RESUMEN
Benthic microbes are key organisms in the oligotrophic Southeastern Mediterranean Sea (SEMS), yet their abundance, activity, and diversity in this rapidly changing basin are not fully understood. We investigated the prokaryotic and microfungal communities throughout years 2018-2020 at 27 stations (6-1900 m water depths, down to 20 cm below the sediment surface), in two transects with distinct downslope transport regimes, and along the eutrophic coastline. We estimated microbial abundance with flow cytometry, secondary production as leucine assimilation, and sequenced marker genes (the 16S rRNA and internal transcribed spacer) to assess diversity indices. The highest abundance (0.21 × 108 cells gr-1 sediment) was estimated at slope stations where we assumed substantial transport rates and found an accumulation of organic carbon. Secondary production was the highest nearshore (12 ± 4 ng C gr-1 h-1), and markedly declined offshore (0.5 ± 0.9 ng C gr-1 h-1). Populations of archaea (dominant Nitrososphaeria and Nanoarchaeia) and diverse bacteria were stable over three years, and taxonomic composition was dictated mainly by depth gradients. Saprotrophic and pathotrophic microfungi Ascomycota (70% ± 23%) and Basidiomycota (16% ± 18%) were prevalent, whereas parasitic chytrids were abundant nearshore. Our results highlight the role of downslope transport, which enriched the typical deep-sea communities with anaerobic lineages, in shaping microbial populations near the continental slope.
Asunto(s)
Archaea , Bacterias , Archaea/genética , Bacterias/genética , Sedimentos Geológicos/microbiología , Mar Mediterráneo , ARN Ribosómico 16S/genéticaRESUMEN
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, РСÐ-126 and РСÐ-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.
Asunto(s)
Bioensayo/métodos , Dioxinas/toxicidad , Peces , Sedimentos Geológicos/análisis , Hígado/química , Contaminantes Químicos del Agua/análisis , Animales , Benzofuranos/farmacocinética , Benzofuranos/toxicidad , Dioxinas/farmacocinética , Monitoreo del Ambiente , Genes Reporteros , Sedimentos Geológicos/química , Océano Índico , Israel , Mar Mediterráneo , Bifenilos Policlorados/análisis , Bifenilos Policlorados/farmacocinética , Bifenilos Policlorados/toxicidad , Extractos de Tejidos/análisis , Extractos de Tejidos/química , Extractos de Tejidos/toxicidad , Contaminantes Químicos del Agua/farmacocinética , Contaminantes Químicos del Agua/toxicidadRESUMEN
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.
Asunto(s)
Monitoreo del Ambiente/métodos , Perfilación de la Expresión Génica , Hígado/metabolismo , Dorada , Animales , Secuencia de Bases , Cartilla de ADN , Hibridación de Ácido Nucleico , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
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.
Asunto(s)
Astacoidea/crecimiento & desarrollo , Astacoidea/genética , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Animales , Astacoidea/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la PolimerasaRESUMEN
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.
Asunto(s)
Astacoidea/metabolismo , Proteínas de Unión al Calcio/biosíntesis , Calcio/metabolismo , Proteínas de la Matriz Extracelular/biosíntesis , Matriz Extracelular/metabolismo , Regulación de la Expresión Génica/fisiología , Estructuras Animales/metabolismo , Animales , Astacoidea/genética , Secuencia de Bases , Proteínas de Unión al Calcio/genética , Clonación Molecular , Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/genética , Perfilación de la Expresión Génica , Masculino , Datos de Secuencia Molecular , Muda/fisiología , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
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.
Asunto(s)
Cadmio/toxicidad , Metalotioneína/análisis , Metalotioneína/inmunología , Perciformes/metabolismo , Contaminantes del Agua/toxicidad , Actinas/genética , Actinas/metabolismo , Animales , Biomarcadores , Cadmio/metabolismo , Ecosistema , Monitoreo del Ambiente , Ensayo de Inmunoadsorción Enzimática/métodos , Hígado/química , Metalotioneína/biosíntesis , Metalotioneína/genética , Perciformes/genética , Proteínas Recombinantes/inmunología , Contaminantes del Agua/metabolismoRESUMEN
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 downregulated, 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.