A new method of Laminaria japonica strain selection and sporeling raising by the use of gametophyte clones

One-celled female and male gametophytes of three Laminaria japonica strains were isolated, cultured and gametophyte clones were formed. A technique combining strain selection with sporeling raising by the use of these female and male gametophyte clones was studied. Experiments on 9 different crossing combinations was conducted in November of 1997 in Qingdao, P. R. China. The main economic characteristics, frond length and fresh weight, of sporophytes of different crossing combinations were measured. F-1 sporophytes of No. 2 showed a higher fresh weight and longer length, therefore, No. 2 (Wh860 + x Lid) was selected as a good combination. Its parental female and male gametophyte clones are being mass cultured for sporeling production. By this method, the time needed for strain selection was shortened from 5-6 to 2 years. As compared with the routine method of sporeling raising by the collection of zoospores, the time of sporeling raising of this method decrease by 50%, and the production cost is also reduced by 50%. It is believed that this method will be labour and time saving and a more economic way for strain selection and sporeling raising in L. japonica cultivation industry.

Review of genetic engineering of Laminaria japonica (Laminariales, Phaeophyta) in China

Progress has been made in establishing a genetic transformation model for Laminaria japonica (Phaeophyta, Laminariales). The model includes introduction of foreign genes by biolistic bombardment, use of promoter SV40 to drive gene expression, algal regeneration by parthenogenesis and selection by chloramphenicol or hygromycin.

Size and time of day affect the response of postlarvae and early juvenile grooved tiger prawns Penaeus semisulcatus De Haan (Decapoda:Penaeidae) to natural and artificial seagrass in the laboratory

Juvenile tiger prawns (Penaeus semisulcatus De Haan and P. esculentus Haswell) show a strong association with vegetated habitats and are rarely caught on non-vegetated areas. This pattern of distribution may be caused by postlarvae selecting vegetation when they settle, or to differences in post-settlement mortality in different habitats. In this study, we examined whether the postlarvae and early juvenile stages of P. semisulcatus would distinguish between seagrass (Zostera capricorni Aschers) without epiphytes, artificial seagrass and bare substratum in the laboratory. The responses of prawns reared from the egg to different stages of postlarval and juvenile development were tested to determine whether, and when, each size class showed a response to a particular habitat. Five size classes of postlarvae (average carapace lengths [CL] of 1.2, 1.4, 1.6, 1.7 and 2.0 mm) were offered a choice between Z. capricorni and bare sand. Small size classes of postlarvae either did not respond to Z. capricorni (1.2 and 1.6 mm CL), or were more abundant on bare substratum than Z. capricorni. In contrast, the largest size classes of postlarvae (1.7 and 2.0 mm CL) were more abundant on Z. capricorni during the day but not at night. The behaviour of postlarvae changed markedly at a size of 1.7 mm CL (22 days from the first nauplius): smaller postlarvae frequently swam in the water column; 1.7 and 2.0 mm CL postlarvae spent much more. time resting on the substrate and perched on seagrass leaves. This size at which postlarvae first respond to seagrass during the day, and show mainly benthic behaviour, is similar to the size at which they are found on shallow seagrass beds in northern Australia. Large postlarvae (2.7 mm CL) and juveniles (4.1 mm CL) both were more abundant on artificial seagrass than bare sand during the day but not at night, indicating that they respond to structured habitats. When large postlarvae (2.4 mm CL) and juveniles (3.5 mm CL) were offered a choice between Z. capricorni without epiphytes and artificial seagrass, they were more abundant on the Z. capricorni, which suggests that chemical cues from seagrass may explain some of the responses of P. semisulcatus to seagrass. (C) 1997 Elsevier Science B.V.

Translocation of assimilates in Undaria and its cultivation in China

Undaria cultivation on a commercial scale began in China only in the last decade. Today, Undaria pinnatifida is the main species under cultivation concentrated in two provinces, Liaoning and Shandong. The annual production in the early nineties was 8000-13 000 tons dry weight, which is two or three times the pre-1980 figures. The raft cultivation method maintaining the alga at the desired depths generally ensures the light saturated rate of photosynthesis on clear days, and enhances production. Under the cultivated condition, the calculated annual primary productivity of this alga is 160 g C m(-2) y(-1). Translocation of C-14-labelled photoassimilates in rapidly growing sporophyte of Undaria pinnatifida was studied in the open sea. Samples from different parts of the blade with counterparts exposed to tracer ((NaHCO3)-C-14) showed that the translocation that occurred mainly from the tip of the blade to the growing region had obvious source-sink relationship. It took 20 minutes to translocate the labelled photoassimilates from the epidermis, via cortex, to the medulla of the midrib, where rates of translocation averaging 42-48 cm h(-1) were observed in the open sea. Production experiments of tip-cutting of the blades showed an increased production of 9%.

Integrated cultivation of the red alga Kappaphycus alvarezii and the pearl oyster Pinctada martensi

The effect of simultaneously cultivating the pearl oyster Pinctada martensi and the red alga Kappaphycus alvarezii on growth rates of both species was investigated in laboratory and field studies conducted from December 1993 to June 1995. The two study sites were in subtidal areas 100 km apart off the east coast of Hainan Island, China. Pearl oysters were cultivated in the center of an algal farm and red alga was cultivated in the center of the pearl oyster farm. These field experiments showed higher growth rates of both P. martensi and K. alvarezii in a co-culture system than in a monospecies culture system. Laboratory studies showed that the algae removed nitrogenous wastes released by pearl oysters. Algae treated with pearl oyster wastes grew much faster than those without oyster wastes. Algae treated with the seawater to which NH4Cl, NaNO3 and NaNO2 were added grew at the same rate as those treated with natural seawater containing oyster nitrogenous wastes, suggesting that enhanced growth of algae in the co-culture system was largely due to nitrogenous metabolites of the pearl oysters. In the co-culture, growth of pearl oysters was positively influenced by the presence of rapidly growing algae but when seawater temperature decreased below 20 degrees C, the algae grew slowly and there was no measurable benefit of mixed culture to either algae or pearl oyster.

SOME ASPECTS OF THE GROWTH OF GRACILARIA-TENUISITIPITATA IN POND CULTURE

The effect of temperature, salinity, nitrogen, culture density and depth on the growth of Gracilaria tenuistipitata were investigated between April 1985 and March 1986 in outdoor ponds in Guangxi Province, South China. The mean annual growth rate was 2.4% per day. Under favourable temperatures of 20-30-degrees-C, daily growth rate may reach as high as 3.3%. Salinity had an obvious effect on growth and photosynthesis and growth peaked at 21 parts per thousand, with a broad plateau between 7-27 parts per thousand. Growth experiments showed that a total nitrogen (NH4-N plus NO3-N) concentration of 4 muM was sufficient to enable the plants to maintain a daily growth rate of 2.7%. The best growth of the plant was obtained at a culture density of 0.5-1 kg M-2 and a culture depth of 30 cm in the pond.

Seeding nets with neutral spores of the red alga Porphyra umbilicalis (L.) Kutzing for use in integrated multi-trophic aquaculture (IMTA)

Nets in traditional Porphyra mariculture are seeded with conchospores derived from the conchocelis phase, and spend a nursery period in culture tanks or calm coastal waters until they reach several centimeters in length. Some species of Porphyra can regenerate the foliose phase directly through asexual reproduction, which suggests that the time, infrastructure, and costs associated with conchocelis culture might be avoided by seeding nets with asexual spores. Here, we present work from a short-term mariculture study using nets seeded with asexual spores (neutral spores) of a native Maine species of Porphyra. Porphyra umbilicalis (L.) Kutzing was selected for this proof of concept research because of its reproductive biology, abundance across seasons in Maine, and evidence of its promise as a mariculture crop. We studied the maturation, release, and germination of the neutral spores to develop an appropriate seeding protocol for nets, followed by development of a nursery raceway to provide an easily manipulated environment for the seeded nets. Neutral spores were produced throughout the year on the central Maine coast,however, there was a temporal variability in the number and survival of released neutral spores, depending upon thallus position in the intertidal zone. Small thalli were strictly vegetative, but most thalli reproduced by neutral spores- sexual reproduction was absent. Neutral spores germinated quickly at 10 and 15 'C, but germination was delayed at 5 degrees C. Unlike some algal zygotes and spores, neutral spores of R umbilicalis required light to germinate; however, irradiances of 25 and 100 mu mol photons M-2 S-1 were equally sufficient for germination. Rafts of seeded nets were deployed in Cobscook Bay, Maine, at two distances from salmon aquaculture pens and at a control site on a nearby, fallow aquaculture site (no salmon). There was no difference in nitrogen content of harvested thalli; however, both the density and the surface area of harvested thalli were different among the sites. The possible causes of these differences are discussed in the context of potential use of P umbilicalis in IMTA. (C) 2007 Elsevier B.V. All rights reserved.

Development of rRNA and rDNA-targeted probes for fluorescence in situ hybridization to detect Heterosigma akashiwo (Raphidophyceae)

Heterosigma akashiwo (Hada) is a fragile, fish-killing alga. Efforts to understand and prevent blooms due to this harmful species to mitigate the impact on aquaculture require the development of methods for rapid and precise identification and quantification, so that adequate warning of a harmful algal bloom may be given. Here, we report the development and application of rRNA and rDNA-targeted oligonucleotide probes for fluorescence in situ hybridization (FISH) to aid in the detection and enumeration of H. akashiwo. The designed probes were species specific, showing no cross-reactivity with four common HAB causative species: Prorocentrum micans Ehrenberg, P. minimum (Pavillard) Schiller, Alexandrium tarmarense (Lebour) Balech, and Skeletonema costatum (Greville) Cleve, or with four other microalgae, including Gymnodinium sp. Stein, Platy-monas cordiformis (Karter) Korsch, Skeletonema sp.1 Greville and Skeletonema sp.2. The rRNA-targeted probe hybridized to cytoplasmic rRNA, showing strong green fluorescence throughout the whole cell, while cells labeled by rDNA-targeted probe exhibited exclusively fluorescent nucleus. The detection protocols were optimized and could be completed within an hour. For rRNA and rDNA probes, about a corresponding 80% and 70% of targeted cells could be identified and quantified during the whole growth circle, despite the inapparent variability in the average probe reactivity. The established FISH was proved promising for specific, rapid, precise, and quantitative detection of H. akashiwo. (C) 2008 Elsevier B.V. All rights reserved.

Diverse tetracycline resistant bacteria and resistance genes from coastal waters of Jiaozhou Bay

Environmental microbiology investigation was carried out in Jiaozhou Bay to determine the source and distribution of tetracycline-resistant bacteria and their resistance mechanisms. At least 25 species or the equivalent molecular phylogenetic taxa in 16 genera of resistant bacteria could be identified based on 16S ribosomal deoxyribonucleic acid sequence analysis. Enterobacteriaceae, Pseudomonadaceae, and Vibrionaceae constituted the majority of the typical resistant isolates. Indigenous estuarine and marine Halomonadaceae, Pseudoalteromonadaceae, Rhodobacteraceae, and Shewanellaceae bacteria also harbored tetracycline resistance. All the six resistance determinants screened, tet(A)-(E) and tet(G), could be detected, and the predominant genes were tet(A), tet(B), and tet(G). Both anthropogenic activity-related and indigenous estuarine or coastal bacteria might contribute to the tet gene reservoir, and resistant bacteria and their molecular determinants may serve as bioindicators of coastal environmental quality. Our work probably is the first identification of tet(E) in Proteus, tet(G) in Acinetobacter, tet(C) and tet(D) in Halomonas, tet(D) and tet(G) in Shewanella, and tet(B), tet(C), tet(E), and tet(G) in Roseobacter.

Discovery of genes involved in defense/immunity functions in a haemocytes cDNA library from Fenneropenaeus chinensis by ESTs annotation

Crustacean haemocytes play important roles in the host immune response including recognition, phagocytosis, melanization, cytotoxicity and inter-cellular signal communication. Expressed sequence tags (ESTs) analysis is proved to be an efficient approach not only for gene discovery, but also for gene expression profiles performance. In order to further understand the innate immune system and defense mechanisms of Chinese shrimp at molecular level, complementary DNA library is constructed from the haemocyte tissue of Fenneropenaeus chinensis. A total of 2371 cDNA clones are successfully sequenced and the average sequence length is 460 bp. About 50% are identified as orthologs of known genes from other organisms by BLASTx and BLASTn program. By sequences comparability and analysis, 34 important genes including 177 ESTs are identified that may be involved in defense or immune functions in shrimp based on the known knowledge. These genes are categorized into five categories according to their putative functions in shrimp immune system: 13 genes are different types of antimicrobial peptides (AMP, penaeidin, antilipopolysaccharide factor, etc.), and their proportion is about 3 8%; 11 genes belong to prophenoloxidase system (prophenoloxidase, serine proteinase, serine proteinase inhibitor, etc.), and their proportion is about 32%; five genes have high homology with clotting protein (lectin, transglutaminase, etc), and their proportion is about 15%; three genes may be involved in inter-cell signal communication (peroxinectin, integrin), and their proportion is about 9%; two genes have been identified to be chaperone proteins (Hsc70, thioredoxin peroxidase), and their proportion is about 6%. These EST sequences enrich our understanding of the immune genes of F chinensis and will help farther experimental research into immune factors and improve our knowledge of the immune mechanisms of shrimp. (c) 2007 Elsevier B.V. All rights reserved.

Study on early-stage development of conchospore in Porphyra yezoensis Ueda

Porphyra yezoensis Ueda (Rhodophyta) is a seaweed of economic importance with a typical dimorphic life cycle consisting of a leafy gametophyte and a filamentous sporophyte. Recently, it has been recognized as a model system for fundamental and applied studies in marine biological sciences. Conchospore, a major spore linking the two distinct multicellular phases in the life cycle, is most widely used in the breeding of P. yezoensis. In this paper, the early-stage development of conchospore, including the attachment and the cell wall formation, was studied with fluorescent reagents staining and Scanning Electron Microscopy detection. Results displayed: (I) the cell wall began to be generated after culturing for 4 h in the attached conchospores; (2) the initially released conchospores were plastids with some filmy, amorphous substance on the surface, and they attached to the fibers firmly via the actively secreted mucilaginous substances after their touch to the fibers; (3) cellulase and pectolase prohibited the attachment of conchospores in the different ways; and (4) only attached conchospores generated cell walls and developed normally, while the suspending ones could not. It indicated that the cellulose played crucial roles in the permanent attachment as the pectin did in the initial attachment. The conchospore attachment seemed to trigger the cell wall formation and the further development. Affects of light on the development of conchospores were also discussed. The results showed that high intensity (200 mu mol.m(-2).s(-1)) and long-wave (>= 580 nm) light facilitated the division rate of conchospores. (C) 2008 Elsevier B.V. All rights reserved.

A prophenoloxidase from the Chinese mitten crab Eriocheir sinensis: Gene cloning, expression and activity analysis

Prophenoloxidase (proPO) is a conserved copper-containing enzyme that plays important roles in immune response of crustaceans and insects. In the present study, the full-length cDNA of a prophenoloxidase (designated EsproPO) was cloned from haemocytes of Chinese mitten crab Eriocheir sinensis by expressed sequence tag (EST) and PCR techniques. The isolated 3549 bp full-length cDNA of EsproPO contained a 2040 bp open reading frame (ORF) encoding a putative proPO protein of 679 amino acids, a 5'-untranslated region (UTR) of 68 bp, and a long 3'-UTR of 1441 bp. Two putative copper-binding sites, a proteolytic activation site, and a complement-like motif (GCGWPQHM) were identified in the deduced amino acid sequence of EsproPO. Homology analysis revealed that EsproPO was highly similar to other proPOs from crustaceans with identities from 52% to 68%. The conserved domains and motifs, and higher similarity with other proPOs suggested that EsproPO was a member of the proPO family. The mRNA expression of EsproPO and PO specific activities in the tissues of hepatopancreas, gill, gonad, muscle, heart, eye and haemocytes were measured by quantitative real-time PCR and colorimetric assay, respectively. The mRNA transcripts of EsproPO and PO specific activities could be detected in all the examined tissues with the highest level both in hepatopancreas. Three peaks of EsproPO mRNA expression were recorded at 2 h, 12 h and 48 h in haemocytes of Chinese mitten crab post Vibrio anguillarum challenge, which was consistent with the temporal profile of PO specific activity. The mRNA expression pattern and the activity fluctuation of EsproPO post V. anguillarum stimulation indicated that it was potentially involved in the acute response against invading bacteria in Chinese mitten crab. (c) 2007 Elsevier Ltd. All rights reserved.

cDNA cloning, characterization and mRNA expression of a pacifastin light chain gene from the Chinese mitten crab Eriocheir sinensis

The pacifastin family, characterized by several conserved arrays of six cysteine residues, is a newly identified serine protease inhibitor (SPI) family discovered uniquely in arthropods and plays important roles in multiple biological processes. In the present study, the full-length cDNA of a pacifastin light chain (designated ESPLC) was cloned from the Chinese mitten crab Eriocheir sinensis by expressed sequence tags (ESTs) and PCR techniques. The 1036 bp ESPLC cDNA contained an 831 bp open reading frame (ORF) encoding a putative pacifastin-related peptide of 276 amino acids, a 5'-untranslated region (UTR) of 67 bp, and a 3'-UTR of 138 bp. Six putative conserved domains sharing a characteristic cysteine array (Cys-Xaa(9-12)-Cys-Asn-Xaa-Cys-Xaa-Cys-Xaa(2-3)-Gly-Xaa(3-4)-Cys-Thr-Xaa(3)-Cys) were identified in the deduced amino acid sequence of ESPLC. The conservation of these PLDs (pacifastin light chain domains) and the relative higher similarity of ESPLC to other pacifastin-related precursors suggested that ESPLC was a member of pacifastin family. The mRNA transcripts of ESPLC were found to be higher expressed in hepatopancreas, gill and haemolymph than in gonad, muscle and heart, with the highest expression level in hepatopancreas. The ESPLC mRNA expression in haemolymph of Chinese mitten crab was up-regulated at 2 h and 12 h after challenged with Listonella anguillarum. The tissue distribution and temporal characteristics of ESPLC mRNA expression, similar to that of prophenoloxidase gene in E. sinensis, suggested that ESPLC was potentially involved in the response against invading bacteria, with the possibility that it functioned in the prophenoloxidase system in E sinensis. (C) 2008 Elsevier Ltd. All rights reserved.

Molecular cloning, characterization and expression of heat shock protein 90 gene in the haemocytes of bay scallop Argopecten irradians

Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone that plays key roles in the folding, maintenance of structural integrity and regulation of a subset of cytosolic proteins. In the present study, the cDNA of Argopecten irradians HSP90 (designated AiHSP90) was cloned by the combination of homology cloning and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of AiHSP90 was of 2669 bp, including an open reading frame (ORF) of 2175 bp encoding a polypeptide of 724 amino acids with predicted molecular weight of 83.08 kDa and theoretical isoelectric point of 4.81. BLAST analysis revealed that AiHSP90 shared high similarity with other known HSP90s, and the five conserved amino acid blocks defined as HSP90 protein family signatures were also identified in AiHSP90, which indicated that AiHSP90 should be a cytosolic member of the HSP90 family. Fluorescent real-time quantitative PCR was employed to examine the expression pattern of AiHSP90 mRNA in haemocytes of scallops challenged by Gram-negative bacteria Vibrio anguillarum and Gram-positive bacteria Micrococcus luteus. In both bacterial challenged groups, the relative expression level of AiHSP90 transcript was up-regulated and reached maximal. level at 9 h after injection, and then dropped progressively to the original level at about 48 h post challenge. The results indicated that AiHSP90 was potentially involved in the immune responses against bacteria challenge in scallop A. irradian. (c) 2007 Elsevier Ltd. All rights reserved.

Phylogeography of the Northern Atlantic species Chondrus crispus (Gigartinales, Rhodophyta) inferred from nuclear rDNA internal transcribed spacer sequences

Eighteen isolates of the red algae Chondrus crispus were collected from Northern Atlantic sites, together with C. ocellatus, C. yendoi and C. pinnulatus from the North Pacific. The nuclear rDNA internal transcribed spacer (ITS) was sequenced and compared, spanning both the ITS regions and the 5.8S rRNA gene. Percentage of nucleotide variation for C. crispus ranged from 0.3% to 4.0%. Phylogenetic analyses were performed using maximum parsimony (MP), neighbor-joining (NJ) and minimum evolution methods. They showed that two main clades existed within the C. crispus samples examined and that suggested C. crispus had a single Atlantic origin. The clustering however did not follow the geographic origin. We hypothesized that the current distribution of C. crispus populations might be a result of three main factors: temperature boundaries, paleoclimate and paleoceanography. ITS data exhibited abundant molecular information not only for phylogeographical investigation but also for systematics studies.