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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 1007 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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

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 4× 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. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

The present study sought to quantitatively characterize the live and dead benthic foraminifera communities of the deep southeastern Levantine basin of the Mediterranean Sea (33.4º–31.7 ºN, 31.3º–34.9 ºE; 100–1900 m water depth) and their relationships to environmental conditions. Box corer samples were collected at 50 sites between June and July 2013. The foraminiferal macrofauna (> 250 µm) were enumerated and identified (76% to the species level). Six live foraminiferal assemblages were identified, inhabiting six biotopes, the shelf margin (SM), two upper continental slopes (UCS1 and UCS2), the lower continental slope (LCS), the eastern bathyal plain (EBP) and the western bathyal plain (WBP). The dead communities were divided into four biotopes, generally compatible with the live ones, excluding the UCS2 and the EBP. The foraminiferal density in the various live biotopes was relatively stable across the studied area, excluding the UCS2 and EBP, unlike the density of the dead shells, which increased with depth. The number of taxa per biotope was estimated by rarefaction curves and compared to the observed numbers, with a decreasing number of live taxa with water depth. The alpha-diversity, which was evaluated in relation to the number of sampled individuals, reached an asymptote in all biotopes, with very low values in the WBP. The within-biotope heterogeneity was evaluated by the average Chao-Sørensen similarity index and by a beta-diversity index (exp(gamma diversity) - exp(alpha diversity)), revealing variable heterogeneities in both assemblages. Water depth, sediment grain size mode, CaCO 3 (wt%), and clay fraction (wt%) were jointly but feebly correlated with live faunal composition. [ABSTRACT FROM AUTHOR]

Abstract: Copper transporting ATPase, ATP7A, is an ATP dependent copper pump present in all vertebrates, critical for the maintenance of intracellular and whole body copper homeostasis. Effects of copper treatment on ATP7A gene expression in fibroblast cells (SAF1) of the sea bream (Sparus aurata) were investigated by qRT-PCR and by a medium density microarray from a closely related species, striped sea bream (Lithognathus mormyrus). To discriminate between the effects of Cu and other metals, SAF1 cells were exposed to sub-toxic levels of Cu, Zn and Cd. Expression of Cu homeostasis genes copper transporter 1 (CTR1), Cu ATPase (ATP7A), Cu chaperone (ATOX1) and metallothionein (MT) together with the oxidative stress markers glutathione reductase (GR) and Cu/Zn superoxide dismutase (CuZn/SOD) were measured 0, 4 and 24 hours post-exposure by qRT-PCR. Microarray was conducted on samples from 4 hours post Cu exposure. Cu, Zn and Cd increased MT and GR mRNA levels, while only Cu increased ATP7A mRNA levels. Microarray results confirmed the effects of Cu on ATP7A and MT and in addition showed changes in the expression of genes involved in protein transport and secretion. Results suggest that ATP7A may be regulated at the transcriptional level directly by Cu and by a mechanism that is different from that exerteted by metals on MT genes. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

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 μm). A linear correlation was found between these two parameters at larger oocyte diameter (>150 μm). VTG level reached a maximum of 204 fmol/pmol 18S RNA or 49 fmol/μg RNA. The results demonstrate the wide dynamic range of the established real-time PCR assay. [Copyright &y& Elsevier]