Mitochondrial gene sequences useful for species identification of western North Atlantic Ocean sharks

Molecular-based approaches for shark species identification have been driven largely by issues specific to the fishery. In an effort to establish a more comprehensive identification data set, we investigated DNA sequence variation of a 1.4-kb region from the mitochondrial genome covering partial sequences from the 12S rDNA, 16S rDNA, and the complete valine tRNA from 35 shark species from the Atlantic fishery. Generally, within-species variability was low in relation to interspecific divergence because species haloptypes formed monophyletic groups. Phylogenetic analyses resolved ordinal relationships among Carcharhiniformes and Lamniformes, and revealed support for the families Sphyrnidae and Triakidae (within Carcharhiniformes) and Lamnidae and Alopidae (within Lamniformes). The combination of limited intraspecific variability and sufficient between-species divergence indicates that this locus is suitable for species identification.

PCR-based assay for detection of four coral pathogens

Several microorganisms have been identified as pathogenic agents responsible for various outbreaks of coral disease. Little has been learned about the exclusivity of a pathogen to given disease signs. Most pathogens have only been implicated within a subset of corals, leaving gaps in our knowledge of the host range and geographic extent of a given pathogen. PCR-based assays provide a rapid and inexpensive route for detection of pathogens. Pathogen-specific 16S rDNA primer sets were designed to target four identified coral pathogens: Aurantimonas coralicida, Serratia marcescens, Vibrio shilonii, and Vibrio coralliilyticus. Assays detected the presence of targets at concentrations of less than one cell per microliter. The assay was applied to 142 coral samples from the Florida Keys, Puerto Rico, and U.S. Virgin Islands as an in situ specificity test. Assays displayed a high-level of specificity, seemingly limited only by the resolution of the 16S rDNA.

A survey on relation of morphological, molecular and phylogenetic structure of zoanthids of the islands located in the Hormoz Strait (Hormoz, Qeshm, Larak, Hengam)

The order Zoantharia (Zoanthids) is one of the most neglected orders of cnidarians in the Persian Gulf. The present study aims to investigate the biodiversity of this order with morphological and molecular examination in the Persian Gulf. For this purpose, 123 colonies of zoanthids with variety of shape and colors have been collected from intertidal and shallow water zone of four islands, i. e. Hengam, Qeshm, Larak and Hormoz. After sampling, morphological characteristics of each specimen were recorded based on in situ photographs. Then DNA was extracted using the cetyl trimethyl ammonium bromide (CTAB) method. Both mitochondrial 16S ribosomal DNA (mt 16S rDNA) and cytochrome oxidase subunit I (COI) gene fragments were amplified and sequenced. The results of preliminary morphological identification integrated with two mitochondrial markers sequencing demonstrated the presence of five different species in this region; Zoanthus sansibaricus, Palythoa mutuki, Palythoa cf. mutuki, Palythoa tuberculosa and Neozoanthus persicus?. Although at first sight, morphological properties were not successful to delineate zoanthid species, they become reliable criteria to identify and delineate species in field studies after molecular identification.

Bacterial and pathological study of caudal fin rot in brood stocks of Salmo trutta caspius from the propagation and breeding center of Shahid Bahonar of Kelardasht region

In order to study caudal fin rot with emphasis on Aeromonas hydrophila and Pseudomonas fluorescens in Salmo trutta caspius from the salmonids propagation and breeding center of Shahid Bahonar of kelardasht region, One hundred and eighty brood stocks having fin damage symptoms were chosen. Two bacterial samples from each fish were cultured on Aeromonas and Pseudomonas specific media. Biochemical tests, API2OE identification system and antibiogram test using six antibiotic disks were performed for diagnosing isolates bacteria and finding suitable antibiotic. Thirty samples from caudal fin of damaged fishes were fixed in 10% formalin and 51.tm microscopic sections were prepared using standard scatological methods and then stained by Haematoxylin-Eosin staining method to observe the pathological changes and also Maccallum-Goodpasture staining method to observe the bacterial colonies. In second stage of the study, bacterial samples were taken from thirty brood stocks using similar method at the first stage of sampling. For isolation and biochemical diagnosis of Aeromonas and Pseudormonas genus, the samples were analyzed by molecular research included PCR amplification (using 16S rDNA genes of the genus pseudomonas and 16S-23S rDNA intergenic spacer of the genus Aeromonas) and restriction analysis by four restriction enzymes for each genus. The results of biochemical tests showed that isolated bacteria were belonged to Aeromonas caviae and Aeromonas hydrophila (subspecies anaerogenes), Pseudomonas fluorescens, Pseudomonas putida and Pseudomonas alcaligenes while the results of API2OE identification system showed that the isolated bacteria belonged to Aeromonas hydrophila, Pseudomonas fluorescens, Pseudomonas putida and Pseudomonas aeruginosa. Restriction analysis of Aeromonas samples with Hin6l, Csp6I, Taql, and Tasl revealed three samples were different from others while restriction analysis of Pseudomonas samples with Alul, Hinfl, Rsal, and Trull showed at least five species or biovars. The results of antibiogram test showed all Aeromonas samples were sensitive to Trimethoprim, Chloramphenicol and Nitrofurazone, mostly to Nalidixic acid and Chloramphenicol, while most of samples were resistant to Erythromycin and Oxytetracycline. Pseudomonas samples were only sensitive to Nitrofurazone and mostly resistant to Oxytetracycline, Nalidixic acid, Erythromycin, Trimethoprim and Chloramphenicol. The results of light microscope study showed hyperplasia and spongiosis of the malpigian cells of epidermis, increasing of melanin pigments underlying epidermis; sever necrosis in both epidermis and dermis and also sloughing the epidermis in some cases. Occurrence of clefts through the epithelium, neovascularization, hyperemia and mild inflammatory response in dermis and separation of the fin rays also were observed. No bacterial colonies were found in the sections.

Identification of larval sea basses (Centropristis spp.) using ribosomal DNA-specific molecular assays

The identification of sea bass (Centropristis) larvae to species is difficult because of similar morphological characters, spawning times, and overlapping species ranges. Black sea bass (Centropristis striata) is an important fishery species and is currently considered to be overfished south of Cape Hatteras, North Carolina. We describe methods for identifying three species of sea bass larvae using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) assays based on species-specific amplification of rDNA internal transcribed spacer regions. The assays were tested against DNA of ten other co-occurring reef fish species to ensure the assay's specificity. Centropristis larvae were collected on three cruises during cross-shelf transects and were used to validate the assays. Seventy-six Centropristis larva were assayed and 69 (91%) were identified successfully. DNA was not amplified from 5% of the larvae and identification was inconclusive for 3% of the larvae. Those assays can be used to identify sea bass eggs and larvae and will help to assess spawning locations, spawning times, and larval dispersal.

The impact of molecular biology on assessment of water quality: advantages and limitations of current techniques

The advent of molecular biology has had a dramatic impact on all aspects of biology, not least applied microbial ecology. Microbiological testing of water has traditionally depended largely on culture techniques. Growing understanding that only a small proportion of microbial species are culturable, and that many microorganisms may attain a viable but non-culturable state, has promoted the development of novel approaches to monitoring pathogens in the environment. This has been paralleled by an increased awareness of the surprising genetic diversity of natural microbial populations. By targeting gene sequences that are specific for particular microorganisms, for example genes that encode diagnostic enzymes, or species-specific domains of conserved genes such as 16S ribosomal RNA coding sequences (rrn genes), the problems of culture can be avoided. Technical developments, notably in the area of in vitro amplification of DNA using the polymerase chain reaction (PCR), now permit routine detection and identification of specific microorganisms, even when present in very low numbers. Although the techniques of molecular biology have provided some very powerful tools for environmental microbiology, it should not be forgotten that these have their own drawbacks and biases in sampling. For example, molecular techniques are dependent on efficient lysis and recovery of nucleic acids from both vegetative forms and spores of microbial species that may differ radically when growing in the laboratory compared with the natural environment. Furthermore, PCR amplification can introduce its own bias depending on the nature of the oligonucleotide primers utilised. However, despite these potential caveats, it seems likely that a molecular biological approach, particularly with its potential for automation, will provide the mainstay of diagnostic technology for the foreseeable future.

Differenzierung von Kammmuscheln durch DNA-Analyse

Abstract In the last years scallops have reached a considerable popularity and the import of scallops into the EU has increased about 20 % over the last fi ve years from some 50.000 t to nearly 63.000 t in the year 2010. Scallops are fi shed or farmed, and traded as fresh or deep frozen product. Recently investigation of scallop products of various origins by determining the species using molecular biological techniques showed that the species had been mislabelled in a considerable proportion of samples. Determination of the species was performed by PCR-based DNA-analysis of mitochondrial DNA followed by (i) sequencing the PCR product and (ii) comparison of the DNA sequence with entries in GenBank using BLAST. The deduced sequences of the analysed samples were considerably different from each other allowing the unambiguous assignment of samples to a certain species. Kurzfassung Die Nachfrage von Kammmuscheln in der EU hat in den letzten fünf Jahren erheblich zugenommen. Der Import stieg von knapp 53.000 t im Jahr 2005 um 20% auf annähernd 63.000 t im Jahr 2010. Gehandelt werden Kammmuscheln sowohl als frische als auch als Tiefkühlware aus Wildfängen und Aquakultur. Untersuchungen von Kammmuschel-Proben aus verschiedenen Ursprungsländern und Bestimmung der Spezies auf molekularbiologischer Basis zeigten, dass ein erheblicher Anteil der Proben falsch deklariert war. Die Bestimmung der Spezies erfolgte durch Vervielfältigung eines Abschnitts des 16S rRNA Gens durch Polymerase- Kettenreaktion (PCR), anschließender Sequenzanalyse der PCR-Produkte und Vergleich der DNA Sequenzen untereinander und mit Dateneintragungen in GenBank. Die DNA-Sequenzen der ermittelten Abschnitte der 16S rRNA der Proben unterschieden sich erheblich voneinander und erlaubten eine eindeutige Zuordnung zu jeweils einer Spezies.

A key to selected rockfishes (Sebastes spp.) based on mitochondrial DNA restriction fragment analysis

Larval and juvenile rockfishes (Sebastes spp.) are difficult to identify using morphological characters. We developed a key based on sizes of restriction endonuclease fragments of the NADH dehydrogenase-3 and -4 (ND3/ND4) and 12S and 16S ribosomal RNA (12S/16S) mitochondrial regions. The key makes use of variation in the ND3/ND4 region. Restriction endonuclease Dde I variation can corroborate identifications, as can 12S/16S variation. The key, based on 71 species, includes most North American taxa, several Asian species, and Sebastolobus alascanus and Helicolenus hilgendorfi that are closely related to rockfishes. Fifty-eight of 71 rockfish species in our database can be distinguished unequivocally, using one to five restriction enzymes; identities of the remaining species are narrowed to small groups: 1) S. polyspinis, S. crameri, and S. ciliatus or variabilis (the two species could not be distinguished and were considered as a single species) ; 2) S. chlorostictus, S. eos, and S. rosenblatti; 3) S. entomelas and S. mystinus; 4)S. emphaeus, S. variegatus, and S. wilsoni; and 5) S. carnatus and S. chrysomelas.

Genetic diversity in wild stocks of the giant freshwater prawn (Macrobrachium rosenbergii): implications for aquaculture and conservation

The giant freshwater prawn (Macrobrachium rosenbergii) is cultured widely around the world but little is known about the levels and patterns of genetic diversity in either wild or cultured stocks. Studies have suggested that genetic diversity may be relatively low in some cultured stocks due to the history of how they were founded and subsequent exposure to repeated population bottlenecks in hatcheries. In contrast, wild stocks have an extensive distribution that extends from Southern Asia across Southeast (SE) Asia to the Pacific region. Therefore, wild stocks could be an important resource for genetic improvement of culture stocks in the future. Understanding the extent and patterns of genetic diversity in wild giant freshwater prawn stocks will assist decisions about the direction future breeding programs may take. Wild stock genetic diversity was examined using a 472 base-pair segment of the 16S rRNA gene in 18 wild populations collected from across the natural range of the species. Two major clades ("eastern" and "western") were identifi ed either side of Huxley’s line, with a minimum divergence of 6.2 per cent, which implies separation since the Miocene period (5-10 MYA). While divergence estimates within major clades was small (maximum 0.9 per cent), evidence was also found for population structuring at a lower spatial scale. This will be examined more intensively with a faster evolving mtDNA gene in the future.

Threatened corals provide underexplored microbial habitats

Contemporary in-depth sequencing of environmental samples has provided novel insights into microbial community structures, revealing that their diversity had been previously underestimated. Communities in marine environments are commonly composed of a few dominant taxa and a high number of taxonomically diverse, low-abundance organisms. However, studying the roles and genomic information of these “rare” organisms remains challenging, because little is known about their ecological niches and the environmental conditions to which they respond. Given the current threat to coral reef ecosystems, we investigated the potential of corals to provide highly specialized habitats for bacterial taxa including those that are rarely detected or absent in surrounding reef waters. The analysis of more than 350,000 small subunit ribosomal RNA (16S rRNA) sequence tags and almost 2,000 nearly full-length 16S rRNA gene sequences revealed that rare seawater biosphere members are highly abundant or even dominant in diverse Caribbean corals. Closely related corals (in the same genus/family) harbored similar bacterial communities. At higher taxonomic levels, however, the similarities of these communities did not correlate with the phylogenetic relationships among corals, opening novel questions about the evolutionary stability of coral-microbial associations. Large proportions of OTUs (28.7–49.1%) were unique to the coral species of origin. Analysis of the most dominant ribotypes suggests that many uncovered bacterial taxa exist in coral habitats and await future exploration. Our results indicate that coral species, and by extension other animal hosts, act as specialized habitats of otherwise rare microbes in marine ecosystems. Here, deep sequencing provided insights into coral microbiota at an unparalleled resolution and revealed that corals harbor many bacterial taxa previously not known. Given that two of the coral species investigated are listed as threatened under the U.S. Endangered Species Act, our results add an important microbial diversity-based perspective to the significance of conserving coral reefs.

Microbial community analysis of Acropora palamata mucus swabs, water and sediment samples from Hawksnest Bay, St. John, U.S. Virgin Islands

Colonies of the scleractinian coral Acropora palmata, listed as threatened under the US Endangered Species Act in 2006, have been monitored in Hawksnest Bay, within Virgin Islands National Park, St. John, from 2004 through 2010 by scientists with the US Geological Survey, National Park Service, and the University of the Virgin Islands. The focus has been on documenting the prevalence of disease, including white band, white pox (also called patchy necrosis and white patches), and unidentified diseases (Rogers et al., 2008; Muller et al., 2008). In an effort to learn more about the pathologies that might be involved with the diseases that were observed, samples were collected from apparently healthy and diseased colonies in July 2009 for analysis. Two different microbial assays were performed on Epicentre Biotechnologies DNA swabs containing A. palmata coral mucus, and on water and sediment samples collected in Hawksnest Bay. Both assays are based on polymerase chain reaction (PCR) amplification of portions of the small rRNA gene (16S). The objectives were to determine 1) if known coral bacterial pathogens Serratia marcescens (Acroporid Serratiosis), Vibrio coralliilyticus (temperature-dependent bleaching, White Syndrome), Vibrio shiloi (bleaching, necrosis), and Aurantimonas coralicida (White Plague Type II) were present in any samples, and 2) if there were any differences in microbial community profiles of each healthy, unaffected or diseased coral mucus swab. In addition to coral mucus, water and sediment samples were included to show ambient microbial populations. In the first test, PCR was used to separately amplify the unique and diagnostic region of the 16S rRNA gene for each of the coral pathogens being screened. Each pathogen test was designed so that an amplified DNA fragment could be seen only if the specific pathogen was present in a sample. A positive result was indicated by bands of DNA of the appropriate size on an agarose gel, which separates DNA fragments based on the size of the molecule. DNA from pure cultures of each of the pathogens was used as a positive control for each assay.

Hybridization between two serranids, the coney (Cephalopholis fulva) and the creole-fish (Paranthias furcifer), at Bermuda

Intergeneric hybridization between the epinepheline serranids Cephalopholis fulva and Paranthias furcifer in waters off Bermuda was investigated by using morphological and molecular characters. Putative hybrids, as well as members of each presumed parent species, were analyzed for 44 morphological characters and screened for genetic variation at 16 nuclear allozyme loci, two nuclear (n)DNA loci, and three mitochondrial (mt)DNA gene regions. Four of 16 allozyme loci, creatine kinase (CK-B*), fumarase (FH*), isocitrate dehydrogenase (ICDH-S*), and lactate dehydrogenase (LDH-B*), were unique in C. fulva and P. furcifer. Restriction fragments of two nuclear DNA intron regions, an actin gene intron and the second intron in the S7 ribosomal protein gene, also exhibited consistent differences between the two presumed parent species. Restriction fragments of three mtDNA regions—ND4, ATPase 6, and 12S/16S ribosomal RNA—were analyzed to identify maternal parentage of putative hybrids. Both morphological data and nuclear genetic data were found to be consistent with the hypothesis that the putative hybrids were the result of interbreeding between C. fulva and P. furcifer. Mean values of 38 morphological characters were different between presumed parent species, and putative hybrids were intermediate to presumed parent species for 33 of these characters. A principal component analysis of the morphological and meristic data was also consistent with hybridization between C. fulva and P. furcifer. Thirteen of 15 putative hybrids were heterozygous at all diagnostic nuclear loci, consistent with F1 hybrids. Two putative hybrids were identified as post-F1 hybrids based on homozygosity at one nuclear locus each. Mitochondrial DNA analysis showed that the maternal parent of all putative hybrid individuals was C. fulva. A survey of nuclear and mitochondrial loci of 57 C. fulva and 37 P. furcifer from Bermuda revealed no evidence of introgression between the parent species mediated by hybridization.

Genetic characterization of three Ompok species using mitochondrial DNA sequences

Partial sequences of cytochrome b (Cyt b) and 16S ribosomal RNA (16S rRNA) mitochondrial genes were used for species identification and estimating phylogenetic relationship among three commercially important Ompok species viz. O. Pabda, O. pabo and O. bimaculatus. The sequence analysis of Cyt b (1118bp) and 16S rRNA (569 & 570bp) genes revealed that O. pabda, O. pabo & 0. bimaculatus were genetically distinct species and they exhibited identical phylogenetic relationship. The present study discussed usefulness of mtDNA genes (Cyt b & 16S rRNA) in resolving taxonomic ambiguity and estimating phylogenetics relationship.

Study of inter species diversity and population structure by molecular genetic method in Iranian Artemia

Artemia is a small crustacean that adapted to live in brine water and has been seen in different brine water sources in Iran. Considering the importance of genetic studies manifest inter population differences in species, to estimate genetic structure, detect difference at molecular level and separate different Artemia populations of Iran, also study of phylogenic relationships among them, samples of Artemia were collected from nine region: Urmia lake in West Azerbaijan, Shoor and Inche-Borun lakes in Golestan, Hoze-Soltan and Namak lakes in Qom, Maharloo and Bakhteghan lakes in Fars, Nough pool in Kerman and Mighan pool in Markazi and DNA extracted by phenol-chloroform method. Primers designed on a ribosomal fragment (16s rRNA) of mt DNA sequence and PCR was done. Digestion of the 1566 bp segment PCR product by 10 restriction endonuclease (Alu I, EcoR I, Eco47 I, Hae III, Hind III, Hinf I, Mbo I, Msp I, Rsa I, TaqI) showed 25 different haplotypes: 9 in Urmia, 4 in Shoor and Inche- Borun, 1 in Namak and Hoze-Soltan, 3 in Mighan, 1 in Bakhtegan Maharlo, 3 in Maharloo and 4 in Nough. Measurement of haplotype and nucleotide diversity intra population and nucleotide diversity and divergence inter populations and evolutionary distance between haplotypes showed a high diversity in mitochondrial genome of Artemia in studied regions whose results are similar to those explained for highly geographic expansion organism. In addition, results showed considerable heterogeneity between different populations and there are enough evidences in haplotypic level for separation of studied samples and division of Iranian Artemia to seven populations including Urmia, Shoor and Inche-Borun, Hoze-Soltan and Namak, Maharloo, Bakhteghan, Nough and Mighan. Phylogenetic analysis of the 16S rRNA data set resulted strict consensus and neighbor joining distance trees, demonstrated that all samples were monophyletic and parthenogenetic form derivation from bisexual populations and genetically high resemblance to those of A. urmiana. Study of 270 specimens from different region showed the genus Artemia in Iran clustered into three clades including: 1- Shoor, Inche-Burun, Hoze-Soltan, Namak, Bakhtegan and Maharloo 2- Nough and Mighan 3- Urmia. Totally, obtained results indicated to ability of used techniques for study of inter species diversity, population structure, reveal of phylogenic relationship and dividing of different populations of Artemia in Iran.