Effects of Intertidal Position on Metabolism and Behavior in the Acorn Barnacle, Balanus glandula.

The intertidal zone is characterized by persistent, tidally-driven fluctuations in both abiotic (e.g., temperature, oxygen, and salinity) and biotic (e.g., food availability and predation) factors, which make this a physiologically challenging habitat for resident organisms. The relative magnitude and degree of variability of environmental stress differ between intertidal zones, with the most extreme physiological stress often being experienced by organisms in the high intertidal. Given that so many of the constantly shifting parameters in this habitat are primary drivers of metabolic rate (e.g., temperature, [O2], and food availability), we hypothesized that sessile conspecifics residing in different tidal zones would exhibit distinct "metabolic phenotypes," a term we use to collectively describe the organisms' baseline metabolic performance and capacity. To investigate this hypothesis, we collected acorn barnacles (Balanus glandula) from low, mid, and high intertidal positions in San Luis Obispo Bay, CA, and measured a suite of biochemical (whole-animal citrate synthase (CS) and lactate dehydrogenase (LDH) activity, and aerial [D-lactate]), physiological (O2 consumption rates), morphological (body size), and behavioral (e.g., cirri beat frequency and percentage of time operculum open) indices of metabolism. We found tidal zone-dependent differences in B. glandula metabolism that primarily related to anaerobic capacity, cirral activity patterns, and body size. Barnacles from the low intertidal tended to have a greater capacity for anaerobic metabolism (i.e., increased LDH activity and increased baseline [D-lactate]), have reduced cirral beating activity-and presumably reduced feeding-when submerged, and be smaller in size compared to conspecifics in the high intertidal. We did not, however, see any D-lactate accumulation in barnacles from any tidal height throughout 96 h of air exposure. This trend indicates that the enhanced capacity of low intertidal barnacles for anaerobic metabolism may have evolved to support metabolism during more prolonged episodes of emersion or during events other than emersion (e.g., coastal hypoxia and predation). There were also no significant differences in CS activity or baseline O2 consumption rates (in air or seawater at 14°C) across tidal heights, which imply that aerobic metabolic capacity may not be as sensitive to tidal position as anaerobic processes. Understanding how individuals occupying different shore heights differ in their metabolic capacity becomes increasingly interesting in the context of global climate change, given that the intertidal zone is predicted to experience even greater extremes in abiotic stress.

Interactions of long-term food ration variation and short-term fasting on insulin-like growth factor-1 (IGF-1) pathways in copper rockfish (Sebastes caurinus).

Variation in food intake affects somatic growth by altering the expression of hormones in the somatotropic endocrine axis including insulin-like growth factor-1 (IGF-1). Here, we examined IGF-1 pathway responses to long- and short-term variation in food availability in copper rockfish (Sebastes caurinus), a nearshore Pacific rockfish important for commercial and recreational fisheries. Juvenile copper rockfish were raised under differing ration amounts (3% or 9% mass feed·g-1 fish wet mass·day-1) for 140 d to simulate 'long-term' feeding variation, after which some fish from both rations were fasted for 12 d to generate 'short-term' conditions of food deprivation. Rockfish on the 9% ration treatment grew more quickly than those on the 3% ration and were larger in mass, length, and body condition (k) after 152 d. Fish on the 9% ration had higher blood glucose than those on the 3% ration, with fasting decreasing blood glucose in both ration treatments, indicating that both long-term and short-term feed treatments altered energy status. Plasma IGF-1 was higher in rockfish from the 9% ration than those in the 3% ration and was also higher in fed fish than fasted fish. Additionally, plasma IGF-1 related positively to individual variation in specific growth rate (SGR). The positive association between IGF-1 and SGR showed discordance in fish that had experienced different levels of food and growth over the long-term but not short-term, suggesting that long-term nutritional experience can influence the relationship between IGF-1 and growth in this species. Rockfish on the 3% ration showed a lower relative abundance of gene transcripts encoding igf1 in the liver, but higher hepatic mRNAs for IGF binding proteins igfbp1a and igfbp1b. Fasting similarly decreased the abundance of igf1 mRNAs in the liver of fish reared under both the 9% and 3% rations, while concurrently increasing mRNAs encoding the IGF binding proteins igfbp1a, -1b, and -3a. Hepatic mRNAs for igfbp2b, -5a, and -5b were lower with long-term ration variation (3% ration) and fasting. Fish that experienced long-term reduced rations also had higher mRNA levels for igfbp3a, -3b, and IGF receptors isoforms A (igf1rA) and B (igf1rB) in skeletal muscle, but lower mRNA levels for igf1. Fasting increased muscle mRNA abundance for igfbp3a, igf1rA, and igf1rB, and decreased levels for igfbp2a and igf1. These data show that a positive relationship between circulating IGF-1 and individual growth rate is maintained in copper rockfish even when that growth variation relates to differences in food consumption across varying time scales, but that long- and short-term variation in food quantity can shift basal concentrations of circulating IGF-1 in this species.

Endocrine and metabolic impacts of warming aquatic habitats: differential responses between recently isolated populations of a eurythermal desert pupfish.

Temperatures of inland aquatic habitats are increasing with climate change, and understanding how fishes respond physiologically to thermal stress will be crucial for identifying species most susceptible to these changes. Desert fishes may be particularly vulnerable to rising temperatures because many species occupy only a fraction of their historical range and occur in habitats with already high temperatures. Here, we examined endocrine and metabolic responses to elevated temperature in Amargosa pupfish, Cyprinodon nevadensis amargosae. We studied C. n. amargosae from two habitats with distinct thermal conditions: the Amargosa River, which experiences diurnally and seasonally variable temperatures (0.2-40°C); and Tecopa Bore, a spring and marsh fed by hot groundwater (47.5°C) from an artesian borehole. These allopatric populations differ in morphology, and prior evidence suggests that temperature might contribute to these differences via altered thyroid hormone (TH) regulation of morphological development. Here, we document variation in hepatic iodothyronine deiodinase type 2 (dio2) and type 3 (dio3) and TH receptor ß (trß) gene transcript abundance between the Amargosa River and Tecopa Bore wild populations. Fish from these populations acclimated to 24 or 34°C retained differences in hepatic dio2, dio3 and trß mRNAs and also varied in transcripts encoding the TH membrane transporters monocarboxylate transporter 8 (mct8) and organic anion-transporting protein 1c1 (oatp1c1). Tecopa Bore pupfish also exhibited higher dio2 and trß mRNA levels in skeletal muscle relative to Amargosa River fish. Muscle citrate synthase activity was lower at 34°C for both populations, whereas lactate dehydrogenase activity and lactate dehydrogenase A-chain (ldhA) transcripts were both higher and 3,5,3'-triiodothryonine responsive in Tecopa Bore pupfish only. These findings reveal that local population variation and thermal experience interact to shape how pupfish respond to elevated temperatures, and point to the need to consider such interactions in management actions for desert fishes under a changing climate.

Effect of 4-nonylphenol on the immune response of the Pacific oyster Crassostrea gigas following bacterial infection with Vibrio campbellii.

The xenoestrogen 4-nonylphenol (NP) is a ubiquitous aquatic pollutant and has been shown to impair reproduction, development, growth and, more recently, immune function in marine invertebrates. We investigated the effects of short-term (7 d) exposure to low (2 µg l-1) and high (100 µg l-1) levels of NP on cellular and humoral elements of the innate immune response of Crassostrea gigas to a bacterial challenge. To this end, we measured 1) total hemocyte counts (THC), 2) relative transcript abundance of ten immune-related genes (defh1, defh2, bigdef1, bigdef2, bpi, lysozyme-1, galectin, C-type lectin 2, timp, and transglutaminase) in the hemocytes, gill and mantle, and 3) hemolymph plasma lysozyme activity, following experimental Vibrio campbellii infection. Both low and high levels of NP were found to repress a bacteria-induced increase in THC observed in the control oysters. While several genes were differentially expressed following bacterial introduction (bigdef2, bpi, lysozyme-1, timp, transglutaminase), only two genes (bpi in the hemocytes, transglutaminase in the mantle) exhibited a different bacteria-induced expression profile following NP exposure, relative to the control oysters. Independently of infection-status, exposure to NP also altered mRNA transcript abundance of several genes (bpi, galectin, C-type lectin 2) in naïve, saline-injected oysters. Finally, plasma lysozyme activity levels were significantly higher in low dose NP-treated oysters (both naïve and bacteria challenged) relative to control oysters. Combined, these results suggest that exposure to ecologically-relevant (low) and extreme (high) levels of NP can alter both cellular and humoral elements of the innate immune response in C. gigas, an aquaculture species of global economic importance.

Effect of hypercapnic hypoxia and bacterial infection (Vibrio campbellii) on protein synthesis rates in the Pacific whiteleg shrimp, Litopenaeus vannamei.

Estuarine species frequently encounter areas of simultaneously low dissolved O2 (hypoxia) and high CO2 (hypercapnia). Organisms exposed to hypoxia experience a metabolic depression that serves to decrease ATP utilization and O2 demand during stress. This downregulation is typically facilitated by a reduction in protein synthesis, a process that can be responsible for up to 60% of basal metabolism. The added effects of hypercapnia, however, are unclear. Certain decapods also exhibit a metabolic depression in response to bacterial challenges, leading us to hypothesize that protein synthesis may also be reduced during infection. In the present study, we examined the effects of hypoxia (H), hypercapnic hypoxia (HH), and bacterial infection (Vibrio campbellii) on tissue-specific (muscle and hepatopancreas) fractional protein synthesis rates (ks) in Litopenaeus vannamei. We observed a significant decrease in ks in muscle after 24 h exposure to both H and HH, and in hepatopancreas after 24 h exposure to HH. Thus ks is responsive to changes in O2, and the combined effect of hypercapnic hypoxia on ks is more severe than hypoxia alone. These reductions in ks appear to be driven by changes in RNA translational efficiency (kRNA), and not RNA capacity (Cs). Bacterial infection, however, had no significant effect on ks in either tissue. These results suggest that crustaceans reduce metabolic demand during environmental hypoxia by reducing global protein synthesis, and that this effect is magnified when hypercapnia is concomitantly present. Conversely, an immune-mediated metabolic depression is not associated with a decrease in overall protein production.

Gene transcripts encoding hypoxia-inducible factor (HIF) exhibit tissue- and muscle fiber type-dependent responses to hypoxia and hypercapnic hypoxia in the Atlantic blue crab, Callinectes sapidus.

Hypoxia inducible factor (HIF) is a transcription factor that under low environmental oxygen regulates the expression of suites of genes involved in metabolism, angiogenesis, erythropoiesis, immune function, and growth. Here, we isolated and sequenced partial cDNAs encoding hif-α and arnt/hif-ß from the Atlantic blue crab, Callinectes sapidus, an estuarine species that frequently encounters concurrent hypoxia (low O(2)) and hypercapnia (elevated CO(2)). We then examined the effects of acute exposure (1h) to hypoxia (H) and hypercapnic hypoxia (HH) on relative transcript abundance for hif-α and arnt/hif-ß in different tissues (glycolytic muscle, oxidative muscle, hepatopancreas, gill, and gonads) using quantitative real-time RT-PCR. Our results indicate that hif-α and arnt/hif-ß mRNAs were constitutively present under well-aerated normoxia (N) conditions in all tissues examined. Further, H and HH exposure resulted in both tissue-specific and muscle fiber type-specific effects on relative hif-α transcript abundance. In the gill and glycolytic muscle, relative hif-α mRNA levels were significantly lower under H and HH, compared to N, while no change (or a slight increase) was detected in oxidative muscle, hepatopancreas and gonadal tissues. H and HH did not affect relative transcript abundance for arnt/hif-ß in any tissue or muscle fiber type. Thus, in crustaceans the HIF response to H and HH appears to involve changes in hif transcript abundance, with variation in hif-α and arnt/hif-ß transcriptional dynamics occurring in both a tissue- and muscle fiber type-dependent manner.

A skeletal muscle model of extreme hypertrophic growth reveals the influence of diffusion on cellular design.

Muscle fibers that power swimming in the blue crab Callinectes sapidus are <80 microm in diameter in juveniles but grow hypertrophically, exceeding 600 microm in adults. Therefore, intracellular diffusion distances become progressively greater as the animals grow and, in adults, vastly exceed those in most cells. This developmental trajectory makes C. sapidus an excellent model for characterization of the influence of diffusion on fiber structure. The anaerobic light fibers, which power burst swimming, undergo a prominent shift in organelle distribution with growth. Mitochondria, which require O2 and rely on the transport of small, rapidly diffusing metabolites, are evenly distributed throughout the small fibers of juveniles, but in the large fibers of adults they are located almost exclusively at the fiber periphery where O2 concentrations are high. Nuclei, which do not require O2, but rely on the transport of large, slow-moving macromolecules, have the inverse pattern: they are distributed peripherally in small fibers but are evenly distributed across the large fibers, thereby reducing diffusion path lengths for large macromolecules. The aerobic dark fibers, which power endurance swimming, have evolved an intricate network of cytoplasmically isolated, highly perfused subdivisions that create the short diffusion distances needed to meet the high aerobic ATP turnover demands of sustained contraction. However, fiber innervation patterns are the same in the dark and light fibers. Thus the dark fibers appear to have disparate functional units for metabolism (fiber subdivision) and contraction (entire fiber). Reaction-diffusion mathematical models demonstrate that diffusion would greatly constrain the rate of metabolic processes without these developmental changes in fiber structure.

The long and winding road: influences of intracellular metabolite diffusion on cellular organization and metabolism in skeletal muscle.

A fundamental principle of physiology is that cells are small in order to minimize diffusion distances for O(2) and intracellular metabolites. In skeletal muscle, it has long been recognized that aerobic fibers that are used for steady state locomotion tend to be smaller than anaerobic fibers that are used for burst movements. This tendency reflects the interaction between diffusion distances and aerobic ATP turnover rates, since maximal intracellular diffusion distances are ultimately limited by fiber size. The effect of diffusion distance on O(2) flux in muscle has been the subject of quantitative analyses for a century, but the influence of ATP diffusion from mitochondria to cellular ATPases on aerobic metabolism has received much less attention. The application of reaction-diffusion mathematical models to experimental measurements of aerobic metabolic processes has revealed that the extreme diffusion distances between mitochondria found in some muscle fibers do not necessarily limit the rates of aerobic processes per se, as long as the metabolic process is sufficiently slow. However, skeletal muscle fibers from a variety of animals appear to have intracellular diffusion distances and/or fiber sizes that put them on the brink of diffusion limitation. Thus, intracellular metabolite diffusion likely influences the evolution of muscle design and places limits on muscle function.

A reaction-diffusion analysis of energetics in large muscle fibers secondarily evolved for aerobic locomotor function.

The muscles that power swimming in the blue crab, Callinectes sapidus, grow hypertrophically, such that in juvenile crabs the cell diameters are <60 microm, whereas fibers of the adult crabs often exceed 600 microm. Thus, as these animals grow, their muscle fibers greatly exceed the surface area to volume ratio and intracellular diffusion distance limits of most cells. Previous studies have shown that arginine phosphate (AP) recovery in the anaerobic (light) fibers, which demonstrate a fiber size dependence on anaerobic processes following contraction, is too slow to be restricted by intracellular metabolite diffusive flux, in spite of the fiber's large size. By contrast, the aerobic (dark) fibers have evolved an intricate network of intracellular subdivisions that maintain an effectively small ;metabolic diameter' throughout development. In the present study, we examined the impact of intracellular metabolite diffusive flux on the rate of post-contractile AP resynthesis in the dark muscle, which has a much higher aerobic capacity than the light muscle. AP recovery was measured for 60 min in adults and 15 min in juveniles following burst contractile activity in dark fibers, and a mathematical reaction-diffusion model was used to test whether the observed aerobic rates of AP resynthesis were fast enough to be limited by intracellular metabolite diffusion. Despite the short diffusion distances and high mitochondrial density, the AP recovery rates were relatively slow and we found no evidence of diffusion limitation. However, during simulation of steady-state contraction, which is an activity more typical of the dark fibers, there were substantial intracellular metabolite gradients, indicative of diffusion limitation. This suggests that high ATP turnover rates may lead to diffusion limitation in muscle even when diffusion distances are short, as in the subdivided dark fibers.

Does intracellular metabolite diffusion limit post-contractile recovery in burst locomotor muscle?

Post-metamorphic growth in the blue crab entails an increase in body mass that spans several orders of magnitude. The muscles that power burst swimming in these animals grow hypertrophically, such that small crabs have fiber diameters that are typical of most cells (<60 microm) while in adult animals the fibers are giant (>600 microm). Thus, as the animals grow, their muscle fibers cross and greatly exceed the surface area to volume ratio (SA:V) and intracellular diffusion distance threshold that is adhered to by most cells. Large fiber size should not impact burst contractile function, but post-contractile recovery may be limited by low SA:V and excessive intracellular diffusion distances. A number of changes occur in muscle structure, metabolic organization and metabolic flux during development to compensate for the effects of increasing fiber size. In the present study, we examined the impact of intracellular metabolite diffusive flux on the rate of post-contractile arginine phosphate (AP) resynthesis in burst locomotor muscle from small and large animals. AP recovery was measured following burst exercise, and these data were compared to a mathematical reaction-diffusion model of aerobic metabolism. The measured rates of AP resynthesis were independent of fiber size, while simulations of aerobic AP resynthesis yielded lower rates in large fibers. These contradictory findings are consistent with previous observations that there is an increased reliance on anaerobic metabolism for post-contractile metabolic recovery in large fibers. However, the model results suggest that the interaction between mitochondrial ATP production rates, ATP consumption rates and diffusion distances yield a system that is not particularly close to being limited by intracellular metabolite diffusion. We conclude that fiber SA:V and O2 flux exert more control than intracellular metabolite diffusive flux over the developmental changes in metabolic organization and metabolic fluxes that characterize these muscles.