Limitations of eDNA analysis for Carcinus maenas abundance estimations.

BACKGROUND: Environmental DNA (eDNA) is an effective tool for the detection and monitoring of presence or absence of rare and invasive species. These techniques have been extended to quantify biomass in vertebrates, particularly in fish species. However, the efficacy of eDNA techniques to quantify biomass in invertebrate species has rarely been examined. This study tested whether eDNA could be used to determine the biomass of the world-wide invasive green crab, Carcinus maenas. In a controlled laboratory study, the relationship between biomass and C. maenas eDNA concentration was examined in the context of different biotic (activity) and abiotic (temperature) parameters. RESULTS: When incubating different numbers of crabs in sterile saltwater for up to 7 days, a relationship between eDNA concentration and biomass was observed at temperatures of 6.7 â„ƒ and 18.7 â„ƒ, but not at 12.8 â„ƒ. Additionally, motor activity, aggression level, time of sampling, and features of organismal decay had significant impact on the concentration of C. maenas eDNA collected. CONCLUSIONS: We show that eDNA concentration did not correlate with biomass, and that biomass, temperature, organismal characteristics, and potentially many more parameters affect shedding and degradation rates for eDNA in this species, thus, impacting the recoverable eDNA concentration. Therefore, eDNA techniques are not likely to provide a reliable signal of biomass in the invasive invertebrate species C. maenas.

Is altered behavior linked to cellular energy regulation in a freshwater mussel (Elliptio complanata) exposed to triclosan?

Environmental stress may alter the bioenergetic balance of organisms by resulting in greater energy investment into detoxification processes, which diverts energy from other biological functions. Here, we examine responses to triclosan (TCS) exposure in a freshwater mussel across multiple biological levels: behavioral (e.g., burrowing and movement activity), organismal (e.g., metabolic rate and heart rate), and subcellular (e.g., gene expression and protein abundance/activity). At the subcellular level, we employed both energetic (i.e., AMP-activated protein kinase (AMPK)) and traditional (i.e., heat shock protein (HSP70), superoxide dismutase (SOD), glutathione-S-transferase (GST)) biomarkers. We found a significant reduction in burrowing and movement behaviors, a 1.8-fold increase in total-AMPK protein abundance, and a 2.8-fold increase in AMPK activity after 21d. GST activity increased after 4d, but not after 21d. Our findings suggest that TCS exposure results in an energetic tradeoff between detoxification at the cellular level and whole-animal activity.

AMP-activated protein kinase is a biomarker of energetic status in freshwater mussels exposed to municipal effluents.

Although biomarkers are frequently used to assess sublethal effects of contaminants, a lack of mechanistic linkages to higher-level effects limits the predictive power of biomarkers. Bioenergetics has been proposed as a framework for linking cellular effects to whole-animal effects. We investigated sublethal effects of exposure to wastewater treatment facility effluent in freshwater mussels in situ, thereby capturing ecologically relevant exposure conditions. Our study focused on the energetic biomarker AMP-activated protein kinase (AMPK), while also considering more traditional biomarkers like heat shock proteins (HSP70), and antioxidant enzymes (i.e., superoxide dismutase (SOD), glutathione-S-transferase (GST)). We examined biomarkers at mRNA and protein levels. Effluent exposure caused a reduction in total-AMPK protein abundance (p=0.05) and AMPK mRNA expression (p=0.02). Conversely, AMPK activity increased at downstream sites by 2.2-fold (p=0.05), indicating increased cellular energy consumption. HSP70 protein abundance was lower at downstream sites (p<0.05), while SOD and GST activity levels significantly increased. By using various biomarkers, we demonstrate that exposure to municipal effluent creates an energetically taxing situation. This is the first study to use AMPK to evaluate the effects of contamination in situ, and our results suggest that energetic biomarkers, like AMPK, complement traditional biomarkers and may help establish functional links between cellular and whole-animal effects.

Energy homeostasis as an integrative tool for assessing limits of environmental stress tolerance in aquatic invertebrates.

Energy balance is a fundamental requirement of stress adaptation and tolerance. We explore the links between metabolism, energy balance and stress tolerance using aquatic invertebrates as an example and demonstrate that using key parameters of energy balance (aerobic scope for growth, reproduction and activity; tissue energy status; metabolic rate depression; and compensatory onset of anaerobiosis) can assist in integrating the effects of multiple stressors and their interactions and in predicting the whole-organism and population-level consequences of environmental stress. We argue that limitations of both the amount of available energy and the rates of its acquisition and metabolic conversions result in trade-offs between basal maintenance of a stressed organism and energy costs of fitness-related functions such as reproduction, development and growth and can set limit to the tolerance of a broad range of environmental stressors. The degree of stress-induced disturbance of energy balance delineates transition from moderate stress compatible with population persistence (pejus range) to extreme stress where only time-limited existence is possible (pessimum range). It also determines the predominant adaptive strategy of metabolic responses (energy compensation vs. conservation) that allows an organism to survive the disturbance. We propose that energy-related biomarkers can be used to determine the conditions when these metabolic transitions occur and thus predict ecological consequences of stress exposures. Bioenergetic considerations can also provide common denominator for integrating stress responses and predicting tolerance limits under the environmentally realistic scenarios when multiple and often variable stressors act simultaneously on an organism. Determination of bioenergetic sustainability at the organism's level (or lack thereof) has practical implications. It can help identify the habitats and/or conditions where a population can survive (even if at the cost of reduced reproduction and growth) and those that are incapable of supporting viable populations. Such an approach will assist in explaining and predicting the species' distribution limits in the face of the environmental change and informing the conservation efforts and resource management practices.

AMP-activated protein kinase (AMPK) in the rock crab, Cancer irroratus: an early indicator of temperature stress.

Exposure of marine invertebrates to high temperatures leads to a switch from aerobic to anaerobic metabolism, a drop in the cellular ATP concentration ([ATP]), and subsequent death. In mammals, AMP-activated protein kinase (AMPK) is a major regulator of cellular [ATP] and activates ATP-producing pathways, while inhibiting ATP-consuming pathways. We hypothesized that temperature stress in marine invertebrates activates AMPK to provide adequate concentrations of ATP at increased but sublethal temperatures and that AMPK consequently can serve as a stress indicator (similar to heat shock proteins, HSPs). We tested these hypotheses through two experiments with the rock crab, Cancer irroratus. First, crabs were exposed to a progressive temperature increase (6 degrees C h(-1)) from 12 to 30 degrees C. AMPK activity, total AMPK protein and HSP70 levels, reaction time, heart rate and lactate accumulation were measured in hearts at 2 degrees C increments. AMPK activity remained constant between 12 and 18 degrees C, but increased up to 9.1(+/-1.5)-fold between 18 and 30 degrees C. The crabs' reaction time also decreased above 18 degrees C. By contrast, HSP70 (total and inducible) and total AMPK protein expression levels did not vary significantly over this temperature range. Second, crabs were exposed for up to 6 h to the sublethal temperature of 26 degrees C. This prolonged exposure led to a constant elevation of AMPK activity and levels of HSP70 mRNA. AMPK mRNA continuously increased, indicating an additional response in gene expression. We conclude that AMPK is an earlier indicator of temperature stress in rock crabs than HSP70, especially during the initial response to high temperatures. We discuss the temperature-dependent increase in AMPK activity in the context of Shelford's law of tolerance. Specifically, we describe AMPK activity as a cellular marker that indicates a thermal threshold, called the pejus temperature, T(p). At T(p) the animals leave their optimum range and enter a temperature range with a limited aerobic scope for exercise. This T(p) is reached periodically during annual temperature fluctuations and has higher biological significance than earlier described critical temperatures, at which the animals switch to anaerobic metabolism and HSP expression is induced.

Relationship between 5-aminoimidazole-4-carboxamide-ribotide and AMP-activated protein kinase activity in the perfused mouse heart.

AMP-activated protein kinase (AMPK) is a cellular energy sensor whose activity responds to AMP concentration ([AMP]). An agent that activates AMPK in cells is 5-aminoimidazole-4-carboxamide-1-riboside (AICA-riboside). Phosphorylated AICA-riboside or AICA-ribotide (ZMP) is an AMP analog. It is generally assumed that ZMP accumulation does not alter [AMP]. Additionally, the effect of AICA-riboside on AMPK activity of the heart is uncertain. Two hypotheses were tested in the isolated mouse heart: 1) sufficient ZMP concentration ([ZMP]) forms to increase AMPK activity, and 2) [ZMP] accumulation increases [AMP]. Perfusion of isolated mouse hearts with Krebs-Henseleit buffer containing 0.15-2 mM AICA-riboside concentration resulted in [ZMP] of 2-8 mM. ZMP accumulation reduced phosphocreatine concentration, which increased cytosolic [AMP]. In hearts with [ZMP] less than approximately 3 mM, in vivo AMPK allosteric activity effects of ZMP were observed; AMPK phosphorylation and [AMP] were not increased. With [ZMP] between 3 and 5 mM, in vitro AMPK activity and phosphorylation increased with unchanged [AMP]. This occurred in hearts perfused with 0.25 mM AICA-riboside for 48 min and 0.5 mM AICA-riboside for 24 min. The [ZMP] resulting in 50% AMPK activity (covalent phosphorylation of AMPK) was 4.1 +/- 0.6 mM. Hearts with [ZMP] >5 mM displayed increased [AMP] and AMPK activity that was not different from hearts with similar [AMP] with no [ZMP]; the half-maximal activity of AMP was 5.6 +/- 1.6 microM. Thus, in mouse hearts, AICA-riboside was metabolized to [ZMP] adequately to increase AMPK activity. Higher [ZMP] also increased cytosolic [AMP], which affects AMPK activity.

Hypoxia and AMP independently regulate AMP-activated protein kinase activity in heart.

The hypothesis was tested that hypoxia increases AMP-activated protein kinase (AMPK) activity independently of AMP concentration ([AMP]) in heart. In isolated perfused rat hearts, cytosolic [AMP] was changed from 0.2 to 16 microM using metabolic inhibitors during both normal oxygenation (95% O2-5% CO2, normoxia) and limited oxygenation (95% N2-5% CO2, hypoxia). Total AMPK activity measured in vitro ranged from 2 to 40 pmol.min(-1).mg protein(-1) in normoxic hearts and from 5 to 55 pmol.min(-1).mg protein(-1) in hypoxic hearts. The dependence of the in vitro total AMPK activity on the in vivo cytosolic [AMP] was determined by fitting the measurements from individual hearts to a hyperbolic equation. The [AMP] resulting in half-maximal total AMPK activity (A0.5) was 3 +/- 1 microM for hypoxic hearts and 28 +/- 13 microM for normoxic hearts. The A0.5 for alpha2-isoform AMPK activity was 2 +/- 1 microM for hypoxic hearts and 13 +/- 8 microM for normoxic hearts. Total AMPK activity correlated with the phosphorylation of the Thr172 residue of the AMPK alpha-subunit. In potassium-arrested hearts perfused with variable O2 content, alpha-subunit Thr172 phosphorylation increased at O2 < or = 21% even though [AMP] was <0.3 microM. Thus hypoxia or O2 < or = 21% increased AMPK phosphorylation and activity independently of cytosolic [AMP]. The hypoxic increase in AMPK activity may result from either direct phosphorylation of Thr172 by an upstream kinase or reduction in the A0.5 for [AMP].

The relationship between AMP-activated protein kinase activity and AMP concentration in the isolated perfused rat heart.

The objective of this study was to define the relationship among AMP-activated protein kinase (AMPK) activity, AMP concentration ([AMP]), and [ATP] in perfused rat hearts. Bromo-octanoate, an inhibitor of beta-oxidation, and amino-oxyacetate, an inhibitor of the malate-aspartate shuttle, were used to modify substrate flux and thus increase cytosolic [AMP]. Cytosolic [AMP] was calculated using metabolites measured by (31)P NMR spectroscopy. Rat hearts were perfused with Krebs-Henseleit solution containing glucose and either no inhibitor, the inhibitors, or the inhibitors plus butyrate, a substrate that bypasses the metabolic blocks. In this way, [AMP] changed from 0.2 to 27.9 microm, and [ATP] varied between 11.7 and 6.8 mm. AMPK activity ranged from 7 to 60 pmol.min(-1).microg of protein(-1). The half-maximal AMPK activation (A(0.5)) was 1.8 +/- 0.3 microm AMP. Measurements in vitro have reported similar AMPK A(0.5) at 0.2 mm ATP, but found that A(0.5) increased 10-20-fold at 4 mm ATP. The low A(0.5) of this study despite a high [ATP] suggests that in vivo the ATP antagonism of AMPK activation is reduced, and/or other factors besides AMP activate AMPK in the heart.