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.

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 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.

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.

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.

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.

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.

Using measurements of muscle cell nuclear RNA with flow cytometry to improve assessment of larval condition of Walleye Pollock (Gadus chalcogrammus)

Nuclear RNA and DNA in muscle cell nuclei of laboratory-reared larvae of Walleye Pollock (Gadus chalcogrammus) were simultaneously measured through the use of flow cytometry for cell-cycle analysis during 2009–11. The addition of nuclear RNA as a covariate increased by 4% the classification accuracy of a discriminant analysis model that used cell-cycle, temperature, and standard length to measure larval condition, compared with a model without it. The greatest improvement, a 7% increase in accuracy, was observed for small larvae (<6.00 mm). Nuclear RNA content varied with rearing temperature, increasing as temperature decreased. There was a loss of DNA when larvae were frozen and thawed because the percentage of cells in the DNA synthesis cell-cycle phase decreased, but DNA content was stable during storage of frozen tissue.

Estimation of RNA/DNA ratio in two north-Indian natural populations of mahseer (Tor tor) and its relationship with growth and hydrobiology

Samples of Tor tor were collected from Bari Reservoir of Udaipur and Narmada River at Hoshangabad (India), in the months of July and November 2005, respectively. Twenty-five samples were collected from each location. Bari Reservoir samples ranged from 17.0 to 24.5 cm in total length and from 75 to 155 g in weight, while Narmada samples ranged from 20.0 to 42.0 cm in length and 90 to 425 g in weight. The nucleic acid content in body muscle of Tor tor and the RNA/DNA ratio were estimated. The age of fishes was estimated by the scale study method and specimens were classified into four age groups. RNA/DNA ratio showed significant linear increase with increase in weight and age till the age of three years after which, the growth rate reduced. The 1-2 year group was the only one common between the two water bodies and a comparison of RNA/DNA ratios showed higher growth rate in Bari Reservoir. The gross primary productivity was also higher in Bari Reservoir being 551 mg cmˉ³ dˉ¹ compared to 404 mg cmˉ³ dˉ¹ observed for Narmada River. The condition factor (K) was found to be higher (1.21) in the fish from the Bari Reservoir compared to those of Narmada River (1.14). The growth rate was higher in females compared to males in >100 g specimens.

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.