Avaliação química e ecotoxicológica para seleção de bioensaios aquáticos sensíveis a efluentes contendo metais

A Resolução CONAMA N 430/2011 exige a utilização de dois bioensaios (dois níveis tróficos) para avaliação ecotoxicológica de efluentes, mas a seleção ao acaso de bioensaios pode permitir lançamentos tóxicos. A sensibilidade dos bioindicadores irá depender da substância tóxica avaliada. Assim, baterias de bioensaios sensíveis devem ser estabelecidas às classes de contaminantes. Na literatura não há estudos que indiquem uma bateria de bioensaios ecotoxicológicos sensíveis para avaliação de efluentes contendo principalmente metais. Esse trabalho teve como objetivo selecionar uma bateria de bioensaios ecotoxicológicos que conjuntamente detectem toxicidade ao maior número de metais isolados e em misturas e que sejam realizados no menor tempo indicado pelas normas de padronização. Foram avaliadas as sensibilidades de seis bioensaios, incluindo três níveis tróficos (produtores, algas: Pseudokirchneriella subcapitata e Chlorella vulgaris; consumidores primários, cladóceros: Daphnia similis e Ceriodaphnia dubia; consumidores secundários, peixes: Poecilia reticulata e Danio rerio), a 10 espécies metálicas individuais (Ag+, Cd2+, Cu+, Cu2+, Cr3+, Cr6+, Pb2+, Ni2+, Zn2+ e Hg2+) e a efluentes reais (siderúrgicos) e simulados em laboratoriais (baseado nos limites máximos permitidos para descarte). Os bioensaios com peixes foram os menos sensíveis, D. rerio não detectou toxicidade em nenhum dos efluentes testados. P. subcapitata foi um bom bioindicador de toxicidade de Cr3+ e D. similis foi o organismo mais sensível a Hg2+. O uso combinado do bioensaio crônico de 72h com C. vulgaris e do bioensaio agudo de 48h com C. dubia garantiu a detecção das menores concentrações dos metais tanto individualmente quanto em efluentes reais e simulados. Apesar de P. subcapitata ser um bom bioindicador da toxicidade de Cr3+, a interação dos metais em misturas tornou C. vulgaris igualmente sensível. Da mesma forma, apesar de D. similis ter sido mais sensível ao Hg2+, o efeito da toxicidade dos efluentes com maiores teores de Hg2+ foi detectado por C. dubia

Uso de geobag como unidade integrante do sistema de tratamento de lixiviado de aterros sanitários: estudo de caso no aterro sanitário de Rio das Ostras, RJ.

O objetivo do estudo foi avaliar o processo de tratamento de lixiviados de aterros sanitários combinado com lodos de fossa séptica com o uso de geobags. Foram realizadas amostragens no Aterro Sanitário de Rio das Ostras, onde foram coletadas amostras do lixiviado bruto, da lagoa de mistura de lodo de fossa séptica e lixiviado que constitui o afluente dos geobags, do efluente dos geobags, da entrada do wetland e da saída do wetland. Todas as amostras foram submetidas a análises físico-químicas (pH, condutividade, DQO, COD, nitrogênio amoniacal, série de sólidos), e ensaio de toxicidade com organismos-teste pertencentes a três níveis tróficos diferentes (Danio rerio, Daphnia similis e Aliivibrio fischeri). Além disso, foi realizado teste de coagulação com o mesmo polímero utilizado no aterro e ensaio de equilíbrio em lote com o lodo do interior do geobag. Os resultados encontrados apontaram para uma eficiência de redução de 90% para o parâmetro DQO, 97% para sólidos suspensos totais e 52% para COD, após o tratamento pelo geobag. Foi observada uma redução na toxicidade para Danio rerio e Daphnia similis quando o lixiviado foi misturado com o lodo de fossa séptica. Para Aliivibrio fischeri, ocorreu redução na toxicidade no efluente do geobag. Esses resultados mostraram que o potencial de utilização do geobag como parte integrante de sistemas de tratamento de lixiviados, considerando-se a especificidade do estudo (tratamento combinado com lodo de fossa séptica), pode contribuir para a solução de um dos principais problemas ambientais oriundos dos aterros sanitários em municípios de médio e pequeno porte.

Caracterização ecotoxicológica e físico-química das águas da Bacia do Rio Morto, Vargem Grande - RJ.

Neste trabalho foi avaliada a qualidade das águas da Bacia do Rio Morto, localizado na Baixada de Jacarepaguá Rio de Janeiro, com base em análise físicoquímicas e ensaios ecotoxicológicos agudos com Danio rerio, Daphnia similis e Aliivibrio fischeri e ensaios ecotoxicológicos crônicos referentes à reprodução com Daphnia similis. Foram comparadas as sensibilidades dos organismos-teste, que pertencem a níveis tróficos diferentes, nos quatro pontos selecionados para coleta de amostras de água no Rio Morto e seus principais tributários: Rio Branco, Rio Sacarrão e canal do Morro do Bruno. Além disso, foi implementado no laboratório o método de ensaio crônico com o microcrustáceo Daphnia similis. As amostras, em sua maioria, apresentaram parâmetros físico-químicos dentro dos limites permitidos pela legislação nacional para a classe de águas doces em que a Bacia estudada está inserida. Não foram observados efeitos agudos nos organismos-teste, não sendo possível o cálculo da CE50 ou CL50, por conseqüência, o FT ficou fixado em 1. No teste agudo para Aliivibrio fischeri, para algumas amostras, foi constatado efeito Hormesis. O mesmo foi verificado em algumas amostras submetidas aos testes crônicos com Daphnia similis.

Comparative Expression of Zebrafish Lats1 and Lats2 and Their Implication in Gastrulation Movements

Large tumor suppressor (Lats) is a Ser/Thr kinase, and it presents an important function in tumor suppression. lats was originally identified in Drosophila and recently in mammals. In mammals, it contains two homologues, lats1 and lats2. In the present study, lats1 and lats2 were characterized from zebrafish (Danio rerio), which is the first report of lats in a nonmammalian vertebrate. The primary structure, genomic organization, and phylogenesis of lats from different species were studied, and the results suggest that lats1 is the direct descendant of invertebrate lats, whereas lats2 is formed by genome duplication. In zebrafish, both lats genes are maternally expressed, while they show distinctly different expression profiles during gastrulation. lats1 is almost ubiquitously expressed through development, and lats2 is more prominently expressed in the non-neural ectoderm region of zebrafish gastrula. Most intriguingly, as revealed by cell tracing and gene expression analysis, morpholino-mediated knockdown of either lats1 or lats2 led to obvious defects of cell migration in gastrulation, indicating the functional significance of lats in gastrulation movements. Developmental Dynamics 238:28502859, 2009. (C) 2009 Wiley-Liss, Inc.

Waterborne exposure to fluorotelomer alcohol 6:2 FTOH alters plasma sex hormone and gene transcription in the hypothalamic-pituitary-gonadal (HPG) axis of zebrafish

Fluorotelomer alcohols (FTOHs) have shown estrogenic activity in vitro and in vivo, but the mechanism of this activity is not known. In this study, 18-week-old zebrafish (Danio rerio) were exposed to 0, 0.03, 0.3 and 3.0 mg/l 1H, 1H, 2H, 2H-perfluorooctan-1-ol (6:2 ETCH) for 7 days, and the effects on plasma sex hormone levels were measured followed by use of real-time PCR to examine selected gene expression in hypothalamic-pituitary-gonadal (HPG) axis and liver. Exposure to 6:2 FTOH significantly increased plasma estradiol (E2) and testosterone (T) levels in both males and females. Furthermore, the ratio of T/E2 was reduced in females while increased in males. In females, the increase of E2 was accompanied by up-regulated hepatic estrogenic receptor alpha (ER alpha) and vitellogenin (VTG1 and VTG3) expression. In males, the elevation of the T level is consistent with the up-regulation of cytochrome P450 c17 alpha-hydroxylase, 17, 20-lase (CYP17) and the down-regulation of cytochrome P450 aromatase A (CYP19A). The present study demonstrated that waterborne exposure to 6:2 FTOH alter plasma sex hormone levels and the ratio of T/E2, as well as the transcriptional profiles of some genes in the HPG axis and liver. The results suggested that FTOHs may disturb fish reproduction through endocrine disrupted activity. (C) 2009 Elsevier B.V. All rights reserved.

Toll-like receptor 4 signaling pathway can be triggered by grass carp reovirus and Aeromonas hydrophila infection in rare minnow Gobiocypris rarus

Toll-like receptor 4 (TLR4) is critical for LPS recognition and cellular responses. It also recognizes some viral envelope proteins. Detection mostly results in the inflammation rather than specific antiviral responses. However, it's unclear in fish. In this report, a TLR4 gene (named as GrTLR4b) was cloned and characterized from rare minnow Gobiocypris rarus. The full length of GrTLR4b cDNA consists of 2766 nucleotides and encodes a polypeptide of 818 amino acids with an estimated molecular mass of 94,518 Da and a predicted isoelectric point of 8.41. The predicted amino acid sequence comprises a signal peptide, six leucine-rich repeat (LRR) motifs, one leucine-rich repeat C-terminal (LRRCT) motif, followed by a transmembrane segment of 23 amino acids, and a cytoplasmic region of 167 amino acids containing one Toll - interleukin 1 - receptor (TIR) motif. It's closely similar to the zebrafish (Danio rerio) TLR4b amino acid sequence with an identity of 77%. Quantitative RT-PCR analysis showed GrTLR4b mRNA was constitutive expression in gill, heart, intestine, kidney, liver, muscle and spleen tissues in healthy animals and up-regulated by viruses and bacteria. After being infected by grass carp reovirus or Aeromonas hydrophila, GrTLR4b expressions were up-regulated from 24 h post-injection and lasted until the fish became moribund (P < 0.05). These data implied that TLR4 signaling pathway could be activated by both viral and bacterial infection in rare minnow. (C) 2009 Elsevier Ltd. All rights reserved.

Identification of a novel gene K23 over-expressed in fish cross-subfamily cloned embryos

A novel gene-K23, differentially expressed in cross-subfamily cloned embryos, was isolated by RACE-PCR technique. It had 2580 base pairs (bp) in length, with a 1,425 bp open reading frame (ORF) encoding a putative protein of 474 amino acids (aa). Bioinformatic analysis indicated that K23 had 22 phosphorylation sites, but it had no signal peptides. Developmental expression analysis in zebrafish showed that K23 transcripts were maternally expressed in ovum and the amount of K23 transcripts increased gradually from zygote to pharyngula period. Subcellular localization analysis revealed that K23 protein was homogeneously distributed both in nuclei and cytoplasm. Taken together, our findings indicate that K23 gene is a novel gene differentially expressed in fish cross-subfamily cloned embryos.

Zebrafish peptidoglycan recognition protein SC (zfPGRP-SC) mediates multiple intracellular signaling pathways

Insect PGRPs can function as bacterial recognition molecules triggering proteolytic and/or signal transduction pathways, with the resultant production of antimicrobial peptides. To explore if zebrafish peptidoglycan recognition protein SC (zfPGRP-SC) has such effects, RNA interference (siRNA) and high-density oligonucleotide microarray analysis were used to identify differentially expressed genes regulated by zfPGRP-SC. The mRNA levels for a set of genes involved in Toll-like receptor signaling pathway, such as TLRs, SARM, MyD88, TRAF6 and nuclear factor (NF)-kappa B2 (p100/p52), were examined by quantitative RT-PCR (QT-PCR). The results from the arrays and QT-PCR showed that the expression of 133 genes was involved in signal transduction pathways, which included Toll-like receptor signaling, Wnt signaling, BMP signaling, insulin receptor signaling, TGF-beta signaling, GPCR signaling, small GTPase signaling, second-messenger-mediated signaling, MAPK signaling, JAK/STAT signaling, apoptosis and anti-apoptosis signaling and other signaling cascades. These signaling pathways may connect with each other to form a complex network to regulate not just immune responses but also other processes such as development and apoptosis. When transiently over-expressed in HEK293T cells, zfPGRP-SC inhibited NF-kappa B activity with and without lipopolysacharide (LPS) stimulation. (C) 2008 Elsevier Ltd. All rights reserved.

Characterization of C-C chemokine receptor subfamily in teleost fish

Chemokines and their receptors play important roles in nervous and immune systems. Little information, however, exists concerning this gene family in teleost fish. In the present Study, 17 C-C chemokine receptors genes were identified from Danio rerio, 9 from Gasterosteus aculeatus, 10 from Oryzius latipes, 8 from Takifugu rubripes and 5 from Tetraodon nigroviridis. Phylogenetic analysis showed that the orthologs to mammalian CCR6, 7, 8, 9 and CCRL1 receptors were evident in zebrafish, but the clear orthologs to mammalian CCR1, 2, 3, 4, 5 and 10 were not found in zebrafish. The gene structure of zebrafish CCR (zfCCR) was further analyzed. The open reading frame of zfCCR3-1, zfCCR3-3, zfCCR6-1, zfCCR6-2, zfCCR8-2 contain one exon, and two exons were identified for zfCCR2-1, zfCCR2-2, zfCCR4 and zfCCRLI-1, three exons for zfCCR3-2, zfCCR5 and zfCCR7, four exons for zfCCR8-1 and zfCCR9-1. The expression analyses showed that in zebrafish, most C-C chemokine receptor genes Were expressed in fertilized eggs and oocytes, and all the receptor genes were expressed in larval stages. The zfCCR2-2, zfCCR3-1, zfCCR4 and zfCCR6-2 genes were expressed in all normal organs examined, whereas not for zfCCR2-1, zfCCR3-3, zfCCR6-1, zfCCR8-1, zfCCR9-2 and zfCCRL1-2. The expression of zfCCR3-2, zfCCR5, zfCCR7, zfCCR9-1 and zfCCRLI-1 were detected in the majority organs. and zfCCR8-2 and zfCCR8-3 detected only in brain. The differential expression pattern of different paralogues in organs may indicate their difference in function, which requires further investigation. (C) 2008 Elsevier Ltd. All rights reserved.

Molecular cloning and expression characterization of ApoC-I in the orange-spotted grouper

Endogenous yolk nutrients are crucial for embryo and larval development in fish, but developmental behavior of the genes that control yolk utilization remains unknown. Apolipoproteins have been shown to play important roles in lipid transport and uptake through the circulation system. In this study, EcApoC-I, the first cloned ApoC-I in teleosts, has been screened from pituitary cDNA library of female orange-spotted grouper (Epinephelus coioides), and the deduced amino acid sequence shows 43.5% identity to one zebrafish (Danio rerio) hypothetical protein similar to ApoC-I, and 21.2%, 21.7%, 22.5%, 20%, and 22.5% identities to Apo C-I of human (Homo sapiens), house mouse (Mus musculus), common tree shrew (Tupaia glis), dog (Canis lupus familiaris) and hamadryas baboon (Papio hamadryas), respectively. Although the sequence identity is low, amphipathic alpha-helices with the potential to bind to lipid were predicted to exist in the EcApoC-I. RT-PCR analysis revealed that it was first transcribed in gastrula embryos and maintained a relatively stable expression level during the following embryogenesis. During embryonic and early larval development, a very high level of EcApoC-I expression was in the yolk syncytial layer, indicating that it plays a significant role in yolk degradation and transfers nutrition to the embryo and early larva. By the day 7 after hatching, EcApoC-I transcripts were observed in brain. In adult, EcApoC-I mRNA was detected abundantly in brain and gonad. In transitional gonads, the EcApoC-I expression is restricted to the germ cells. The data suggested that EcApoC-I might play an important role in brain and gonad morphogenesis and growth.

Gene expression profiles in liver of zebrafish treated with microcystin-LR

Microcystin-LR (MC-LR) is the most frequently studied cyclic heptatoxin produced by cyanobacteria, which has tremendous negative impacts on fish, while its molecular mechanism behind remained unclear at present. Here, Affymetrix Zebrafish GeneChip was used to identify alterations in gene expression of zebrafish (Danio rerio) after MC-LR exposure. Among the 14,900 transcripts in the microarray, 273 genes were differentially expressed, in which 243 genes were elevated and 30 were decreased. According to GOstat analysis, MC-LR mainly influenced the cell cycle and mitogen-activated protein kinases (MAPK) signaling pathways. In addition, many immune-related genes were also influenced. These data suggest that MC-LR could promote tumorigenesis and cause immunotoxicity in fish. (C) 2008 Elsevier B.V. All rights reserved.

Phylogenetic relationships of Cyprinidae (Teleostei : Cypriniformes) inferred from the partial S6K1 gene sequences and implication of indel sites in intron 1

The family Cyprinidae is widely distributed in East Asia, and has the important phylogenetic significance in the fish evolution. In this study, the 5' end partial sequences (containing exon 1, exon 2 and indel 1) of S6K1 gene were obtained from 30 representative species in Cyprinidae and outgroup using PCR amplification and sequencing. The phylogenetic relationships of Cyprinidae were reconstructed with neighbor joining (NJ), maximum parsimony (MP), maximum likelihood (ML), and Bayesian methods. Myxocyprinus asiaticus (Catostomidae) was assigned to the outgroup taxon. Similar phylogenetic relationships within the family Cyprinidae were achieved with the four analyses. Leuciscini and Barbini were monophyletic lineages respectively with the high nodal supports. Leuciscini comprises Hypophthalmichthyinae, Xenocyprinae, Cultrinae, Gobioninae, Acheilognathinae and East Asian species of Leuciscinae and Danioninae. Monophyly of East Asian clade was supported with high nodal support. Barbini comprises Schizothoracinae, Barbinae, Cyprininae and Labeoninae. The monophyletic lineage consisting of Danio rerio, D. myersi, and Rasbora trilineata was basal in the tree. In addition, the large fragment indels in intron 1 were analyzed to improve the understanding of Cyprinidae relationships. The results showed that the large fragment indels were correlated with the relations among species. Some conserved regions in intron 1 were thought to be involved in the functional regulation. However, no correlation was found between sequence variations and species characteristic size.

The c-myc coding DNA sequences of cyprinids (Teleostei : Cypriniformes): Implications for phylogeny

The family Cyprinidae is one of the largest fish families in the world, which is widely distributed in East Asian, with obvious difference in characteristic size among species. The phylogenetic analysis of cyprinid taxa based on the functionally important genes can help to understand the speciation and functional divergench of the Cyprinidae. The c-myc gene is an important gene regulating individual growth. In the present study, the sequence variations of the cyprinid c-myc gene and their phylogenetic significance were analyzed. The 41 complete sequences of the c-myc gene were obtained from cyprinids and outgroups through PCR amplification and clone. The coding DNA sequences of the c-myc gene were used to infer molecular phylogenetic relationships within the Cyprinidae. Myxocyprinus asiaticus (Catostomidae), Misgurnus anguillicaudatus (Cobitidae) and Hemimyzon sinensis (Homalopteridae) were assigned to the outgroup taxa. Phylogenetic analyses using maximum parsimony (MP), maximum likelihood (ML), and Bayesian retrieved similar topology. Within the Cyprinidae, Leuciscini and Barbini formed the monophyletic lineage respectively with high nodal supports. Leuciscini comprises Xeno-cyprinae, Cultrinae, East Asian species of Leuciscinae and Danioninae, Gobioninae and Acheilognathinae, and Barbini contains Schizothoracinae, Barbinae, Cyprininae and Labeoninae. Danio rerio, D. myersi and Rasbora trilineata were supposed to separate from Leuciscinae and Barbini and to form another lineage, The positions of some Danioninae species were still unresolved. Analyses of both amino acid variation with parsimony information and two high variation regions indicated that there is no correlation between variations of single amino acid or high variation regions and characteristic size of cyprinids. In,addition, the species with smaller size were usually found to be basal within clades in the tree, which might be the results of the adaptation to the primitive ecology and survival pressure.

PCR survey of Hox genes in the goldfish Carassius auratus auratus

A tetraploidization event took place in the cyprinid lineage leading to goldfishes about 15 million years ago. A PCR survey for Hox genes in the goldfish Carassius auratus auratus (Actinopterygii: Cyprinidae) was performed to assess the consequences of this genome duplication. Not surprisingly, the genomic organization of the Hox gene clusters of goldfish is similar to that of the closely related zebrafish (Danio rerio). However, the goldfish exhibits a much larger number of recent pseudogenes, which are characterized by indels. These findings are consistent with the hypothesis that dosage effects cause selection pressure to rapidly silence crucial developmental regulators after a tetraploidization event.

Intelectin gene from the grass carp Ctenopharyngodon idella: cDNA cloning, tissue expression, and immunohistochemical localization

The cDNA encoding grass carp intelectin was isolated from a head kidney cDNA library, and termed gcIntL. The deduced amino acid sequence of gcIntL consists of 318 amino acids, and about 55% identical and 74% similar to human intelectin, which is a new type of lectin recognizing galactofuranose, and plays a role in the recognition of bacteria-specific components in animal hosts. The gcIntL gene consists of seven exons and six introns, spacing over approximately 3 kb of genomic sequence. Phylogenetic analysis clearly demonstrated that the gcIntL formed a clade with Danio rerio intelectin and 35 kDa serum lectin. By real-time quantitative RT-PCR analysis, gcIntL transcripts were significantly induced in head kidney, trunk kidney, spleen, and intestine from LPS-stimulated fish. RT-PCR and Western blotting analysis demonstrated that the mRNA and protein of gcIntL gene have the same expression pattern, and both were detected in brain, gill, intestine, head kidney, trunk kidney, spleen, and heart. Furthermore, gcIntL protein could be detected in gill, intestine, trunk kidney, head kidney, spleen, heart, and brain including medulla oblongata and optic lobe, as determined by immunohistochemistry. This is the first report of intelectin expression pattern in fish, and of recombinant gcIntL and polyclonal antibody against gcIntL. (c) 2006 Elsevier Ltd. All rights reserved.