Cellular Energy Allocation Research Articles

Seasonal Bioenergetic Changes in Saccostrea glomerata from Two Age Cohorts Farmed in Clyde River, Nsw, Australia

The study measured the mean and ranges of seasonal changes in whole body energetic values in two age groups of the Sydney rock oyster Saccostrea glomerata from the Clyde River, NSW, Australia. Oysters were collected over four seasons from two aquaculture-relevant age groups (24 and 36 mo) identified as key harvesting ages. Protein, lipid, glycogen energy equivalents, and electron transport system (ETS) activity were determined to identify seasonal changes in energy stores and consumption and to calculate cellular energy allocation. There were clear seasonal trends in energy changes, with glycogen and lipid energy equivalents highest in summer, decreasing to their lowest concentrations in autumn, and then slowly increasing through winter and into spring. Protein energy equivalents and ETS activity demonstrated an opposite trend, with increases in autumn and stable through the other seasons. Cellular energy allocation showed seasonal change, with highest values over summer, gradually decreasing in autumn and winter, and then starting to increase again in spring. These results indicate that seasonal changes in temperature, salinity, and food availability as well as reproductive cycles have a significant influence on S. glomerata energy storage and use. Developing an understanding of energetic responses in bivalves in response to environmental conditions can facilitate a more detailed understanding of growth potential and responses to aquaculture practices such as stocking density. As well, understanding natural energetic fluctuations allows a baseline for the assessment of other field-based stress responses such as anthropogenic contamination in S. glomerata.

English

Under stressful conditions (toxicity), organisms often try to detoxify by mobilizing available energy sources with costs to various metabolic functions, such as growth or reproduction. Cellular energy allocation (CEA) is a methodology used to evaluate the energetic status and which relates with organisms’ overall condition and response to toxic stress. It consists of the integration of the energy reserves available (Ea) and energy consumption (Ec). The effects of different sublethal concentrations (0.828, 0.0828, and 0.00828 mg/l) of cadmium (Cd) was evaluated on the total energy budget of African catfish (Clarias gariepinus) juveniles over 56-day period of exposure. A total of 180 C. gariepinus were exposed under the static renewal assay, and parameters measured were the total energy reserves available (protein, carbohydrate and lipid budgets) and the energy consumption (based on electron transport system activity assay) being further integrated to obtain the CEA. The Bradford method, Phenol-sulphuric acid method and Bligh and Dyer method were used to evaluate the protein, carbohydrate, and lipid contents, respectively of the test animals. Significant changes (p<0.05) in energy reserves and energy consumption were observed upon Cd exposure. Among the three energy reserves obtained, carbohydrate offered the least energy fraction (0.23%), and followed by protein (20.27%). The highest energy fraction was offered by lipid (79.50%). The effect of cadmium brought about a tremendous decrease in Carbohydrate (from 0.04 kj/g in day 7 to 0.01 kj/g in day 28). Lipid always stepped up (from 9.84 kj/g on day 7 to 34.48 jk/g on day 28) to compliment energy loss whenever carbohydrate was exhausted. Protein was least affected (from 1.78 kj/g on day 7 to 1.23 kj/g on day 28) with mild reduction in its reserve. Increased energy consumption was recorded amongst the exposed groups, with the highest concentration (0.828 mg/l) offering the most Ec of 58.54 kj/g on day 56. Significant reduction in CEA occurred across the exposed groups as Ea and Ec dwindled.  These results have shown the adverse effects of Cd on the energy status of C. gariepinus and the sensitive effectiveness of CEA technique in assessing the toxic effects of metallic pollutants on freshwater animals. Key words: Clarias gariepinus, cellular energy allocation, energy available, electron transport system, biomarker, energy metabolism. &nbsp

Additive bioenergetic responses to a pesticide and predation risk in an aquatic insect

Ignoring natural stressors such as predation risk may contribute to the failure of ecological risk assessment of pesticides to protect freshwater biodiversity. To better understand combined effects of multiple stressors, bioenergetic responses are important as these inform about the balance between energy input and consumption, and provide a unifying mechanism to integrate the impact of multiple stressors with different modes of action. We studied in Enallagma cyathigerum damselfly larvae the single and combined effects of exposure to the pesticide chlorpyrifos and predation risk on life history (survival and growth rate) and bioenergetic response variables at the organismal level (assimilation and conversion efficiency) and the cellular level (cellular energy allocation CEA, energy storage Ea, and energy consumption Ec). Chlorpyrifos exposure almost halved the survival of the damselfly larvae, while predation risk had no effect on survival. Both exposure to the pesticide and to predation risk reduced larval growth rates. This was caused by a reduced conversion efficiency under chlorpyrifos exposure, and by a reduced assimilation efficiency under predation risk. Both chlorpyrifos and predation risk reduced the CEA because of a decreased Ea, and for chlorpyrifos also an increased Ec. The lower Ea was driven by reductions in the fat and glycogen contents. Effects of the pesticide and predation risk were consistently additive and for most variables the strongest response was detected when both stressors were present. The absence of any synergisms may be explained by the high mortality and hypometabolism caused by the pesticide. Our results indicate that CEA can be a sensitive biomarker to evaluate effects of not only contaminants but also natural stressors, such as predation risk, and their combined impact on organisms.

Long-term effects of oxytetracycline exposure in zebrafish: A multi-level perspective

Oxytetracycline (OTC) is a broad-spectrum antibiotic widely used in livestock production. Like many other pharmaceuticals, OTC is not completely metabolized by the organism and thus, increasing amounts of the compound are being detected in the aquatic environment. The assessment of the environmental risk of pharmaceuticals is hindered by their very low concentrations and specific modes of action and thus relevant exposure scenarios and sensitive endpoints are needed. Thus, this work aimed to study the long-term effect of OTC exposure in zebrafish (at behavior and biochemical levels) and associated bacterial communities (fish gut and water bacterial communities). Results revealed that at behavioral level, boldness increase (manifested by increased exploratory behavior of a new environment) was observed in fish exposed to low OTC concentrations. Moreover, changes in fish swimming pattern were observed in light periods (increased stress response: hyperactivity and freezing) probably due to photo-sensibility conferred by OTC exposure. Effects at biochemical level suggest that long-term exposure to OTC interfere with cellular energy allocation mainly by reducing lipids levels and increasing energy consumption. Moreover, evidences of oxidative damage were also observed (reduced levels of TG, GST and CAT). The analysis of water and gut microbiome revealed changes in the structure and diversity of bacterial communities potentially leading to changes in communities' biological function. Some of the effects were observed at the lowest concentration tested, 0.1 μg/L which is a concentration already detected in the environment and thus clearly demonstrating the need of a serious ecotoxicological assessment of OTC effects on non-target organisms.

The transcriptional response of the Pacific oyster Crassostrea gigas under simultaneous bacterial and heat stresses

Bacterial infection and heat stress are considered as two major environmental threats for the aquaculture industry of oyster Crassostrea gigas. In the present study, the expression profiles of mRNA transcripts in the hemocytes of oysters under bacterial challenge and heat stress were examined by next-generation sequencing. There were 21,095, 21,957 and 21,141 transcripts identified in the hemocytes of oysters from three groups, respectively, including control group (designated as Con group), Vibrio splendidus challenge group (Bac group), and bacterial and heat stress combined treatment group (BacHeat group). There were 4610, 5093 and 5149 differentially expressed transcripts (DTs) in the three pairwise comparisons Con/Bac, Con/BacHeat and Bac/BacHeat, respectively. The main enriched GO terms in biological process category of the DTs included the metabolic processes, cellular process, response to stimulus and immune system process. The expression patterns of DTs involved in pattern recognition, immune signal transduction and energy metabolic indicated that the immune response to bacterial challenge was disturbed under acute heat stress, which was also confirmed by quantitative real-time PCR. The neuroendocrine immunomodulation, especially the catecholaminergic regulation, played indispensable roles in stress response. The total energy reserves as well as cellular energy allocation (CEA) in hepatopancreas of oysters decreased remarkably especially in BacHeat group, while the energy consumption generally increased, suggesting that the immune defense against the simultaneous stimulation of pathogen and heat stress imposed greater costs on oyster's energy expenditure than a single stressor. These results above indicated that, the heat stress disturbed the normal expression of genes involved in immune response and energy metabolism, accelerated energy consumption and broke the metabolic balance, leading to a decline in resilience to infection and mass mortality of oyster in summer.

Energetic cost derived of short-term starvation on marine crustacean Palaemon elegans

Event Abstract Back to Event Energetic cost derived of short-term starvation on marine crustacean Palaemon elegans Ico Martinez1*, Alicia Herrera1, Inmaculada Herrera1, Daniel R. Bondyale-Juez1, Vanesa Romero-Kutzner1, Mayte Tames-Espinosa1, Theodore T. Packard1 and May Gómez Cabrera1 1 University of Las Palmas de Gran Canaria, Spain Starvation has a major impact on physiological processes in marine plankton and these processes have a major impact on the energy transfer through marine ecosystems. However, quantifying and understanding how starvation changes different aspects of the energy transfer has been retarded because planktologists have largely relied on biomass fractionation to shed light on it. Here, we experiment with the cellular energy allocation approach (CEA index), the respiratory electron transport system activity (ETS), and organic analysis of lipids (Lip), carbohydrates (Carb) and proteins (Prot) to help further our understanding of the energy transfer in the caridean shrimp, Palaemon elegans. We analyzed the effect of 72 hours of starvation using the CEA index that is calculated as the ratio between energy available to energy consumption. It allows us to evaluate the net energy budget (Verslycke and Janssen, 2002). The energy available (Ea) was determined from Lip, Carb, and Prot content, where Ea = Lip + Carb + Prot. Energy consumption (Ec) was calculated from the activity of ETS. All the parameters were transformed into energy equivalents using their respective energy of combustion (Gnaiger, 1983). Proteins were the main energy source (1450.8±355.7 mJ•mgWW-1), followed by lipids (727.2±124.4 mJ•mgWW-1) and carbohydrates (82.1±17.9 mJ•mgWW-1). After 24 hours of starvation, the carbohydrates dropped by 38.2%, a significant decrease. After 48 hours, the proteins had decreased by 36.0%. The lipids were the last to decrease. After 72 hours they had dropped by 42.3%. Adding these organic constituents to calculate the Ea revealed a significant decreasing trend of 34.42% at 72 hours (from 2260.1±485.5 to 1482.3±120.9 mJ•mgWW-1). In contrast, the Ec, as ETS activity, showed a significant increase at 24 hours (from 85.2±16.6 to 158.0±40.4 mJ•h-1•mgWW-1) that stabilized after 72 hours. Overall, the energy balance (Ea/Ec) showed that the energy available was higher than the energy consumption during the experiment. Ea/Ec ratio decreased for the first 24 hours, then remained stable for the next 48 hours. This portrait of energy regulation shows a rapid shift mechanism of P. elegans under sudden adverse conditions. References Gnaiger, E. (1983). ‘Calculation of energetic and biochemical equivalents of respiratory oxygen consumption’, in Polarographic oxygen sensors (Springer), 337–345. Verslycke, T., and Janssen, C. R. (2002). Effects of a changing abiotic environment on the energy metabolism in the estuarine mysid shrimp Neomysis integer (Crustacea: Mysidacea). J. Exp. Mar. Bio. Ecol. 279, 61–72. Keywords: CEA index, Energy available, energy consumption, Palaemon elegans, Starvation Conference: XX Iberian Symposium on Marine Biology Studies (SIEBM XX) , Braga, Portugal, 9 Sep - 12 Sep, 2019. Presentation Type: Poster Presentation Topic: Ecology, Biodiversity and Vulnerable Ecosystems Citation: Martinez I, Herrera A, Herrera I, Bondyale-Juez DR, Romero-Kutzner V, Tames-Espinosa M, Packard TT and Gómez Cabrera M (2019). Energetic cost derived of short-term starvation on marine crustacean Palaemon elegans. Front. Mar. Sci. Conference Abstract: XX Iberian Symposium on Marine Biology Studies (SIEBM XX) . doi: 10.3389/conf.fmars.2019.08.00191 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 14 May 2019; Published Online: 27 Sep 2019. * Correspondence: Mrs. Ico Martinez, University of Las Palmas de Gran Canaria, Las Palmas de Gran Canaria, Spain, ico.martinez@ulpgc.es Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Ico Martinez Alicia Herrera Inmaculada Herrera Daniel R Bondyale-Juez Vanesa Romero-Kutzner Mayte Tames-Espinosa Theodore T Packard May Gómez Cabrera Google Ico Martinez Alicia Herrera Inmaculada Herrera Daniel R Bondyale-Juez Vanesa Romero-Kutzner Mayte Tames-Espinosa Theodore T Packard May Gómez Cabrera Google Scholar Ico Martinez Alicia Herrera Inmaculada Herrera Daniel R Bondyale-Juez Vanesa Romero-Kutzner Mayte Tames-Espinosa Theodore T Packard May Gómez Cabrera PubMed Ico Martinez Alicia Herrera Inmaculada Herrera Daniel R Bondyale-Juez Vanesa Romero-Kutzner Mayte Tames-Espinosa Theodore T Packard May Gómez Cabrera Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Bioenergetics-adverse outcome pathway: Linking organismal and suborganismal energetic endpoints to adverse outcomes.

Adverse outcome pathways (AOPs) link toxicity across levels of biological organization, and thereby facilitate the development of suborganismal responses predictive of whole-organism toxicity and provide the mechanistic information necessary for science-based extrapolation to population-level effects. Thus far AOPs have characterized various acute and chronic toxicity pathways; however, the potential for AOPs to explicitly characterize indirect, energy-mediated effects from toxicants has yet to be fully explored. Indeed, although exposure to contaminants can alter an organism's energy budget, energetic endpoints are rarely incorporated into ecological risk assessment because there is not an integrative framework for linking energetic effects to organismal endpoints relevant to risk assessment (e.g., survival, reproduction, growth). In the present analysis, we developed a generalized bioenergetics-AOP in an effort to make better use of energetic endpoints in risk assessment, specifically exposure scenarios that generate an energetic burden to organisms. To evaluate empirical support for a bioenergetics-AOP, we analyzed published data for links between energetic endpoints across levels of biological organization. We found correlations between 1) cellular energy allocation and whole-animal growth, and 2) metabolic rate and scope for growth. Moreover, we reviewed literature linking energy availability to nontraditional toxicological endpoints (e.g., locomotor performance), and found evidence that toxicants impair aerobic performance and activity. We conclude by highlighting current knowledge gaps that should be addressed to develop specific bioenergetics-AOPs. Environ Toxicol Chem 2019;38:27-45. © 2018 SETAC.

Linking organochlorine exposure to biomarker response patterns in Anurans: a case study of Müller's clawed frog (Xenopus muelleri) from a tropical malaria vector control region.

Organochlorine pesticides are highly persistent in aquatic ecosystems. Amphibians, specifically anurans, play an intricate part in the aquatic food web, and have very permeable skin which makes them prone to bioaccumulation of persistent pollutants. In this study the bioaccumulation of various legacy organochlorine pesticides (OCPs)-including dichlorodiphenyltrichloroethane (DDT), currently used for malaria vector control (MVC)-was assessed along with a set of biomarker responses in Müller's clawed frog Xenopus muelleri collected from the lower Phongolo River floodplain in South Africa. Possible relationships between bioaccumulation and biomarkers (of exposure, oxidative stress biomarkers, and cellular energy allocation) alongside their temporal changes were investigated. The OCP concentrations showed a significant increase over time for the duration of the study. The increase correlated negatively with rainfall from the region. DDT levels were well below expected effects levels with p,p-DDE being the main contributing metabolite. The results of this study indicate OCPs actively accumulate at sub-lethal levels in aquatic frogs from the study area, while showing possible relations towards some of the biochemical stress responses measured. Most notable were negative relationships indicated between p,p-DDE and acetylcholinesterase, malondialdehyde, and carbohydrates and protein energy availability. Levels of DDT were not found to be significantly higher than other legacy pesticides in the frog tissue, although evidence of newly introduced DDT in the frog tissue was found. Further investigation about sub-lethal effects of these pesticides on anurans is required to gain better insight into their full impact on animal livelihood.

Oxidative stress responses and cellular energy allocation changes in microalgae following exposure to widely used human antibiotics

The individual effect of four human antibiotics on the microalgae Raphidocelis subcapitata was investigated following a 120-h exposure. The effects were assessed by analyzing growth, and biochemical parameters related with: 1) antioxidant capacity and oxidative damage by measuring superoxide dismutase (SOD) activity and lipid peroxidation (LPO) levels; and 2) cellular energy allocation (CEA) by quantifying the content in energy reserves, which represents the energy available (Ea), and the electron transport system activity that represents a measure of oxygen and cellular energy consumption (Ec). Growth yield inhibitory concentrations of sulfamethoxazole (18–30%), clarithromycin (28.7%), ciprofloxacin (28%) and erythromycin (17–39%) were found to elicit a considerable increase in Ec, thereby causing a significant decrease in the CEA. The elevated Ec can be a result of the need to respond to oxidative stress occurring under those conditions given the significant increase in SOD activity at these levels. For sulfamethoxazole, erythromycin and ciprofloxacin, the antioxidant responses do not seem to be enough to cope with the reactive oxygen species and prevent oxidative damage, given the elevated LPO levels observed. A stimulatory effect on growth yield was observed (up to 16%) at ciprofloxacin lowest concentration, which highly correlated with the increase in CEA. Based on the no observed effect concentration (NOECs) and/or effective concentration (EC10) results, Ec, SOD and CEA were more sensitive than the classical endpoint of growth rate for all the tested antibiotics. By revealing the antibiotic stress effects in R. subcapitata at the cellular level, this study suggests CEA as a more reliable indicator of the organisms’ physiological status.

Cellular energy allocation analysis of multiple marine bivalves using near infrared spectroscopy

This paper proposes an alternative procedure for calculating Cellular Energy Allocation (CEA) in marine bivalve species. CEA is a measure of the difference in total energy available and energy consumption and is used to assess the health of organism fitness for growth and reproduction. In this study near infra-red spectral (NIRS) quantitative data-driven modelling techniques were applied to total energy available (total protein, lipid and glycogen content) and energy consumption (electron transport system activity (ETS)) of five marine bivalve species (Saccostrea glomerata, Ostrea angasi, Crassostrea gigas, Mytilus galloprovincialis and Anadara trapezia) to produce models for measuring each bioenergetic component and calculating CEA, a biomarker of energetic stress response. Comparison of predicted to measured data showed low prediction errors (RMSEP) and high ratio of predictive error to inter-quartile distance (RPIQ) values. RMSEP for protein, lipid, glycogen and ETS models were 8.7%, 6.8%, 7.6% and 12% of the dynamic range of the validation datasets respectively while RPIQ were 3.6, 5.2, 4 and 3.7 respectively. Conversion of model outputs to energetic equivalents and calculation of total energy stores as per the CEA method demonstrate a close relationship between measured and modelled results (105%±3%). These metrics indicate that the four energy models are accurate, robust and reliable for quantitative application to all five species within the sample variation of the energetic storage measures. Applying NIRS quantitative models using freeze dried tissue eliminates water interference in NIRS capture and provides improved sample homogeneity for both spectral capture and chemical analysis. This technique enables simultaneous analyses of protein, lipid, glycogen and ETS activity to be achieved with a much higher throughput of samples (100–200 samples daily) than conventional energetic storage analysis techniques. In addition, the technique is non-destructive to tissue samples and freeze dried samples can be stored for additional analyses. The value of CEA as a response measure in S. glomerata are included, with examples of changes in CEA over four seasons and the response of oysters to a known metal contamination gradient.

Toxicity of dyes to zebrafish at the biochemical level: Cellular energy allocation and neurotoxicity.

Dyes are widely distributed worldwide, and can be found in wastewaters resulting from industrial or urban effluents. Dyes are of particular concern as contaminants of the aquatic environment, since their toxicity remain poorly understood. Thus, the current study was designed to assess the effects induced by the synthetic azo dye Basic Red 51 (BR51) and by the natural naphthoquinone dye erythrostominone (ERY) on zebrafish early life stages (Danio rerio) at different biological organization levels, i.e., studying how changes in biochemical parameters of important physiological functions (neurotransmission and cellular energy allocation) may be associated with behavior alterations (swimming activity). This approach was also used to assess the effects of ERY after its photodegradation resulting in a colorless product(s) (DERY). Results showed that after 96 h exposure to BR51 and Ery, zebrafish embryos consumed less energy (LOEC = 7.5 mg/L), despite the unaltered levels of available energy (carbohydrates, lipids and proteins). Hence, cellular energy allocation (CEA) was significantly increased. On the other hand, only ERY decreased the acetylcholinesterase activity (LOEC = 15 mg/L). Despite that, zebrafish larvae exposed to both dyes until 144 h were less active. In contrast, DERY did not affect any parameter measured. These results indicate an association between a decrease consumption of energy and decrease swimming activity resulting from an environmental stress condition, independently of the neurotoxicity of the dyes. Degradation of ERY by light prevented all toxic effects previously observed, suggesting a cheap, fast and easy alternative treatment of effluents containing this natural dye. All tools assessed in our current study were sensitive as early-warning endpoints of dyes toxicity on zebrafish early life stages, and suggest that the CEA assay might be useful to predict effects on locomotor activity when cholinergic damage is absent.

Plasticity of energy reserves and metabolic performance of discus fish (Symphysodon aequifasciatus) exposed to low-temperature stress

A 28-day experiment was conducted to investigate the temporal variations in cellular energy allocation (CEA), oxygen consumption rate (OCR) and activities of energy metabolism related enzymes in discus fish (Symphysodon aequifasciatus) exposed to decreased water temperatures. The steady temperature decrease (1°C/day) from control (28°C) to low temperatures (24 or 20°C) significantly reduced the initial energy reserves (mainly lipids and carbohydrates) while significantly increased the energy consumption (respiratory electron transport system (ETS) activity) in the muscle tissue. Accordingly, on day 0 the CEA was slightly reduced with decreased temperatures. After 28days of cold acclimation, however, the initial differences in energy reserves, ETS activity and CEA between treatments were decreased. Concurrently, the OCR was reduced with decreased temperatures, suggesting a decreased metabolic rate. During cold acclimation, however, activities of hepatic alkaline phosphatase (ALP), lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were generally increased with decreased temperatures, indicating a compensation for the decreased metabolic rate. Linear discriminant analysis (LDA) showed that the fish exposed to 24°C only on day 14 was separated from the initial day 0 while those exposed to 20°C on both days 14 and 28 were separated from the initial day 0, with LDH and ALP activities being the two most discriminant factors. Moreover, after cold acclimation the fish exposed to 24°C showed comparable growth rates with those exposed to 28°C. These findings reveal that S. aequifasciatus could recover from the initial energy disturbances resulted from decreased temperatures. The enhanced metabolic activity in response to 24°C was well within the homeostatic range. It might be promising to culture the juvenile S. aequifasciatus at water temperature as low as 24°C.

Adaptive response under multiple stress exposure in fish: From the molecular to individual level

Aquatic systems are subjected to various sources of stress due to global changes, such as increasing temperature and pollution. A major challenge for the next decade will be to evaluate the combined effects of these multiple stressors on organisms and ecosystems. For organisms submitted to chemical, biological or physical stressors, the capacity to set up an efficient adaptive response is a fundamental prerequisite for their long-term survival and performance. In this study, goldfish (Carassius auratus) were subjected to individual and combined pesticide mixtures and increased temperatures to evaluate their adaptive response in multistress conditions from the molecular to the individual level. Fish were exposed for 16 days to a mixture of pesticides at environmental relevant concentrations (S-metolachlor, isoproturon, linuron, atrazine-desethyl, aclonifen, pendimethalin and tebuconazole) and at two temperatures (22 °C and 32 °C). Three major physiological traits of the stress response were measured: the hormonal response (i.e. plasma cortisol), the metabolic balance from molecular to individuals' levels (metabolomics, cellular energy allocation, energy reserves and global condition indexes), and the cellular defense system induction (SOD, CAT and GST). Results show that (1) environmentally relevant concentrations of pesticides lead to significant responses in fish at all biological levels; (2) the metabolic response depends on the nature of stress (thermal vs. chemical); and (3) fish may be unable to set up an efficient adaptive response when chemical and thermal stresses were combined, with adverse outcomes at the individuals’ level.

Effect of Cu and Ni on cellular energy allocation in Enchytraeus albidus.

Effects of nickel and copper on Enchytraeus albidus (Oligochaeta) were investigated using the cellular energy allocation approach. This methodology is used to evaluate the energetic status of an organism and is indicative of its overall condition. Enchytraeids were exposed to the reproduction Effect Concentrations (EC50 and EC90), and the parameters measured were the total energy reserves available (protein, carbohydrate and lipid budgets) and the energy consumption [based on electron transport system activity] which were further integrated to obtain the cellular energy allocation over different periods of exposure (0-2, 2-4 and 4-8 days). Carbohydrates (in comparison to lipids and proteins) were the only energy source mobilized in the case of nickel within 8 days of exposure. For copper exposure, protein budgets were also strongly reduced. Energy consumption increased in a time and dose-dependent way for copper and in the longer exposure period (4-8 days) at the EC90 for Ni exposure, indicating that this is a good biomarker for effects of short-time metal exposure, while cellular energy allocation was only significantly reduced for the EC90 of copper (4-8 days) and EC50 of nickel (2-4 days). The effects of nickel at concentrations causing 50 and 90 % decrease in reproduction were likely not due to the changes in cellular energy allocation within 8 days of exposure.

Energy reserves and cellular energy allocation studies: Should food supply be provided?

Energy budget studies offer important information in terms of organisms' fitness and this has become an increasingly common stress marker. The method is well developed but the issue of food supply (addition or not) during exposure is not consensual. Moreover, chemicals such as cadmium (Cd) are known to affect food uptake, e.g. via feeding inhibition, hence energy budget results could be because of decreased uptake and not direct toxicity. On the other hand, food deprivation can be a stressor itself and bias results. In the present study we compared exposure with (F) and without food (NF) along time (2, 4 and 8days). The Cellular Energy Allocation (CEA) method was used to study the effects of Cd in the soil standard species Enchytraeus albidus (Oligochaeta). In control conditions (un-spiked soil), carbohydrate and lipid budgets were mobilized in NF. When testing Cd, variations in carbohydrates and lipid budgets depend on the presence of food and time of exposure. The main hypothesized mechanisms triggered by Cd exposure were similar (i.e. induction of protein synthesis and increase in energy consumption). Differences between F and NF over time indicate that the process of mobilization/preservation of energy reserves depends on the food/energy intake (e.g. in Cd exposed organisms, mobilization of proteins occurred within 2days when food was present, while in the absence of food carbohydrate and protein budgets were mobilized from 2 to 4days). Comparisons between F-NF studies should not be done directly. Moreover, we recommend exposure without food because it allows a better discrimination of effects (in particular within periods of exposure).

The effects of temperature, soil moisture and UV radiation on biomarkers and energy reserves of the isopod Porcellionides pruinosus

Terrestrial isopods from the species Porcellionides pruinosus were exposed to different ranges of temperature, soil moisture content and doses of UV radiation. For the temperature and soil moisture content experiments, organisms were sampled after 48h, 96h and 14days of exposure, whereas in the UV experiment, they were sampled at the end of the exposure periods, that consisted on a single-pulse with duration ranging from 30min to 8h. For each sampling time the acetylcholinesterase, glutathione S transferases, glutathione peroxidase and catalase activities were determined, as well as lipid peroxidation rate. Energy content (lipids, carbohydrates, proteins) and other energy related parameters: energy available, energy consumption and cellular energy allocation were also determined, along with mortality.The results obtained showed that increases in temperature will affect life traits and specific strategies for isopods to manage their energy budget, in order to handle oxidative stress. It also showed that this species is acclimated to lower moisture scenarios, whereas in case of flood scenarios the turnover point between optimal conditions and mortality is very narrow, which may lead to the local extinction of populations in specific micro-habitats. This study also showed that UV radiation also poses an important stressor for isopods that should be taken in consideration, as the actual doses nowadays present significant negative impact on these organisms.The study also emphasises that the effects of abiotic factors should be included and taken into consideration by policymakers and that the inclusion of abiotic effects in ecotoxicological tests should be included in the analysis of any stressor to improve chemical risk assessment.

MicroRNAs are involved in cadmium tolerance in Daphnia pulex

Daphnia can develop tolerance to cadmium (Cd) after multi-generational exposures. Until now, Cd tolerance in this crustacean was thought to be mainly due to its sequestration via induction of metallothioneins (MTs). Our research supports other studies showing microRNAs (miRNAs) also play a role in this enhanced tolerance. We induced Cd tolerance in Daphnia pulex after exposing them for 25 generations and examined the maintenance of enhanced Cd tolerance under a Cd-free environment for an additional three generations. Acute Cd tolerance as well as long-term effects on population dynamics were measured in selected generations via 48h LC50 tests and 21 d reproductive tests, respectively. Cd tolerance was associated with differential expression of 10 miRNAs (miR-2, miR-33, miR-92, miR-96, miR-153, miR-252, miR-279, miR-283, miR-305 and miR-615). Pathway analysis revealed these miRNAs might increase Cd tolerance by suppressing cellular growth and proliferation by GTPase and cuticle protein pathways, which switch cellular energy allocation to detoxification processes. Moreover, we found increased Cd tolerance is related with induction of MT3 and MT4 and a subsequent downregulation of MT1 and MT3 expression when animals are moved to a Cd-free environment. This is the first study linking aquatic invertebrate miRNAs with induced tolerance to environmental stressors.

오염 퇴적물 평가 기법으로서의 바지락 (Ruditapes philippinarum) 세포내 에너지 할당 (cellular energy allocation, CEA) 적용성 검토

오염 퇴적물 평가 기법으로서의 바지락 (Ruditapes philippinarum) 세포내 에너지 할당 (cellular energy allocation, CEA) 적용성 검토

Thermal stress effects on energy resource allocation and oxygen consumption rate in the juvenile sea cucumber, Holothuria scabra (Jaeger, 1833)

Water temperature is a key factor in aquaculture production of the commercially valuable sea cucumber Holothuria scabra. Knowledge is scarce about actual energetic costs that can be associated with internal acclimatization processes as a response to thermal extremes. In the present study changes in cellular energy allocation, oxygen consumption rate and energy related enzymes' activity (IDH and LDH) were measured in juvenile H. scabra, held at different temperatures: 21, 27 and 33°C. The results showed that the steady temperature change (1°C/day) to both temperature treatments, until reaching the testing temperatures (day 0), clearly affected cellular energy consumption and available energy reserves, measured in the respiratory tree and muscle tissue, respectively. However, 15 and 30days after acclimation, the initial differences in cellular energy allocation between treatments decreased. In contrast to the variations measured in cellular energy allocation, oxygen consumption was highest at 33°C and lowest at 21°C at all three measurement times. Moreover, a significant positive correlation between oxygen consumption rate and temperature was detected at day 15 and day 30. Likewise, a shift from anaerobic to aerobic energy metabolism, indicated by changes in LDH and IDH activities, was observed in the animals from the warm temperature treatment. Results imply that juvenile H. scabra were able to recover from initial disturbances in energy balance, caused by the incremental temperature change of ±6°C. Over the experimental period of 30days, elevated temperature did however, lead to a metabolic shift and more efficient energy turnover, indicated by changes in oxygen consumption rate, LDH and IDH. The synergy of cellular energy allocation and oxygen consumption proved to be a viable indicator to assess the capability of sea cucumbers like H. scabra to cope with extreme temperature conditions. Surprisingly, juvenile H. scabra were able to sustain their energy balance and oxygen consumption rate within the homeostatic range, even at 33°C. Thus, we assume that rearing temperatures of 33°C might be possible, which could improve aquaculture production of H. scabra. However, further research is required to understand the mechanisms and effects of acclimation under aquaculture conditions. Statement of relevanceThe sea cucumber H. scabra is considered a promising aquaculture candidate in the tropics. The commercial interest in H. scabra has led to a great number of attempts to culture this species e.g. in Madagascar, Tanzania, India and Indonesia, where the farming of this species also showed a great potential to increase the livelihood of the local community. Although, considerable research effort has been put into the advancement of the production cycle, fundamental knowledge, e.g. on physiological adjustments due to sub-optimal rearing conditions, remains scarce for H. scabra. Water temperature has been identified as crucial factor, determining production efficiency. In our manuscript we introduce an innovative approach, for the assessment of shifts in the energy budget as well as in metabolic pathways, to detect thermal stress in juvenile H. scabra. Our results show that the combined application of cellular energy allocation (CEA) and measurement of the oxygen consumption rate (OCR) as well as the activity of two metabolic key enzymes: iso-citrate dehydrogenase (IDH) and lactate dehydrogenase (LDH), is a strong analytical tool to evaluate whole animal homeostasis under thermal extremes. To our knowledge this is the first study in which the method of CEA was optimized for the application on isolated muscle and respiratory tree tissue of a sea cucumber, like H. scabra. Hence, our results provide original insights into the thermal acclimation physiology of H. scabra, which provides crucial knowledge for setting up optimal culture conditions for this highly valuable aquaculture candidate.Moreover, our results show that juvenile H. scabra were strongly affected by rapid temperature changes, exhibiting distinct differences in metabolic adjustments as response to short-term cold and warm acclimation, but were able to restore homeostatic conditions at constant cold and warm temperature extremes. Concerning aquaculture practices, these findings imply that CEA might be a good condition indicator to evaluate the overall metabolic status in sea cucumbers. Moreover, rapid temperature fluctuations seem to provoke much higher energetic costs in juvenile H. scabra than constant extreme temperature conditions, which is important to consider in daily management practices.The OCR revealed an enhanced metabolism at warmer temperature conditions, throughout the entire experimental period. Likewise, the warm exposed animals were shifting their energy turnover from a more anaerobic to a more aerobic state, indicated by changes in LDH and IDH activities, and showed increased foraging activity at all times. The combined outcome of CEA, OCR and activities of energy metabolism related enzymes indicates, that the increased metabolic activity in juvenile H. scabra, as response to warmer temperature, was well within the homeostatic range. Thus, contrary to our expectations, a rearing temperature of up to 33°C might presumably be favourable for the aquaculture of H. scabra.

Adaptations of enchytraeids to single and combined effects of physical and chemical stressors

Climate changes are expected to be greatest in the polar and temperate areas, where predictions point to an increase in freeze–thaw events and changes in precipitation, evaporation, and salinity patterns. These events will therefore affect biological activity of the soil compartment that may result in large impacts in ecosystem functioning and dynamics therein. This concern becomes even more important when considering the presence of contaminants due to intense anthropogenic activity, which may lead to synergistic or antagonistic effects and increase or decrease the impact on natural ecosystems. This paper reviews the effect of physical and chemical stressors on enchytraeids, with special emphasis on Enchytraeus albidus because most relevant studies have involved this species. E. albidus is a freeze-tolerant and euryhaline organism and several studies suggest that the absence of salinity may have important (negative) consequences not only for their freeze tolerance ability but also for their reproduction and capacity to deal with the presence of contaminants, such as metals and fungicides. Single and joint effects of constant freezing or freeze–thaw cycles and surfactants, such as 4-nonylphenol, affected negatively E. albidus freeze tolerance by decreasing the levels of cryoprotectants, membrane fluidity and interfering with cellular energy allocation. Because enchytraeids are of ecological significance in many important habitats along the Artic and cold-temperate environments, a reduction in abundance may result in disturbances of the decomposition processes in soils. The knowledge of the biological, physiological, and biochemical limits of enchytraeids to combined effect of physical and chemical stressors are crucial to provide a scientific basis for improving the setting of safety factors when extrapolating from controlled (and optimal) laboratory conditions to natural soil ecosystems. Therefore, there is a need to expand and evolve experiments that more realistically mimic the situation in the field, where interactions between factors are highly relevant. The synergistic or antagonistic interactions identified in the present review may also represent a stepping stone in the evaluation and possible inclusion of natural factors, like cold and salinity, in standardized enchytraeid test guidelines and consequently in risk assessment of chemicals.