Effects of climate change and mixtures of pesticides on the Amazonian fish Colossoma macropomum.

Several studies highlighted the complexity of mixing pesticides present in Amazonian aquatic environments today. There is evidence that indicates that ongoing climate change can alter the pattern of pesticide use, increasing the concentration and frequency of pesticide applications. It is known that the combination of thermal and chemical stress can induce interactive effects in aquatic biota, which accentuates cell and molecular damage. However, considering that the effects of climate change go beyond the increase in temperature the objective of this study was to evaluate the effect of climate change scenarios proposed by 6 th IPCC report and a mixture of pesticides on the tambaqui (Colossoma macropomum). The hypothesis of this study is that the negative effects will be accentuated by the combination of an extreme climate changes scenario and a mixture of pesticides. To test the hypothesis, juvenile tambaqui were exposed to a combination of four pesticides (chlorpyrifos, malathion, carbendazim and atrazine) in two scenarios, one that simulates current environmental conditions and another that predicted the environmental scenario for the year 2100. Fish were subjected to the experimental conditions for 96 h. At the end of the experiment, samples of blood, gills, liver, brain, and muscle were obtained for hematological, genotoxic, biochemical, and histopathological analyses. The results demonstrate that environmentally realistic concentrations of pesticides, when mixed, can alter the biochemical responses of tambaqui. The extreme scenario promotes hematological adjustments, but impairs branchial antioxidant enzymes. There is an interaction between the mixture of pesticides and the extreme scenario, accentuating liver tissue damage, which demonstrates that even increased activity of antioxidant and biotransformation enzymes were not sufficient to prevent liver damage.

Malathion alters the transcription of target genes of the tumour suppressor tp53 and cancerous processes in Colossoma macropomum: Mechanisms of DNA damage response, oxidative stress and apoptosis.

Different classes of pesticides such as fungicides, herbicides, and insecticides, can induce differential expression of genes that are involved in tumorigenesis events in fish, including the expression of tumor suppressor tp53. The degree and duration of the stressful condition is decisive in defining which tp53-dependent pathway will be activated. Herein we evaluate the target genes expression that participates in the regulation pathway of the tumor suppressor tp53 and in the cancerous processes in tambaqui after exposure to malathion. Our hypothesis is that malathion promotes a gene response that is differentially regulated over time, with positive regulation of tp53 target genes related to the apoptotic pathway and a negative regulation of genes that promote antioxidant responses. The fish were exposed to a sublethal concentration of the insecticide for 6 and 48 h. Liver samples were used to analyze the expression of 11 genes using real-time PCR. Overall, the malathion promoted over time increases in tp53 expression and differential expression of tp53 related genes. The exposure resulted in the activation of damage response related genes, caused a positive expression of atm/atr genes. The pro-apoptotic gene bax was up-regulated and the anti-apoptotic bcl2 was down-regulated. Increased expression of mdm2 and sesn1 in the first hours of exposure and no effect on the antioxidant genes sod2 and gpx1 were also observed. We also witnessed an increase in the expression of the hif-1α gene, with no effect on ras proto-oncogene. The extension of this stressful condition accentuated tp53 transcription, and minimized the levels of mdm2, sens1 and bax; however, it down regulated the levels of bcl2 and the bcl2/bax ratio, which indicates the maintenance of the apoptotic response to the detriment of an antioxidant response.

Review of Bacterial Nanocellulose-Based Electrochemical Biosensors: Functionalization, Challenges, and Future Perspectives.

Electrochemical biosensing devices are known for their simple operational procedures, low fabrication cost, and suitable real-time detection. Despite these advantages, they have shown some limitations in the immobilization of biochemicals. The development of alternative materials to overcome these drawbacks has attracted significant attention. Nanocellulose-based materials have revealed valuable features due to their capacity for the immobilization of biomolecules, structural flexibility, and biocompatibility. Bacterial nanocellulose (BNC) has gained a promising role as an alternative to antifouling surfaces. To widen its applicability as a biosensing device, BNC may form part of the supports for the immobilization of specific materials. The possibilities of modification methods and in situ and ex situ functionalization enable new BNC properties. With the new insights into nanoscale studies, we expect that many biosensors currently based on plastic, glass, or paper platforms will rely on renewable platforms, especially BNC ones. Moreover, substrates based on BNC seem to have paved the way for the development of sensing platforms with minimally invasive approaches, such as wearable devices, due to their mechanical flexibility and biocompatibility.

Impact of high temperature, CO2 and parasitic infection on inflammation, immunodepression and programmed cell death in Colossoma macropomum at the transcriptional level.

The production of tambaqui Colossoma macropomum has recently reached a milestone, being considered the main native species produced in South American continental waters. Despite the importance of this fish, its immunity is poorly understood, and global warming could pose severe risks to its health as increasing water temperature leads to an increase in the incidence of parasitic diseases. In an experimental context based on the high-emission scenario of the 5th Intergovernmental Panel on Climate Change (IPCC) report, we evaluated the synergistic effect of exposure to the extreme climate change scenario (RCP8.5) during two exposure periods (7 and 30 days) and two levels of parasitism by monogeneans (low and high). The goal was to understand how the tambaqui immune system will react to this challenge. To achieve this goal, we analyzed the expression of nine immunity-related genes (jak3, stat3, il-10, socs1, casp1, il-1ß, tp53, bcl2, and hif-1α) in the spleen. Our main findings showed downregulation in the jak3/stat3 pathway, genes related to the control of inflammation and apoptosis, in addition to upregulation of proinflammatory genes and those related to pyroptosis during the first 7 days of exposure to the extreme climate scenario, also indicating a stage of immunodepression in these animals. After 30 days of exposure, all genes tended to return to similar levels in the current scenario, possibly due to the decrease in parasite load caused by chronic exposure to the extreme scenario. Our data strongly suggest that the increase in parasitism intensity caused by the extreme climate change scenario is responsible for disturbances in the host's immune system. However, more studies are needed to clarify this poorly understood cascade of events.

Climate change affects the parasitism rate and impairs the regulation of genes related to oxidative stress and ionoregulation of Colossoma macropomum.

Global climate change represents a critical threat to the environment since it influences organismic interactions, such as the host-parasite systems, mainly in ectotherms including fishes. Rising temperature and CO2 are predicted to affect this interaction other and critical physiological processes in fish. Herein, we investigated the effects of different periods of exposure to climate change scenarios and to two degrees of parasitism by monogeneans in the host-parasite interaction, as well as the antioxidant and ionoregulatory responses of tambaqui (Colossoma macropomum), an important species in South American fishing and aquaculture. We hypothesized that temperature and CO2 changes in combination with parasite infection would interfere with the host's physiological processes that are related to oxidative stress and ionoregulation. We experimentally exposed C. macropomum to low and high levels of parasitism in the current and extreme climate scenarios (4.5 °C and 900 ppm CO2 above current levels) for periods of seven and thirty days and we use as analyzed factors; the exposure time, the climate scenario and parasitism level in a 2 × 2 × 2 factorial through a three-way ANOVA as being fish the experimental unit (n = 8). An analysis of gill enzymatic and gene expression profile was performed to assess physiological (SOD, GPx and Na+/K+-ATPase enzymes) and molecular (Nrf2, SOD1, HIF-1α and NKA α1a genes) responses. A clear difference in the parasitism levels of individuals exposed to the extreme climate scenario was observed with a rapid and aggressive increase that was higher after 7 days of exposure though showed a decrease after 30 days. The combination of exposure to the extreme climate change scenario and parasitism caused oxidative stress and osmoregulatory disturbance, which was observed through the analysis of gene expression (Nrf2, SOD1, HIF-1α and NKA α1a) and antioxidant and ionoregulatory enzymes (SOD, GPx and Na+/K+-ATPase) on the host, possibly linked to inflammatory processes caused by the high degree of parasitism. In the coming years, these conditions may result in losses of performance for this species, and as such will represent ecological damage and economical losses, and result in a possible vulnerability in relation to food security.

Temporal exposure to malathion: Biochemical changes in the Amazonian fish tambaqui, Colossoma macropomum.

The main toxicity mechanism of organophosphate insecticides such as malathion is the acetylcholinesterase enzyme inhibition. However, fish responses to organophosphates may vary depending on the activation of different defense mechanisms as well as the length of exposure. As such, the evaluation of acetylcholinesterase activity, in combination with the evaluation of biotransformation and antioxidants enzymes levels, is useful for indicating damage in fish exposed to this insecticide. Moreover, evaluating mitochondrial activity might evidence how the hierarchic responses occur in relation to the length of time that the fish is exposed. Therefore, the aim of our study is to evaluate whether the length of exposure to malathion differentially affects the biochemical responses of tambaqui. Our hypothesis is that the physiological alterations due to exposure are time dependent. Fish were exposed to sublethal concentrations of the insecticide during 6, 12, 24, 36, and 48 h. Contrary to expectations, there was no acetylcholinesterase activity inhibition during the experiment, which indicates an absence of neurotoxicity. Phase II biotransformation mechanism was activated early, especially in the liver. Oxidative damage was evident in the first hours of exposure and was concurrent with the activation of antioxidant enzymes. Mitochondrial bioenergetics were differentially affected by the length of exposure. The data suggest that the tambaqui regulates mitochondrial respiration differently over time, seeking to maintain homeostasis and ATP demand, and ensures the activation of response mechanisms, thus minimizing oxidative damage and avoiding the neurotoxicity of malathion.

Vitrification of canine ovarian tissue using the Ovarian Tissue Cryosystem (OTC) device.

The present study evaluated the effect of Ovarian Tissue Cryosystem (OTC) on follicular morphology and density, as well as on stromal cell density of vitrified canine ovarian tissue. Canine ovarian fragments collected from adult female dogs in stages of the random oestrous cycle were fixed (FC, fresh control) or vitrified (VIT) with an OTC device. After vitrification and warming, the fragments were fixed for histological analysis. Overall, the mean percentage of normal pre-antral follicles decreased after vitrification procedure (FC: 74.5% ± 1.6% vs. VIT: 52.05% ± 1.5%). Although the rates of normal primordial (71.1% ± 1.8%) and secondary (0.7% ± 0.4%) follicles vitrified showed a reduction (p < .05), vitrification using OTC showed considerable preservation of follicles, when compared to the fresh control (81.1% ± 1.5% and 2.3% ± 0.6%, respectively). The mean follicular density was maintained after vitrification (FC: 199.65 ± 12.8 vs. VIT: 199.68 ± 10.8), whereas the stromal cell density decreased in the VIT group. Based on the results, we recommend the use of OTC for vitrification of canine ovarian tissue.

Alpha Lipoic Acid Supplementation Improves Ovarian Tissue Vitrification Outcome: An Alternative to Preserve the Ovarian Function of Morada Nova Ewe.

This study evaluated the effect of adding alpha lipoic acid (ALA) to the vitrification solution of sheep ovarian tissue on 7 days of in vitro culture or 15 days of xenotransplantion. ALA was used at two different concentrations (100 µM: ALA100 and 150 µM: ALA150). Ovarian tissue was evaluated by classical histology (follicular morphology, development, and stromal cell density); immunohistochemistry for forkhead box O3a (FOXO3a); Ki67 (cell proliferation); cluster of differentiation 31 (CD31); and alpha smooth muscle actin (α-SMA). Reactive oxygen species (ROS) levels in ovarian tissue, as well as malondialdehyde (MDA) and nitrite levels in the culture medium, were assessed. Similar percentage of morphologically normal follicles was found in the vitrified ovarian tissue in the presence of ALA100 or ALA150 after in vitro culture or xenotransplantation. Follicular development from all treatments was higher (P < 0.05) than the control group. Moreover, an activation of primordial follicles was observed by FOXO3a. Stromal cell density and immunostaining for Ki67 and CD31 were significantly higher (P < 0.05) in ALA150 vitrified tissue. No difference (P > 0.05) was found in α-SMA between ALA concentrations after in vitro culture or xenograft. ROS levels in the ovarian tissue were similar (P > 0.05) in all treatments, as well as MDA and nitrite levels after 7 days of culture. We concluded that the addition of ALA 150 is able to better preserve the stromal cell density favoring granulosa cell proliferation and neovascularization.

Equine ovarian tissue xenografting: impacts of cooling, vitrification, and VEGF.

Ovarian tissue transplantation methods using cooled and cryopreserved samples have been attractive options for fertility preservation in animal models and humans. The aim of this study was to evaluate the impact of previous exposure to cooling, cryopreservation, and VEGF on the overall efficiency of equine ovarian tissue after heterotopic xenotransplantation in mice. The end points evaluated were follicular morphology and development, follicular and stromal cell densities, angiogenesis (i.e. the density of new and mature blood vessels), collagen types I and III fiber densities, and total fibrosis. Ovaries of adult mares were harvested after ovariectomy, and ovarian fragments were xenografted in the i.p. wall of BALB nude mice. Ten types of treatments involving different combinations of cooling, cryopreservation, xenografting procedures, and VEGF exposure were compared. The novel aspect of this study was the use of equine ovarian tissue xenotransplantation in mice, challenging the fragments with different combinations of treatments. The main findings were (i) cooling but not cryopreservation was effective in preserving the follicular morphology, (ii) a greater percentage of developing follicles but lower follicular and stromal cell densities were observed after ovarian tissue engraftment, (iii) exposure to VEGF increased new and mature vessels in cryopreserved-transplanted tissue, and (iv) an appropriate balance in the collagen types I and III fiber ratio in cooling-transplanted tissue was observed after exposure to VEGF. This study contributes to advancing knowledge in the preservation of ovarian tissue after cooling-cryopreservation and transplantation aiming to be applied to genetically superior/valuable horses, livestock, endangered animals, and, possibly, humans. LAY SUMMARY: Due to ethical limitations involving humans, the female horse (mare) has recently emerged as an alternative model for reproductive comparisons with women to optimize fertility restoration using ovarian tissue transplantation techniques. This study determined if ovarian tissue from donor mares (n = 3), exposed or not to vascular endothelial growth factor (VEGF) before transplantation, better survives for 7 days after transplantation into mouse hosts (n = 12). Tissues submitted to different combinations of cooling, freezing, and transplanting treatments, along with control groups, were evaluated using the parameters morphology, development, the density of immature eggs (follicles), the density of supportive (stromal) cells, collagen protein proportions, and density of blood vessels. Frozen-thawed treatments had lower percentages of normal follicles. Exposure to VEGF increased blood vessel densities in frozen tissue and favored adequate collagen levels in cooled-transplanted treatments. In conclusion, VEGF exposure seems to be beneficial for mare ovarian tissue transplantation and warrants further investigation.

Ecophysiology, genotoxicity, histopathology, and gene responses of naphthalene injected Colossoma macropomum (Cuvier, 1818) exposed to hypoxia.

The present study aimed to evaluate the biological responses of Colossoma macropomum to naphthalene injection and subsequent hypoxia exposure, emphasizing the expression of the tumor suppressor gene tp53. Tambaquis were intraperitoneally injected with naphthalene (50 mg/kg) and, after 96 hours, the fish were transferred to respirometry chambers and, submitted to progressive hypoxia for the determination of critical PO2. In a subsequent experiment, the fish received an intraperitoneal injection of naphthalene and were kept for 96 hours under normoxia. Successively, fish were challenged with acute hypoxia (PO2

Effects of partially saturated conditions on the metabolically active microbiome and on nitrogen removal in vertical subsurface flow constructed wetlands.

Nitrogen dynamics and its association to metabolically active microbial populations were assessed in two vertical subsurface vertical flow (VF) wetlands treating urban wastewater. These VF wetlands were operated in parallel with unsaturated (UVF) and partially saturated (SVF) configurations. The SVF wetland exhibited almost 2-fold higher total nitrogen removal rate (5 g TN m-2 d-1) in relation to the UVF wetland (3 g TN m-2 d-1), as well as a low NOx-N accumulation (1 mg L-1 vs. 26 mg L-1 in SVF and UVF wetland effluents, respectively). After 6 months of operation, ammonia oxidizing prokaryotes (AOP) and nitrite oxidizing bacteria (NOB) displayed an important role in both wetlands. Oxygen availability and ammonia limiting conditions promoted shifts on the metabolically active nitrifying community within 'nitrification aggregates' of wetland biofilms. Ammonia oxidizing archaea (AOA) and Nitrospira spp. overcame ammonia oxidizing bacteria (AOB) in the oxic layers of both wetlands. Microbial quantitative and diversity assessments revealed a positive correlation between Nitrobacter and AOA, whereas Nitrospira resulted negatively correlated with Nitrobacter and AOB populations. The denitrifying gene expression was enhanced mainly in the bottom layer of the SVF wetland, in concomitance with the depletion of NOx-N from wastewater. Functional gene expression of nitrifying and denitrifying populations combined with the active microbiome diversity brought new insights on the microbial nitrogen-cycling occurring within VF wetland biofilms under different operational conditions.

Unraveling the active microbial populations involved in nitrogen utilization in a vertical subsurface flow constructed wetland treating urban wastewater.

The dynamics of the active microbial populations involved in nitrogen transformation in a vertical subsurface flow constructed wetland (VF) treating urban wastewater was assessed. The wetland (1.5m2) operated under average loads of 130gCODm-2d-1 and 17gTNm-2d-1 in Period I, and 80gCODm-2d-1 and 19gTNm-2d-1 in Period II. The hydraulic loading rate (HLR) was 375mmd-1 and C/N ratio was 2 in both periods. Samples for microbial characterization were collected from the filter medium (top and bottom layers) of the wetland, water influent and effluent at the end of Periods I (Jun-Oct) and II (Nov-Jan). The combination of qPCR and high-throughput sequencing (NGS, MiSeq) assessment at DNA and RNA level of 16S rRNA genes and nitrogen-based functional genes (amoA and nosZ-clade I) revealed that nitrification was associated both with ammonia-oxidizing bacteria (AOB) (Nitrosospira) and ammonia-oxidizing archaea (AOA) (Nitrososphaeraceae), and nitrite-oxidizing bacteria (NOB) such as Nitrobacter. Considering the active abundance (based in amoA transcripts), the AOA population revealed to be more stable than AOB in both periods and depths of the wetland, being less affected by the organic loading rate (OLR). Although denitrifying bacteria (nosZ copies and transcripts) were actively detected in all depths, the denitrification process was low (removal of 2gTNm-2d-1 for both periods) concomitant with NOx-N accumulation in the effluent. Overall, AOA, AOB and denitrifying bacteria (nosZ) were observed to be more active in bottom than in top layer at lower OLR (Period II). A proper design of OLR and HLR seems to be crucial to control the activity of microbial biofilms in VF wetlands on the basis of oxygen, organic-carbon and NOx-N forms, to improve their capacity for total nitrogen removal.