Integrative data system for monitoring biota and natural habitats in the Israeli Eastern Mediterranean marine environment.

An integrative data system for monitoring the biota of the Mediterranean waters of Israel as well as selected records from adjacent Levantine basin regions is presented here, aimed at providing data and research tools for long-term bio-geographic and ecological studies and more important, providing background data for assisting governmental regulators to establish educated habitat-oriented environmental policy. The system relies on the geographic information system (GIS) online map-based platform and contains at present the following components: biotic database of ~ 170,000 recorded sampling events; uniform habitat maps of 63 benthic habitats and 2 pelagic ones, constructed using relevant bathymetric features and biotic community compositions; bathymetric hill-shade map; depth contours; raster depth grid and human interference map. Other informative auxiliary maps are planned to be added (e.g., map of potential pockmark sites, detailed maps of tiny carbonate crust nolls and more). A number of 883 cited documents were listed by us for potential extraction of sampling efforts, most of them are available to us as PDFs  and are available also to the users, excluding copyright-protected ones. Forty-three major projects were depicted in addition to a variety of small studies (e.g., university theses). Thirty-five sampling devices were documented and described, and 3187 species-level identifications were already recorded. In addition, the system provides access to description of sampling devices and pictures of species and seascapes. New data is continuously deposited to the system and the system is flexible, allowing future addition of new types of information. The system site is accessible through the link: https://experience.arcgis.com/experience/40e86605ff4d4e5096ed2c901fec2a2f .

Monitoring long-term spatial and temporal trends of the infaunal community characteristics along the shallow waters of the Mediterranean coast of Israel.

Sandy sediment and its infauna were annually sampled along the shallow waters of the Israeli coast during the 2005-2016 period, as a part of the Israeli National Environmental Program framework, aiming to detect anthropogenic interference in that province by monitoring changes in the species composition, abundance, and diversity of the infaunal communities and in accompanied abiotic parameters: the levels of total organic carbon and a series of heavy metals and the site-specific grain size distribution. The > 250-µm fraction of the fauna was segregated from the sampled sediment and was identified to species or higher taxonomic level. Three spatial biotopes were determined based on their unique faunal composition, Haifa Bay, Haifa harbor, and the southern coast. Species homogeneity among samples of each biotope was evaluated. Temporal and spatial changes of the species composition, abundance, and diversity were calculated for each biotope, mostly revealing random annual fluctuations. Only two minor temporal trends were observed: two spatially identical and temporally different faunal communities in the southern coast biotope, distinguishing the 2005-2007 and 2008-2016 periods, and a slight increase in the number of species across time in the two Haifa Bay provinces. Total organic carbon was highly correlated to the faunal composition with the highest organic carbon levels in the Haifa harbor biotope. The biotopes' mutually occurring abundant species were sufficient to determine biotope borders and the contribution of intermittently sampled rare species, including the zoogeographically Indo-Pacific originated ones was feeble, important only to identify species migration and faunistics. Practically, three sampling sites along the Israeli shallow soft substrate, corresponding to the defined spatial biotopes, are sufficient to monitor the effect of environmental changes. Seasonal sampling twice a year is recommended as well as more accurate species identification using molecular taxonomy.

A crayfish molar tooth protein with putative mineralized exoskeletal chitinous matrix properties.

Some crustaceans possess exoskeletons that are reinforced with calcium carbonate. In the crayfish Cherax quadricarinatus, the molar tooth, which is part of the mandibular exoskeleton, contains an unusual crystalline enamel-like apatite layer. As this layer resembles vertebrate enamel in composition and function, it offers an interesting example of convergent evolution. Unlike other parts of the crayfish exoskeleton, which is periodically shed and regenerated during the molt cycle, molar mineral deposition takes place during the pre-molt stage. The molar mineral composition transforms continuously from fluorapatite through amorphous calcium phosphate to amorphous calcium carbonate and is mounted on chitin. The process of crayfish molar formation is entirely extracellular and presumably controlled by proteins, lipids, polysaccharides, low-molecular weight molecules and calcium salts. We have identified a novel molar protein termed Cq-M15 from C. quadricarinatus and cloned its transcript from the molar-forming epithelium. Its transcript and differential expression were confirmed by a next-generation sequencing library. The predicted acidic pI of Cq-M15 suggests its possible involvement in mineral arrangement. Cq-M15 is expressed in several exoskeletal tissues at pre-molt and its silencing is lethal. Like other arthropod cuticular proteins, Cq-M15 possesses a chitin-binding Rebers-Riddiford domain, with a recombinant version of the protein found to bind chitin. Cq-M15 was also found to interact with calcium ions in a concentration-dependent manner. This latter property might make Cq-M15 useful for bone and dental regenerative efforts. We suggest that, in the molar tooth, this protein might be involved in calcium phosphate and/or carbonate precipitation.

Different transcription regulation routes are exerted by L- and D-amino acid enantiomers of peptide hormones.

Conversion of one or more amino acids in eukaryotic peptides to the D-enantiomer configuration is catalyzed by specific L/D-peptide isomerases and it is a poorly investigated post-translational modification. No common modified amino acid or specific modified position has been recognized, and mechanisms underlying changes in the peptide function provided by this conversion are not widely studied. The 72 amino acid crustacean hyperglycemic hormone (CHH) in Astacidea crustaceans exhibits a co-existence of two peptide enantiomers with either D- or L-phenylalanine as their third residue. It is a pleiotropic hormone regulating several physiological processes in different target tissues and along different time scales. CHH enantiomers differently affect time courses and intensities of examined processes. The short-term effects of the two isomers on gene expression were examined in the hepatopancreas, gills, hemocytes and muscles of the astacid Pontastacus leptodactylus. Gene expression in muscles and hemocytes was not affected by either of the isomers. Two modes of action for CHH were elucidated in the hepatopancreas and the gills: specific gene induction in both organs by D-CHH, and targeted attenuation caused by both enantiomers in the gills. Consequently, a two-receptor system is proposed for conveying the effect of the two CHH isomers.

Expression of cytoskeletal and molt-related genes is temporally scheduled in the hypodermis of the crayfish Procambarus clarkii during premolt.

The rigid crustacean exoskeleton, the cuticle, is composed of the polysaccharide chitin, structural proteins and mineral deposits. It is periodically replaced to enable growth and its construction is an energy-demanding process. Ecdysis, the shedding event of the old cuticle, is preceded by a preparatory phase, termed premolt, in which the present cuticle is partially degraded and a new one is formed underneath it. Procambarus clarkii (Girard 1852), an astacid crustacean, was used here to comprehensively examine the changing patterns of gene expression in the hypodermis underlying the cuticle of the carapace at seven time points along ~14 premolt days. Next generation sequencing was used to construct a multi-tissue P. clarkii transcript sequence assembly for general use in a variety of transcriptomic studies. A reference transcriptome was created here in order to perform digital transcript expression analysis, determining the gene expression profiles in each of the examined premolt stages. The analysis revealed a cascade of sequential expression events of molt-related genes involved in chitin degradation, synthesis and modification, as well as synthesis of collagen and four groups of cuticular structural genes. The new description of major transcriptional events during premolt and the determination of their timing provide temporal markers for future studies of molt progress and regulation. The peaks of the expression of the molt-related genes were preceded by expression peaks of cytoskeletal genes that are hypothesized to be essential for premolt progress through regulating protein synthesis and/or transport, probably by remodeling the cytoskeletal structure.

Deep-sea meiofaunal communities in the south-eastern Levantine basin and their shaping factors - Morphological-taxonomy-free metabarcoding approach.

The <3% dissimilar Amplicon Sequence Variant (ASV) clusters of the 18S-V4 barcode were used as species-proxies for the evaluation of ASV composition and ASV diversity indices characterizing the hitherto poorly investigated meiofaunal communities of the south-eastern part of the Levantine basin. Accompanied by abundance measurements, the relationships of these characteristics with sedimentary and bottom terrain parameters were interpreted. The construction of community composition profiles, namely ASVs' list and their estimated abundances, was done using our previously established procedure (Harbuzov et al., 2022, Marine Genomics 65, 100980), combining metabarcoding with sample reads normalization by the abundance of hard-bodied meiofaunal taxa. The study province included the 54-1418 m depth range, across vertical sub-bottom horizons ranging 0-17 cm. Oxygen, hydrogen sulfide and methane concentrations in the pore water, as well as sediment grain size spectra and sedimentary protein and carbohydrate levels, were measured, followed by an evaluation of their involvement in the shaping of the meiofaunal communities' characteristics. Community composition was generally site-and-horizon dependent and its abundance decreased with increasing bottom depth and across sub-bottom horizons, typical to benthic habitats which are nourished by organic carbon from the euphotic zone. The relatively sharply inclined continental slope bottom located in the northern part of the Israeli coast was an exception. Its meiofaunal community characteristics were speculated to be affected by intensive sediment mixing and lateral transport of food from the shelf, in addition to the effect of the euphotic zone-originated food sources.

Hemocyanin with phenoloxidase activity in the chitin matrix of the crayfish gastrolith.

Gastroliths are transient extracellular calcium deposits formed by the crayfish Cherax quadricarinatus von Martens on both sides of the stomach wall during pre-molt. Gastroliths are made of a rigid chitinous organic matrix, constructed as sclerotized chitin-protein microfibrils within which calcium carbonate is deposited. Although gastroliths share many characteristics with the exoskeleton, they are simpler in structure and relatively homogeneous in composition, making them an excellent cuticle-like model for the study of cuticular proteins. In searching for molt-related proteins involved in gastrolith formation, two integrated approaches were employed, namely the isolation and mass spectrometric analysis of proteins from the gastrolith matrix, and 454-sequencing of mRNAs from both the gastrolith-forming and sub-cuticular epithelia. SDS-PAGE separation of gastrolith proteins revealed a set of bands at apparent molecular masses of 75-85 kDa; mass spectrometry data matched peptide sequences from the deduced amino acid sequences of seven hemocyanin transcripts. This assignment was then examined by immunoblot analysis using anti-hemocyanin antibodies, also used to determine the spatial distribution of the proteins in situ. Apart from contributing to oxygen transport, crustacean hemocyanins were previously suggested to be involved in several aspects of the molt cycle, including hardening of the new post-molt exoskeleton via phenoloxidation. The phenoloxidase activity of gastrolith hemocyanins was demonstrated. It was also noted that hemocyanin transcript expression during pre-molt was specific to the hepatopancreas. Our results thus reflect a set of functionally versatile proteins, expressed in a remote metabolic tissue and dispersed via the hemolymph to perform different roles in various organs and structures.

Crustacean oxi-reductases protein sequences derived from a functional genomic project potentially involved in ecdysteroid hormones metabolism - a starting point for function examination.

A transcriptomic assembly originated from hypodermis and Y organ of the crustacean Pontastacus leptodactylus is used here for in silico characterization of oxi-reductase enzymes potentially involved in the metabolism of ecdysteroid molting hormones. RNA samples were extracted from male Y organ and its neighboring hypodermis in all stages of the molt cycle. An equimolar RNA mix from all stages was sequenced using next generation sequencing technologies and de novo assembled, resulting with 74,877 unique contigs. These transcript sequences were annotated by examining their resemblance to all GenBank translated transcripts, determining their Gene Ontology terms and their characterizing domains. Based on the present knowledge of arthropod ecdysteroid metabolism and more generally on steroid metabolism in other taxa, transcripts potentially related to ecdysteroid metabolism were identified and their longest possible conceptual protein sequences were constructed in two stages, correct reading frame was deduced from BLASTX resemblances, followed by elongation of the protein sequence by identifying the correct translation frame of the original transcript. The analyzed genes belonged to several oxi-reductase superfamilies including the Rieske non heme iron oxygenases, cytochrome P450s, short-chained hydroxysteroid oxi-reductases, aldo/keto oxireductases, lamin B receptor/sterol reductases and glucose-methanol-cholin oxi-reductatses. A total of 68 proteins were characterized and the most probable participants in the ecdysteroid metabolism where indicated. The study provides transcript and protein structural information, a starting point for further functional studies, using a variety of gene-specific methods to demonstrate or disprove the roles of these proteins in relation to ecdysteroid metabolism in P. leptodactylus.

Amplicon sequence variant-based meiofaunal community composition revealed by DADA2 tool is compatible with species composition.

The present study is aimed at implementing the morphological identification-free amplicon sequence variant (ASV) concept for describing meiofaunal species composition, while strongly indicating reasonable compatibility with the underlying species. A primer pair was constructed and demonstrated to PCR amplify a 470-490 bp 18S barcode from a variety of meiofaunal taxa, high throughput sequenced using the Illumina 300 × 2 bps platform. Sixteen 18S multi-species HTS assemblies were created from meiofaunal samples and merged to one assembly of ~2,150,000 reads. Five quality scores (q = 35, 30, 25, 20, 15) were implemented to filter five 18S barcode assemblies, which served as inputs for the DADA2 software, ending with five reference ASV libraries. Each of these libraries was clustered, applying 3% dissimilarity threshold, revealed an average number of 1.38 ±â€¯0.078 ASVs / cluster. Hence, demonstrating high level of ASV uniqueness. The libraries which were based on q ≤ 25 reached a near-asymptote number of ASVs which together with the low average number of ASVs / cluster, strongly indicated fair representation of the actual number of the underlying species. Hence, the q = 25 library was selected to be used as metabarcoding reference library. It contained 461 ASVs and 342-3% clusters with average number of 1.34 ±â€¯1.036 ASV / cluster and their BLASTN annotation elucidated a variety of expected meiofaunal taxa. The sixteen assemblies of sample-specific paired reads were mapped to this reference library and sample ASV profiles, namely the list of ASVs and their proportional copy numbers were created and clustered.

A protein involved in the assembly of an extracellular calcium storage matrix.

Gastroliths, the calcium storage organs of crustaceans, consist of chitin-protein-mineral complexes in which the mineral component is stabilized amorphous calcium carbonate. To date, only three proteins, GAP 65, gastrolith matrix protein (GAMP), and orchestin, have been identified in gastroliths. Here, we report a novel protein, GAP 10, isolated from the gastrolith of the crayfish Cherax quadricarinatus and specifically expressed in its gastrolith disc. The encoding gene was cloned by partial sequencing of the protein extracted from the gastrolith matrix. Based on an assembled microarray cDNA chip, GAP 10 transcripts were found to be highly (12-fold) up-regulated in premolt gastrolith disc and significantly down-regulated in the hypodermis at the same molt stage. The deduced protein sequence of GAP 10 lacks chitin-binding domains and does not show homology to known proteins in the GenBank data base. It does, however, have an amino acid composition that has similarity to proteins extracted from invertebrate and ascidian-calcified extracellular matrices. The GAP 10 sequence contains a predicted signal peptide and predicted phosphorylation sites. In addition, the protein is phosphorylated and exhibits calcium-binding ability. Repeated daily injections of GAP 10 double strand RNA to premolt C. quadricarinatus resulted in a prolonged premolt stage and in the development of gastroliths with irregularly rough surfaces. These findings suggest that GAP 10 may be involved in the assembly of the gastrolith chitin-protein-mineral complex, particularly in the deposition of amorphous calcium carbonate.

Potential of the hepatic transcriptome expression profile of the striped seabream (Lithognathus mormyrus) as an environmental biomarker.

The potential of the hepatic transcriptome expression profile evaluated in a sentinel feral fish to serve as an environmental biomarker was examined. Expression profiles of Lithognathus mormyrus individuals were exhibited using cDNA microarray and were related to the set of exposure conditions at their sites and dates of collection. Expression profiles of individual fish were reasonably clustered according to the fish samples. In addition, several sample-specific gene clusters were determined, designated sample gene signatures. The selection procedure for future optimal reference RNA is discussed. The relationship between transcriptome expression and fish samples indicated a potential for using the former as an environmental biomarker.

Quantitative immunochemical evaluation of fish metallothionein upon exposure to cadmium.

Efficient implementation of an environmental biomarker requires multi-annual comparability over a wide geographical range. The present study improved the comparability of a quantitative competitive metallothionein (MT) enzyme-linked-immuno-sorbent-assay (ELISA) in the sentinel fish Lithognathus mormyrus by introducing to the assay recombinant MT and beta-actin standards. Commercial antibodies for cod MT and mammalian actin were implemented. In addition, a sensitive anti L. mormyrus MT antibody was produced, adequate only for solid phase immunochemical assays. Cadmium was applied to the fish through injection and feeding to serve as a testing platform of the ELISA. The results demonstrated high potential protective capacity of the liver against toxic levels of transition metals through increasing MT levels. MT transcript levels were evaluated also from fish sampled at polluted and relatively clean natural sites, indicating applicability of MT as biomarker of exposure to a multi-factorial pollution, in comparison to its low revealed sensitivity to controlled cadmium exposure.

Estimating the efficiency of fish cross-species cDNA microarray hybridization.

Using an available cross-species cDNA microarray is advantageous for examining multigene expression patterns in non-model organisms, saving the need for construction of species-specific arrays. The aim of the present study was to estimate relative efficiency of cross-species hybridizations across bony fishes, using bioinformatics tools. The methodology may serve also as a model for similar evaluations in other taxa. The theoretical evaluation was done by substituting comparative whole-transcriptome sequence similarity information into the thermodynamic hybridization equation. Complementary DNA sequence assemblages of nine fish species belonging to common families or suborders and distributed across the bony fish taxonomic branch were selected for transcriptome-wise comparisons. Actual cross-species hybridizations among fish of different taxonomic distances were used to validate and eventually to calibrate the theoretically computed relative efficiencies.

Transcript and protein environmental biomarkers in fish--a review.

The levels of contaminant-affected gene products (transcripts and proteins) are increasingly utilized as environmental biomarkers, and their appropriate implementation as diagnostic tools is discussed. The required characteristics of a gene product biomarker are accurate evaluation using properly normalized absolute units, aiming at long-term comparability of biomarker levels over a wide geographical range and among many laboratories. Quantitative RT-PCR and competitive ELISA are suggested as preferred evaluation methods for transcript and protein, respectively. Constitutively expressed RNAs or proteins which are part of the examined homogenate are suggested as normalizing agents, compensating for variable processing efficiency. Essential characterization of expression patterns is suggested, providing reference values to be compared to the monitored levels. This comparison would enable estimation of the intensity of biological effects of contaminants. Contaminant-independent reference expression patterns should include natural fluctuations of the biomarker level. Contaminant-dependent patterns should include dose response to model contaminants chronically administered in two environmentally-realistic routes, reaching extreme sub-lethal affected levels. Recent studies using fish as environmental sentinel species, applying gene products as environmental biomarkers, and implementing at least part of the depicted methodologies are reviewed.

Dioxin-like biological activity of organic extracts from sediments and fish livers sampled along the Israeli Mediterranean and Red Sea coasts.

This study provides, for the first time, a baseline evaluation of dioxin-like biological activity in sediments and fish sampled in- and adjacent to anchorages along the Mediterranean and Red Sea coasts of Israel. It indicates the effect of past pollution, still present in the sediments of older Israeli harbors, with putative contribution of still existing sources of pollution. A commercial reporter gene bioassay was used to evaluate the biological activity of dioxin-like compounds extracted from the samples. HRGC/HRMS analysis of several samples contributed a profile of dioxin-like compounds in sediments and fish. The results point out 1,2,3,4,6,7,8-HeptaCDD, 2,3,4,6,7,8-HexaCDF, 1,2,3,4,6,7,8-HeptaCDF, РСВ-126 and РСВ-118 as major contributors to the dioxin-like activity in sediments. It indicates polychlorinated biphenyls non-selective absorption in fish livers, in contrary to a biased accumulation of poorly chlorinated and more potent dibenzodioxins and dibenzofurans.

The eyestalk transcriptome of red swamp crayfish Procambarus clarkii.

The red swamp crayfish (Procambarus clarkii, Girard 1852) is among the most economically important freshwater crustacean species, and it is also considered one of the most aggressive invasive species worldwide. Despite its commercial importance and being one of the most studied crayfish species, its genomic and transcriptomic layout has only been partially studied. Illumina RNA-sequencing was applied to characterize the eyestalk transcriptome and identify its most characterizing genes. A collection of 83,170,732 reads from eyestalks was obtained using Illumina paired-end sequencing technology. A de novo assembly was performed with the Trinity assembly software generating 119,255 contigs (average length of 1,007 bp) and identifying the first sequenced transcriptome in this species. The eyestalk is a major site for the production of neurohormones and controls a variety of physiological functions such as osmotic regulation, molting, epidermal color patterns and reproduction. Hence, its transcriptomic characterization is interesting and potentially instrumental to the elucidation of genes which have not been comprehensively described yet. Moreover, the availability of such a large amount of information supported the characterization of molecular families which have never been described before. The P. clarkii eyestalk transcriptome reported here provides a resource for improving the knowledge of the still incompletely defined neuroendocrinology of this species and represents an important source of data for all the interested carcinologists.

Evaluating molar CYP1A level in fish hepatic microsomes by competitive ELISA using recombinant membrane-free CYP1A standard protein.

Fish cytochrome P4501A (CYP1A) is a widely accepted environmental biomarker, detecting biological effects of several xenobiotic groups present in aquatic environments, when evaluated in target tissues of a biosensor species. However, appropriate utilization of its protein level as a routine environmental diagnostic tool requires evaluation of properly normalized molar levels, mitigating comparison among different laboratories, during a multi-annual time scale and over a variety of tested populations of the biosensor species. A competitive enzyme-linked immunosorbent assay (ELISA) was developed for determination of CYP1A of the striped sea bream, Lithognathus mormyrus, using our previously described antibody raised to a trout CYP1A synthetic peptide, and a recombinant L. mormyrus CYP1A as a competitor. The L. mormyrus CYP1A-cDNA was cloned and modified by truncating its 5' hydrophobic membrane anchor, as well as by addition of 4x histidine tag, permitting its partial purification on a nickel-NTA column. The modified cDNA was ligated into the PCWOri+ vector and heterologously produced in Escherichia coli as a cytosolic, membrane-free protein, retaining its immuno-affinity with the anti-CYP1A antibody in the presence of the detergent Triton X-100. The detergent was added to the ELISA solution during the competitive step, rendering the microsomal CYP1A more accessible to the antibody. ELISA components, including coated levels of the modified standard CYP1A, and the concentrations of the Triton X-100, CYP1A-specific antibody, and the range of dissolved CYP1A standard protein, were optimized. Hypothesized immuno-affinity differences between the microsomal and the recombinant CYP1As, and among microsomal samples, as well as assay accuracy, were examined and discussed. This ELISA can serve for more efficient utilization of fish CYP1A as a pollution biomarker, and also as a model for establishing competitive ELISAs aimed at quantification of many different microsomal P450 proteins.

Quantitative evaluation of hepatic cytochrome P4501A transcript, protein, and catalytic activity in the striped sea bream (Lithognathus mormyrus).

Hepatic cytochrome P4501A (CYP1A) expression was partially characterized in the striped sea bream (Lithognathus mormyrus) from the Mediterranean coast of Israel as part of the process of establishing the CYP1A gene as an environmental biomarker. Reverse transcription-competitive polymerase chain reaction, competitive enzyme-linked immunosorbent assay, and ethoxyresorufin O-deethylase (EROD) assay were used for evaluating transcript, protein, and catalytic activity levels, respectively, in absolute units. Highest elucidated transcript, protein, and catalytic activity levels were 0.264 +/- SD 0.084 fmol/microg total RNA, 0.88 +/- 0.52 pmol/microg total protein, and 1.11 +/- 0.52 pmol resorufin/min/microg total protein, respectively, and the lower levels were 0.009 +/- 0.007 fmol/microg total RNA, 0.17 +/- 0.08 pmol/microg total protein, and 0.11 +/- 0.06 pmol resorufin/min/microg total protein, respectively, demonstrating substantial induction potential. All alternate pairs of seven examined field samples, revealing a transcript-level ratio higher than 1.7, also demonstrated a significant difference between their transcript levels, indicating a potential to detect relatively small biomarker changes (1.7-fold) caused by environmental effects. Simultaneous triple measurements of transcript, protein, and catalytic activity were carried out in individuals from two field samples and during a 318-d decay experiment. Fish from the field samples revealed significant alternate bivariate correlation between transcript, protein, and enzymatic activity. Conflicting results were found when analyzing the decay experiment, in which both protein and catalytic activity levels decreased significantly to basal levels, in contrast to no significant change in transcript levels throughout the experiment. No significant difference was observed between males and females regarding the levels of CYP1A transcript, protein, and EROD.

Application of real-time PCR for quantitative determination of hepatic vitellogenin transcript levels in the striped sea bream, Lithognathus mormyrus.

The striped sea bream (Lithognathus mormyrus) has been recently introduced as a bioindicator fish species in Mediterranean coastal habitats. The purpose of this study was to apply a real-time PCR assay for the determination of absolute levels of hepatic VTG transcript in this fish, identifying minimal and maximal levels and establishing the relationship between VTG RNA levels and ovarian development. A partial VTG cDNA was cloned from hepatic RNA of a striped sea bream female. Specific primers were designed based on its sequence and used for PCR and also for in vitro synthesis of partial VTG RNA standard. Hepatic VTG transcript levels were quantified by real-time PCR, using serially diluted VTG RNA standards for construction of a calibration curve and equation. VTG RNA levels were normalized to total RNA or 18S ribosomal RNA (determined by real-time PCR). VTG RNA was hardly detected in the liver of males, or females with small oocytes (diameter < 100 microm). A linear correlation was found between these two parameters at larger oocyte diameter (> 150 microm). VTG level reached a maximum of 204 fmol/pmol 18S RNA or 49 fmol/microg RNA. The results demonstrate the wide dynamic range of the established real-time PCR assay.

Quantifying fish metallothionein transcript by real time PCR for its utilization as an environmental biomarker.

The study is aimed at introducing the hepatic level of metallothionein transcript in the fish Lithognathus mormyrus as environmental biomarker, including: (a). establishing real time PCR procedure for the evaluation of metallothionein and 18S ribosomal RNA transcript levels, (b). examining the suitability of two alternate normalization factors, 18S- and total RNA, (c). partially characterizing hepatic metallothionein transcript expression--(1). in two samples of feral fish, aimed at determining within-sample variability, (2). during a 318 days depuration experiment aimed at determining basal transcript level, (3). after cadmium injection aimed at determining maximal induced level. Hepatic transcript levels of cytochrome p4501A measured in the same individuals and published elsewhere [Environ. Toxicol. Chem. 22 (2003)], were re-analysed concurrent with the metallothionein ones. 18S rRNA was chosen as normalizing agent of choice, compensating for demonstrated partial RNA degradation in part of the preparations. Minimal and maximal metallothionein transcript levels were determined, 61+/-47 and 2159+/-905 atomol/pmol 18S rRNA, respectively. Within-sample variability of the two feral fish samples, or of similarly treated experimental fish groups, expressed as percentage of the standard deviation from the average transcript level, ranged between 41% and 80%, and as low as 3.8-fold difference between pairs of feral or experimental groups was statistically significant.