Showing papers in "Comparative Biochemistry and Physiology B in 2019"

Effects of high-fat diet on growth performance, lipid accumulation and lipid metabolism-related MicroRNA/gene expression in the liver of grass carp (Ctenopharyngodon idella).

Abstract: An 8 week experiment was conducted to evaluate the effects of dietary fat on growth and on the accumulation of lipids and the expression of lipid metabolism-related microRNAs (miRNAs) and genes in grass carp (Ctenopharyngodon idella). Two diets (normal fat diet (NFD), 60 g/kg lipid content; high fat diet (HFD), 160 g/kg lipid content) were fed to triplicate groups of 35 fish [initial weight of (40.0 ± 0.5) g]. The results showed that increased dietary fat did not lead to significant differences in the feed conversion rate (FCR) and specific growth rate (SGR) (P > .05), but the HFD significantly increased the hepatosomatic index (HSI) (P C) level was significantly increased in the HFD group (P

The effect of temperature on Triclosan and Lead exposed mussels.

Abstract: Interest on the effects of emerging contaminants over aquatic organisms has increased in the last years. Nonetheless, the toxic action of classical natural and anthropogenically-driven metals has also to be monitored, especially because they reflect real environmental situations. For that, in the present study we focused on the effects on the marine mussel Mytilus galloprovincialis of the personal care product Triclosan (TCS) and Lead (Pb), as toxic metal, under separate and co-exposure situations at environmentally relevant concentrations: TCS (1 μg/L) and Pb (50 μg/L). The consideration of an additional factor such as an increase in ambient temperature was also included to provide a forecasted scenario of climate change: from the ambient temperature at actual conditions (17 °C) to a predicted warming situation (22 °C). Water chemical characterization and some physical properties and bioaccumulation of TCS and Pb in mussels at the end of the experiment (28 days) was considered. The parameters followed up comprise the energy related system production (electron transport system) and glycogen and protein reserves. Antioxidant enzymatic defences towards reactive oxygen species (ROS) and the consequences of ROS damage over endogenous lipids (LPO) and proteins (PC). Overall the results suggested only particular responses to chemical exposures at 17 °C whereas at 22 °C the detoxification machinery was set up and this prevented the occurrence of LPO. Nonetheless, PC formation occurred under Pb and TCS + Pb co-exposure at the highest temperature. Due to the complexity of the study: 4 chemical conditions, 2 temperatures and 10 biomarkers considered, a principal component ordination (PCO) analysis was included. The results of this integrative analysis confirmed a clear effect of the temperature, more responsiveness to drugs at 22 °C and in all likelihood due to Pb presence.

A complete enzymatic capacity for long-chain polyunsaturated fatty acid biosynthesis is present in the Amazonian teleost tambaqui, Colossoma macropomum

Abstract: In vertebrates, the essential fatty acids (FA) that satisfy the dietary requirements for a given species depend upon its desaturation and elongation capabilities to convert the C18 polyunsaturated fatty acids (PUFA), namely linoleic acid (LA, 18:2n-6) and α-linolenic acid (ALA, 18:3n-3), into the biologically active long-chain (C20-24) polyunsaturated fatty acids (LC-PUFA), including arachidonic acid (ARA, 20:4n-6), eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3). Recent studies have established that tambaqui (Colossoma macropomum), an important aquaculture-produced species in Brazil, is a herbivorous fish that can fulfil its essential FA requirements with dietary provision C18 PUFA LA and ALA, although the molecular mechanisms underpinning such ability remained unclear. The present study aimed at cloning and functionally characterizing genes encoding key desaturase and elongase enzymes, namely fads2, elovl5 and elovl2, involved in the LC-PUFA biosynthetic pathways in tambaqui. First, a fads2-like desaturase was isolated from tambaqui. When expressed in yeast, the tambaqui Fads2 showed Δ6, Δ5 and Δ8 desaturase capacities within the same enzyme, enabling all desaturation reactions required for ARA, EPA and DHA biosynthesis. Moreover, tambaqui possesses two elongases that are bona fide orthologs of elovl5 and elovl2. Their functional characterization confirmed that they can operate towards a variety of PUFA substrates with chain lengths ranging from 18 to 22 carbons. Overall our results provide compelling evidence that demonstrates that all the desaturase and elongase activities required to convert LA and ALA into ARA, EPA and DHA are present in tambaqui within the three genes studied herein, i.e. fads2, elovl5 and elovl2.

Molecular identification and expression profiles of four splice variants of Sex-lethal gene in Cherax quadricarinatus.

Abstract: Cherax quadricarinatus, as one of the world's most valuable freshwater shrimp species, has received extensive attention in recent years. As males grow larger and faster than females, development of the sex control breeding techniques is of great interest, but knowledge on sex determination and differentiation in C. quadricarinatus remains poorly unknown. Sxl (Sex-lethal) is an important gene in the sexual differentiation regulatory hierarchy. It reflects the ratio of sex chromosomes to autosomes into molecule changes and directs sex-specific splicing forms of precursor mRNA. In the present study, the full-length cDNA sequences of four Sxl splice variants were identified from C. quadricarinatus, designated as CqSxl1, CqSxl2, CqSxl3 and CqSxl4, respectively. Sequence analysis determined different splicing sites near the translation termination region of four Sxl transcript isoforms. Two highly conserved classical RRM domains were found according to predicted secondary structures of Sxl proteins. mRNA expression of CqSxl in different tissues, developmental stage of embryos and testes were investigated by real-time quantitative PCR. Among four isoforms, CqSxl3 showed tissue specificity with higher expression levels in testis than in ovary. CqSxl1 and CqSxl4 were found widely expressed in various tissues and CqSxl2 was almost undetectable. In early developmental stages, the expression levels of CqSxl1/3/4 gradually increased along with embyonic development. In addition, CqSxl genes presented the higher transcript levels in the early stage of testis development. Furthermore, CqSxl3 silencing induced a significant decrease of the transcript of Cq-IAG, an androgenic hormone-encoding gene responsible for masculine development. These data indicate that CqSxl3 might be involved in male sex determination in C. quadricarinatus. Our study will contribute to understanding the mechanism of sex determination in C. quadricarinatus, and also can provide theoretical guidance for establishing a sex control technology.

The effect of micronutrient supplementation on growth and hepatic metabolism in diploid and triploid Atlantic salmon (Salmo salar) parr fed a low marine ingredient diet

Abstract: The effects of low marine ingredient diets supplemented with graded levels (L1, L2, L3) of a micronutrient package (NP) on growth and metabolic responses were studied in diploid and triploid salmon parr. Diploids fed L2 showed significantly improved growth and reduced liver, hepatic steatosis, and viscerosomatic indices, while fish fed L3 showed suppressed growth rate 14 weeks post feeding. In contrast, dietary NP level had no effect on triploid performance. Whole body mineral composition, with exception of copper, did not differ between diet or ploidy. Whole fish total AAs and N-metabolites showed no variation by diet or ploidy. Free circulating AAs and white muscle N-metabolites were higher in triploids than diploids, while branch-chained amino acids were higher in diploids than triploids. Diploids had higher whole body α-tocopherol and hepatic vitamins K1 and K2 than triploids. Increased tissue B-vitamins for niacin and whole-body folate with dietary NP supplementation were observed in diploids but not triploids, while whole body riboflavin was higher in diploids than triploids. Hepatic transcriptome profiles showed that diploids fed diet L2 was more similar to that observed in triploids fed diet L3. In particular, sterol biosynthesis pathways were down-regulated, whereas cytochrome P450 metabolism was up-regulated. One‑carbon metabolism was also affected by increasing levels of supplementation in both ploidies. Collectively, results suggested that, for optimised growth and liver function, micronutrient levels be supplemented above current National Research Council (2011) recommendations for Atlantic salmon when fed low marine ingredient diets. The study also suggested differences in nutritional requirements between ploidy.

Oxygen-dependent distinct expression of hif-1α gene in aerobic and anaerobic tissues of the Amazon Oscar, Astronotus crassipinnis.

Abstract: The aquatic habitats of the Amazon basin present dramatic variation of oxygen level, and, to survive such changes, many aquatic animals developed biochemical and physiological adaptations. The advanced teleost Astronotus crassipinnis (Perciformes) is a fish tolerant to hypoxia and known to endure such naturally variable environments. Hypoxia-Inducible factor-1α (hif-1α) is among the most important and studied genes related to hypoxia-tolerance, maintaining regular cellular function and controlling anaerobic metabolism. In the present work, we studied hif-1α expression and related it to changes in metabolic pathways of Astronotus crassipinnis exposed to 1, 3 and 5 h of hypoxia, followed by 3 h of recovery. The results show that A. crassipinnis depresses aerobic metabolic under hypoxia, with a decrease in glycolysis and oxidative enzyme activities, and increases its anaerobic metabolism with an increase in LDH activity coupled with a decrease in oxygen consumption, which indicates an increase in anaerobic capacity. In addition, the animal differentially regulates hif-1α gene in each tissue studied, with a positive relationship to its metabolic profile, suggesting that hif-1α might be one of the most important induction factors that regulate hypoxia tolerance in this species.

Expression and transcriptional regulation of gsdf in spotted scat (Scatophagus argus).

Abstract: Gonadal soma-derived factor (Gsdf) is critical for testicular differentiation and early germ cell development in teleosts. The spotted scat (Scatophagus argus), with a stable XX-XY sex-determination system and the candidate sex determination gene dmrt1, provides a good model for understanding the mechanism of sex determination and differentiation in teleosts. In this study, we analyzed spotted scat gsdf tissue distribution and gene expression patterns in gonads, as well as further analysis of transcriptional regulation. Tissue distribution analysis showed that gsdf was only expressed in testis and ovary. Real-time PCR showed that both gsdf and dmrt1 were expressed significantly higher in testes at different phases (phase III, IV and V) compared to ovaries at phase II, III and IV, while gsdf was expressed significantly higher in phase II ovaries than those of phase III and IV. Western blot analysis also showed that Gsdf was more highly expressed in the testis than ovary. Immunohistochemistry analysis showed that Gsdf was expressed in Sertoli cells surrounding spermatogonia in the testis, while it was expressed in the somatic cells surrounding the oogonia of the ovary. Approximately 2.7 kb of the 5′ upstream region of gsdf was cloned from the spotted scat genomic DNA and in silico promoter analysis revealed the putative transcription factor binding sites of Dmrt1 and Sf1. The luciferase reporter assay, using the human embryonic kidney cells, demonstrated that Dmrt1 activated gsdf expression in a dose-dependent manner in the presence of Sf1 in spotted scat. These results suggest that Gsdf could play a role in regulating the development of spermatogonia and oogonia, and also participate in male sex differentiation by acting as a downstream gene of Dmrt1 in spotted scat.

Dietary replacement of fish-meal impaired protein synthesis and immune response of juvenile Pacific white shrimp, Litopenaeus vannamei at low salinity.

Abstract: An 8-week feeding trial was conducted to evaluate the effect of fish-meal replacement on growth performance, protein synthesis and immune response of juvenile Pacific white shrimp, Litopenaeus vannamei reared at low salinity (7‰). Five isonitrogenous and isolipidic diets were formulated to contain graded levels (25, 20, 15, 10 and 5%) of fish-meal. High quality alternative solutions were performed, crystalline amino acids, phytase, mannan oligosaccharides and some micro-nutrients were supplemented in the low fish-meal diets. Each diet was randomly assigned to triplicate tanks, each tank with 30 shrimp (mean weight 0.3 g), the shrimp were fed 3 times a day. Weight gain and survival were decreased with the decreasing dietary fish meal levels. When dietary fish-meal decreased, the gene expression of TOR, Raptor and eIF4E2 in hepatopancreas were decreased with the decreasing fish meal levels, eIF4E2 in intestine was decreased while 4E-BP was increased with the decreasing fish meal levels. The mRNA level of SOD in hepatopancreas decreased, and the expression of GPx and CAT increased with the decreasing FM levels. The Toll pathway was affected by dietary FM levels, the expression of Toll2, TNFSF, MyD88, Rho and p38 in intestine were increased with the decreasing FM levels. The results indicated that at low salinity condition, fish meal level lower than 15% would inhibit the protein synthesis and harm to the health of shrimp.

Oxidative stress and biomarker responses in the Atlantic halibut after long term exposure to elevated CO2 and a range of temperatures

Abstract: CO2 emissions from human activities are increasing, resulting in greater rates of change in the oceans, exceeding any other event in geological and historical records over the past 300 million years. Oceans are warming and pH levels are decreasing. Marine organisms will need to respond to multiple stressors and the potential consequences of global change-related effects in fish needs to be investigated. Fish are affected by many biotic and abiotic environmental variables, including temperature and CO2 fluctuations, and it is therefore critical to investigate how these variables may affect physiological and biochemical processes. We investigated the effects of elevated CO2 levels (pH of 8.0, which served as a control, or 7.6, which is predicted for the year 2100) combined with exposure to different temperatures (5, 10, 12, 14, 16, and 18 °C) in the Atlantic halibut (Hippoglossus hippoglossus) during a three month experiment. Since regulation of reactive oxygen species (ROS) is crucial for physiological processes the focus was on the antioxidant defense system and we assessed the effects on catalytic activities of antioxidant enzymes (SOD, CAT, GR, GST, GPx). In addition we also analyzed effects on cholinesterase enzymes (AChE and BChE), and CYP1A enzyme activities (EROD). The treatments resulted in oxidative stress, and damage was evident in the form of protein carbonyls which were consistently higher in the elevated CO2-treated fish at all temperatures. Analyses of antioxidant enzymes did not show the same results, suggesting that the exposure to elevated CO2 increased ROS formation but not defences. The antioxidant defense system was insufficient, and the resulting oxidative damage could impact physiological function of the halibut on a cellular level.

Assessment of muscular energy metabolism and heat shock response of the green abalone Haliotis fulgens (Gastropoda: Philipi) at extreme temperatures combined with acute hypoxia and hypercapnia.

Abstract: The interaction between ocean warming, hypoxia and hypercapnia, suggested by climate projections, may push an organism earlier to the limits of its thermal tolerance window. In a previous study on juveniles of green abalone (Haliotis fulgens), combined exposure to hypoxia and hypercapnia during heat stress induced a lowered critical thermal maximum (CTmax), indicated by constrained oxygen consumption, muscular spams and loss of attachment. Thus, the present study investigated the cell physiology in foot muscle of H. fulgens juveniles exposed to acute warming (18 °C to 32 °C at +3 °C day-1) under hypoxia (50% air saturation) and hypercapnia (~1000 μatm PCO2), alone and in combination, to decipher the mechanisms leading to functional loss in this tissue. Under exposure to either hypoxia or hypercapnia, citrate synthase (CS) activity decreased with initial warming, in line with thermal compensation, but returned to control levels at 32 °C. The anaerobic enzymes lactate and tauropine dehydrogenase increased only under hypoxia at 32 °C. Under the combined treatment, CS overcame thermal compensation and remained stable overall, indicating active mitochondrial regulation under these conditions. Limited accumulation of anaerobic metabolites indicates unchanged mode of energy production. In all treatments, upregulation of Hsp70 mRNA was observed already at 30 °C. However, lack of evidence for Hsp70 protein accumulation provides only limited support to thermal denaturation of proteins. We conclude that under combined hypoxia and hypercapnia, metabolic depression allowed the H. fulgens musculature to retain an aerobic mode of metabolism in response to warming but may have contributed to functional loss.

Molecular cloning of IL-6, IL-10, IL-11, IFN-ɤ and modulation of pro- and anti-inflammatory cytokines in cobia (Rachycentron canadum) after Photobacterium damselae subsp. piscicida infection.

Abstract: Photobacterium damselae subsp. piscicida (P. damselae subsp. piscicida) is the agent of Photobacteriosis, a serious fish disease that produces an acute infection and high mortality in farmed cobia. It has been proved that regulation of pro- and anti-inflammatory cytokines play a central role in initiation of proper inflammatory responses against bacterial infection. Here we have analyzed the expression of pro-inflammatory cytokines (IL-1β, TNF-α, IL-6, IL-8 and IFN-ɤ) and anti-inflammatory cytokines (IL-10 and IL-11) in spleen and head kidney during acute P. damselae subsp. piscicida infection of cobia. Our data revealed that cytokines tested showed distinct patterns of expression. While TNF-α and IL-8 showed a decay pattern of expression, IL-1β response was quite late. Moreover, P. damselae subsp. piscicida infection induced the simultaneous expressions of pro-inflammatory (IL-6, IFN-ɤ) and anti-inflammatory (IL-10, IL-11) cytokines. Together these results indicate the innate immunity of cobia is actively suppressed by P. damselae subsp. piscicida.

Phoenixin: Expression at different ovarian development stages and effects on genes ralated to reproduction in spotted scat, Scatophagus argus.

Abstract: Phoenixin (Pnx), a recently discovered neuropeptide, has been implicated in reproduction. Pnx mainly exists in two active isoforms, phoenixin-14 (Pnx-14) and phoenixin-20 (Pnx-20). However, little is known about the functions of Pnx in teleosts. To determine the roles of Pnx in the regulation of reproduction in Scatophagus argus, the physiological characterization of the Pnx was analyzed. During ovary development, the expression of pnx in phase IV was higher than in phase II and III in the hypothalamus. In the pituitary, pnx expression was highest in phase IV, moderate in phase III, and lowest in phase II. When hypothalamus and pituitary fragments were cultured in vitro with Pnx-14 and Pnx-20 (10 nM and 100 nM) for 6 h, the expression of GnRHR (gonadotropin releasing hormone receptor), lh (luteinizing hormone) and fsh (follicular stimulating hormone) in the pituitary increased significantly, except GnRH (gonadotropin releasing hormone) in the hypothalamus. Similarly, the expression of GnRHR, lh and fsh in the pituitary increased significantly after injecting S. argus with Pnx-14 and Pnx-20 (10 ng/g and 100 ng/g body weight (bw)), except GnRHR and fsh treated with 10 ng/gbw Pnx-20 in the pituitary and GnRHs in the hypothalamus. These results indicate that Pnx may not only stimulate the reproduction of the S. argus through the hypothalamic-pituitary-gonadal (HPG) axis, but also directly through the pituitary.

A comparison of the nutritional physiology and gut microbiome of urban and rural house sparrows (Passer domesticus).

Abstract: Urbanization influences food quality and availability for many wild species, but our knowledge of the consequences urbanization has on the nutritional physiology of these animals is currently limited. To fill this gap, we captured House Sparrows (Passer domesticus) from rural and urban environments and hypothesized that increased access to human refuse in urban areas may significantly alter the gut microbiome and nutritional physiology of Sparrows. While there were no significant differences in circulating triglycerides or free glycerol concentrations between populations, urban birds had significantly greater blood glucose concentrations, which suggests greater circulating glucagon concentrations, accessibility to carbohydrates, and/or higher rates of gluconeogenesis in an urban setting. Rural birds had significantly more plasma uric acid, suggesting that they may metabolize more proteins or experience lower inflammation than urban birds. Rural birds also had significantly higher liver free glycerol concentrations, indicating that they metabolize more fat than urban birds. There were no significant differences in the relative abundance of gut microbial taxa at the phyla level between the two populations, but linear discriminant analysis effect size (LEfSe) showed that urban House Sparrows were more enriched with class- and order-level microbes from the phylum Proteobacteria, which are implicated in several mammalian intestinal and extra-intestinal diseases. These findings demonstrate that urbanization significantly alters the nutritional physiology and the composition of the gut microbiome of House Sparrows.

High level of dietary soybean oil affects the glucose and lipid metabolism in large yellow croaker Larimichthys crocea through the insulin-mediated PI3K/AKT signaling pathway

Abstract: The present study was conducted to investigate the metabolic responses of glucose and lipid in large yellow croaker Larimichthys crocea (initial weight, 36.80 ± 0.39 g) to high level of dietary soybean oil. Three isonitrogenous (46% crude protein) and isolipidic (13% crude lipid) experimental diets were designed, with 100% fish oil (FO), 50% fish oil and 50% soybean oil (FS) and 100% soybean oil (SO), respectively. After a 12-week growth trial, the results showed that compared with FO group, contents n-6 PUFAs increased while the n-3 PUFAs decreased significantly both in liver and muscle in FS and SO groups. Concentrations of blood glucose, leptin, free fatty acid and total triglyceride reached the highest values in SO group, while blood insulin showed no significant difference among all groups. The gene expressions of insulin receptor substrate-2, glucose-6-phosphatase, phosphoenolpyruvate carboxykinase, fatty acid synthetase, and lipoprotein lipase increased, and the insulin receptor substrate-1, phosphotidylinsositol-3-kinase (PI3K), hexokinase, glycogen synthetase and glucose transporter 2 in liver decreased significantly in SO group. Meanwhile, the phosphorylation of protein kinase B (AKT) also decreased significantly in this group. These results suggested that high level of dietary soybean oil depressed PI3K/AKT signaling pathway, and then affected glucose and lipid metabolism by glycolysis, gluconeogenesis, glucose transportation, glycogenesis and lipogenesis.

Myoglobin as a versatile peroxidase: Implications for a more important role for vertebrate striated muscle in antioxidant defense.

Abstract: Myoglobins (Mb) are ubiquitous proteins found in striated muscle of nearly all vertebrate taxa. Although their function is most commonly associated with facilitating oxygen storage and diffusion, Mb has also been implicated in cellular antioxidant defense. The oxidized (Fe3+) form of Mb (metMB) can react with hydrogen peroxide (H2O2) to produce ferrylMb. FerrylMb can be reduced back to metMb for another round of reaction with H2O2. In the present study, we have shown that horse skeletal muscle Mb displays peroxidase activity using 2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) and 3,3',5,5'-tetramethylbenzidine (TMB) as reducing substrates, as well as the biologically-relevant substrates NADH/NADPH, ascorbate, caffeic acid, and resveratrol. We have also shown that ferrylMb can be reduced by both ethanol and acetaldehyde, which are known to accumulate in some vertebrate tissues under anaerobic conditions, such as anoxic goldfish and crucian carp, implying a potential mechanism for ethanol detoxification in striated muscle. We found that metMb peroxidase activity is pH-dependent, increasing as pH decreases from 7.4 to 6.1, which is biologically relevant to anaerobic vertebrate muscle when incurring intracellular lactic acidosis. Finally, we found that metMb reacts with hypochlorite in a heme-dependent fashion, indicating that Mb could play a role in hypochlorite detoxification. Taken together, these data suggest that Mb peroxidase activity might be an important antioxidant mechanism in vertebrate cardiac and skeletal muscle under a variety of physiological conditions, such as those that might occur in contracting skeletal muscle or during hypoxia.

Temperature-induced changes in body lipid composition affect vulnerability to oxidative stress in Daphnia magna.

Abstract: Optimizing physiological functions at different temperatures includes shifts in the lipid composition of ectothermic animals. These shifts may be associated with changes in lipid peroxidation in response to oxidative stress, because lipids differ in their susceptibility to oxidative damage. Polyunsaturated fatty acids (PUFA) are particular prone to peroxidation. Here, we analyzed changes in the fatty acid composition, cholesterol content and the level of oxidative damage as thiobarbituric reactive substances (TBARS) in Daphnia magna as a function of acclimation temperature. The total fatty acid content was highest in cold-acclimated animals. The relative share of most PUFA decreased with increasing acclimation temperature. In contrast, the contribution of saturated and monounsaturated fatty acids (SFA and MUFA) increased with acclimation temperature, although the latter to a lower extent. Cholesterol content remained unchanged. The level of oxidative damage was lowest in animals reared at warm temperatures, most likely reflecting their lowest content of PUFA. Heat exposure (1 h at 33 °C) caused the highest increase in lipid peroxidation in cold-acclimated animals, containing more PUFA. Our data suggest that cold-induced adjustments in the body lipid composition increase the vulnerability of zooplankton to heat-induced oxidative stress. In particular, animals performing diel vertical migration may be highly susceptible to temperature-induced lipid damage.

Hypoxia enhances blood O2 affinity and depresses skeletal muscle O2 consumption in zebrafish (Danio rerio)

Abstract: Zebrafish (Danio rerio) are widely used animal models. Nevertheless, the mechanisms underlying hypoxia tolerance in this species have remained poorly understood. In the present study, we have determined the effects of hypoxia on blood-O2 transport properties and mitochondrial respiration rate in permeabilized muscle fibres of adult zebrafish exposed to either 1) a gradual decrease in O2 levels until fish lost equilibrium (~1 h, acute hypoxia), or 2) severe hypoxia (PO2 ∼ 15 Torr) for 48 h (prolonged hypoxia). Acute, short-term hypoxia caused an increase in hemoglobin (Hb) O2 affinity (decrease in P50), due to a decrease in erythrocyte ATP after erythrocyte swelling. No changes in isoHb expression patterns were observed between hypoxic and normoxic treatments. Prolonged hypoxia elicited additional reponses on O2 consumption: lactate accumulated in the blood, indicating that zebrafish relied on glycolysis for ATP production, and mitochondrial respiration of skeletal muscle was overall significantly inhibited. In addition, male zebrafish had higher hypoxia tolerance (measured as time to loss of equilibrium) than females. The present study contributes to our understanding of the adaptive mechanisms that allow zebrafish, and by inference other fish species, to cope with low O2 levels.

Regulation of triglyceride synthesis by estradiol in the livers of hybrid tilapia (Oreochromis niloticus ♀ × O. aureus ♂).

Abstract: Estradiol (E2) is a sex steroid hormone that modulates multiple physiological processes in teleosts. The aim of this study was to explore the role of E2 in the hepatic lipid metabolism of hybrid tilapia. The hybrid tilapias were injected with different concentrations of E2 (0 mg/kg, 10 mg/kg, 25 mg/kg and 50 mg/kg) and ICI 182,780 (ICI) (35 mg/kg) (an E2 receptor antagonist). Subsequently, the liver lipid depositions were analyzed by tissue sections with oil red O staining. Serum total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL) and nonesterified fatty acids (NEFAs) were assayed from the fish in different groups. Genes related to very low-density lipoprotein (VLDL) assembly, lipoprotein lipase and lipoprotein receptors, fatty acid uptake and triacylglycerol metabolism were determined by quantitative RT-PCR. The results showed that 50 mg/kg E2 injections enlarged the lipid droplets significantly. Simultaneously, the E2 injections tended to upregulate TC, TG, LDL, and HDL in the serum. The 50 mg/kg E2 group showed a significantly higher expression of the VLDL assembly genes but depressed levels of LDLR and LRP1. In addition, FABP3, FABP11a and DGAT2 were significantly elevated, while CD36 and ACO1 decreased in the 50 mg/kg E2 injection. The ICI injection inhibited the expression of MTP, LPL, LRP1, CD36, FABP11a, ACO1 and FAS in tilapia livers. These results demonstrated that by stimulating the expression of genes associated with the VLDL assembly, inhibiting lipoprotein lipase and lipoprotein receptor-related genes and promoting the rate-limiting enzyme in the synthesis of the TG, E2 induced deposition of lipids in the livers of hybrid tilapia. Overall, the results suggest a role for E2 in fish lipid metabolisms that provide new clues to illustrate the sex steroid function in energy metabolism in livers.

Evaluation of baseline haemolymph biochemistry, volume and total body energetics to determine an accurate condition index in the black tiger shrimp, Penaeus monodon.

Abstract: Crustaceans are exposed to a range of environmental factors that can impact their condition, physiological function and growth. Condition indices are broadly defined as the extent to which stored nutrient reserves allow normal physiological function and growth, but can also represent more than nutrition alone. There is currently no reliable indicator to non-destructively measure shrimp physiological or nutritional condition. In this study, haemolymph and biochemical indices were benchmarked against a range of physiological status condition indicators, including haemolymph biochemistry parameters and carcass traits, in fed and unfed sub-adult black tiger shrimp (Penaeus monodon) and across the moult cycle. Results based on fluorescent marker injection and analysis of recovered amounts showed that haemolymph volume was elevated immediately after the moult, but decreased significantly within 2 days and remained stable for the remainder of the inter-moult period. Brix index (Brix) strongly correlated with haemolymph biochemical, shrimp condition and carcass composition indices. These included haemolymph volume, total protein and triglycerides, as well as gross energy, hepatosomatic index (HSI) and body weight gain per moult. Overall, results demonstrated that Brix was the simplest, fastest and most cost-effective indicator to accurately and non-destructively estimate physiological condition in P. monodon. Given the correlations with a broad range of indices, Brix may represent a more holistic estimate of condition, incorporating multifactorial aspects of shrimp condition including moult cycle and nutrition. Additionally, the baseline measurements of metabolites across the moult and under starvation conditions improves our fundamental understanding of overall condition in P. monodon.

Identification of the alternative oxidase gene and its expression in the copepod Tigriopus californicus.

Abstract: In addition to the typical electron transport system (ETS) in animal mitochondria responsible for oxidative phosphorylation, in some species there exists an alternative oxidase (AOX) pathway capable of catalyzing the oxidation of ubiquinol and the reduction of oxygen to water. The discovery of AOX in animals is recent and further investigations into its expression, regulation, and physiological role have been hampered by the lack of a tractable experimental model organism. Our recent DNA database searches using bioinformatics revealed an AOX sequence in several marine copepods including Tigriopus californicus. This species lives in tidepools along the west coast of North America and is subject to a wide variety of daily environmental stresses. Here we verify the presence of the AOX gene in T. californicus and the expression of AOX mRNA and AOX protein in various life stages of the animal. We demonstrate that levels of the AOX protein increase in T. californicus in response to cold and heat stress compared to normal rearing temperature. We predict that a functional AOX pathway is present in T. californicus, propose that this species will be a useful model organism for the study of AOX in animals, and discuss future directions for animal AOX research.

Chitin deacetylase 1 and 2 are indispensable for larval–pupal and pupal–adult molts in Heortia vitessoides (Lepidoptera: Crambidae)

Abstract: Heortia vitessoides Moore is a notorious defoliator of Aquilaria sinensis (Lour.) Gilg trees. Chitin deacetylases (CDAs) catalyze the N-deacetylation of chitin, which is a crucial process for chitin modification. Here, we identified and characterized HvCDA1 and HvCDA2 from H. vitessoides. HvCDA1 and HvCDA2 possess typical domain structures of CDAs and belong to the Group I CDAs. HvCDA1 and HvCDA2 were highly expressed before and after the larval–larval molt. In addition, both exhibited relatively high mRNA expression levels during the larval–pupal molt, the pupal stage, and the pupal–adult molt. HvCDA1 and HvCDA2 transcript expression levels were highest in the body wall and relatively high in the larval head. Significant increases in the HvCDA1 and HvCDA2 transcript expression levels were observed in the larvae upon exposure to 20-hydroxyecdysone. RNA interference-mediated HvCDA1 and HvCDA2 silencing significantly inhibited HvCDA1 and HvCDA2 expression, with abnormal or nonviable phenotypes being observed. Post injection survival rates of the larvae injected with dsHvCDA1 and dsHvCDA2 were 66.7% and 46.7% (larval–pupal) during development and 23.0% and 6.7% (pupal–adult), respectively. These rates were significantly lower than those of the control group insects. Our results suggest that HvCDA1 and HvCDA2 play important roles in the larval–pupal and pupal–adult transitions and represent potential targets for the management of H. vitessoides.

Developmental dynamics of myogenesis in Pacific oyster Crassostrea gigas.

Abstract: Pacific oyster (Crassostrea gigas) is a sessile bivalve living in the intertidal zone It has become an attractive model for developmental studies due to its metamorphic transition from a mobile planktonic larvae to a sessile adults To determine the effect of metamorphosis on muscle development in oyster larvae, we characterized myogenesis during larval development and metamorphosis by phalloidin staining which labels filamentous actin filaments Our data revealed a dynamic pattern of myogenesis during embryonic and larval development It appears that simple "U-shaped" muscle ring first developed at the trochophore stage This was followed by a more complex musculature including an anterior adductor, velum ventral retractors at the veliger stage, and the addition of posterior adductors and foot retractors at the veliger and pediveliger stages During metamorphosis, muscle structures in the anterior adductor, velum retractors and ventral retractors were degenerated At the same time, mantle and gill musculature appeared and became the primary muscle system in juveniles together with the posterior adductor In addition, indirect immunofluorescence with the monoclonal antibody against C gigas muscle proteins (myosin heavy chains (MYHC) and α-actinin) were used to monitor changes in the developing musculature at different larval stages The immunofluorescence staining results of muscle proteins were consistent with phalloidin staining The expression locations of two muscle proteins were similar and mainly located in larval velum retractor and adductor muscle The α-actinin expression positions were located in Z-lined of velum striated retractors Data from these studies provide a comprehensive description of myogenesis in C gigas embryos and larvae Moreover, our data showed that metamorphosis has a significant impact on remolding the musculature after transition from a mobile planktonic larvae to a sessile mollusk, associated with certain muscle group degradation

Sex biased expression of anti-Mullerian hormone (amh) gene in a live bearing fish, Gambusia holbrooki: Evolutionary implications and potential role in sex differentiation

Abstract: The amh, a member of transforming growth factor-β (TGF-β) family, is known to play a critical role in vertebrate male sex differentiation, with its paralogue/s evolving to determine sex in few heterogametic (XX/XY) teleosts. However, it remains relatively unexplored in the reproductively unique live bearing teleosts. Therefore, this study comparatively examined the structure and content of G. holbrooki amh as well as characterised its expression. A paralogous Y-specific amh (amhy) was not detected, suggesting an unlikely role in sex determination. Two transcripts (1.4 and 1.5 kb) were detected in adults: the larger (1.5 kb) retaining intron 5, coding for a truncated AMH-N and no TGF-β domain. The small (1.4 kb) transcript, had both domains intact and clustered with members of Poeciliidae. In contrast to other vertebrates, a higher conservation between the N- rather than the C- terminus of amh in Poeciliidae was observed, suggesting an adaptation that may be unique to live bearing teleosts. The amh expression was 6 times higher in brain of both sexes and testis compared with ovaries (p = .001). Intriguingly, female splenic tissues showed 10 times higher expression (p = .006) and such female bias splenic expression has not been reported in any teleosts. Ontogenic expression was 25 times higher in male embryos at gastrulation stage (p = .001), much earlier than those reported in egg-laying teleosts. Such heightened expression in male embryos suggests a repressive role associated with proliferation and migration of primordial germ cells (PGCs) that are known to occur earlier at blastulation in teleosts—potentially influencing gonadal fate.

Molecular cloning, phylogenetic analysis and functional characterisation of an Elovl7-like elongase from a marine crustacean, the orange mud crab (Scylla olivacea).

Abstract: The capacity of crustaceans to biosynthesise long-chain polyunsaturated fatty acids has yet to be fully defined, due to the lack of evidence on the functional activities of enzymes involved in desaturation or elongation of fatty acid substrates. We report here the cloning and in vitro functional analysis of an elongase from the orange mud crab, Scylla olivacea. Sequence and phylogenetic analysis placed the elovl close to the vertebrate Elovl1 and Elovl7 clade, which is distinct from the other remaining five Elovl families. The elongase was also clustered together with several elongases from crustaceans and insects. This elongase showed activities towards 16:1n-7, and at lower rate, linoleic acid (18:2n-6) and linolenic acid (18:3n-3). To our knowledge this is the first description of a functional enzyme involved in biosynthesis of long-chained polyunsaturated fatty acids in a crustacean species. Expression of the S. olivacea elovl7-like mRNA was prominent in stomach, intestine and gill tissues, due to the need to regulate the permeability of epithelial tissue through modification of fatty acid compositions. The implication of our findings, in terms of ability of Crustacea phylum to biosynthesise polyunsaturated fatty acids is discussed.

Expression patterns and ligand binding characterization of Plus-C odorant-binding protein 14 from Adelphocoris lineolatus (Goeze).

Abstract: Odorant-binding proteins (OBPs) can bind and transport hydrophobic odorants across the sensillum lymph to the olfactory receptors (ORs) and play crucial roles in insect chemosensory systems. Although the ligand spectra of classical OBPs have been extensively characterized, little is known about OBPs in the Plus-C subgroup. Here, we focus on AlinOBP14, a Plus-C OBP from the hemipteran mirid bug pest Adelphocoris lineolatus (Goeze). Quantitative real-time PCR experiments suggest that AlinOBP14 is ubiquitously expressed at different developmental stages but is highly expressed in the adult head, the non-chemosensory organ. Fluorescence-based competitive binding assays show that β-ionone, nerolidol, farnesol and insect juvenile hormone III (JHIII) strongly bind to AlinOBP14. No significant internal binding pocket is predicted by homology modeling. Instead, the long N-terminal and C-terminal regions and parts of several α-helixes form a cupped cavity to accommodate ligands. Molecular docking reveals that the four potential ligands have distinct binding orientations, implying different roles of the N-terminal extension in ligand recognition. This hypothesis is further confirmed via a ligand binding assay in which the recombinant N-terminal mutant AlinOBP14 displays comparable binding affinities for β-ionone and trans, trans-farnesol but decreased binding affinities for nerolidol and JHIII. Thus, our current study is the first to characterize the ligand binding spectra of a Plus-C OBP in hemipteran insect species and reveals that N-terminal extensions could be required for its recognition of putative ligands.

Photoperiod affects blunt snout bream (Megalobrama amblycephala) growth, diel rhythm of cortisol, activities of antioxidant enzymes and mRNA expression of GH/IGF-I.

Abstract: Juvenile blunt snout bream Megalobrama amblycephala were reared under three photoperiods, 8 L: 16D, 12 L: 12D and 16 L: 8D (L: light; D: dark) for over 8 weeks. The results showed that growth performance was significantly enhanced by long photoperiod. Contrary to feed conversion ratio, final length, final weight, weight gain and feed intake increased significantly as illumination time increased from 8 h daily to 16 h daily. Low size heterogeneity and whole-body lipid content in fish exposed to long photoperiod were also observed. Both relative mRNA expression level of GH and IGF-I increased as illumination time increased from 8 h to 16 h daily. The lowest value of plasma cortisol was observed at the middle the photophase while the highest value was observed at the transition between the day and night span. Hepatic MDA content significantly increased as illumination time increased from 8 h daily to 16 h daily. The activities of hepatic catalase and glutathione peroxidase were lowest in fish exposed to 16 L: 8D photoperiod and significantly lower than that in fish exposed to 8 L: 16D. These results indicate that photoperiod manipulation may not only improve growth performance but also reduce size heterogeneity. However, prolonged photoperiod could cause chronic stress since plasma cortisol level was higher in the long photoperiod group, leading to an increasing oxidative stress.

Developmental dynamics of myogenesis in Yesso Scallop Patinopecten yessoensis.

Abstract: The development of bivalves has been extensively studied over the last 150 years. Despite this, the developmental dynamics of myogenesis in bivalves remains largely unknown, particularly at the early developmental stages. In the present study, we investigate the characteristics of muscle development of Yesso scallop Patinopecten yessoensis by phalloidin staining, light, electron and confocal microscopy. Myoblasts containing chaotic myofilaments are initially found in the blastocoel of trochophore, and become more organized during the transformation from trochophore into veliger. This is followed by a well-structured musculature including an anterior adductor, velum retractors and ventral retractors at the early veliger stage. With development into late veliger, larval muscle system is composed of the branched velum retractors and ventral retractors, anterior and posterior adductors. The most striking change for pediveliger is the development of foot retractor and mantle related muscles at this stage. During metamorphosis, the retractor muscles and anterior adductor undergo the irreversible shrink until vanishing completely towards the end of larval life, which coincide with the growth of foot retractor and mantle margin. All retractor muscles are found to be composed of striated fibres, whereas the adductor muscles have both smooth and striated components. The present study provides new evidences on the dynamic pattern of myogenesis during embryonic and larval development in scallops, which will greatly improve our understanding of scallop myogenesis and provide the basis for comparative analysis of muscle development in bivalves.

Effects of glucocorticoids on lipid metabolism and AMPK in broiler chickens' liver.

Abstract: Adenosine monophosphate-activated protein kinase (AMPK) plays a pivotal role in the regulation of carbohydrate, lipid, and protein metabolism in animals. In this study, we examined whether any cross talk exists between glucocorticoids and AMPK in the regulation of the liver bile acid biosynthesis pathway. Dexamethasone treatment decreased the growth performance of broiler chickens. The liver mRNA levels of fatty acid transport protein (FATP-1), farnesoid X receptor (FXR), AMPK alpha 1 subunit (AMPKα1), and glucocorticoid receptor were significantly upregulated in DEX-treated broilers; the gene expression of liver cholesterol 7 alpha-hydroxylase (CYP7A1) was significantly downregulated. The protein level of liver CYP7A1 was significantly decreased by DEX treatment at both 24 and 72 h, while the protein level of p-AMPK/ t-AMPK stayed unchanged. In the in vitro cultured hepatocytes, compound C pretreatment blocked the increase in CYP7A1 protein level by DEX and significantly suppressed FATP-1, SREBP-1c, FXR, and CYP7A1 gene expression stimulated by DEX. Compound C treatment significantly reduces the protein level of p-AMPK, and the combination of compound C and DEX significantly reduces the protein level of t-AMPK. Thus, glucocorticoids affected liver AMPK and the bile acid synthesis signal pathway, and AMPK might be involved in the glucocorticoid effect of liver bile acid synthesis.

Mitochondrial membranes in cardiac muscle from Antarctic notothenioid fishes vary in phospholipid composition and membrane fluidity.

Abstract: Antarctic notothenioid fishes are highly stenothermal, yet their tolerance for warming is species-dependent. Because a body of literature points to the loss of cardiac function as underlying thermal limits in ectothermic animals, we investigated potential relationships among properties of ventricular mitochondrial membranes in notothenioids with known differences in both cardiac mitochondrial metabolism and organismal thermal tolerance. Fluidity of mitochondrial membranes was quantified by fluorescence depolarization for the white-blooded Chaenocephalus aceratus and the red-blooded Notothenia coriiceps. In these same membranes, lipid compositions and products of lipid peroxidation, the latter of which can disrupt membrane order, were analyzed in both species and in a second icefish, Pseudochaenichthys georgianus. Mitochondrial membranes from C. aceratus were significantly more fluid than those of the more thermotolerant species N. coriiceps (P < .0001). Consistent with this, ratios of total phosphatidylethanolamine (PE) to total phosphatidylcholine (PC) were lower in membranes from both species of icefishes, compared to those of N. coriiceps (P < .05). However, membranes of N. coriiceps displayed a greater unsaturation index (P < .0001). No differences among species were found in membrane products of lipid peroxidation. With rising temperatures, greater contents of PC in mitochondrial membranes from ventricles of icefishes are likely to promote membrane hyperfluidization at a lower temperature than for cardiac mitochondrial membranes from the red-blooded notothenioid. We propose that physical and chemical properties of the mitochondrial membranes may contribute to some of the observed differences in thermal sensitivity of physiological function among these species.

Nonadditive and allele-specific expression of insulin-like growth factor 1 in Nile tilapia (Oreochromis niloticus, ♀) × blue tilapia (O. aureus, ♂) hybrids.

Abstract: Hybrid Nile tilapia (Oreochromis niloticus, ♀) × blue tilapia (O. aureus, ♂) is a widely cultured tilapia variety due to its growth vigor compared to the parent species. As a peptide hormone, insulin-like growth factor 1 (IGF-1) plays a critical role in regulating somatic growth. The present study focuses on the expression characteristics of IGF-1 in hybrid tilapia. The cloned complete open reading frame of IGF-1 in hybrid tilapia is 549 bp in length, encoding a protein of 182 amino acids. The deduced protein is highly similar to that of Nile tilapia and blue tilapia. IGF-1 was found to be primarily expressed in the liver and muscle in the hybrid; lower expression levels were found in other tissues such as the intestine, spleen, and head-kidney. Increased mRNA expression was observed in the liver and muscle of the hybrid compared to Nile tilapia and blue tilapia, indicating a nonadditive expression pattern in the hybrid. An IGF-1 SNP site (397 site: C in Nile tilapia, G in blue tilapia) for differentiating the Nile tilapia or blue tilapia subgenome in hybrids was identified. Pyrosequencing analysis of the liver transcriptome indicated that most of the hybrids (9 of 10 individuals) predominantly expressed the G allele, demonstrating bias of the blue tilapia subgenome. The present study provides novel data indicating, for the first time, overall gene expression of IGF-1 and allele-specific expression in hybrid tilapia.