Emergent properties of free-living nematode assemblages exposed to multiple stresses.

Biological communities are currently facing multi-stressor scenarios whose ecological impacts are challenging to estimate. In that respect, considering the complex nature of ecosystems and types and interaction among stressors is mandatory. Microcosm approaches using free-living nematode assemblages can effectively be used to assess complexity since they preserve the interactions inherent to complex systems when testing for multiple stress effects. In this study, we investigated the interaction effects of three stress factors, namely i-metallic mixture of Cu, Pb, Zn, and Hg (control [L0], low, [L1] and high [L2]), ii- CO2-driven acidification (pH 7.6 and 8.0), and iii- temperature rise (26 and 28 °C), on estuarine free-living nematode assemblages. Metal contamination had the greatest influence on free-living nematode assemblages, irrespective of pH and temperature scenarios. Interestingly, whilst the most abundant free-living nematode genera showed significant decreases in their densities when exposed to contamination, other, less abundant, genera were apparently favored and showed significantly higher densities in contaminated treatments. The augmented densities of tolerant genera may be attributed to indirect effects resulting from the impacts of toxicity on other components of the system, indicating the potential for emergent effects in response to stress. Temperature and pH interacted significantly with contamination. Whilst temperature rise had potentialized contamination effects, acidification showed the opposite trend, acting as a buffer to the effects of contamination. Such results show that temperature rise and CO2-driven acidification interact with contamination on coastal waters, highlighting the importance of considering the intricate interplay of these co-occurring stressors when assessing the ecological impacts on coastal ecosystems.

Integrative ecotoxicity evaluation of Cd, Cu, Zn and Ni in aquatic animals reveals high tolerance of Artemia franciscana.

Heavy metals pose a significant threat to animals in aquatic environments due to the adverse effects they exert. Species of the genus Artemia have been described as heavy metal tolerant, but the sensitivity/tolerance range for these species has not been established. In the present study, the toxicity of Cd, Cu, Zn and Ni as reported in the ECOTOX and Web of Science databases was examined for Artemia franciscana and compared with other species and taxonomic groups using an integrative ecotoxicity evaluation. The hazard concentration for 5% of the species (HC5) of acute toxicity tests (24-96 h), obtained through a species sensitivity distribution (SSD) indicated that Cu (0.02 mg/L) and Cd (0.03 mg/L) were the metals with the highest toxicity to aquatic animals followed by Zn (0.15 mg/L) and Ni (0.23 mg/L). In addition to the higher hazard of Cu and Cd to aquatic animals, the comparison of acute LC50 values for A. franciscana indicates lower toxicity of Cd followed by Cu, Zn, and Ni (200.0, 14.5, 9.5, and 0.6 mg/L, respectively). Using the SSD and physiological sensitivity (S) approaches, it was demonstrated that A. franciscana is relatively tolerant to Cd (SSD= HC99; S = 2.21), Cu (SSD= HC97; S = 2.00), Zn (SSD= HC90; S = 1.29) and Ni (SSD= HC83; S = 0.96) compared with other species and taxonomic groups. It appears that tolerance to the metals Cd, Cu, Zn and Ni is unique to the family Artemiidae within the order Anostraca, as the families Streptocephalidae and Thamnocephalidae are not tolerant (have negative S values). Our study confirmed that as expected, A. franciscana presents higher tolerance to Cd, Cu, Zn, and Ni than other aquatic animals. Our findings confirm that A. franciscana can be used as a model organism to understand mechanisms involved in tolerance to heavy metals, mainly Cd and Cu, which are considered highly toxic to other animals.

Differential Effects of Atrazine on Chlorophyceae Species and Association with Morphology, Photosynthesis, Chlorophyll Content, and Glutathione-S-Transferase Activity.

Atrazine is a herbicide widely used in the control of weeds in crops such as corn, sugar cane, and sorghum. It is often found in aquatic environments, where it can potentially endanger nontarget organisms such as microalgae. The present study evaluated atrazine toxicity to seven different species of Chlorophyceae and the tolerance of the species to the herbicide was related to morphological, photosynthetic, chlorophyll-a content and the activity of the glutathione-S-transferase enzyme (GST). The comparison of median effect concentration (EC50) values for growth inhibition indicates higher toxicity of atrazine for Pseudopediastrum boryanum and Desmodesmus communis, intermediate toxicity for Ankistrodesmus densus, Chlamydomonas puliminiorfes, and Raphidocelis subcapitata, and lower toxicity for Kirchneriella lunaris and Ankistrodesmus falcatus (EC50: 38, 42, 66, 103, 248, 1004, and 1585 µg L-1 atrazine, respectively). Principal component analysis (PCA) with algal characteristics suggested that the atrazine-sensitive algae P. boryanum and D. communis were positively associated with photosynthetic levels and negatively associated with GST activity and chlorophyll-a concentration. The PCA also suggested that the atrazine-tolerant algae A. falcatus and K. lunaris were positively associated with morphological parameters, where the larger the cell size, the more tolerant. Although it is difficult to associate a single characteristic of algae as the key factor determining the tolerance to atrazine, results presented in this work indicate that the cell area, the photosynthetic parameters (mainly saturating irradiance), chlorophyll-a content, and the biotransformation by GST in combination may be potential predictors for the differential tolerance of Chlorophyceae species to the herbicide. Environ Toxicol Chem 2022;41:1675-1685. © 2022 SETAC.

Glutathione S-transferase activity in aquatic macrophytes and halophytes and biotransformation potential for biocides.

Glutathione S-transferase (GST) participates in the biotransformation of many xenobiotics including biocides. Its activity in plants is generally associated with their phytoremediation capabilities. Biocides have been used in agriculture and antifouling paints and they represent risks for the aquatic environment. The present study aimed to: (1) evaluate the basal GST activity in roots, stems, and leaves from thirteen plants (eleven aquatic macrophytes and two halophytes) collected at South Brazil wetlands; (2) estimate the biotransformation potential of Nothoscordum gracile for five biocides using competitive kinetic assays with 1-chloro-2,4-dinitrobenzene (CDNB), a typical GST substrate. The N. gracile, Spartina alterniflora and Cakile maritima presented the highest GST activities among the tested plants. The Lineweaver-Burk plot obtained from the GST competitive kinetic assays confirmed that the biocides chlorothalonil, 4,5-dichloro-N-octyl-3(2H)-isothiazolone (DCOIT), dichlofluanid, and diuron, but not irgarol, compete with the substrate CDNB for GST. Chlorothalonil and DCOIT showed the lowest IC20 values (11.1 and 10.6 µM, respectively), followed by dichlofluanid (38.6 µM) and diuron (353.1 µM). The inhibition of GST-CDNB activity by 100 nM biocide was higher for chlorothalonil, DCOIT, and dichlofluanid (46.5, 49.0, and 45.1%, respectively) than for diuron (6.5%) and irgarol (2.2%). The present study indicates plant species that have significant GST activity and could be potentially used for phytoremediation. The competitive kinetic tests suggest that among the five biocides that were tested, chlorothalonil, DCOIT, and dichlofluanid are probably preferred for biotransformation via GST in plant.

A scientometric analysis of ecotoxicological studies with the herbicide atrazine and microalgae and cyanobacteria as test organisms.

Atrazine (ATZ) is one of the most widely used herbicides in the world. A scientometric study was conducted to analyze the evolution of research on ATZ. The study also looked at the use of microalgae and cyanobacteria as biological models for toxicity tests during the period from 1959 to 2019, in the category of toxicology of Web of Science. The results show an increase in the number of scientific publications mainly in the USA, Canada, and China. The majority of papers was published in journals with high impact factors, demonstrating the relevance of such studies. About 83% of the studies aimed to evaluate the effect of ATZ on non-target organisms. From those, 7.5% included microalgae and cyanobacteria. The majority of them worked with chlorophyceae to perform toxicity bioassays of ATZ and analyze its sublethal effects. The gaps identified by this analysis included a small number of collaborations between research groups from different countries; the number of studies with terrestrial organisms, which are larger in comparison to aquatic organisms; and the fact that none of the studies with ATZ and microalgae was performed in the field. These findings can point out to researchers and funding agencies the gaps in knowledge on the toxic effects of ATZ and guide the development of new research projects as well as environmental policies.

Environmental contamination alters the intestinal microbial community of the livebearer killifish Phalloceros caudimaculatus.

Intestinal microbiota perform important functions for the health of fishes. Knowing the microbial composition and evaluating the possible effects caused by anthropogenic pollution in the intestinal microbiota of fish populations might represent an important step in defining microbial biomarkers for water pollution. This study evaluated the impact of environmental contamination on the gut microbiota of the livebearer killifish Phalloceros caudimaculatus. The 16S survey using the V4 region of the 16S rRNA gene was used to characterize and compare the microbiota of two P. caudimaculatus populations from streams with different levels of environmental contamination in Rio Grande, RS, Brazil. Twelve bacterial operational taxonomic units (OTUs) (around one-third of the total) were shared between both fish populations. They represent the core microbiota of the gut in this species. The dominant phyla were Protebacteria and Firmicutes, with more than 80% of relative abundance. The dominant genus was Burkholderia with more than 35% of the relative abundance irrespective of the environmental condition. We detected a lower microbial diversity (Shannon index and observed OTUs) in fish from the polluted stream compared to the reference stream. The PERMANOVA analysis showed that the intestinal microbial communities from fish living in the polluted stream were distinct from those found in the reference stream (p < 0.05). Finally, we identified Luteolibacter, Methylocaldum and Rhodobacter genera, which correlated strongly with the polluted stream. These taxa might represent potential microbial biomarkers of exposure to environmental contaminants in the guts of fish. Confirmation of these findings in other polluted environments might allow the development of a microbiota-based screening approach for environmental evaluation in ecotoxicological studies in aquatic ecosystems.

The effects of Microcystis aeruginosa cells lysate containing microcystins on physiological and molecular responses in the nematode Caenorhabditis elegans.

Microcystins (MCs) are potent toxins produced by environmental cyanobacterial blooms. The present study evaluated the effects of a Microcystis aeruginosa cyanobacterial lysate containing 0.1, 1, and 10 µg L-1 MC-LR equivalent in the C. elegans Bristol N2 wild-type and the effects caused by equivalent concentrations of a MC-LR standard. The lysate was prepared from a culture of toxic strain (RST9501) originated from the Patos Lagoon Estuary (RS, Brazil). The minimal concentration necessary to cause significant effects in C. elegans under exposure to M. aeruginosa lysate or to MC-LR standard were, respectively, 10 and 0.1 µg L-1 MC-LR equivalent for growth and 10 and 1 µg L-1 MC-LR equivalent for fertility. Reproduction (ie, brood size) was only affected by the exposure to 10 µg L-1 MC-LR standard and was not affected by the lysate. The nematodes that were exposed to lysate containing 1 µg L-1 MC-LR equivalent or MC-LR were also analyzed for pharyngeal pumping and gene expression using RT-qPCR. The worms' rhythmic contractions of the pharynx were similarly affected by the lysate containing 1 µg L-1 of MC-LR equivalent and the MC-LR standard. The MC-LR standard caused down-regulation of genes related to growth (daf-16), fertility (spe-10), and biotransformation (gst-2). This is the first study to evaluate the effects of a toxic cyanobacterial lysate using the C. elegans model. This study suggests the organism as a potential biotest to evaluate toxicity of natural waters containing M. aeruginosa cells and to environmental risk assessment associated to cyanobacterial bloom events.

Validation of Housekeeping Genes as Internal Controls for the Study of the Effects of Microcystin-LR in Zebrafish by Real-Time PCR.

Microcystin-leucine arginine (MC-LR) is a natural toxin produced by cyanobacterial blooms that causes severe liver damage in fish. It is crucial to investigate if housekeeping genes are affected by MC-LR in zebrafish, to permit the adequate evaluation of gene expression by RT-qPCR. We evaluated the gene expression stability (M value) and regulation by chemical treatment (using E-Ct and E-ΔCt) to validate the use of eight housekeeping genes in fish exposed to 0, 0.31, and 6.10 µg L-1 MC-LR for 24 h. We suggest the use of the combination of ß-actin1, b2m, and arnt2 in the liver and ß-actin1, 18S rRNA, and arnt2 in gills as housekeeping genes. The evaluation of gene regulation following MC-LR exposure denoted a strong repression of 18S rRNA (17- and 10-fold decrease) and tbp (10- and 2-fold decrease) and induction of ef-1α (8- and 14-fold increase) in the liver of zebrafish exposed to 0.31 and 6.10 µg L-1 MC-LR, respectively. This is the first study showing that housekeeping genes commonly used in gene expression could be affected in the liver by environmentally relevant concentrations of MC-LR. The study validates adequate housekeeping genes that could be used in toxicological studies with MC-LR in zebrafish.

Effect of a toxic Microcystis aeruginosa lysate on the mRNA expression of proto-oncogenes and tumor suppressor genes in zebrafish.

Cyanobacterial blooms of Microcystis aeruginosa represent a significant risk to the environment and have become a worldwide concern. M. aeruginosa can produce the hepatotoxins microcystins (MCs) with potential for tumor promotion. The present study evaluated the time-dependent effects in the transcription of tumor-related genes in the zebrafish, Danio rerio, exposed to dilutions of a M. aeruginosa lysate containing 3.5 and 54.6 µg L-1 MCs. We used a cultured M. aeruginosa strain, RST 9501, which contains mainly the variant [D-Leu1] MC-LR and originated from the Patos Lagoon Estuary (RS, Brazil). The exposure caused short-term repression of tumor suppressor genes and long-term repression of proto-oncogenes. These responses were more evident for p53 that was repressed with exposure for 6, 24 and 96 h, and fosab and myca that were consistently repressed with exposure for 384 h, when fish were exposed to both M. aeruginosa lysate dilutions, compared to controls (p < 0.05). The suppressor genes, baxa and gadd45α, and the proto-oncogene, junba, were suppressed mainly at 96 h, where both dilutions of the lysate caused repression compared to controls (p < 0.05). The p53 gene was the only gene to be induced; this occurred in fish exposed to lysate containing 3.5 µg L-1 for 384 h. This is the first study to show that M. aeruginosa containing an environmentally relevant concentration of [D-Leu1] MC-LR could cause time-dependent repression of proto-oncogenes and tumor suppressor genes in fish. The results suggest that short-term repression of tumor suppressor genes could participate in the mechanism of tumor promotion caused by M. aeruginosa in fish.

The herbicide atrazine affects sperm quality and the expression of antioxidant and spermatogenesis genes in zebrafish testes.

The herbicide atrazine (ATZ) is used worldwide in the control of annual grasses and broad-leaved weeds. The present study evaluated sperm quality parameters in zebrafish Danio rerio after 11-day exposure to nominal ATZ concentrations of 2, 10, and 100 µg L-1. All ATZ concentrations caused a decrease in motility, mitochondrial functionality, and membrane integrity, as measured using conventional microscopy or fluorescence microscopy with specific probes. The DNA integrity of sperm was not affected. The levels of expression of genes related to spermatogenesis, antioxidant defenses, and DNA repair were also investigated using RT-qPCR. The ATZ caused transcriptional repression of the spermatogenesis-related genes SRD5A2 and CFTR, the antioxidant defense genes SOD2 and GPX4B, and the DNA repair gene XPC. This is the first study to show that environmentally relevant concentrations of ATZ significantly affect the sperm quality in fish, possibly resulting in reduced fertility rates. In addition, we showed that the repression of genes related to spermatogenesis and cellular defense could be part of the mechanisms involved in the ATZ toxicity in the testes of male fish.

Cytochrome P450 1A mRNA in the guppy Phalloceros caudimaculatus and response to beta-naphthoflavone and environmental samples.

The cytochrome P450 1A (CYP1A) mRNA is induced by environmental contaminants such as PAHs, PCBs and dioxins. The present study cloned the CYP1A transcript from the guppy Phalloceros caudimaculatus, which represents a potential fish for toxicological studies in South America. The newly identified CYP1A encodes a protein with 521 amino acids that shared 96-70% identity with other fishes. The characterization of organ- and time-dependent induction of CYP1A using RT-qPCR was evaluated after waterborne exposure to beta-naphthoflavone (BNF; 1µM). The minimum exposure time that elicited significant CYP1A induction was 1h for liver, gill, gut, brain, anal fin and fingerlings; 2h for dorsal fin; and 4h for kidney and tail fin. CYP1A tended to reach peak induction in the first few hours (4h-8h) of experiment in most organs, although levels remained induced until the end of the experiment (96h). Validation of CYP1A use in environmental sample was performed by exposing P. caudimaculatus to elutriate made from sediment of three streams located in adjacent areas of the Patos Lagoon Estuary (RS, Brazil). CYP1A in liver, gills and anal fin was induced by elutriate made from urban (S1) and industrial (S2) sites; and not induced by a reference site located 22 Km from potential contaminant sources, suggesting that environmental contamination plays a role in this induction. The results suggest that fins could be used for CYP1A biomarker analysis and employed in non-lethal biopsy methods for environmental monitoring. The responsiveness of the newly identified CYP1A to BNF and elutriate indicates that the guppy P. caudimaculatus could be used for environmental toxicology investigations in South American environments.

Responses of the CYP1A biomarker in Jenynsia multidentata and Phalloceros caudimaculatus and evaluation of a CYP1A refractory phenotype.

The level of cytochrome P450 1A (CYP1A) in fish is used as a typical environmental biomarker for the presence of organic contaminants. We used RT-qPCR to investigate CYP1A mRNA levels in the liver, gill and gonopodium of guppies Jenynsia multidentata and Phalloceros caudimaculatus in wetlands within the Rio Grande city (RG) which is under the influence of the Patos Lagoon Estuary (RS, Brazil). The CYP1A mRNA levels evaluated in fish liver from two locations that receive non-treated wastewater effluents (S3 and S4) and another locations near an oil refinery (S6) and an industrial complex (S7), were higher than in locations remote from those sites (S1, S2 and S5). The sum of 16 priority PAHs in sediment confirmed high levels in S4 and S6 (3914.0 and 4414.0 ng g(-1) dw, respectively) comparing to S7>S2>S3>S5>S1 (119.3, 66.3, 62.8, 16.4 and 1.7 ng g(-1) dw). J. multidentata from sites S1 to S4 that were transferred to the laboratory exhibited CYP1A induction after 24 h waterborne exposure to 1 µM betanaphtoflavone (BNF) in all organs compared to controls, except in the liver of fish from site S4. This lack of CYP1A induction by BNF indicates a CYP1A refractory phenotype in guppy. Although this characteristic possibly involves the alteration in AHR signaling or control, the mechanism of resistance is unknown. The present study provides information about the use of the use of CYP1A in South American guppies as an useful biomarker tool for environmental contamination studies.

Regulation of pregnane-X-receptor, CYP3A and P-glycoprotein genes in the PCB-resistant killifish (Fundulus heteroclitus) population from New Bedford Harbor.

Killifish survive and reproduce in the New Bedford Harbor (NBH) in Massachusetts (MA), USA, a site severely contaminated with polychlorinated biphenyls (PCBs) for decades. Levels of 22 different PCB congeners were analyzed in liver from killifish collected in 2008. Concentrations of dioxin-like PCBs in liver of NBH killifish were ∼400 times higher, and the levels of non-dioxin-like PCBs ∼3000 times higher than in killifish from a reference site, Scorton Creek (SC), MA. The NBH killifish are known to be resistant to the toxicity of dioxin-like compounds and to have a reduced aryl hydrocarbon receptor (AhR) signaling response. Little is known about the responses of these fish to non-dioxin-like PCBs, which are at extraordinarily high levels in NBH fish. In mammals, some non-dioxin-like PCB congeners act through nuclear receptor 1I2, the pregnane-X-receptor (PXR). To explore this pathway in killifish, a PXR cDNA was sequenced and its molecular phylogenetic relationship to other vertebrate PXRs was determined. Killifish were also collected in 2009 from NBH and SC, and after four months in the laboratory they were injected with a single dose of either the dioxin-like PCB 126 (an AhR agonist) or the non-dioxin-like PCB 153 (a mammalian PXR agonist). Gills and liver were sampled three days after injection and transcript levels of genes encoding PXR, cytochrome P450 3A (CYP3A), P-glycoprotein (Pgp), AhR2 and cytochrome P450 1A (CYP1A) were measured by quantitative PCR. As expected, there was little effect of PCB exposure on mRNA expression of AhR2 or CYP1A in liver and gills of NBH fish. In NBH fish, but not in SC fish, there was increased mRNA expression of hepatic PXR, CYP3A and Pgp upon exposure to either of the two PCB congeners. However, basal PXR and Pgp mRNA levels in liver of NBH fish were significantly lower than in SC fish. A different pattern was seen in gills, where there were no differences in basal mRNA expression of these genes between the two populations. In SC fish, but not in NBH fish, there was increased mRNA expression of branchial PXR and CYP3A upon exposure to PCB126 and of CYP3A upon exposure to PCB153. The results suggest a difference between the two populations in non-AhR transcription factor signaling in liver and gills, and that this could involve killifish PXR. It also implies possible cross-regulatory interactions between that factor (presumably PXR) and AhR2 in liver of these fish.

Biochemical biomarkers in barnacles Balanus improvisus: pollution and seasonal effects.

Biochemical biomarkers were evaluated in the barnacle Balanus improvisus (Crustacea: Cirripedia) sampled from both polluted and reference sites in the Patos Lagoon Estuary, Southern Brazil. During winter, higher glutathione S-transferase (GST) activity was recorded in the barnacles from the polluted sites, indicating environmental exposure to contaminants. Relatively low lipid peroxide levels (LPO) were also observed in barnacles from polluted sites, indicating that oxidative stress by lipid peroxidation was not a major threat in barnacles from those sites. Seasonal differences in the GST and total oxyradical scavenging capacity (TOSC) could have contributed to the low LPO levels in the summer relative to the levels in the winter. Catalase activity and metallothionein levels were not affected by contamination or seasonality. The seasonal changes observed in biomarker responses were paralleled by the differences in temperature, which could have affected physiological responses, including the balance between pro-oxidants and antioxidants.

Beta-naphthoflavone-inducedCYP1A expression in the guppy Jenynsia multidentata: Time-dependent response, anesthetic MS-222 effect and fin analysis.

Cytochrome P450 1A (CYP1A) expression in fish is used as a biomarker of exposure to organic contaminants, such PAHs, PCBs and dioxins, in the aquatic environment. South American guppy fish Jenynsia multidentata were exposed to the prototypical aryl hydrocarbon receptor (AHR) agonist beta-naphthoflavone (BNF; 1µM) and the fins were biopsied to characterize different aspects of CYP1A induction. RTq-PCR was used to quantify CYP1A mRNA levels in fish tissues. CYP1A induction in the gill, liver and anal fin (gonopodium) occurred within the first hour of waterborne exposure to BNF and persisted throughout 2, 4, 8, 24, 48 and 96h compared to controls (DMSO vehicle; p<0.05). The organ-specific temporal pattern of induction was marked by mRNA levels consistently augment as duration of exposure increases and tend to a sustained induction from 24h to 96h for gill and liver (∼15-fold and ∼50-fold over control, respectively). In gonopodium, there was a maximum CYP1A mRNA level at 4h (∼34-fold over control). Basal CYP1A mRNA levels and its induction following BNF exposure were not affected by administration of a chemical anesthetic (fish immersion in 100mgl(-1) MS-222 for 2-5min) in the gill, liver, gonopodium, dorsal or tail fin (p<0.05). In an ex vivo assay, in which small pieces of biopsied fins were exposed to BNF for 4h, high CYP1A induction was observed in the tail and gonopodium (∼49-fold and ∼69-fold, respectively) but not in the dorsal fin compared to controls. To our knowledge, this is the first study to show that a 1h waterborne exposure to an AHR agonist is sufficient to cause CYP1A induction in fish organs and fins. The present study added new information to the field regarding the use of MS-222 as an anesthetic on fish and the analysis of biopsied fins as an alternative non-lethalex vivo assay for evaluating the CYP1A biomarker in fish. This observation could be useful for planning fish toxicological bioassays and biomonitoring studies on the aquatic environments in South America.

Salinity-dependent copper accumulation in the guppy Poecilia vivipara is associated with CTR1 and ATP7B transcriptional regulation.

Copper (Cu) accumulation and regulation of key-genes involved in Cu homeostasis were evaluated in freshwater- and saltwater-acclimated guppies Poecilia vivipara. Fish were exposed (96h) to environmentally relevant concentrations of dissolved Cu (0, 5.0, 9.0 and 20.0µg/L). In freshwater guppies, gill and liver Cu accumulation was dependent on Cu concentration in the exposure medium. In saltwater guppies, this dependence was observed only in the gut. These findings indicate that Cu accumulation was salinity- and tissue-dependent. Key genes involved in Cu metabolism were sequenced for the first time in P. vivipara. Transcripts coding for the high-affinity copper transporter (CTR1) and copper-transporting ATPase (ATP7B) were identified using polymerase chain reaction (PCR) and gene sequencing. The full-length CTR1 open reading frame (1560bp) and a partial ATP7B (690bp) were discovered. Predicted amino acid sequences shared high identities with the CTR1 of Fundulus heteroclitus (81%) and the ATP7B of Sparus aurata (87%). Basal transcriptional levels addressed by RT-qPCR in control fish indicate that CTR1 and ATP7B was highly transcribed in liver of freshwater guppies while CTR1 was highly transcribed in gut of saltwater guppies. This could explain the higher Cu accumulation observed in liver of freshwater guppies and in gut of saltwater guppies, because CTR1 is involved in Cu uptake. Reduced gill mRNA expression of CTR1 was observed in freshwater guppies exposed to 20.0µg/L Cu and in saltwater guppies exposed to 5.0µg/L Cu. In turn, reduced mRNA expression of gut ATP7B was observed in freshwater and salt water guppies exposed to 9.0 and 20.0µg/L Cu. Liver CTR1 and ATP7B transcription were not affected by Cu exposure. These findings suggest that gill CTR1 and gut ATP7B are down-regulated to limit Cu absorption after exposure to dissolved Cu, while liver CTR1 and ATP7B levels are maintained to allow Cu storage and detoxification. In conclusion, findings reported here indicate that Cu accumulation in the euryhaline guppy P. vivipara is tissue specific and dependent on water salinity. They also suggest that Cu homeostasis involves a differential transcriptional regulation of the newly identified Cu transporters, CTR1 and ATP7B.

Identification and expression of multiple CYP1-like and CYP3-like genes in the bivalve mollusk Mytilus edulis.

Various sequencing projects over the last several years have aided the discovery of previously uncharacterized invertebrate sequences, including new cytochrome P450 genes (CYPs). Here we present data on the identification and characterization of two CYP1-like and three CYP3-like genes from the bivalve mollusk Mytilus edulis, and assess their potential as biomarkers based on their responses to several known vertebrate aryl hydrocarbon receptor (AHR) agonists. Quantitative real-time PCR was used to measure CYP transcript levels in digestive gland, labial palps, adductor muscle, gill, foot, and different regions of the mantle. Levels of both CYP1-like genes were highest in digestive gland, whereas labial palps had the highest expression levels of the three CYP3-like genes followed by digestive gland and outer margin of the mantle. Mussels were exposed by injection to the AHR agonists, ß-naphthoflavone (BNF; 25 µg g(-1)), 3,3',4,4',5-polychlorinated biphenyl (PCB126; 2 µg g(-1)), or 6-formylindolo[3,2-b]carbazole (FICZ; 0.1 µg g(-1)), or to Aroclor 1254 (a mixture of PCBs; 50 µg g(-1)) for 24 h, followed by CYP expression analysis. There was no statistically significant change in expression of either of the CYP1-like genes after exposure to the various AHR agonists. The CYP3-like-1 gene was significantly up-regulated by BNF in gill tissues and the CYP3-like-2 gene was up-regulated in digestive gland by PCB126 and in gill tissue by BNF. These results suggest that distinct mechanisms of CYP gene activation could be present in M. edulis, although the importance of the CYP1-like and CYP3-like genes for xenobiotic and endogenous lipids biotransformation requires additional investigation.

Basal and 3-methylcholanthrene-induced expression of cytochrome P450 1A, 1B and 1C genes in the Brazilian guppy, Poecilia vivipara.

In fish there are four cytochrome P450 (CYP1) subfamilies: CYP1A, CYP1B, CYP1C, and CYP1D. Here we cloned Poecilia vivipara CYP1A, with an inferred amino acid sequence 91% identical to CYP1A from the killifish Fundulus heteroclitus, another member of the Cypriniformes, and an important model in ecotoxicology. In addition, we examined the expression of CYP1A, CYP1B1, and CYP1C1 by qPCR in liver, gill, and intestine of adult P. vivipara injected with 3-methylcholanthrene (3-MC) or held in clean water (control group) for 24h. All three tissues examined showed basal expression of the three CYP1 genes. CYP1A was most strongly expressed in the liver, while CYP1B1, and CYP1C1 were most strongly expressed in the gill and intestine respectively. 3-MC induced CYP1A, CYP1B1, and CYP1C1 significantly (20-120-fold) in the three organs, consistent with the regulation of CYP1A, CYP1B1 and CYP1C1 via the aryl hydrocarbon receptor. Validation of CYP1 gene biomarkers in fish collected from a contaminated urban mangrove environment was confirmed with significant induction of CYP1A and CYP1C1 in gills (10-15-fold) and CYP1B1 in liver (23-fold), relative to fish from a control site. The responsiveness of these CYP1 genes indicates P. vivipara is suitable as a model for environmental toxicology studies and environmental assessment in Brazil.

Biomarkers of organic contamination in the South American fish Poecilia vivipara and Jenynsia multidentata.

South American cyprinodontiform fish are potential candidates to be used as model biomarker species of exposure in environmental toxicology. The aim of this study was to identify molecular and biochemical biomarkers of pollution using Poecilia vivipara (Poecilidae) and Jenynsia multidentata (Anablepidae). Partial nucleotide sequences for cytochrome P-450 1A (CYP1A), a classical biomarker of exposure to organic contaminants in fish, were identified in P. vivipara and J. multidentata (approximately 650 nucleotides) using degenerated primers and polymerase chain reaction (PCR). These sequences shared approximately 90% identity in the predicted amino acid sequence with the corresponding CYP1A region of Fundulus heteroclitus. Real-time quantitative PCR (RT-qPCR) analysis confirmed that CYP1A transcription was markedly induced in the liver and gills of J. multidentata (approximately185-fold and 20-fold, respectively) and P. vivipara (122-fold and 739-fold, respectively) 24 h after exposure to 1 µM synthetic CYP1A inducer ß-naphthoflavone (BNF). At 24 h after injection with 1 µg/g environmental carcinogenic contaminant benzo[a]pyrene (BaP), a decreased total antioxidant capacity against peroxyl radicals was observed both in liver of J. multidentata and gills of P. vivipara. BaP injection in both fish did not produce changes in lipid peroxide (thiobarbituric acid-reactive substances, TBARS) levels, suggesting an absence of an oxidative stress condition. The newly identified CYP1A may thus serve as general biomarker of exposure to organic contaminant in future studies using P. vivipara and J. multidentata. Data also indicate the importance of species-specific differences in biomarker responses in these South American cyprinodontiform fish, suggesting distinct resistance/susceptibility properties to polycyclic aromatic hydrocarbons.

Identification of differentially transcribed genes in shrimp Litopenaeus vannamei exposed to osmotic stress and challenged with WSSV virus.

The effects of hyposmotic stress and white spot syndrome virus (WSSV) challenge in expression was studied in the marine shrimp Litopenaeus vannamei. Messenger RNA from gills of shrimp submitted to osmotic stress was isolated to identify genes differentially expressed through the suppressive subtractive hybridization (SSH) method. Two subtractive libraries forward and two reverse were constructed to identify up and down-regulated genes under these conditions. About 192 clones were sequenced, of which 46 genes were identified. These genes encode proteins corresponding to a wide range of biological roles, including defense, cell signaling, electron transfer, cell proliferation and differentiation, apoptosis, intermediary metabolism, cytoskeleton and digestion. Among the identified genes, 19 were up-regulated and 27 were down-regulated in the animals kept at a lower ion concentration. We evaluated the expression of eight genes by RT-qPCR in shrimp submitted to hyposmotic conditions with and without WSSV challenge. The SSH enabled the identification of genes that are influenced by hyposmotic stress. A significant up-regulation was observed in lectin-C, QM, TGF beta inducible nuclear protein 1, ciclophilin, malate dehydrogenase, mitochondrial ATP synthase F chain and ATP synthase subunit 9 precursor transcripts. However, the expression of these genes in L. vannamei was not affected by WSSV infection both at isosmotic and hyposmotic conditions.