Chromosomal localization of 5S rDNA in Chinese shrimp (Fenneropenaeus chinensis): a chromosome-specific marker for chromosome identification

Chinese shrimp (Fenneropenaeus chinensis) is an economically important aquaculture species in China. However, cytogenetic and genomic data is limited in the organism partly because the chromosomes are difficult to isolate and analyze. In this study, fluorescence in-situ hybridization (FISH) was used to identify the chromosomes of F. chinensis. The 5S ribosomal RNA gene (rDNA) of F. chinensis was isolated, cloned and then used as a hybridization probe. The results show that the 5S rDNA was located on one pair of homologous chromosomes in F. chinensis. In addition, triploid shrimp were used to evaluate the feasibility of chromosome identification using FISH and to validate the method. It was confirmed that 5S rDNA can be used as a chromosome-specific probe for chromosome identification in F. chinensis. The successful application of FISH in F. chinensis shows that chromosome-specific probes can be developed and this finding will facilitate further research on the chromosomes of penaeid shrimps.     

Molecular cytogenetic of the Amoy croaker,A rgyrosomus amoyensis(Teleostei,Sciaenidae)

The family Sciaenidae is remarkable for its species richness and economic importance. However, the cytogenetic data available in this fish group are still limited, especially those obtained using fl uorescence in situ hybridization(FISH). In the present study, the chromosome characteristics of a sciaenid species, Argyrosomus amoyensis, were examined with several cytogenetic methods, including dual-FISH with 18 S and 5 S rDNA probes, and a self-genomic in situ hybridization procedure(Self-GISH). The karyotype of A. amoyensis comprised 2 n=48 acrocentric chromosomes. A single pair of nucleolar organizer regions(NORs) was located at the proximal position of chromosome 1, which was positive for silver nitrate impregnation(AgNO_3) staining and denaturation-propidium iodide(DPI) staining but negative for Giemsa staining and 4',6-diamidino-2-phenylindole(DAPI) staining, and was confi rmed by FISH with 18 S rDNA probes. The 5 S rDNA sites were located at the centromeric region of chromosome 3. Telomeric FISH signals were detected at all chromosome ends with dif ferent intensities, but internal telomeric sequences(ITSs) were not found. Self-GISH resulted in strong signals distributed at the centromeric regions of all chromosomes. C-banding revealed not only centromeric heterochromatin, but also heterochromatin that located on NORs, in interstitial and distal telomeric regions of specifi c chromosomes. These results suggest that the karyotype of Amoy croaker was relatively conserved and primitive. By comparison with the reported cytogenetic data of other sciaenids, it can be deduced that although the karyotypic macrostructure and chromosomal localization of 18 S rDNA are conserved, the distribution of 5 S rDNA varies dynamically among sciaenid species. Thus, the 5 S rDNA sites may have different evolutionary dynamics in relation to other chromosomal regions, and have the potential to be ef fective cytotaxonomic markers in Sciaenidae.     

Chromosomal mapping of 5S and 18S-5.8S-25SrRNA genes in Saccharina japonica(Phaeophyceae) as visualized by dual-color fluorescence in situ hybridization

It has been reported that there was a linkage of 5 S rRNA gene to 18 S-5.8 S-25 S rRNA gene in a few of species in Ochrophyta.In regard to the usual two positions of linked 5 S rDNA to the 3' end of 25 S rDNA,two pairs of primers were designed for amplification to verify this linkage of two genes in a kelp cultivar of Saccharina japonica,one of species in Ochrophyta.This result supplemented the previous report that 5 S rDNA was unlinked to 25 S rDNA in this kelp.In order to simultaneously visualize this unlinkage of two genes,dual-color fluorescence in situ hybridization(FISH) technique was applied to the cytogenetics of S.japonica.Dual-color FISH images showed that two and four hybridization signals were present in the kelp gametophyte and sporophyte,respectively,metaphase nuclei hybridized simultaneously with the labeled probes of 18 S rDNA and 5 S rDNA.Both haploid and diploid karyotypes in decreasing length of chromosomes showed that 18 S-5.8 S-25 S rDNA was localized at the interstitial region of Chromosome 23,whereas 5 S rDNA resided at the sub-telomeric region of Chromosome 27.These karyotypes suggested that the kelp nuclear genome had only one locus of each rRNA gene,and their loci on different chromosomes indicated the physical unlinkage of 5 S rDNA to 18 S-5.8 S-25 S rDNA in this kelp.Therefore,dual-color FISH seems to be a powerful technique for the discrimination and pairing of chromosomes featured in both small size and nearly identical shape in S.japonica.     

Application of rRNA probes and fluorescence in situ hybridization for rapid detection of the toxic dinoflagellate Alexandrium minutum

The dinoflagellate Alexandrium minutum is often associated with harmful algal blooms (HABs). This species consists of many strains that differ in their ability to produce toxins but have similar morphology, making identification difficult. In this study, species-specific rRNA probes were designed for whole-cell fluorescence in situ hybridization (FISH) to distinguish A. minutum from two phylogenetic clades. We acquired the complete SSU to LSU rDNA sequences (GenBank accession numbers JF906989-JF906999) of 11 Alexandrium strains and used these to design rRNA targeted oligonucleotide probes. Three ribotype-specific probes, M-GC-1, M-PC-2, and M-PC-3, were designed. The former is specific for the GC clade ("Global clade") of A. minutum, the majority of which have been found non-toxic, and the latter two are specific for the PSP (paralytic shellfish poisoning)-producing PC clade ("Pacific clade"). The specificity of these three probes was confirmed by FISH. All cells in observed fields of view were fluorescently labeled when probes and target species were incubated under optimized FISH conditions. However, the accessibility of rRNA molecules in ribosomes varied among the probe binding positions. Thus, there was variation in the distribution of positive signals in labeled cells within nucleolus and cytosol (M-GC-1, M-PC-3), or just nucleolus (M-PC-2). Our results provide a methodological basis for studying the biogeography and population dynamics of A. minutum, and providing an early warning of toxic HABs.     

The Complete Sequence of Mitochondrial COⅡ Gene of Fenneropenaeus chinensis and Its Applicability as a Marker for Phylogenetic Analysis

The complete mitochondrial cytochrome oxidase subunit Ⅱ (COⅡ) gene of Penaeinae shrimp Fenneropenaeus chinensis was cloned and sequenced. The gene is 688 bp in length and codes for 229 amino acids. It shows 83.2%, 87.0% and 83.8% sequence similarity to Marsupenaeus Japonicus, Penaeus monodon and Farfantepenaeus notialis, respectively. The A+T content of the whole gene and that at the third position of codons are 64.7% and 78.2%, respectively. The phylogenetic relationship between F. chinensis and three other species representing genera Farfanatepenaeus, Marsupenaeus and Penaeus was analyzed. Results showed that the genetic distances among the four taxa ranged from 0.144 0 to 0.200 5, exceeding those estimated with COⅠ and partial 16S rRNA gene sequences among Marsupenaeus, Litopenaeus and Melicertus, and being therefore larger than the value among subgenera. It has been suggested that the COⅡ gene has a faster evolutionary rate than that of the COⅠ gene and partial 16S rRNA gene and could be used for phylogenetic analysis at genus or species level. The results of the present study indicated that Farfantepenaeus, Fenneropenaeus, Marsupenaeus and Penaeus are at a higher phylogenetic level than subgenus, which supports the opinion of the elevation of phylogenetic status of the four subgenera to genus level.     

Hematological Changes m White Spot Syndrome Virus-Infected Shrimp, Fenneropenaeus chinensis (Osbeck)

The pathological changes of hemocytes in the haemolymph and hepatopancreas were examined in experimentally and naturally WSSV (white spot syndrome virus) infected Fenneropenaeus chinensis. The results showed that the pathological manifesta- tions of hemocytes were similar among moribund shrimps infected via injection, feeding and by nature. Firstly, the total hemocyte counts (THCs) in WSSV-infected shrimp were significantly lower than those in healthy shrimp. Secondly, necrotic, broken and dis- integrated cells were often observed, and a typical hematolysis was present in the haemolymph smear of WSSV-infected shrimp. Thirdly, necrosis and typical apoptosis of hemocytes were detected with TEM in the peripheral haemolymph of WSSV-infected shrimp. Hyalinocytes and semi-granulocytes with masses of WSSVs in their nuclei often appeared, whereas no granular bemocytes with WSSV were found in the hepatopancreas of moribund infected shrimps. All our results supported that hemocytes were the main target cells of WSSV, and hyalinocytes and semigranular hemocytes seemed to be more favorable for WSSV infection in F. chinensis.     

PCR-DGGE analysis of intestinal bacteria and effect of Bacillus spp.on intestinal microbial diversity in kuruma shrimp(Marsupenaeus japonicus)

In this study,the intestinal microbiota of kuruma shrimp(Marsupenaeus japonicus) was examined by molecular analysis of the 16S rDNA to identify the dominant intestinal bacteria and to investigate the effects of Bacillus spp.on intestinal microbial diversity.Samples of the intestines of kuruma shrimp fed normal feed and Bacillus spp.amended feed.PCR and denaturing gradient gel electrophoresis(DGGE) analyses were then performed on DNA extracted directly from the guts.Population fingerprints of the predominant organisms were generated by DGGE analysis of the universal V3 16S rDNA amplicons,and distinct bands in the gels were sequenced.The results suggested that the gut of kuruma shrimp was dominated by Vibrio sp.and uncultured gamma proteobacterium.Overall,the results of this study suggest that PCR-DGGE is a possible method of studying the intestinal microbial diversity of shrimp.     

Partial Sequence Analysis of Mitochondrial COI Gene of the Chinese Shrimp, Fenneropenaeus Chinensis

Eight hundred and thirty eight base pair fragment of mitochondrial COI gene of wild and cultured populations (CP1, CP4, CP5 and CP6) of Fenneropenaeus chinensis was amplified and sequenced. The A, T, G and C contents of the sequence were 235bp (28.0% ),.307bp (36.6%), 138bp (16.5%) and 158bp (18.9%), respectively. Furthermore, 556bp fragment of the sequence was used to discuss the phylogenetic relationship among 14 Penaeidae species using Alpheus armillatus as the outgroup. From the molecular phylogenetic tree constructed by neighbor-joining method, we obtained three large shrimp groups: Farfantepenaeus, Litopenaeus and Fenneropenaeus group. The results also indicated that there were a closer genetic relationships between F. aztecus and F. paulensis, L. schmitti and L. setiferus, F. indicus and F. merguiensis, and the genus Farfantepenaeus was closer to Litopenaeus.     

Identification of two Skeletonema costatum-like diatoms by fluorescence in situ hybridization

A harmful algae bloom (HAB) is a dense aggregation of algae in a marine or aquatic environment that can result in significant environmental problems. To forecast the occurrence of HAB, development of a rapid and precise detection method is urgently required. In this study, two Skeletonema costatum-like diatoms (SK-1 and SK-2), were identified morphologically under a light microscope, and detected using fluorescent in situ hybridization (FISH). Strain SK-1 was isolated from a frequently HAB affected area of the East China Sea, and strain SK-2 from an aquatic farm in Qingdao, China. Fluorescent DNA probes were designed that were complementary to the ITS sequence (including 5.8S rDNA) of strain SK-1. After hybridization, strong green fluorescence was observed in cells of strain SK-1 under an epifluorescence microscope; however, no such fluorescence was observed with strain SK-2, which indicates that probes hybridized only the DNA of the target strain, SK-1, in species-specific manner, and that the two strains do not belong to a same species. This finding was confirmed by ITS sequence analysis. The FISH technique used in this study was sensitive, simple, and rapid, and is a promising tool for detecting target HAB species in natural environments.     

PCR-DGGE analysis of intestinal bacteria and effect of Bacillus spp. on intestinal microbial diversity in kuruma shrimp (Marsupenaeus japonicus)

In this study, the intestinal microbiota of kuruma shrimp (Marsupenaeus japonicus) was examined by molecular analysis of the 16S rDNA to identify the dominant intestinal bacteria and to investigate the effects of Bacillus spp. on intestinal microbial diversity. Samples of the intestines of kuruma shrimp fed normal feed and Bacillus spp. amended feed. PCR and denaturing gradient gel electrophoresis (DGGE) analyses were then performed on DNA extracted directly from the guts. Population fingerprints of the predominant organisms were generated by DGGE analysis of the universal V3 16S rDNA amplicons, and distinct bands in the gels were sequenced. The results suggested that the gut of kuruma shrimp was dominated by Vibrio sp. and uncultured gamma proteobacterium. Overall, the results of this study suggest that PCR-DGGE is a possible method of studying the intestinal microbial diversity of shrimp.     

The Complete Sequence of Mitochondrial COII Gene of Fenneropenaeus chinensis and Its Applicability as a Marker for Phylogenetic Analysis

The complete mitochondrial cytochrome oxidase subunit II (COII) gene of Penaeinae shrimp Fenneropenaeus chinen- sis was cloned and sequenced. The gene is 688 bp in length and codes for 229 amino acids. It shows 83.2%, 87.0% and 83.8% sequence similarity to Marsupenaeus japonicus, Penaeus monodon and Farfantepenaeus notialis, respectively. The A T content of the whole gene and that at the third position of codons are 64.7% and 78.2%, respectively. The phylogenetic relationship between F. chinensis and three other species representing genera Farfanatepenaeus, Marsupenaeus and Penaeus was analyzed. Results showed that the genetic distances among the four taxa ranged from 0.144 0 to 0.200 5, exceeding those estimated with COI and partial 16S rRNA gene sequences among Marsupenaeus, Litopenaeus and Melicertus, and being therefore larger than the value among subgenera. It has been suggested that the COII gene has a faster evolutionary rate than that of the COI gene and partial 16S rRNA gene and could be used for phylogenetic analysis at genus or species level. The results of the present study indicated that Farfantepenaeus, Fenneropenaeus, Marsupenaeus and Penaeus are at a higher phylogenetic level than subgenus, which supports the opinion of the elevation of phylogenetic status of the four subgenera to genus level.     

Chromosome Behavior of Heat Shock Induced Triploid in Fenneropenaeus Chinensis

INTRODUCTIONTriploidanimalsareusefulbecausetheyaresterile ,characterizedbypositivegrowthinthere productiveseason ,usefulfortesting ,forintroductionofnon nativespecies ,andforprotectionofde velopedstrains.Theinductionoftriploidyhadbeenreportedinmanyaquaculturespecies .Triploidshavebeensuccessfullyinducedinshrimpusingtemperatureshockorchemicalshock (CB ,6 DMAP)in 4species :Sicyoniaingentis (Xiangetal.,1 991 ) ,Fenneropenaeuschinensis (Xiangetal.,1 992 ;Daietal.,1 993;Baoetal.,1 993;Li…     

A Study on the Contribution of Different Food Sources to Shrimp Growth in an Intensive Fenneropenaeus chinensis Pond

Stable isotope methods can be used to determine the food sources and prey items of aquatic organisms accurately andreliably.This study examined the relative contribution of artificial foods (the formulated feed and Artemia) and natural foods toshrimp growth in an intensive Fenneropenaeus chinensis pond by using carbon and nitrogen stable isotopes.The results showed that the nutrition utilization efficiency of the harvested shrimp was low,only 33.18% of feed nitrogen and 21.73% of feed carbon beingconverted to shrimp flesh.Our stable isotope results showed that the shrimp obtained nutrition for maximum growth from artificialfoods,whose contribution was 93.5%,with the remaining attributed to the natural foods.However,there was 0.94 t harvested shrimpderived from natural foods (the rest of 13.56t harvested shrimp derived from artificial foods) in lha intensive pond with a shrimpproduction of 14.50 tha-1.Therefore,unit area shrimp production can be increased by increasing the contribution proportion of natu-ral foods in intensive shrimp farming.     

Compound-specific isotopes of fatty acids as indicators of trophic interactions in the East China Sea ecosystem

The composition and compound-specific isotopes of fatty acids were studied within food webs in the East China Sea.Lipid-normalized stable carbon isotopes of total organic carbon had a good correlation with trophic level.Variations in fatty acid compositions among different species were observed but were unclear.Different dietary structures could be traced from molecular isotopes of selected fatty acids in the Shiba shrimp(Matapenaeus joyneri),the coastal mud shrimp(Solenocera crassicomis) and the northern Maoxia shrimp(Acetes chinensis).Both M.joyneri and S.crassicomis are mainly benthos feeders,while A.chinensis is a pelagic species,although they have a similar fatty acid composition.There was a good correlation for isotopes of arachidonic acid(C20:4n6;ARA) and docosahexaenoic acid(C22:6n3;DHA)among pelagic species from higher trophic levels.The isotopic compositions of DHA in benthic species were more negative than those of pelagic species at the same trophic level.The fact that the diet of benthic species contains more degraded items,the carbon isotopes of which are derived from a large biochemical fraction,may be the reason for this variation.A comparative study of benthic and pelagic species demonstrated the different carbon sources in potential food items and the presence of a more complex system at the watersediment interface.     

Development of an in situ loop-mediated isothermal amplification technique for chromosomal localization of DNA sequences

In situ loop-mediated isothermal amplification (in situ LAMP) combines in situ hybridization and loop-mediated isothermal amplification (LAMP) techniques for chromosomal localization of DNA sequences. In situ LAMP is a method that is generally more specific and sensitive than conventional techniques such as fluorescence in situ hybridization (FISH), primed in situ labeling (PRINS), and cycling primed in situ labeling (C-PRINS). Here, we describe the development and application of in situ LAMP to identify the chromosomal localization of DNA sequences. To benchmark this technique, we successfully applied this technique to localize the major ribosomal RNA gene on the chromosomes of the Zhikong scallop (Chlamys farreri).     

REQUIREMENTS OF SHRIMP, PENAEUS CHINENSIS O''''SBECK FOR POTASSIUM, SODIUM, MAGNESIUM AND IODINE

Potassium, sodium, magnesium and iodine requirements of shrimp, Penaeus chinensis were studied.Orthogonal design was employed in this experiment. The composition of the basal diet consisted of fishmeal, peanut cake, corn meal, soybean cake, wheat bran, vitamin mix and mineral mix, and supple-mentations of potassium, sodium, magnesium and iodine in the basal diet were made according to theL_9(3~4) orthogonal table. The results indicatal that iodine supplementation improved growth of the shrimpsignificantly and raised survival very significanly, iodine requirement of the shrimp was 0.003%, sodiumrequirement was 0.87% or less; and that 1.1-1.3% potassium and 0.18-0.38% magnesium in the dietwere proper nutrition supplements for the shrimp.     

SELENIUM REQUIREMENT OF SHRIMP PENAEUS CHINENSIS

Penaeus chinensis were reared in fibreglass tanks for the study of their selenium requirements. The shrimp were fed semipurified diets containing graded levels of selenium, and weight gains, activities of glutathione peroxidase (GSH-Px) and selenium contents in muscle and hepatopancreas were determined. Weight gain and GSH-Px activity were the highest when the shrimp were fed diet containing 20 mg/kg selenium; Good linear correlation was found between GSH-Px activities and selenium contents in the diets, and the number of healthy shrimp. The experiment showed that 20 mg/kg selenium in the diet is optimal for the shrimp and that GSH-Px activity can be an important biochemical index of the selenium nutrition status of the animal.     

Archaeal diversity and abundance within different layers of summer sea-ice and seawater from Prydz Bay,Antarctica

Fluorescence in-situ hybridization （FISH） and 16S rRNA gene clone library analyses were used to determine the abundance and diversity of archaea in Prydz Bay, Antarctica. Correlation analysis was also performed to assess links between physicochemical parameters and archaeal abundance and diversity within the sea-ice. Samples of sea-ice and seawater were collected during the 26th Chinese National Antarctic Research Expedition. The results of FISH showed that archaea were relatively abundant within the top layer of the sea-ice, and correlation analysis suggested that the concentration of 4NH＋ might be one of the main factors underlying this distribution pattern. However, using 16S rRNA gene libraries, archaea were not detected in the top and middle layers of the sea-ice. All archaeal clones obtained from the bottom layer of the sea-ice were grouped into the Marine Group I Crenarchaeota while the archaeal clones from seawater were assigned to Marine Group I Crenarchaeota, Marine Group II Euryarchaeota, and Marine Group III Euryarchaeota. Overall, the ifndings of this study showed that the diversity of archaea in the sea-ice in Prydz Bay was low.