Discovery of the genes in response to white spot syndrome virus (WSSV) infection in Fenneropenaeus chinensis through cDNA microarray

We used microarray technology to study differentially expressed genes in white spot syndrome virus (WSSV)-infected shrimp. A total of 3136 cDNA targets, including 1578 unique genes from a cephalothorax cDNA library and 1536 cDNA clones from reverse and forward suppression subtractive hybridization (SSH) libraries of Fenneropenaeus chinensis, plus 14 negative and 8 blank control clones, were spotted onto a 18 x 18 mm area of NH2-modified glass slides. Gene expression patterns in the cephalothorax of shrimp at 6 h after WSSV injection and moribund shrimp naturally infected by WSSV were analyzed. A total of 105 elements on the arrays showed a similar regulation pattern in artificially infected shrimp and naturally infected moribund shrimp; parts of the results were confirmed by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The up-regulated expression of immune-related genes, including heat shock proteins (HSP70 and HSP90), trehalose-phosphate synthase (TPS), ubiquitin C, and so forth, were observed when shrimp were challenged with WSSV. Genes including myosin LC2, ATP synthase A chain, and arginine kinase were found to be down-regulated after WSSV infection. The expression of housekeeping genes such as actin, elongation factor, and tubulin is not stable, and so these genes are not suitable as internal standards for semiquantitative RT-PCR when shrimp are challenged by WSSV. As a substitute, we found that triosephosphate isomerase (TPI) was an ideal candidate of interstandards in this situation.

Characterization and mapping of expressed sequence tags isolated from a subtracted cDNA library of Litopenaeus vannamei injected with white spot syndrome virus

A large number of polymorphic simple sequence repeats (SSRs) or microsatellites are needed to develop a genetic map for shrimp. However, developing an SSR map is very time-consuming, expensive, and most SSRs are not specifically linked to gene loci of immediate interest. We report here on our strategy to develop polymorphic markers using expressed sequence tags (ESTs) by designing primers flanking single or multiple SSRs with three or more repeats. A subtracted cDNA library was prepared using RNA from specific pathogen-free (SPF) Litopenaeus vannamei juveniles (similar to 1 g) collected before (0) and after (48 h) inoculation with the China isolate of white spot syndrome virus (WSSV). A total of 224 clones were sequenced, 194 of which were useful for homology comparisons against annotated genes in NCBI nonredundant (nr) and protein databases, providing 179 sequences encoded by nuclear DNA, 4 mitochondrial DNA, and 11 were similar to portions of WSSV genome. The nuclear sequences clustered in 43 groups, 11 of which were homologous to various ESTs of unknown function, 4 had no homology to any sequence, and 28 showed similarities to known genes of invertebrates and vertebrates, representatives of cellular metabolic processes such as calcium ion balance, cytoskeleton mRNAs, and protein synthesis. A few sequences were homologous to immune system-related (allergens) genes and two were similar to motifs of the sex-lethal gene of Drosophila. A large number of EST sequences were similar to domains of the EF-hand superfamily (Ca2+ binding motif and FRQ protein domain of myosin light chains). Single or multiple SSRs with three or more repeats were found in approximately 61 % of the 179 nuclear sequences. Primer sets were designed from 28 sequences representing 19 known or putative genes and tested for polymorphism (EST-SSR marker) in a small test panel containing 16 individuals. Ten (53%) of the 19 putative or unknown function genes were polymorphic, 4 monomorphic, and 3 either failed to satisfactorily amplify genomic DNA or the allele amplification conditions need to be further optimized. Five polymorphic ESTs were genotyped with the entire reference mapping family, two of them (actin, accession #CX535973 and shrimp allergen arginine kinase, accession #CX535999) did not amplify with all offspring of the IRMF panel suggesting presence of null alleles, and three of them amplified in most of the IRM F offspring and were used for linkage analysis. EF-hand motif of myosin light chain (accession #CX535935) was placed in ShrimpMap's linkage group 7, whereas ribosomal protein S5 (accession #CX535957) and troponin I (accession #CX535976) remained unassigned. Results indicate that (a) a large number of ESTs isolated from this cDNA library are similar to cytoskeleton mRNAs and may reflect a normal pathway of the cellular response after im infection with WSSV, and (b) primers flanking single or multiple SSRs with three or more repeats from shrimp ESTs could be an efficient approach to develop polymorphic markers useful for linkage mapping. Work is underway to map additional SSR-containing ESTs from this and other cDNA libraries as a plausible strategy to increase marker density in ShrimpMap.

Diversity of Both the Cultivable Protease-Producing Bacteria and Their Extracellular Proteases in the Sediments of the South China Sea

Protease-producing bacteria are known to play an important role in degrading sedimentary particular organic nitrogen, and yet, their diversity and extracellular proteases remain largely unknown. In this paper, the diversity of the cultivable protease-producing bacteria and their extracellular proteases in the sediments of the South China Sea was investigated. The richness of the cultivable protease-producing bacteria reached 10(6) cells/g in all sediment samples. Analysis of the 16S rRNA gene sequences revealed that the predominant cultivated protease-producing bacteria are Gammaproteobacteria affiliated with the genera Pseudoalteromonas, Alteromonas, Marinobacter, Idiomarina, Halomonas, Vibrio, Shewanella, Pseudomonas, and Rheinheimera, with Alteromonas (34.6%) and Pseudoalteromonas (28.2%) as the predominant groups. Inhibitor analysis showed that nearly all the extracellular proteases from the bacteria are serine proteases or metalloproteases. Moreover, these proteases have different hydrolytic ability to different proteins, reflecting they may belong to different kinds of serine proteases or metalloproteases. To our knowledge, this study represents the first report of the diversity of bacterial proteases in deep-sea sediments.

Modulatory effects of ammonia-N on the immune system of Penaeus japonicus to virulence of white spot syndrome virus

To study response to white spot syndrome virus (WSSV) under ammonia stress, Penaeus japonicus were exposed to 5 mg l(-1) ammonia-N and challenged orally with WSSV (NW). Controls consisted of an ammonia-N-exposed control group (N), a WSSV-challenged positive control group (W), and an untreated control group (control). Immune parameters measured were total haemocyte count (THC), haemocyte phagocytosis, plasma protein content and haemolymph enzymatic activities for prophenoloxidase (proPO), alkaline phosphatase (ALP), and nitric oxide synthase (NOS). THC and plasma protein had downward trends with time in all treatment groups (NW, N, and W) in contrast to the untreated control group (control). The percentage phagocytosis, NOS activity, and ALP and proPO activity of W and NW decreased initially then increased from 6 to 78 h (except for NOS and ALP, from 6 to 54 h) before declining thereafter until the end of the experiment. Compared with untreated controls (control), there was a downward trend for all measured parameters in the treatment groups (N, NW, and W), but the degree was W > NW > N. WSSV was detected at 78 h postchallenge in both W and NW. In conclusion, 5 mg l(-1) ammonia-N reduced the immunocompetence of P japonicus and may have decreased the virulence of WSSV (C) 2004 Elsevier B.V. All rights reserved.

Digestive gut structure and activity of protease, amylase, and alkaline phosphatase in Calanus sinicus during summer in the Yellow Sea and the East China Sea

Calanus sinicus aggregate at the depth of 40-60 m (ambient temperature is 16 degreesC) in the waters of the continental shelf of the Yellow Sea during summer. in animals found in near shore regions, there are changes in digestive gut cells structure, digestive enzyme activity (protease, amylase), and tissue enzyme (alkaline phosphatase (ALP)), which may represent adaptations by this cold-water animal to a sharp seasonal increase in temperature of 6-23 degreesC. The activities of the digestive enzymes (protease and amylase) are very low in animals at stations near the estuary of Yangtse River, whereas they are relatively high in animals at stations in the central Yellow Sea, During summer, B-cells of the intestine and the villi intestinalis disappear in animals that do not feed at stations near the estuary of the Yangtse River. Respiration rates were undetectable or quite low during summer in C. sinicus from stations near the estuary of the Yangtse River, whereas they were relatively high at stations in the central Yellow Sea. Based upon the morphological characteristics of the digestive gut structure, enzyme levels, respiration rates, and the distribution of C. sinicus, we concluded that C. sinicus might be dormant during summer in the near shore areas of the East China Sea while remaining active in the central Yellow Sea. (C) 2002 Elsevier Science B.V. All rights reserved.

Studies on the transmission of WSSV (white spot syndrome virus) in juvenile Marsupenaeus japonicus via marine microalgae

We studied the possible role that marine microalgae may play during the outbreaks of WSS (white spot syndrome). In order to elucidate the possibility of marine microalgae carrying WSSV (white spot syndrome virus), six marine microallgae (Isochr.vsis galbana, Skeletonema costatum, Chlorella sp., Heterosigma akashiwo, Scrippsiella trochoidea, Dunaliella salina) were co-cultured with adult Marsupenaeus japollicus infected with WSSV and were assayed daily by nested-PCR to study whether they could carry WSSV. Further experiments were conducted to investigate whether the virus carried by microalgae could re-infect juvenile M. japonicus. Results showed that all of the experimental microalgae, except H. akashiwo could carry WSSV, and among them, Chlorella sp. and S. trochoidea had the strongest WSSV-carrying ability. Unlike other invertebrate carriers of WSSV, the WSSV detections in microalgae, which were positive after I and 3 days, were negative after 10 days of incubation. WSSV detection results in juvenile M. japonicus showed that the juvenile shrimp were re-infected by co-cultured Chlorella sp., although the juvenile M. japonicus carried so small an amount of WSSV that it could only be detected by nested-PCR. The results of this experiment suggest that microalgae might be one possible horizontal transmission pathway for WSSV. Further research, however, is required to better understand the factors behind the different carrying abilities and virus-carrying mechanisms of different microalgae. (c) 2007 Elsevier Inc. All rights reserved.

Studies on nitric oxide synthase activity in haemocytes of shrimps Fenneropenaeus chinensis and Marsupenaeus japonicus after white spot syndrome virus infection

The nitric oxide synthase (NOS) activity in the haemocytes of shrimps Fenneropenaeus chinensis (Osbeck) and Marsupenaeus japonicus (Bate) was Studied after white spot syndrome virus (WSSV) infection to determine its characteristics in response to virus infection. First, the NOS activity in haemocytes of shrimps was determined by the means of NBT reduction and changes in cell conformation. And the variations of NOS activity in shrimps after challenge with WSSV intramuscularly were evaluated through the analysis Of L-citrulline and total nitrite/nitrate (both as NO derivates) concentrations. The result showed that NOS activity in the haemocytes of F chinensis increased slightly from 0 to 12 h postchallenge, indicated by the variations Of L-Citrulline (from 11.15 +/- 0.10 to 12.08 +/- 0.64 mu M) and total nitrite/nitrate concentrations (from 10.45 +/- 0.65 to 12.67 +/- 0.52 mu M). Then it decreased sharply till the end of the experiment (84 h postchallenge), the concentrations Of L-Citrulline and total nitrite/nitrate at 84 It were 1.58 +/- 0.24 and 2.69 +/- 0.70 mu M, respectively. The LPS-stimulated NOS activity kept constant during the experiment. However, in M. japonicus, the NOS activity kept increasing during the first 72 It postchallenge, the concentrations Of L-Citrulline and total nitrite/nitrate increased from 7.82 +/- 0.77 at 0 h to 10.79 +/- 0.50 mu M at 72 h, and from 8.98 +/- 0.43 at 0 h to 11.20 +/- 0.37 mu M at 72 h, respectively. Then it decreased till the end of the experiment (216 h postchallenge), and the concentrations of L-Citrulline and total nitrite/nitrate at 216 h were 5.66 +/- 0.27 and 4.68 +/- 0.16 mu M, respectively. More importantly, an apparent increase of I-PS-stimulated NOS activity was observed in M japonicus at 48 h postchallenge, which was about 4 times higher than that in the control group of health shrimps. In correspondence with the difference of NOS activity between the two species of shrimps, the Cumulative mortalities of the shrimps were also different. All shrimps of F. chinensis in the mortality experiment died in 66 h, much more quickly than M. japonicus, Whose accumulative mortality reached 100% after 240 h. Data here reported let us hypothesize that NOS activity in the haemocytes of shrimps F chinensis and M. japonicus responses to WSSV infection differently, and this might be one of the reasons for the different susceptibility of F chinensis and M. japonicus to WSSV infection. (c) 2005 Elsevier Inc. All rights reserved.

Identification of a silicatein(-related) protease in the giant spicules of the deep-sea hexactinellid Monorhaphis chuni

Silicateins, members of the cathepsin L family, are enzymes that have been shown to be involved in the biosynthesis/condensation of biosilica in spicules from Demospongiae (phylum Porifera), e. g. Tethya aurantium and Suberites domuncula. The class Hexactinellida also forms spicules from this inorganic material. This class of sponges includes species that form the largest biogenic silica structures on earth. The giant basal spicules from the hexactinellids Monorhaphis chuni and Monorhaphis intermedia can reach lengths of up to 3 m and diameters of 10 mm. The giant spicules as well as the tauactines consist of a biosilica shell that surrounds the axial canal, which harbours the axial filament, in regular concentric, lamellar layers, suggesting an appositional growth of the spicules. The lamellae contain 27 kDa proteins, which undergo post-translational modification (phosphorylation), while total spicule extracts contain additional 70 kDa proteins. The 27 kDa proteins cross-reacted with anti-silicatein antibodies. The extracts of spicules from the hexactinellid Monorhaphis displayed proteolytic activity like the silicateins from the demosponge S. domuncula. Since the proteolytic activity in spicule extracts from both classes of sponge could be sensitively inhibited by E-64 (a specific cysteine proteinase inhibitor), we used a labelled E-64 sample as a probe to identify the protein that bound to this inhibitor on a blot. The experiments revealed that the labelled E-64 selectively recognized the 27 kDa protein. Our data strongly suggest that silicatein(-related) molecules are also present in Hexactinellida. These new results are considered to also be of impact for applied biotechnological studies.

Factors affecting the protease activity of venom from jellyfish Rhopilema esculentum Kishinouye

In this paper, the effects of some chemical and physical factors such as temperature, pH values, glycerol, and divalent metal cations on the protease activity of venom from jellyfish, Rhopilema esculentum Kishinouye, were assayed. Protease activity was dependent on temperature and pH values. Zn2+, Mg2+, and Mn2+ in sodium phosphate buffer (0.02 M, pH 8.0) could increase protease activity. Mn2+ had the best effects among the three metal cations and the effect was about 20 times of that of Zn2+ or Mg2+ and its maximal protease activity was 2.3 x 10(5) U/mL. EDTA could increase protease activity. PMSF had hardly affected protease activity. O-Phenanthroline and glycerol played an important part in inhibiting protease activity and their maximal inhibiting rates were 87.5% and 82.1%, respectively. (c) 2005 Elsevier Ltd. All rights reserved.

H5N1 avian influenza re-emergence of Lake Communication Qinghai: phylogenetic and antigenic analyses of the newly isolated viruses and roles of migratory birds in virus circulation

Highly pathogenic avian influenza H5N1 virus has swept west across the globe and caused serious debates on the roles of migratory birds in virus circulation since the first large-scale outbreak in migratory birds of Lake Qinghai, 2005. In May 2006, another outbreak struck Lake Qinghai and six novel strains were isolated. To elucidate these QH06 viruses, the six isolates were subjected to whole-genome sequencing. Phylogenetic analyses show that QH06 viruses are derived from the lineages of Lake Qinghai, 2005. Five of the six novel isolates are adjacent to the strain A/Cygnus olor/Croatia/1/05, and the last one is related to the strain A/duck/Novosibirsk/ 02/05, an isolate of the flyway. Antigenic analyses suggest that QH06 and QH05 viruses are similar to each other. These findings implicate that QH06 viruses of Lake Qinghai may travel back via migratory birds, though not ruling out the possibility of local circulation of viruses of Lake Qinghai.

Detecção do melon yellowing associated virus (MYaV) em áreas produtoras de melão na Região Nordeste.

2009

Ocorrência do Barley/Cereal yellow dwarf virus e seus vetores em cereais de inverno no Rio Grande do Sul, Santa Catarina, Paraná e Mato Grosso do Sul em 2008.

2009

Ocorrência do Barley/Cereal yellow dwarf virus e seus vetores em cereais de inverno no Rio Grande do Sul em 2007.

2008

Comparison of comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry and gas chromatography-mass spectrometry for the analysis of tobacco essential oils

A sample of tobacco essential oil was analyzed using gas chromatography-mass spectrometry (GUMS)and comprehensive two-dimensional gas chromatography coupled to a time-of-flight mass spectrometry (GC x GC/TOFMS), respectively. In the GUMS analysis, serially coupled columns were used. By comparing the GUMS results with GC x GC/TOFMS result,,, many more components in the essential oil could be found within the two-dimensional separation space of GC x GC. The quantitative determination of components in the essential oil was performed by GC x GC with flame ionization detection (FID), using a method of multiple internal standards calibration, (c) 2005 Elsevier B.V. All rights reserved.

Quantitative determination of compounds in tobacco essential oils by comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry

A quantitative analysis of the individual compounds in tobacco essential oils is performed by comprehensive two-dimensional gas chromatography (GC x GC) combined with flame ionization detector (FID). A time-of-flight mass spectrometer (TOF/MS) was coupled to GC x GC for the identification of the resolved peaks. The response of a flame ionization detector to different compound classes was calibrated using multiple internal standards. In total, 172 compounds were identified with good match and 61 compounds with high probability value were reliably quantified. For comparative purposes, the essential oil sample was also quantified by one-dimensional gas chromatography-mass spectrometry (GC/MS) with multiple internal standards method. The results showed that there was close agreement between the two analysis methods when the peak purity and match quality in one-dimensional GC/MS are high enough. (c) 2005 Elsevier B.V. All rights reserved.