Effects of acute temperature or salinity stress on the immune response in sea cucumber, Apostichopus japonicus

Invertebrates are increasingly raised in mariculture, where it is important to monitor immune function and to minimize stresses that could suppress immunity. The activities of phagocytosis, superoxide dismutase (SOD), catalase (CAT), myeloperoxiclase (MPO), and lysozyme (LSZ) were measured to evaluate the immune capacities of the sea cucumber, Apostichopus japonicus, to acute temperature changes (from 12 degrees C to 0 degrees C, 8 degrees C, 16 degrees C, 24 degrees C, and 32 degrees C for 72 h) and salinity changes (from 30 parts per thousand to 20 parts per thousand, 25 parts per thousand, and 35 parts per thousand for 72 h) in the laboratory. Phagocytosis was significantly affected by temperature increases in 3 h, and by salinity (25 parts per thousand and 35 parts per thousand) changes in 1 h. SOD activities decreased significantly in 0.5 h to 6 h samples at 24 degrees C. At 32 degrees C, SOD activities decreased significantly in 0.5 h and 1 h exposures, and obviously increased for 12 h exposure. CAT activities decreased significantly at 24 degrees C for 0.5 h exposure, and increased significantly at 32 degrees C in 3 h to 12 h exposures. Activities of MPO increased significantly at 0 degrees C in 0.5 h to 6 In exposures and at 8 degrees C for 1 h. By contrast, activities of MPO decreased significantly in 24 degrees C and 32 degrees C treatments. In elevated-temperature treatments, activities of LSZ increased significantly except at 32 degrees C for 6 h to 12 h exposures. SOD activity was significantly affected by salinity change. CAT activity decreased significantly after only 1 h exposure to salinity of 20 parts per thousand.. Activities of MPO and LSZ showed that A. japonicus tolerates limited salinity stress. High-temperature stress had a much greater effect on the immune capacities of A. japonicus than did low-temperature and salinity stresses. Crown Copyright (C) 2008 Published by Elsevier Inc. All rights reserved.

Immune condition of Apostichopus japonicus during aestivation

Aestivation is an indispensable state in the life history of sea cucumbers, Apostichopus japonicus. The immune characteristics of the coelomic fluid of A. japonicus, were investigated during aestivation. Samples were collected between July and November 2006 from a coastal pond located off the Yellow Sea in Jiaonan, Shandong Province, China. The total coelomocytes counts (TCC), total superoxide dismutase (T-SOD), catalase (CAT), myeloperoxidase (MPO), and lysozyme (IZM) in the coelomic fluid were measured. The activities of catecholamines, [adrenaline (AD), noradrenaline (NA), and dopamine (DOP)] were estimated. TCC decreased from July to September, indicating weakness of the cellular immune activity at that time. Activities of SOD, CAT, MPO, and LZM changed significantly from July to October. Catecholamines AD and NA in coelomic fluid were significantly higher on August 21 and November 27. There was no significant variation in DOP during the sampling period. Thus, immune characters in coelomic fluid of A. japonicus changed significantly during aestivation. Water temperature was significantly and negatively correlated with TCC, and salinity was significantly and positively correlated with AD and NA. The mechanism of aestivation in A. japonicus is complex and might not be attributed only to environmental changes, such as temperature and salinity, as shown in previous studies. (C) 2008 Elsevier B.V. All rights reserved.

Survival, growth and immune activity of scallop Chlamys farreri cultured at different depths in Haizhou Bay (Yellow Sea, China) during hot season

Survival, growth and immune response of the scallop, Chlamys farreri, cultured in lantern nets at five different depths (2, 5, 10, 15, and 20 m below the sea surface) were studied in Haizhou Bay during the hot season (summer and autumn) of 2007. Survival and growth rates were quantified bimonthly. Immune activities in hemolymph (superoxide dismutase (SOD) and acid phosphatase (ACP)) were measured to evaluate the health of scallops at the end of the study. Environmental parameters at the five depths were also monitored during the experiment. Mortalities mainly occurred during summer. Survival of scallops suspended at 15 m (78.0%) and 20 m (86.7%) was significantly higher than at 2 m (62.9%), 5 m (60.8%) or 10 m (66.8%) at the end of the study. Mean shell height grew significantly faster at 10 m (205.0 mu m/d) and 20 m (236.9 mu m/d) than at 2, 5 or 15 m in summer (July 9 to September 1); however, shell growth rate at 20 m was significantly lower than at the other four depths in autumn (September 2 to November 6). In contrast to summer, scallops at 5 m grew faster (262.9 mu m/d) during autumn. The growth of soft tissue at different depths showed a similar trend to the shell. Growth rates of shell height and soft tissue were faster in autumn than in summer, with the exception of shell height at 20 m. SOD activity of scallops increased with depth, and ACP activity was significantly higher at 15 and 20 m than at other depths, which suggests that scallops were healthier near the bottom. Factors explaining the depth-related mortality and growth of scallops are also discussed. We conclude that the mass mortality of scallop, C. farreri, during summer can be prevented by moving the culture area to deeper water and yield can be maximized by suspending the scallops in deep water during summer and then transferring them to shallow water in autumn.

The unique skeleton of siliceous sponges (Porifera; Hexactinellida and Demospongiae) that evolved first from the Urmetazoa during the Proterozoic: a review

Sponges (phylum Porifera) had been considered as an enigmatic phylum, prior to the analysis of their genetic repertoire/tool kit. Already with the isolation of the first adhesion molecule, galectin, it became clear that the sequences of sponge cell surface receptors and of molecules forming the intracellular signal transduction pathways triggered by them, share high similarity with those identified in other metazoan phyla. These studies demonstrated that all metazoan phyla, including Porifera, originate from one common ancestor, the Urmetazoa. The sponges evolved prior to the Ediacaran-Cambrian boundary (542 million years ago [myr]) during two major "snowball earth events", the Sturtian glaciation (710 to 680 myr) and the Varanger-Marinoan ice ages (605 to 585 myr). During this period the ocean was richer in silica due to the silicate weathering. The oldest sponge fossils (Hexactinellida) have been described from Australia, China and Mongolia and are thought to have existed coeval with the diverse Ediacara fauna. Only little younger are the fossils discovered in the Sansha section in Hunan (Early Cambrian; China). It has been proposed that only the sponges possessed the genetic repertoire to cope with the adverse conditions, e.g. temperature-protection molecules or proteins protecting them against ultraviolet radiation. The skeletal elements of the Hexactinellida (model organisms Monorhaphis chuni and Monorhaphis intermedia or Hyalonema sieboldi) and Demospongiae (models Suberites domuncula and Geodia cydonium), the spicules, are formed enzymatically by the anabolic enzyme silicatein and the catabolic enzyme silicase. Both, the spicules of Hexactinellida and of Demospongiae, comprise a central axial canal and an axial filament which harbors the silicatein. After intracellular formation of the first lamella around the channel and the subsequent extracellular apposition of further lamellae the spicules are completed in a net formed of collagen fibers. The data summarized here substantiate that with the finding of silicatein a new aera in the field of bio/inorganic chemistry started. For the first time strategies could be formulated and experimentally proven that allow the formation/synthesis of inorganic structures by organic molecules. These findings are not only of importance for the further understanding of basic pathways in the body plan formation of sponges but also of eminent importance for applied/commercial processes in a sustainable use of biomolecules for novel bio/inorganic materials.

The complete mitochondrial genome of the ridgetail white prawn Exopalaemon carinicauda Holthuis, 1950 (Crustacean: Decapoda: Palaemonidae) revealed a novel rearrangement of tRNA genes

The complete mitochondrial (mt) DNA sequence was determined for a ridgetail white prawn, Exopalaemon carinicauda Holthuis, 1950 (Crustacea: Decopoda: Palaemonidae). The mt genome is 15,730 bp in length, encoding a standard set of 13 protein-coding genes, 2 ribosomal RNA genes, and 22 transfer RNA genes, which is typical for metazoans. The majority-strand consists of 33.6% A, 23.0% C, 13.4% G, and 30.0% T bases (AT skew = 0.057: GC skew = -0.264). A total of 1045 bp of non-coding nucleotides were observed in 16 intergenic regions,,including a major A+ T rich (79.7%) noncoding region (886 bp). A novel translocation of tRNA(Pro) and tRNA(Thr) was found when comparing this genome with the pancrustacean ground pattern indicating that gene order is not conserved among caridean mitochondria. Furthermore, the rate of Ka/Ks in 13 protein-coding genes between three caridean species is Much less than 1, which indicates a strong Purifying selection within this group. To investigate the phylogenetic relationship within Malacostraca, phylogenetic trees based oil Currently available malacostracan complete mitochondrial sequences were built with the maximum likelihood and Bayesian models. All analyses based oil nucleotide and amino acid data strongly support the monophyly of Decapoda. The Penaeidae, Reptantia, Caridea, and Meiura clades were also recovered as monophyletic groups with Strong Statistical Support. However, the phylogenetic relationships within Pleocyemata are unstable, as represented by the inclusion or exclusion of Caridea. (C) 2009 Elsevier B.V. All rights reserved.

Chromosomal mapping of 5S ribosomal RNA genes in the eastern oyster, Crassostrea virginica gmelin by fluorescence in situ hybridization

Chromosomal location of the 5S ribosomal RNA gene was studied in the eastern oyster, Crassostrea virginica Gmelin. using fluorescence in situ hybridization (FISH). Metaphase chromosomes were obtained from early embryos, and the FISH probe was made by PCR (polymerase chain reaction) amplification of the 5S rRNA gene and labeled by incorporation of digoxigenin-1 1-dUTP during PCR. Hybridization was detected with fluorescein-labeled antidigoxigenin antibodies. Two pairs of FISH signals were observed on metaphase chromosomes. Karyotypic analysis showed that the 5S rRNA gene cluster is interstitially located on short arms of chromosomes 5 and 6. On chromosome 5, the 5S rRNA genes were located immediately next to the centromere, whereas on chromosome 6, they were located approximately half way between the telomere and the centromere. Chromosomes of C. virginica are difficult to identify because of their similarities in size and arm ratio, and the chromosomal location of 5S rRNA genes provides unambiguous identification of chromosomes 5 and 6. Previous studies have mapped the major rRNA gene cluster (18S-5.8S-28S) to chromosome 2. and this study shows that the 5S rRNA gene cluster is not linked to the major rRNA genes and duplicated during evolution.

Cloning and expression analysis of a HSP70 gene from Pacific abalone (Haliotis discus hannai)

Heat shock protein 70 (HSP70), the primary member of HSPs that are responsive of thermal stress, is found in all multicellular organisms and functions mostly as molecular chaperon. The inducible HSP70 cDNA cloned from Pacific abalone (Haliotis discus hannai) using rapid amplification of cDNA ends (RACE), was highly homologous to other HSP70 genes. The full-length cDNA of the Pacific abalone HSP70 was 2631 bp, consisting of a 5'-terminal untranslated region (UTR) of 90 bp, a 3'-terminal UTR of 573 by with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame of 1968 bp. The HSP70 cDNA encoded a polypeptide of 655 amino acids with an ATPase domain of 382 amino acids, the substrate peptide binding domain of 161 amino acids and a C-terminus domain of 112 amino acids. The temporal expression of HSP70 was measured by semi-quantitative RT-PCR after heat shock and bacterial challenge. Challenge of Pacific abalone with heat shock or the pathogenic bacteria Vibrio anguillarum resulted in a dramatic increase in the expression of HSP70 mRNA level in muscle, followed by a recovery to normal level after 96 h. Unlike the muscle, the levels of HSP70 expression in gills reached the top at 12 h and maintained a relatively high level compared with the control after thermal and bacterial challenge. The upregulated mRNA expression of HSP70 in the abalone following heat shock and infection response indicates that the HSP70 gene is inducible and involved in immune response. (c) 2006 Elsevier Ltd. All rights reserved.

Chromosomal mapping of major ribosomal rRNA genes in the hard clam (Mercenaria mercenaria) using fluorescence in situ hybridization

Karyotype and chromosomal location of the major ribosomal RNA genes were studied in the hard clam (Mercenaria mercenaria Linnaeus) using fluorescence in situ hybridization (FISH). Metaphase chromosomes were obtained from early embryos. Internal transcribed spacers (ITS) between major RNA genes were amplified and used as FISH probes. The probes were labeled with digoxigenin-11-dUTP by polymerase chain reaction and detected with fluorescein-labeled anti-digoxigenin antibodies. FISH with the ITS probes produced two to four signals per nucleus or metaphase. M. mercenaria had a haploid number of 19 chromosomes with a karyotype of seven metacentric, four metacentric or submetacentric, seven submetacentric, and one submetacentric or subtelocentric chromosomes (7M + 4M/SM + 7SM + 1SM/ST). Two ITS loci were observed: one located near the centromere on the long arm of Chromosome 10 and the other at the telomere of the short arm of Chromosome 12. FISH signals on Chromosome 10 are strong and consistent, while signals on Chromosome 12 are variable. This study provides the first karyotype and chromosomal assignment of the major RNA genes in M. mercenaria. Similar studies in a wide range of species are needed to understand the role of chromosomal changes in bivalve evolution.

Chromosomal mapping of the major ribosomal RNA genes in the dwarf surfclam (Mulinia lateralis Say)

Chromosomal location of the major ribosomal RNA genes (rRNA) were studied in the dwarf surfclam (Mulinia lateralis, Say) using fluorescence in situ hybridization (FISH). FISH probes for the rRNA genes were made by polymerase chain reaction (PCR), labeled with digoxigenin-11-dUTP and detected with fluorescein-labeled antidigoxigenin antibodies. Mulinia lateralis had a diploid number of 38 chromosomes and all chromosomes were telocentric. FISH with the rRNA probe produced positive and consistent signals on two pairs of chromosomes: Chromosome 15 with a relative length of 4.6% and Chromosome 19, the shortest chromosome. Both loci were telomeric. The rRNA location provides the first physical landmark of the M. lateralis genome.

生物钟基因mPer2调控节律性免疫功能的机制研究

This project was carried out with the aims to investigate the mechanism of circadian immune regulation by one of the core Clock gene, mPer2. To achieve this, we selected mPer2 knock out (mPer2-/-) mice as the optimal animal model. Two different approaches were performed. In the first approach, we injected WT or mPer2-/- mice with an equal dosage of lipopolysaccharide (LPS), and systematically measured serum corticosterone induction, expression of core Clock genes, as well as a key enzyme for corticosterone metabolism (mStAR) in adrenal gland. We found that the acute induction of corticosterone and mStAR were closely associated with the circadian immune response to LPS. Besides, real time quantitative PCR (q-PCR) and luciferase assay consistently showed that mStAR is a Clock controlled gene in adrenal gland, where its expression is negatively influenced by mPer2. In the second approach, expression level and circadian manner of 11 cytotoxicity regulation genes in WT or mPer2-/- mice bone marrow were measured by q-PCR in order to explore the candidate genes which could mediate the circadian immune regulation by mPer2. We found that expression level of Ly49C, Ly49I, and Nkg2d was significant down-regulated in mPer2-/- mice. Further, we found that daily expression of Ly49C and Nkg2d fluctuated in a circadian manner in WT mice, where these rhythms were disrupted in mPer2-/- mice. Thus, it was suggested that these two cytotoxic genes were two clock controlled genes whose circadian expression were regulated by mPer2. Taken together, our results suggested that corticosterone, mStAR, Ly49C, and Nkg2d were four candidate molecules that may mediate the circadian immune response regulation by mPer2.

Análise da variabilidade intra-espécie dos genes pld e rpoB de Corynebacterium pseudotuberculosis por meio de marcadores PCR-RFLP.

Corynebacterium pseudotuberculosis é o agente etiológico da linfadenite caseosa em ovinos e caprinos, uma doença infectocontagiosa crônica caracterizada por abscessos em linfonodos superficiais ou internos. Os animais acometidos podem apresentar lesões internas ou externas, o que representa perdas econômicas graves na caprinovinocultura, como o comprometimento da produção de lã em ovinos, deficiência reprodutiva, condenação da carne ou morte do animal. Uma vez estabelecida a infecção no rebanho, torna-se difícil sua erradicação. Com o objetivo de avaliar a variabilidade genética intra-espécie em C. pseudotuberculosis, 31 isolados provenientes de seis municípios do Estado de Pernambuco, sendo 23 de caprinos e oito de ovinos, tiveram os seus DNAs genômicos extraídos e analisados por reação em cadeia da polimerase e análise de polimorfismos por tamanho de fragmentos de restrição (PCR-RFLP). Os locis escolhidos foram os genes pld e rpoB, cujas seqüências dos oligonucleotídeos amplificaram fragmentos de 924 e 447 pares de bases (pb), respectivamente. Os produtos da amplificação foram digeridos com as enzimas Pst I e Msp I (gene pld) e HpyCh4 IV e Msp I (gene rpoB). Os fragmentos de restrição foram corridos em gel de poliacrilamida a 15%, os quais foram corados com brometo de etídeo. Para o gene pld, digerido com a enzima Pst I, foram obtidos fragmentos de 566, 195, 91 e 72 pb; e com a enzima Msp I, fragmentos de 395, 369, 108 e 52 pb. O gene rpoB digerido com a enzima HpyCh4 IV apresentou fragmentos de 235, 126 e 86 pb; e com a enzima Msp I apresentou fragmentos de 222, 93, 78 e 54 pb. O padrão de bandas obtido para os 31 isolados de C. pseudotuberculosis, analisados neste estudo, foi monomórfico, não havendo, portanto, polimorfismo entre os diferentes isolados, independentes da espécie hospedeira ou da área geográfica estudada, o que corrobora com o padrão homogêneo da infecção para a região.

A potencialidade de Agropyron, espécie afim ao trigo cultivado, como fonte de introgressão de genes agronomicamente importantes.

2001

Efetividade de genes de resistência de trigo a oídio, safra 2005.

2006

Efetividade de genes de resistência de trigo a oídio, safra 2007.

2008

Identificação e caracterização funcional de genes em estudos comparativos dos genomas de Mycosphaerella graminicola e Mycosphaerella fijiensis.

2008