRNA expression levels of SSHsp70 under different stress conditions. The obviously high levels of SSHsp70 transcript were in hemocyte, heart, hepatopancreas and gill, whereas low levels were detected in muscle, eyestalk, stomach, and gut. In different temperature treatments, the expression levels of SSHsp70 in low or high temperatures were higher than those in temperate temperature. In pathogen challenge treatments, the mRNA expression level of SSHsp70 reached a maximum level after 18 h and then dropped progressively. In different salt concentration treatments, the mRNA expression level of SSHsp70 had a minimum level at 25‰ salt concentration and high expression levels at high or low salt concentration. In different nitrite concentration treatments, the mRNA expression level of SSHsp70 increased progressively with the increase of nitrite concentration. The results confirmed Hsp70 could be used as a tool for evolution and phylogenetic analysis, a kind of potential biomarker, and a disease resistance factor used in application.

Cell loss by apoptosis is involved in the intestinal degeneration that occurs during aestivation in the sea cucumber Apostichopus japonicus.

The sea cucumber Apostichopus japonicus (Selenka) commonly undergoes aestivation in response to high water temperatures. This process is accompanied by tissue regression and body mass reduction. Previous studies have suggested that apoptosis may play a role in the tissue remodeling that occurs during aestivation, although this has not definitively been shown. To investigate this hypothesis, the present study used A. japonicus as a model organism to examine cell loss through apoptosis in intestinal degeneration during aestivation. Apostichopus japonicus individuals were collected from Yellow Sea (N 36° 05' 44.87″, E 120° 31' 58.51″), China in April 2016 and split into two groups. Aestivation was induced in the experimental group by incubation at 25°C. This resulted in a significant decrease in body mass and increased evidence of intestinal degeneration in hematoxylin and eosin, Hoechst 33342, and in situ TUNEL analyses of tissue sections. Along with further Hoechst 33342 analysis using intestinal cell smears, these results showed that A. japonicus intestinal cell apoptosis occurred soon after the initial temperature increase, with most apoptotic events completing within 20days. Transcriptional quantification of the Ajcaspase-8 (CASP8) and Ajcaspase-3 (CASP3) apoptotic genes demonstrated that their expression was significantly elevated at the beginning of the experiment but was decreased at later stages of aestivation. The results of this study strongly suggest that apoptosis is involved in the intestinal regression of A. japonicus during aestivation, and play important role in understanding fundamental cellular events in tissue regression under environmental stress.

Ocean acidification adversely influences metabolism, extracellular pH and calcification of an economically important marine bivalve, Tegillarca granosa.

Oceanic uptake of CO2 from the atmosphere has significantly reduced surface seawater pH and altered the carbonate chemistry within, leading to global Ocean Acidification (OA). The blood clam, Tegillarca granosa, is an economically and ecologically significant marine bivalve that is widely distributed along the coastal and estuarine areas of Asia. To investigate the physiological responses to OA, blood clams were exposed to ambient and three reduced seawater pH levels (8.1, 7.8, 7.6 and 7.4) for 40 days, respectively. Results obtained suggest that OA suppresses the feeding activity and aerobic metabolism, but elevates proteins catabolism of blood clams. OA also causes extracellular acidosis and decreases haemolymph Ca2+ concentration. In addition, our data also suggest that OA impairs the calcification process and inner shell surface integrity. Overall, OA adversely influences metabolism, acid-base status and calcification of blood clams, subsequently leading to a decrease in the fitness of this marine bivalve species.

Chloroplast genome of one brown seaweed, Saccharina japonica (Laminariales, Phaeophyta): its structural features and phylogenetic analyses with other photosynthetic plastids.

The chloroplast genome sequence of one brown seaweed, Saccharina japonica, was fully determined. It is characterized by 130,584 base pairs (bp) with a large and a small single-copy region (LSC and SSC), separated by two copies of inverted repeats (IR1 and IR2). The inverted repeat is 5015 bp long, and the sizes of SSC and LSC are 43,174 bp and 77,378 bp, respectively. The chloroplast genome of S. japonica consists of 139 protein-coding genes, 29 tRNA genes, and 3 ribosomal RNA genes. One intron was found in one tRNA-Leu gene in the chloroplast genome of S. japonica. Four types of overlapping genes were identified, ycf24 overlapped with ycf16 by 4 nucleotides (nt), ftrB overlapped with ycf12 by 6 nt, rpl4 and rpl23 overlapped by 8 nt, finally, psbC overlapped with psbD by 53 nt. With two sets of concatenated plastid protein data, 40-protein dataset and 26-protein dataset, the chloroplast phylogenetic relationship among S. japonica and the other photosynthetic species was evaluated. We found that the chloroplast genomes of haptophyte, cryptophyte and heterokont were not resolved into one cluster by the 40-protein dataset with amino acid composition bias, although it was recovered with strong support by the 26-protein dataset.

Characterization and expression analysis of hsp70 gene from Ulva prolifera J. Agardh (Chlorophycophyta, Chlorophyceae).

In the Yellow Sea of China, large-scale green tides have broken out consecutively from 2007 to 2011. Ulva prolifera, the causative species of green tide, showed great ability to acclimate to adverse circumstance. To explore the mechanisms of rapid growth and stress resistance during the bloom, we characterized and analyzed hsp70 from U. prolifera. The results showed that hsp70 gene had 6 exons and 5 introns. The promoter-like region contained multiple cis-acting elements. The transcription of hsp70 was up-regulated by UV irradiation, heat treatment and salinities induction, but less influenced by desiccation. In vitro expression of HSP70 protein and western blot was also conducted, and the recombinant protein will be used in detecting the interaction between HSP70 and related functional proteins in the future. The study suggested that hsp70 could be used in prediction of stress tolerance in algae and monitoring environmental changes.

Molecular cloning and expression analysis of a cytosolic Hsp70 gene from Laminaria japonica (Laminariaceae, Phaeophyta).

In this study, a full-length cytosolic heat shock protein 70 complementary DNA (cDNA) of Laminaria japonica (designated as LJHsp70) was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) coupled with rapid amplification of cDNA ends. The full length of LJHsp70 cDNA was 2,918 bp, with a 5' untranslated region of 248 bp, a 3' untranslated region of 696 bp, and an open reading frame of 1,974 bp encoding a polypeptide of 657 amino acids with an estimated molecular mass of 72.03 kDa and an estimated isoelectric point of 4.97. There was highly repeated sequence of CAA in 5' untranslated region of LJHsp70. The result of phylogenetic tree of Hsp70s, the BLAST program, analysis and cytosolic Hsp70-specific motif of LJHsp70 verified that the cloned LJHsp70 belonged to cytosolic Hsp70 family. Three typical Hsp70 signature motifs were detected in LJHsp70 by InterPro analysis. Under different stress conditions, messenger RNA (mRNA) expression levels of LJHsp70 were quantified by quantitative RT-PCR. To L. japonica sporophytes kept in different temperatures for 1 h, the expression level of LJHsp70 at 30 degrees C was highest and twofold higher than that at 10 degrees C. To L. japonica sporophytes kept at 25 degrees C for different times, the mRNA expression level of LJHsp70 reached a maximum level after 7 h and then dropped progressively. The expression level of LJHsp70 at 0 or 5 per thousand salt concentration for 2 h was twofold higher than that at 30 per thousand salt concentration for 2 h. The results showed that LJHsp70 may be a kind of potential biomarker used to monitor environment conditions.

GENETIC MAPPING OF THE LAMINARIA JAPONICA (LAMINARALES, PHAEOPHYTA) USING AMPLIFIED FRAGMENT LENGTH POLYMORPHISM MARKERS(1).

To establish a molecular-marker-assisted system of breeding and genetic study for Laminaria japonica Aresch., amplified fragment length polymorphism (AFLP) was used to construct a genetic linkage map of L. japonica featuring 230 progeny of F2 cross population. Eighteen primer combinations produced 370 polymorphic loci and 215 polymorphic loci segregated in a 3:1 Mendelian segregation ratio (P ≤ 0.05). Of the 215 segregated loci, 142 were ordered into 27 linkage groups. The length of the linkage groups ranged from 6.7 to 90.3 centimorgans (cM) with an average length of 49.6 cM, and the total length was 1,085.8 cM, which covered 68.4% of the estimated 1,586.9 cM genome. The number of mapped markers on each linkage group ranged from 2 to 12, averaging 5.3 markers per group. The average density of the markers was 1 per 9.4 cM. Based on the marker density and the resolution of the map, the constructed linkage map can satisfy the need for quantitative trait locus (QTL) location and molecular-marker-assisted breeding for Laminaria.

Purification and characterization of agarases from a marine bacterium Vibrio sp. F-6.

Marine bacterium Vibrio sp. F-6, utilizing agarose as a carbon source to produce agarases, was isolated from seawater samples taken from Qingdao, China. Two agarases (AG-a and AG-b) were purified to a homogeneity from the cultural supernatant of Vibrio sp. F-6 through ammonium sulfate precipitation, Q-Sepharose FF chromatography, and Sephacryl S-100 gel filtration. Molecular weights of agarases were estimated to be 54.0 kDa (AG-a) and 34.5 kDa (AG-b) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH values for AG-a and AG-b were about 7.0 and 9.0, respectively. AG-a was stable in the pH range of 4.0-9.0 and AG-b was stable in the pH range of 4.0-10.0. The optimum temperatures of AG-a and AG-b were 40 and 55 degrees C, respectively. AG-a was stable at temperature below 50 degrees C. AG-b was stable at temperature below 60 degrees C. Zn(2+), Mg(2+) or Ca(2+) increased AG-a activity, while Mn(2+), Cu(2+) or Ca(2+) increased AG-b activity. However, Ag(+), Hg(2+), Fe(3+), EDTA and SDS inhibited AG-a and AG-b activities. The main hydrolysates of agarose by AG-a were neoagarotetraose and neoagarohexaose. The main hydrolysates of agarose by AG-b were neoagarooctaose and neoagarohexaose. When the mixture of AG-a and AG-b were used, agarose was mainly degraded into neoagarobiose.