Showing papers in "Comparative Biochemistry and Physiology A-molecular & Integrative Physiology in 2011"

CO2 induced seawater acidification impacts sea urchin larval development I: Elevated metabolic rates decrease scope for growth and induce developmental delay

Abstract: Anthropogenic CO(2) emissions are acidifying the world's oceans. A growing body of evidence is showing that ocean acidification impacts growth and developmental rates of marine invertebrates. Here we test the impact of elevated seawater pCO(2) (129Pa, 1271 atm) on early development, larval metabolic and feeding rates in a marine model organism, the sea urchin Strongylocentrotus purpuratus. Growth and development was assessed by measuring total body length, body rod length, postoral rod length and posterolateral rod length. Comparing these parameters between treatments suggests that larvae suffer from a developmental delay (by ca. 8%) rather than from the previously postulated reductions in size at comparable developmental stages. Further, we found maximum increases in respiration rates of +100% under elevated pCO(2), while body length corrected feeding rates did not differ between larvae from both treatments. Calculating scope for growth illustrates that larvae raised under high pCO(2) spent an average of 39 to 45% of the available energy for somatic growth, while control larvae could allocate between 78 and 80% of the available energy into growth processes. Our results highlight the importance of defining a standard frame of reference when comparing a given parameter between treatments, as observed differences can be easily due to comparison of different larval ages with their specific set of biological characters.

Assessing the development and application of the accelerometry technique for estimating energy expenditure.

Abstract: A theoretically valid proxy of energy expenditure is the acceleration of an animal's mass due to the movement of its body parts. Acceleration can be measured by an accelerometer and recorded onto a data logging device. Relevant studies have usually derived a measure of acceleration from the raw data that represents acceleration purely due to movement of the animal. This is termed ‘overall dynamic body acceleration’ (ODBA) and to date has proved a robust derivation of acceleration for use as an energy expenditure proxy. Acceleration data loggers are generally easy to deploy and the measures recorded appear robust to slight variation in location and orientation. This review discusses important issues concerning the accelerometry technique for estimating energy expenditure and ODBA; deriving ODBA, calibrating ODBA, acceleration logger recording frequencies, scenarios where ODBA is less likely to be valid, and the power in recording acceleration and heart rate together. While present evidence suggests that ODBA may not quantify energy expenditure during diving by birds and mammals, several recent studies have assessed changes in mechanical work in such species qualitatively through variation in ODBA during periods of submergence. The use of ODBA in field metabolic studies is likely to continue growing, supported by its relative ease of use and range of applications.

The heart rate method for estimating metabolic rate: review and recommendations.

Abstract: Under most circumstances heart rate (f(H)) is correlated with the rate of oxygen consumption (VO(2)) and hence the rate of energy expenditure or metabolic rate (MR). For over 60 years this simple principle has underpinned the use of heart rate to estimate metabolic rate in a range of animal species and to answer questions about their physiology, behaviour and ecology. The heart rate method can be applied both quantitatively and qualitatively. The quantitative approach is a two-stage process where firstly f(H) and MR are measured simultaneously under controlled conditions and a predictive calibration relationship derived. Secondly, measurements of heart rate are made and converted to estimates of MR using the calibration relationship. The qualitative approach jumps directly to the second stage, comparing estimates of f(H) under different circumstances and drawing conclusions about MR under the assumption that a relationship exists. This review describes the range of studies which have adopted either the quantitative or qualitative approach to estimating the MR of birds, mammals and reptiles. Studies have tended to focus on species, states and questions which are hard to measure, control or define using other techniques. For example, species studied include large, wide-ranging species such as ungulates, marine predators, and domestic livestock while research questions have concerned behaviours such as flight, diving and the effects of stress. In particular, the qualitative approach has applied to circumstances and/or species where it may be hard or impossible to derive a calibration relationship for practical reasons. The calibration process itself can be complex and a number of factors such as body mass, activity state and stress levels can affect the relationship between f(H) and VO(2). I recommend that a quantitative approach be adopted wherever possible but that this may entail deriving a calibration relationship which is practical and applicable, rather than the most accurate possible. I conclude with a series of recommendations for the application and development of this method.

Metabolic rate and oxidative stress in insects exposed to low temperature thermal fluctuations.

Abstract: Fluctuating temperatures are a predominant feature of the natural environment but their effects on ectotherm physiology are not well-understood. The warm periods of fluctuating thermal regimes (FTRs) provide opportunities for repair leading to increased survival, but there are also indications of negative effects of warm exposure. In this study, we examined respiration and oxidative stress in adult Alphitobius diaperinus exposed to FTRs and to constant low temperatures. We hypothesized that cold exposure will cause oxidative stress and that FTRs would reduce the amount of chill injuries, via activation of the antioxidant system. We measured V˙CO2, activities of super oxide dismutase (SOD), amounts of total (GSHt) and oxidized glutathione (GSSG) during cold and warm periods of FTRs. Increased severity of cold exposure caused a decrease in the glutathione pool. SOD levels increased during the recovery period in the more severe FTR. The antioxidant response was sufficient to counter the reactive oxygen species production, as the GSH:GSSG ratio increased. We conclude that cold stress causes oxidative damage in these beetles, and that a warm recovery period activates the antioxidant system allowing repair of cold-induced damage, leading to the increased survival previously noted in beetles exposed to fluctuating versus constant temperatures.

Heat tolerance and its plasticity in Antarctic fishes.

Abstract: The adaptive radiation of the Antarctic notothenioid ancestral benthic fish stock within the chronic freezing waters of the Southern Ocean gave rise to five highly cold adapted families. Their stenothermy, first observed from several high-latitude McMurdo Sound species, has been of increasing recent interest given the threat of rising polar water temperatures from global climate change. In this study we determined the heat tolerance in a geographically diverse group of 11 Antarctic species as their critical thermal maximum (CTMax). When acclimatized to their natural freezing water temperatures, environmental CTMaxs ranged from 11.95 to 16.17 °C, well below those of fishes endemic to warmer waters. There was a significant regional split, with higher CTMaxs in species from the more northerly and thermally variable Seasonal Pack-ice Zone. When eight of the Antarctic species were warm acclimated to 4 °C all showed a significant increase over their environmental CTMaxs, with several showing plasticity comparable in magnitude to some far more eurythermal fishes. When the accrual of heat tolerance during acclimation was followed in three high-latitude McMurdo Sound species, it was found to develop slowly in two of them, which was correlated with their low metabolic rates.

CO2 induced seawater acidification impacts sea urchin larval development II: Gene expression patterns in pluteus larvae

Abstract: Extensive use of fossil fuels is leading to increasing CO2 concentrations in the atmosphere and causes changes in the carbonate chemistry of the oceans which represents a major sink for anthropogenic CO2 As a result, the oceans' surface pH is expected to decrease by ca 04 units by the year 2100, a major change with potentially negative consequences for some marine species Because of their carbonate skeleton, sea urchins and their larval stages are regarded as likely to be one of the more sensitive taxa In order to investigate sensitivity of pre-feeding (2days post-fertilization) and feeding (4 and 7days post-fertilization) pluteus larvae, we raised Strongylocentrotus purpuratus embryos in control (pH 81 and pCO2 41Pa eg 399μatm) and CO2 acidified seawater with pH of 77 (pCO2 134Pa eg 1318μatm) and investigated growth, calcification and survival At three time points (day 2, day 4 and day 7 post-fertilization), we measured the expression of 26 representative genes important for metabolism, calcification and ion regulation using RT-qPCR After one week of development, we observed a significant difference in growth Maximum differences in size were detected at day 4 (ca 10% reduction in body length) A comparison of gene expression patterns using PCA and ANOSIM clearly distinguished between the different age groups (two-way ANOSIM: Global R=1) while acidification effects were less pronounced (Global R=0518) Significant differences in gene expression patterns (ANOSIM R=0938, SIMPER: 43% difference) were also detected at day 4 leading to the hypothesis that differences between CO2 treatments could reflect patterns of expression seen in control experiments of a younger larva and thus a developmental artifact rather than a direct CO2 effect We found an up regulation of metabolic genes (between 10%and 20% in ATP-synthase, citrate synthase, pyruvate kinase and thiolase at day 4) and down regulation of calcification related genes (between 23% and 36% in msp130, SM30B, and SM50 at day 4) Ion regulation was mainly impacted by up regulation of Na+/K+-ATPase at day 4 (15%) and down regulation of NHE3 at day 4 (45%) We conclude that in studies in which a stressor induces an alteration in the speed of development, it is crucial to employ experimental designs with a high time resolution in order to correct for developmental artifacts This helps prevent misinterpretation of stressor effects on organism physiology

Flow-through respirometry applied to chamber systems: pros and cons, hints and tips.

Abstract: Flow-through respirometry is a powerful, accurate methodology for metabolic measurement that is applicable to organisms spanning a body mass range of many orders of magnitude. Concentrating on flow-through respirometry that utilizes a chamber to contain the experimental animals, we describe the most common flow measurement and control methodologies (push, pull and stop-flow) and their associated advantages and disadvantages. Objective methods for calculating air flow rates through the chamber, based on the body mass and taxon of the experimental organism, are presented. Techniques for removing the effect of water vapor dilution, including the direct measurement of water vapor pressure and mathematical compensation for its presence, are described and evaluated, as are issues surrounding the analysis of one or both of the respiratory gases (oxygen and carbon dioxide), and issues related to the mathematical correction of wash-out phenomena (response correction). Two important biomedical applications of flow-through respirometry (metabolic phenotyping and room calorimetry) are discussed in detail, and we conclude with a list of suggestions aimed primarily at investigators starting out in applying flow-through respirometry.

Aerobic training stimulates growth and promotes disease resistance in Atlantic salmon (Salmo salar).

Abstract: Improving fish robustness is of utmost relevance to reducing fish losses in farming. Although not previously examined, we hypothesized that aerobic training, as shown for human studies, could strengthen disease resistance in Atlantic salmon (Salmo salar). Thus, we exercised salmon pre-smolts for 6 weeks at two different aerobic training regimes; a continuous intensity training (CT; 0.8 bl s− 1) and an interval training (IT; 0.8 bl s− 1 16 h and 1.0 bl s− 1 8 h) and compared them with untrained controls (C; 0.05 bl s− 1). The effects of endurance training on disease resistance were evaluated using an IPN virus challenge test, while the cardiac immune modulatory effects were characterized by qPCR and microarray gene expression analyses. In addition, swimming performance and growth parameters were investigated. Survival after the IPN challenge was higher for IT (74%) fish than for either CT (64%) or C (61%) fish. While both CT and IT groups showed lower cardiac transcription levels of TNF-α, IL-1β and IL-6 prior to the IPN challenge test, IT fish showed the strongest regulation of genes involved in immune responses and other processes known to affect disease resistance. Both CT and IT regimes resulted in better growth compared with control fish, with CT fish developing a better swimming efficiency during training. Overall, interval aerobic training improved growth and increased robustness of Atlantic salmon, manifested by better disease resistance, which we found was associated with a modulation of relevant gene classes on the cardiac transcriptome.

An experimental test of the role of environmental temperature variability on ectotherm molecular, physiological and life-history traits: implications for global warming.

Abstract: Global climate change is one of the greatest threats to biodiversity; one of the most important effects is the increase in the mean earth surface temperature. However, another but poorly studied main characteristic of global change appears to be an increase in temperature variability. Most of the current analyses of global change have focused on mean values, paying less attention to the role of the fluctuations of environmental variables. We experimentally tested the effects of environmental temperature variability on characteristics associated to the fitness (body mass balance, growth rate, and survival), metabolic rate (VCO 2 ) and molecular traits (heat shock protein expression, Hsp70), in an ectotherm, the terrestrial woodlouse Porcellio laevis. Our general hypotheses are that higher values of thermal amplitude may directly affect life-history traits, increasing metabolic cost and stress responses. At first, results supported our hypotheses showing a diversity of responses among characters to the experimental thermal treatments. We emphasize that knowledge about the cellular and physiological mechanisms by which animals cope with environmental changes is essential to understand the impact of mean climatic change and variability. Also, we consider that the studies that only incorporate only mean temperatures to predict the life-history, ecological and evolutionary impact of global temperature changes present important problems to predict the diversity of responses of the organism. This is because the analysis ignores the complexity and details of the molecular and physiological processes by which animals cope with environmental variability, as well as the life-history and demographic consequences of such variability.

Estimating metabolic heat loss in birds and mammals by combining infrared thermography with biophysical modelling

Abstract: Infrared thermography (IRT) is a technique that determines surface temperature based on physical laws of radiative transfer. Thermal imaging cameras have been used since the 1960s to determine the surface temperature patterns of a wide range of birds and mammals and how species regulate their surface temperature in response to different environmental conditions. As a large proportion of metabolic energy is transferred from the body to the environment as heat, biophysical models have been formulated to determine metabolic heat loss. These models are based on heat transfer equations for radiation, convection, conduction and evaporation and therefore surface temperature recorded by IRT can be used to calculate heat loss from different body regions. This approach has successfully demonstrated that in birds and mammals heat loss is regulated from poorly insulated regions of the body which are seen to be thermal windows for the dissipation of body heat. Rather than absolute measurement of metabolic heat loss, IRT and biophysical models have been most useful in estimating the relative heat loss from different body regions. Further calibration studies will improve the accuracy of models but the strength of this approach is that it is a non-invasive method of measuring the relative energy cost of an animal in response to different environments, behaviours and physiological states. It is likely that the increasing availability and portability of thermal imaging systems will lead to many new insights into the thermal physiology of endotherms.

Strategies for maintaining Na⁺ balance in zebrafish (Danio rerio) during prolonged exposure to acidic water.

Abstract: article i nfo + uptake Na + efflux The objective of the present study was to characterize the capacity of zebrafish (Danio rerio) to regulate whole body Na + levels during exposure to acidic (pH 38-40) water Exposure to acidic water significantly affected the mRNA levels of 14 claudin and two occludin isoforms, tight junction proteins thought to be involved in regulating paracellular efflux Despite these changes, Na + efflux as well as uptake of polyethylene glycol (PEG), a marker for paracellular pathway, was persistently elevated during the 2-week period of acid exposure, although there was a transient recovery between 12- and 72-h Pre-exposing fish to acidic water for 2 weeks failed to attenuate the increase in Na + efflux associated with acute exposure to acidic water of low (Ca 2+ ) However, during recovery in water of circumneutral pH following exposure to acidic water, normal rates of Na + efflux were restored within 5 h The rate of Na + uptake was significantly elevated between 4 and 7 days of exposure to acidic water; the increase was associated with significant increases in maximal Na + uptake capacity (JMAXNa +) and affinity constant (KM) These results demonstrate that in acidic water, zebrafish maintain their whole body Na + balance primarily by

The effects of temperature on metabolic interaction between digestion and locomotion in juveniles of three cyprinid fish (Carassius auratus, Cyprinus carpio and Spinibarbus sinensis).

Abstract: To test whether the effects of temperature on the metabolic mode changed among different fish species, we investigated the specific dynamic action (SDA) and swimming performance of fasting and fed fish at 15 and 25°C in three juvenile Cyprinidae fish species: goldfish (Carassius auratus), common carp (Cyprinus carpio) and qingbo (Spinibarbus sinensis). Both taxon and temperature had significant effects on the resting oxygen consumption rate (M˙O(rest)), SDA and swimming performance (p<0.05). In addition, the effect of temperature differed significantly among the different species (interaction effect, p<0.05). Under the low temperature condition, digestion had no effect on either critical swimming speed (U(crit)) or the active MO(2) (MO(active)) for all fish species (additive metabolic mode). When the temperature was increased from 15 to 25°C, the metabolic scope (MS) for digestion increased approximately 182, 49 and 17%, and the MS for locomotion increased approximately 129, 58 and 138% in goldfish, common carp and qingbo, respectively. The total metabolic demands for both digestion and locomotion (i.e., the sum of digestive MS and locomotive MS) increased approximately 143, 56 and 112% in goldfish, common carp and qingbo, respectively. The total MS for both digestion and locomotion (the difference between MO(active) in fed fish and MO(rest) in fasting fish) increased approximately 106, 58 and 78% in goldfish, common carp and qingbo, respectively. Thus, the MS for locomotion in fed goldfish decreased due to the large increase in digestive function at the high temperature, and the U(crit) of fed goldfish decreased by 11% compared to that of fasting fish (p<0.05) (digestion-priory metabolic mode). The metabolic mode of qingbo changed to locomotion-priority mode, as illustrated by the large increase in locomotive MS in response to the increase in temperature. In the common carp, temperature had no effect on metabolic mode as illustrated by the parallel increases in cardio-respiratory capacity and metabolic capacity of digestive and locomotive organs. A discussion on the changes in metabolic mode in response to temperature and its possible relationship with the metabolic characteristics of a given fish species was also documented in this paper.

Ecophysiological responses to temperature of the "killer shrimp" Dikerogammarus villosus: is the invader really stronger than the native Gammarus pulex?

Abstract: With global climate changes, biological invasions are considered to be one of the main causes of the decline of freshwater biodiversity. In this context, predicted increases in global temperature may alter the geographical distributions of native and invasive species. The purpose of our study was to examine the metabolic, behavioral and physiological responses to short-term temperature acclimation of two widely distributed species (the most successful European invader, Dikerogammarus villosus, and its main victim, Gammarus pulex), in order to estimate the potential effect of global warming on its invasion of freshwater ecosystems. Our results show that D. villosus is more vulnerable to high temperatures than G. pulex. The native species seems to be best adapted to intermediate temperatures (10-20°C) with a possibility of adjustment to "extreme" temperatures (5-27°C), whereas the "killer shrimp" D. villosus seems best adapted to lower temperatures (5-10°C) with a limited possibility of adjustment above 20°C. In the light of our results, global warming is likely to be less favorable to the invasive species. However, D. villosus showed reduced metabolic and activity rates, associated with higher glycogen content. This adaptive strategy was interpreted as having functional advantages, allowing D. villosus to successfully invade harsh and/or unpredictable biotopes. In addition, our results show that glycogen stores may be used as a powerful indicator of the optimal thermal window for aquatic ectotherms.

Leptin reduces Atlantic salmon growth through the central pro-opiomelanocortin pathway

Abstract: article Leptin (Lep) is a key factor for the energy homeostasis in mammals, but the available data of its role in teleosts are not conclusive. There are large sequence differences among mammalian and teleost Lep, both at the gene and protein level. Therefore, in order to characterize Lep function in fish, the use of species-specific Lep is crucial. In this study, the cDNA sequence of salmon leptin a1 (lepa1) was used to establish a production protocol for recombinant salmon LepA1 (rsLepA1) in Escherichia coli, that enabled a final yield of 1.7 mg pure protein L �1 culture. The effects of 20-day administration of rsLepA1 on growth and brain neuroendocrine peptide gene expression (npy, cart, agrp (-1 and -2), pomc (-a1, -a2, -a2s, and -b)) were studied in juvenile, immature Atlantic salmon (96.5±2.1 g) fed a commercial diet to satiation. Intraperitoneal osmotic pumps were used to deliver rsLepA1 at four different concentrations (calculated pumping rates were 0, 0.1, 1.0 and 10 ng g

Drosophila melanogaster larvae fed by glucose and fructose demonstrate difference in oxidative stress markers and antioxidant enzymes of adult flies.

Abstract: Activities of antioxidant and associated enzymes, and oxidative stress markers were assessed in newly enclosed adult fruit flies Drosophila melanogaster developed on diets with 4 and 10% glucose or fructose. In fly males, 10% fructose promoted higher content of protein carbonyls and catalase activity, but lower superoxide dismutase (SOD) activity than 4%, while in females—lower levels of high molecular mass thiols (H-SH). Females at all diets had virtually the same level of lipid peroxides, low-molecular-mass thiols, catalase, and superoxide dismutase activities. Fed with 4% fructose and glucose males demonstrated 24 and 26% lower H-SH level than females, respectively. On diets with 4% glucose, 10% glucose and fructose females had 32, 26 and 27% lower catalase activity than respective males, and 1.3–1.5-fold lower glucose-6-phosphate dehydrogenase activity on glucose-containing diets. Strong positive correlations between H-SH level and G6PD activity, as well as between catalase and G6PDH activity were found. These results suggest that type and concentration of dietary carbohydrate affect antioxidant defense in fruit flies. It also substantially depends on fly sex, comprising presumably levels of protein carbonyls and lipid peroxides, as well as catalase and SOD activities in males and G6PDH activity in females.

Direct animal calorimetry, the underused gold standard for quantifying the fire of life.

Abstract: Direct animal calorimetry, the gold standard method for quantifying animal heat production (HP), has been largely supplanted by respirometric indirect calorimetry owing to the relative ease and ready commercial availability of the latter technique Direct calorimetry, however, can accurately quantify HP and thus metabolic rate (MR) in both metabolically normal and abnormal states, whereas respirometric indirect calorimetry relies on important assumptions that apparently have never been tested in animals with genetic or pharmacologically-induced alterations that dysregulate metabolic fuel partitioning and storage so as to promote obesity and/or diabetes Contemporary obesity and diabetes research relies heavily on metabolically abnormal animals Recent data implicating individual and group variation in the gut microbiome in obesity and diabetes raise important questions about transforming aerobic gas exchange into HP because 99% of gut bacteria are anaerobic and they outnumber eukaryotic cells in the body by ∼ 10-fold Recent credible work in non-standard laboratory animals documents substantial errors in respirometry-based estimates of HP Accordingly, it seems obvious that new research employing simultaneous direct and indirect calorimetry (total calorimetry) will be essential to validate respirometric MR phenotyping in existing and future pharmacological and genetic models of obesity and diabetes We also detail the use of total calorimetry with simultaneous core temperature assessment as a model for studying homeostatic control in a variety of experimental situations, including acute and chronic drug administration Finally, we offer some tips on performing direct calorimetry, both singly and in combination with indirect calorimetry and core temperature assessment

Effects of high environmental ammonia on branchial ammonia excretion rates and tissue Rh-protein mRNA expression levels in seawater acclimated Dungeness crab Metacarcinus magister

Abstract: In the present study of the marine Dungeness crabs Metacarcinus magister , the long term effects of high environmental ammonia (HEA) on hemolymph ammonia and urea concentrations, branchial ammonia excretion rates and mRNA expression levels of the crustacean Rh-like ammonia transporter (RhMM), H + -ATPase (subunit B), Na + /K + -ATPase (α-subunit) and Na + /H + -exchanger (NHE) were investigated. Under control conditions, the crabs’ hemolymph exhibited a total ammonia concentration of 179.3 ± 14.5 μmol L −1 , while urea accounted for 467.2 ± 33.5 μmol L −1 , respectively. Both anterior and posterior gills were capable of excreting ammonia against a 16-fold inwardly directed gradient. Under control conditions, mRNA expression levels of RhMM were high in the gills in contrast to very low expression levels in all other tissues investigated, including the antennal gland, hepatopancreas, and skeletal muscle. After exposure to 1 mmol L −1 NH 4 Cl, hemolymph ammonia increased within the first 12 h to ca. 500 µmol L −1 and crabs were able the keep this hemolymph ammonia level for at least 4 days. During this initial period, branchial RhMM and H + -ATPase (subunit B) mRNA expression levels roughly doubled. After 14 days of HEA exposure, hemolymph ammonia raised up to environmental levels, whereas urea levels increased by ca. 30%. At the same time, whole animal ammonia and urea excretion vanished. Additionally, branchial RhMM, H + -ATPase, Na + /K + -ATPase and NHE mRNA levels decreased significantly after long term HEA exposure, whereas expression levels of RhMM in the internal tissues increased substantially. Interestingly, crabs acclimated to HEA showed no mortality even after 4 weeks of HEA exposure. This suggests that M. magister possesses a highly adaptive mechanism to cope with elevated ammonia concentrations in its body fluids, including an up-regulation of an Rh-like ammonia transporter in the internal tissues and excretion or storage of waste nitrogen in a so far unknown form.

Comparative seawater performance and deformity prevalence in out-of-season diploid and triploid Atlantic salmon (Salmo salar) post-smolts.

Abstract: The use of sterile triploid stock in the Atlantic salmon (Salmo salar, L) farming industry is the only commercially available means to prevent the ecological impact of domesticated escapees. This study compared the seawater (SW) performance and deformity prevalence of diploid and triploid post-smolts from 2 full-sib families produced out-of-season. Triploids completed smoltification 4 weeks earlier and at a significantly higher body-weight. Growth and survival in SW were not significantly affected by ploidy. The incidence of external deformities, dominated by jaw malformation, was ~12% in triploids and below 5% in diploids. Vertebral deformities were more prevalent in the fastest growing triploid family only. Heart morphometry differed between ploidies which may relate to a higher cardiac workload in triploids. No clear alteration of the gill apparatus was detected. The most significant detrimental effect of triploidy was on the rate and severity of cataract that were observed from August onward (50% and 92% of diploids and triploids respectively affected after 1-year in SW). At that time, cataracts were diagnosed by histological examinations as irreversible with a probable osmotic origin which could arise from factors such as water quality, nutritional deficiencies or thermal variations. This study warrants further research aiming at adapting rearing practices to the needs of triploid stocks as to improve their performance and welfare.

Effects of LED light spectra on oxidative stress and the protective role of melatonin in relation to the daily rhythm of the yellowtail clownfish, Amphiprion clarkii

Abstract: The present study aimed to test the effects of melatonin on oxidative stress in the yellowtail clownfish, Amphiprion clarkii, as produced by light emitting diodes (LEDs): red, green, and blue. We investigated the effects of the different LEDs on oxidative stress by measuring the mRNA expression of arylalkylamine N-acetyltransferase (AANAT2), the expression and activities of antioxidant enzymes (superoxide dismutase, SOD (EC 1.15.1.1); and catalase, CAT (EC 1.11.1.6)), and plasma H2O2 and plasma melatonin levels. In red light, the expression of AANAT2, SOD, and CAT mRNA was significantly higher than those under the other light spectra. SOD and CAT activities and plasma H2O2 and melatonin levels were also significantly higher for the red spectra than those for the other light spectra. These results indicate that red light induces oxidative stress. To investigate the effects of melatonin on oxidative stress, we injected melatonin into live fish (in vivo) or treated cultured pineal organ (in vitro) with melatonin. We found that AANAT2, SOD, and CAT mRNA expression levels, SOD and CAT activities, and plasma H2O2, lipid peroxidation (LPO) and melatonin levels were significantly lower than those for the controls. Therefore, our results indicate that red light induces oxidative stress and melatonin plays the role of a strong antioxidant in yellowtail clownfish.

The presence of multiple phenoloxidases in Caribbean reef-building corals.

Abstract: The melanin-synthesis pathways, phenoloxidase (PO) and laccases, are staple components of invertebrate immunity and have been shown to be vital in disease resistance. The importance of this pathway in immunity is a consequence of the release of oxygen radicals with cytotoxic effects and the production of insoluble melanin, which aids in the encapsulation of pathogens and parasites. Recently, melanization has been demonstrated as a critical immune response in several coral systems, although the biochemical components have not been thoroughly investigated. Coral diseases are posing a serious threat to coral reef survival, necessitating a full understanding of resistance mechanisms. In this study, we take a comparative approach to probe potential pathway components of melanin-synthesis in seven species from four different families of healthy Caribbean reef-building corals. Using different quinone substrates, we tested for the activity of the POs catecholase and cresolase, as well as laccase activity in each coral species. Since many invertebrate POs demonstrate some dependence on cations such as copper, calcium and magnesium, we treated the coral extracts with the chelators EDTA and EGTA to test the reliance of coral catecholase on these cations. The activity of the antioxidants peroxidase, superoxide dismutase and catalase was also tested in each coral and correlated to PO activity. All corals had demonstrable catecholase, cresolase and laccase activities, but only catecholase and cresolase activities varied significantly among species. Catecholase activity in each coral species was reduced by treatment with EDTA and EGTA, although some coral species were less affected than the others. Overall, these data show remarkable heterogeneity among the seven coral species of boulder-like reef building Caribbean coral. These differences may originate from the level of investment of each coral species into immunity and may explain disease ecology on the reef.

Patterns of gastric evacuation, digesta characteristics and pH changes along the gastrointestinal tract of gilthead sea bream (Sparus aurata L.) and European sea bass (Dicentrarchus labrax L.).

Abstract: A comparative study of gastric evacuation rates (GERs) and digesta content, moisture and pH values along the gastrointestinal tract was performed between gilthead sea bream and European sea bass. In order to distinguish species-specific differences from diet-elicited effects, all parameters were determined under either a fishmeal diet or a carob seed germ meal diet that contained high levels of total and soluble non-starch polysaccharides. GERs were significantly different between species and they were not affected by diet. Similarly, species-specific patterns were revealed in the distribution of digesta and water content along the gastrointestinal tract. In sea bream, stomach digesta and water content decreased with time, whereas in sea bass stomach retained the highest digesta and water content throughout the sampling period. The anterior and distal intestine exhibited the lowest accommodating capacities of digesta and water in either species. Overall, sea bream performed stomach digestion at lower hydration levels and higher pH compared with sea bass. Diet affected stomach moisture in both species and pH of stomach digesta in sea bass and of all intestinal sections in sea bream. The results obtained indicated that water and inorganic ion exchanges through the gut may differentiate between the species and warrant further investigation.

Effect of fasting on body composition and responses to stress in sunshine bass.

Abstract: The integrated responses of the hormonal regulation of growth and stress in sunshine bass ( Morone chrysops X Morone saxatilis ) as regulated by feed deprivation were investigated. Groups of fish were fed 1.5% of the body weight per day or offered no feed for 4 weeks. Another group of fish was not fed for 3 weeks and feed was offered during the fourth week. Fish in each group were sampled immediately before or after a 15-min low water confinement stressor after each week of the experiment. Liver mass and liver glycogen content were decreased after one week of fasting and remained low until the end of the study. However, both recovered after a week of refeeding. Intraperitoneal fat was significantly lower after two weeks of fasting and did not recover after a week of refeeding. None of these components were affected by confinement stress. Plasma glucose in unstressed fish was generally unaffected by fasting or refeeding; however, plasma glucose increased after confinement stress in fed but not in fasted fish. The cortisol stress response was unaltered by fasting and remained robust. Plasma IGF-I generally decreased in fasted fish but was not significantly lower than fed fish until the fourth week. A week of refeeding did not restore plasma IGF-I concentrations. Plasma IGF-I concentrations were higher in confinement stressed fed fish after two and four weeks but were unchanged in the fourth week. There was no change in the plasma IGF-I concentrations in fasted or refed fish due to the stress. Liver weight and liver glycogen were essentially depleted after 2 weeks of fasting. The reduction of liver glycogen greatly reduced the glucose response to stress; however, the cortisol stress response was maintained for at least four weeks of fasting. Intraperitoneal fat was decreased very little after 4 weeks of fasting. Plasma IGF-I concentrations were reduced only after 3 weeks of fasting.

The expression patterns of heat shock genes and proteins and their role during vertebrate's development.

Abstract: Highly evolutionary conserved heat shock proteins (HSPs) act as molecular chaperones in regulation of cellular homeostasis and promoting survival. Generally they are induced by a variety of stressors whose effect could be disastrous on the organism, but they are also widely constitutively expressed in the absence of stress. Varied HSP expressions seem to be very essential in the critical steps of embryonic and extra-embryonic structures formation and may correspond to cell movements, proliferation, morphogenesis and apoptosis, which occur during embryonic development. While our knowledge of detailed HSP expression patterns is in constant progress, their functions during embryonic development are not yet fully understood. In the paper, we review available data on HSP expression and discuss their role during vertebrate development.

Allometric estimation of metabolic rates in animals.

Abstract: The relationship between body mass (M) and metabolic rate (MR) typically accounts for most (>90%) of the inter-specific variation in MR. As such, when measurement of a species of interest is not possible, its MR can often be predicted using M. However, choosing an appropriate relationship to make such predictions is critical, and the choice is complicated by ongoing debate about the structure of the relationship between M and MR. The present study examines a range of methods including ordinary least squares (OLS), reduced major axis (RMA), and phylogenetically-informed (PI) approaches for estimating log(MR) from log(M), as well as non-linear approaches for estimating the relationship between MR and M without the need for log-transformation. Using data for the basal metabolic rates of mammals, it is shown that RMA regression overestimates the scaling exponent of MR (b, where MR=aM(b)), suggesting that OLS regression is appropriate for these data. PI approaches are preferred over non-PI ones, and the best estimates of log(MR) are obtained by including information on body temperature, climate, habitat, island endemism, and use of torpor in addition to log(M). However, the use of log-transformed data introduces bias into estimates of MR, while the use of non-linear regression underestimates MR for small mammals. This suggests that no single relationship is appropriate for describing the relationship between MR and M for all mammals, and that relationships for more narrow taxonomic groups or body mass ranges should be used when predicting MR from M.

Bioenergetic components of reproductive effort in viviparous snakes: costs of vitellogenesis exceed costs of pregnancy.

Abstract: article i nfo Reproductive effort has been defined as the proportion of an organism's energy budget that is allocated to re- production over a biologically meaningful time period. Historically, studies of reproductive bioenergetics considered energy content of gametes, but not costs of gamete production. Although metabolic costs of vitel- logenesis (MCV) fundamentally reflect the primary bioenergetic cost of reproductive allocation in female reptiles, the few investigations that have considered costs of reproductive allocation have focused on meta- bolic costs of pregnancy (MCP) in viviparous species. We define MCP as energetic costs incurred by pregnant females, including all costs of maintaining gestation conditions necessary for embryogenesis. MCP by our def- inition do not include fetal costs of embryogenesis. We measured metabolic rates in five species of viviparous snakes (Agkistrodon contortrix, Boa constrictor, Eryx colubrinus, Nerodia sipedon, and Thamnophis sirtalis) dur- ing vitellogenesis and pregnancy in order to estimate MCV and MCP. Across all species, MCV were responsible for 30% increases in maternal metabolism. Phylogenetically-independent contrasts showed that MCV were significantly greater in B. constrictor than in other species, likely because B. constrictor yolk energy content was greater than that of other species. Estimates of MCP were not significantly different from zero in any spe- cies. In viviparous snakes, MCV appear to represent significant bioenergetic expenditures, while MCP do not. We suggest that MCV, together with yolk energy content, represent the most significant component of rep- tilian reproductive effort, and therefore deserve greater attention than MCP in studies of reptilian reproduc- tive bioenergetics.

Molecular characterization and expression patterns of Spodoptera litura heat shock protein 70/90, and their response to zinc stress.

Abstract: Heat shock proteins (hsps) are highly evolutionarily conserved, whose expressions are altered with varying degrees in response to stress factors. Here, hsp70 and hsp90 cDNA were cloned from Spodoptera litura, and two hsps comprised corresponding family protein sequences, signature sequences and EEVD motif. They showed high homology to their counterparts in other species, and the phylogenetic analysis of hsp90 was consistent with the known insect classification. The hsp70/90 transcripts were present in S. litura midgut, cuticle and fat body, of which the highest levels of both were detected in fat body. The expression levels of both were different during four life stages, and the lowest and highest levels were detected in fifth instar larvae and adults, respectively. The hsp70 expression was induced significantly after the insects were exposed to 150 mg Zn/kg, but the down-regulation in other treatments showed significantly negative correlation with Zn accumulations in the body. The hsp90 expression was induced in all treated pupae and 150-450 mg Zn/kg treated larvae, while inhibited effects were observed in larvae exposed to 600-750 mg Zn/kg. Our results showed the induced response of S. litura hsp90 to Zn was more sensitive than hsp70, whereas inhibited response of hsp70 was much stronger than hsp90.

Phylogenetic constraints on digesta separation: Variation in fluid throughput in the digestive tract in mammalian herbivores

Abstract: The relevance of the mean retention time (MRT) of particles through the gastrointestinal tract (GIT) is well understood and MRTparticleGIT is an important parameter in digestion models. Solute markers have been used to estimate MRTsoluteGIT (or ‘fluid passage’) in animals, but the relevance of this measure is less evident and is usually sought in its relation to MRTparticleGIT. The ratio between the two measures indicates the degree of ‘digesta washing’, with little washing occurring at ratios of 1, aborad washing at ratios N1 (where the solute marker travels faster than the particle marker), and orad (retrograde) washing at ratios b1 (where the solute marker travels slower than the particle marker). We analysed digesta washing in a dataset of 98 mammalian species including man of different digestion types (caecum, colon and nonruminant foregut fermenters, and ruminants), controlling for phylogeny; a subset of 72 species allowed testing for the influence of food intake level. The results indicate that MRTsoluteGIT and the degree of digesta washing are related to digestion type, whereas variation in MRTparticleGIT is influenced mainly by effects of body mass and food intake. Thus, fluid throughput and digesta washing emerge as important correlates of digestive anatomy. Most importantly, primates appear constrained to little digesta washing compared to non-primate mammalian herbivores, regardless of their digestion type. These results may help explain the absence of primates from certain herbivore niches and represent a drastic example of a physiologic limitation in a phylogenetic group. Moreexperimental research is required to illuminate relative benefits and costs of digesta washing.

Developmental energetics of zebrafish, Danio rerio.

Abstract: Using zebrafish (Danio rerio) as a case study, we show that the maturity concept of Dynamic Energy Budget (DEB) theory is a useful metric for developmental state. Maturity does not depend on food or temperature contrary to age and to some extent length. We compile the maturity levels for each developmental milestone recorded in staging atlases. The analysis of feeding, growth, reproduction and aging patterns throughout the embryo, juvenile and adult life stages are well-captured by a simple extension of the standard DEB model and reveals that embryo development is slow relative to adults. A threefold acceleration of development occurs during the larval period. Moreover we demonstrate that growth and reproduction depend on food in predictable ways and their simultaneous observation is necessary to estimate parameters. We used data on diverse aspects of the energy budget simultaneously for parameter estimation using the covariation method. The lowest mean food intake level to initiate reproduction was found to be as high as 0.6 times the maximum level. The digestion efficiency for Tetramin™ was around 0.5, growth efficiency was just 0.7 and the value for the allocation fraction to soma (0.44) was close to the one that maximizes ultimate reproduction.

Dynamic gene expression of GH/PRL-family hormone receptors in gill and kidney during freshwater-acclimation of Mozambique tilapia.

Abstract: In teleosts, prolactin (PRL) and growth hormone (GH) act at key osmoregulatory tissues to regulate hydromineral balance. This study was aimed at characterizing patterns of expression for genes encoding receptors for the GH/PRL-family of hormones in the gill and kidney of Mozambique tilapia (Oreochromis mossambicus) during freshwater (FW)-acclimation. Transfer of seawater (SW)-acclimated tilapia to FW elicited rapid and sustained increases in plasma levels and pituitary gene expression of PRL177 and PRL188; plasma hormone and pituitary mRNA levels of GH were unchanged. In the gill, PRL receptor 1 (PRLR1) mRNA increased markedly after transfer to FW by 6h, while increases in GH receptor (GHR) mRNA were observed 48 h and 14 d after the transfer. By contrast, neither PRLR2 nor the somatolactin receptor (SLR) was responsive to FW transfer. Paralleling these endocrine responses were marked increases in branchial gene expression of a Na+/Cl- cotransporter and a Na+/H+ exchanger, indicators of FW-type mitochondrion-rich cells (MRCs), at 24 and 48 h after FW transfer, respectively. Expression of Na+/K+/2Cl- cotransporter, an indicator of SW-type MRCs, was sharply down-regulated by 6h after transfer to FW. In kidney, PRLR1, PRLR2 and SLR mRNA levels were unchanged, while GHR mRNA was up-regulated from 6h after FW transfer to all points thereafter. Collectively, these results suggest that the modulation of the gene expression for PRL and GH receptors in osmoregulatory tissues represents an important aspect of FW-acclimation of tilapia.

Is there an influence of body mass on digesta mean retention time in herbivores? A comparative study on ungulates

Abstract: The relation between body mass (BM) and digesta mean retention time (MRT) in herbivores was the focus of several studies in recent years. It was assumed that MRT scaled with BM 0.25 based on the isometric scaling of gut capacity (BM 1.0 ) and allometric scaling of energy intake (BM 0.75 ). Literature studies that tested this hypothesis produced conflicting results, arriving sometimes at higher or lower exponents than the postulated 0.25. This study was conducted with 8 ruminants ( n = 2–6 per species) and 6 hindgut fermenting species/breeds ( n = 2–6, warthog n = 1) with a BM range of 60–4000 kg. All animals received a ration of 100% grass hay with ad libitum access. Dry matter intake was measured and the MRT was estimated by the use of a solute and a particle (1–2 mm) marker. No significant scaling of MRT particle with BM was observed for all herbivores (32 BM 0.04 , p = 0.518) and hindgut fermenters (32 BM 0.00 , p = 1.00). The scaling exponent for ruminants only showed a tendency towards significance (29 BM 0.12 , p = 0.071). Ruminants on average had an MRT particle 1.61-fold longer than hindgut fermenters. Whereas an exponent of 0.25 is reasonable from theoretical considerations, much lower exponents were found in this and other studies. The energetic benefit of increasing MRT is by no means continuous, since the energy released from a given food unit via digestion decreases over time. The low and non-significant scaling factors for both digestion types suggest that in ungulates, MRT is less influenced by BM (maximal allometric exponent ≤ 0.1) than often reported.