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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. [ABSTRACT FROM AUTHOR]

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 alsofound 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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]

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. [ABSTRACT FROM AUTHOR]

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 - 1 4 premolt days. Next generation sequencing was used to construct a multitissue 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 analysisrevealed 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. [ABSTRACT FROM AUTHOR]

Highlights: [•] Transcript assembly of crustacean epithelium was constructed and characterized. [•] Proteins potentially related to ecdysteroid hormones metabolism were characterized. [•] Identifying potential 3-beta-SDRs related to ecdysteroid modification. [•] Identifying potential 3-oxo-delta-4 steroid 5-beta reductases related to ecdysteroidogenesis. [•] Identifying 7-DHCR rarely demonstrated in arthropods. [ABSTRACT FROM AUTHOR]

The crustacean Hyperglycemic Hormone (cHH) is a neuropeptide present in many decapods. Two different chiral isomers are simultaneously present in Astacid crayfish and their specific biological functions are still poorly understood. The present study is aimed at better understanding the potentially different effect of each of the isomers on the hepatopancreatic gene expression profile in the crayfish Pontastacus leptodactylus, in the context of short term hyperglycemia. Hence, two different chemically synthesized cHH enantiomers, containing either L- or D-Phe3, were injected to the circulation of intermolt females following removal of their X organ-Sinus gland complex. The effects triggered by the injection of the two alternate isomers were detected after one hour through measurement of circulating glucose levels. Triggered changes of the transcriptome expression profile in the hepatopancreas were analyzed by RNA-seq. A whole transcriptome shotgun sequence assembly provided the assumedly complete transcriptome of P. leptodactylus hepatopancreas, followed by RNA-seq analysis of changes in the expression level of many genes caused by the application of each of the hormone isomers. Circulating glucose levels were much higher in response to the D-isoform than to the L-isoform injection, one hour from injection. Similarly, the RNA-seq analysis confirmed a stronger effect on gene expression following the administration of D-cHH, while just limited alterations were caused by the L-isomer. These findings demonstrated a more prominent short term effect of the D-cHH on the transcription profile and shed light on the effect of the D-isomer on specific functional gene groups. Another contribution of the study is the construction of a de novo assembly of the hepatopancreas transcriptome, consisting of 39,935 contigs, that dramatically increases the molecular information available for this species and for crustaceans in general, providing an efficient tool for studying gene expression patterns in this organ. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The study was aimed at examining the effects of tert-butyl hydroperoxide (tBHP) on hepatic transcriptome expression patterns of the teleost fish Lithognathus mormyrus. tBHP is an organic hydro-peroxide, widely used as a model pro-oxidant. It generates the reactive oxygen species (ROS) tert-butoxyl and tert-butylperoxyl. Complementary DNAs of tBHP-treated vs control fish were applied onto a previously produced cDNA microarray of ∼ 1500 unique sequences. The effects of the tBHP application were demonstrated by leukocyte infiltration into the liver and by differential expression of various genes, some already known to be involved in ROS-related responses. Indicator genes of putative ROS effects were: aldehyde dehydrogenase 3A2, Heme oxygenase and the hemopexin-like protein. Putative indicators of transendothelial leukocyte migration and function were: p22phox, Rac1 and CD63-like genes. Interestingly, 7-dehydrocholesterol reductase was significantly down-regulated in response to all treatments. Several non-annotated genes revealed uniform directions of differential expression in response to all treatments. [ABSTRACT FROM AUTHOR]

Abstract: In the crustacean Cherax quadricarinatus, alterations of multi-transcript expression patterns between intermolt and late premolt stages were identified in the hypodermis and in the gastrolith disk via a cDNA microarray. The gastrolith disk is a specialized epithelium forming the gastroliths at premolt. The gastroliths are deposits of calcium carbonate derived from the digested cuticle contributing the mineral to the newly formed exoskeleton at postmolt. The late premolt stage was characterized by a dramatic general up-regulation of genes in the gastrolith disk. This phenomenon is explained by the gastrolith disk function rapid formation of the relatively large gastrolith during a short period of time. Besides genes of general importance for this dramatic change, three genes related to the chitin–protein–mineral structure were identified. The cDNA and the deduced protein of the novel one of them, the chitin deacetylase 1 (Cq-CDA1) was fully characterized and its resemblance to already characterized structural proteins of the gastrolith matrix was described. Cq-CDA1 characteristics strongly indicate its participation in the gastrolith construction, although its protein product was not identified yet in the gastrolith. In addition, many differentially expressed genes with unknown function were elucidated. An unexpected milder down-regulation was observed in the hypodermis. [Copyright &y& Elsevier]

The expression of the vitellogenin gene of the red-claw crayfish Cherax quadricarinatus (CqVg) was previously demonstrated in male crayfish during an endocrinologically induced molt cycle. The hypothesis that this expression is under the direct control of ecdysteroids was tested in this study both in vivo and in vitro. Unlike vitellogenin of insects, CqVg was not found to be ecdysteroid-responsive. Thus, a multigenic approach was employed for the identification of other hepatopancreatic ecdysteroid-responsive genes by a cDNA microarray. For the purposes of this study, a multi-parametric molt-staging technique, based on X-ray detection of gastrolith growth, was developed. To identify ecdysteroid-responsive genes during premolt, the molt cycle was induced by two manipulations, 20-hydroxyecdysone administration and X-organ-sinus gland complex removal; both resulted in significant elevation of ecdysteroids. Two clusters of affected genes (129 and 122 genes, respectively) were revealed by the microarray. It is suggested that only genes belonging to similarly responsive (up- or downregulated) gene clusters in both manipulations (102 genes) could be considered putative ecdysteroid-responsive genes. Some of these ecdysteroid-responsive genes showed homology to genes controlling chitin metabolism, proteases and other cellular activities, while 56.8% were unknown. The majority of the genes were downregnlated, presumably by an energetic shift of the hepatopancreas prior to ecdysis. The effect of 20-hydroxyecdysone on representative genes from this group was confirmed in vitro using a hepatopancreas tissue culture. This approach for ecdysteroid-responsive gene identification could also be implemented in other tissues for the elucidation of ecdysteroid-specific signaling pathways during the crustacean molt cycle. [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]

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/µg total RNA, 0.88 ± 0.52 pmol/µg total protein, and 1.11 ± 0.52 pmol resorufin/min/µg total protein, respectively, and the lower levels were 0.009 ± 0.007 fmol/µg total RNA, 0.17 ± 0.08 pmol/µg total protein, and 0.11 ± 0.06 pmol resorufin/min/µg total protein, respectively, demonstrating substantial induction potential. All alternate pairs of seven examined field samples, revealing a transcriptlevel 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. [ABSTRACT FROM AUTHOR]

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]

The major component of the animal egg yolk is the lipoglycoprotein vitellin, derived from its precursor vitellogenin (VTG), which is produced species-specifically in decapod crustaceans in the hepatopancreas and/or in the ovary of reproductive females. Previous studies on Procambarus clarkii vitellogenesis report the existence of two single VTGs. Here, from a multiple tissue transcriptome including ovaries and hepatopancreas of P. clarkii, we characterized four different VTG and two VTG-like transcriptomes encoding for the discoidal lipoprotein-high density lipoprotein/β-glucan binding protein (dLp/HDL-BGBP). The relative expression of the various genes was evaluated by quantitative Real-Time PCR in both the ovary and hepatopancreas of females at different reproductive stages (from immature until fully mature oocytes). These studies revealed tissue-specificity and a reproductive stage related expression for the VTGs and a constitutive expression in the hepatopancreas of dLp/HDL-BGBP independent from the reproductive stage. This study may lead to more detailed study of the vitellogenins, their transcription regulation, and to the determination of broader patterns of expression present in the female hepatopancreas and ovary during the vitellogenesis. These findings provide a starting point useful for two different practical aims. The first is related to studies on P. clarkii reproduction, since this species is highly appreciated on the market worldwide. The second is related to the study of new potential interference in P. clarkii reproduction to delay or inhibit the worldwide spread of this aggressively invasive species. [ABSTRACT FROM AUTHOR]

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 crustacean Hyperglycemic Hormone (cHH) is a neuropeptide present in many decapods. Two different chiral isomers are simultaneously present in Astacid crayfish and their specific biological functions are still poorly understood. The present study is aimed at better understanding the potentially different effect of each of the isomers on the hepatopancreatic gene expression profile in the crayfish Pontastacus leptodactylus, in the context of short term hyperglycemia. Hence, two different chemically synthesized cHH enantiomers, containing either L- or D-Phe(3), were injected to the circulation of intermolt females following removal of their X organ-Sinus gland complex. The effects triggered by the injection of the two alternate isomers were detected after one hour through measurement of circulating glucose levels. Triggered changes of the transcriptome expression profile in the hepatopancreas were analyzed by RNA-seq. A whole transcriptome shotgun sequence assembly provided the assumedly complete transcriptome of P. leptodactylus hepatopancreas, followed by RNA-seq analysis of changes in the expression level of many genes caused by the application of each of the hormone isomers. Circulating glucose levels were much higher in response to the D-isoform than to the L-isoform injection, one hour from injection. Similarly, the RNA-seq analysis confirmed a stronger effect on gene expression following the administration of D-cHH, while just limited alterations were caused by the L-isomer. These findings demonstrated a more prominent short term effect of the D-cHH on the transcription profile and shed light on the effect of the D-isomer on specific functional gene groups. Another contribution of the study is the construction of a de novo assembly of the hepatopancreas transcriptome, consisting of 39,935 contigs, that dramatically increases the molecular information available for this species and for crustaceans in general, providing an efficient tool for studying gene expression patterns in this organ.