Sustainable aquaculture and aquatic resources management

RSS Fish Physiology and Biochemistry

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Comparing the effects of different dietary organic acids on the growth, intestinal short-chain fatty acids, and liver histopathology of red hybrid tilapia ( Oreochromis sp.) and potential use of these as preservatives

Published on 26/3/2017

Abstract

Dietary organic acids are increasingly being investigated as a potential means of improving growth and nutrient utilization in aquatic animals. A 9-week study was performed to compare equal amounts (2%) of different organic acids (sodium butyrate, acetate, propionate, or formate) on the growth, muscle proximate composition, fatty acid composition, cholesterol and lipid peroxidation, differential cell counts, plasma biochemistry, intestinal short-chain fatty acid (SCFA) level, and liver histopathology to red hybrid tilapia (Oreochromis sp.) (initial mean weight of 2.87 g). A second experiment was performed to determine their effects on lipid peroxidation and trimethylamine (TMA) when added at 1% to tilapia meat and left out for 24 h. The results of the first experiment showed no treatment effect to growth, feeding efficiencies, or muscle fatty acid composition, but all dietary organic acids significantly decreased intestinal SCFA. Dietary butyrate and propionate significantly decreased muscle lipid peroxidation compared to the control group, but the dietary formate treatment had the lowest lipid peroxidation compared to all treatments. Muscle crude protein and lipid in tilapia fed the formate diet were significantly lower and higher, respectively, and showed evidence of stress based on the differential cell counts, significantly higher plasma glucose and liver glycogen, as well as inflammatory responses in the liver. Although a potential benefit of dietary organic acids was a reduction to lipid peroxidation, this could be accomplished post-harvest by direct additions to the meat. In addition, inclusions of butyrate and propionate to tilapia meat significantly decreased TMA, which might be a more cost-effective option to improve the shelf life of tilapia products.

Protein degradation systems in the skeletal muscles of parr and smolt Atlantic salmon Salmo salar L. and brown trout Salmo trutta L.

Published on 25/3/2017

Abstract

Although protein degradation limits the rate of muscle growth in fish, the role of proteolytic systems responsible for degrading myofibrillar proteins in skeletal muscle is not well defined. The study herein aims to evaluate the role of calpains (calcium-activated proteases) and proteasomes (ATP-dependent proteases) in mediating muscle protein turnover at different life stages in wild salmonids. Protease activities were estimated in Atlantic salmon (Salmo salar L.) and brown trout (Salmo trutta L.) parr and smolts from the Indera River (Kola Peninsula, Russia). Calpain and proteasome activities in Atlantic salmon skeletal muscles were lower in smolts as compared with parr. Reduced muscle protein degradation accompanying Atlantic salmon parr-smolt transformation appeared to provide intense muscle growth essential for a minimum threshold size achievement that is required for smoltification. Calpain and proteasome activities in brown trout parr and smolts at age 3+ did not significantly differ. However, calpain activity was higher in smolts brown trout 4+ as compared with parr, while proteasome activity was lower. Results suggest that brown trout smoltification does not correspond with intense muscle growth and is more facultative and plastic in comparison with Atlantic salmon smoltification. Obtained data on muscle protein degradation capacity as well as length-weight parameters of fish reflect differences between salmon and trout in growth and smoltification strategies.

DNA integrity-protecting and survival-promoting activity of serotonergic system in sturgeon juveniles and sazans

Published on 19/3/2017

Abstract

The study concerns evaluation of engagement of serotonergic system in the mechanisms of antimutagenic protection and survival under adverse conditions. It is shown that under long-term exposure to oil and industrial pollution, simultaneous sharp increase of mutations level in the erythrocytes and downregulation of serotonin-modulating anticonsolidation protein (SMAP; Mekhtiev 2000) in the livers of the sturgeon juveniles (Acipenser gueldenstaedtii persicus) is observed. Mutation level was evaluated by the micronucleus analysis and SMAP level—by the indirect ELISA-test utilizing anti-SMAP immunoglobulins. Intramuscular administration of SMAP leads to significant decrease of micronucleus amount in the erythrocytes of the sturgeon juveniles exposed to sediments polluted with PAH and heavy metals. Pre-conditioning of sazans by low dose of insecticide actara (100 mg/l) leads to upregulation of SMAP and to survival of all the animals in the experimental group, kept under high concentration of actara (400 mg/l), while all animals in the control group succumbed under these conditions. I.m. administration of SMAP prior to putting the sazans into the water containing high levels of actara (400 mg/l), in contrast to controls, leads to their total survival. Participation of serotonergic system in the mechanisms of antimutagenic protection and survival promotion under damaging conditions is concluded.

Effects of fasting, temperature, and photoperiod on preproghrelin mRNA expression in Chinese perch

Published on 19/3/2017

Abstract

Preproghrelin, a gut/brain peptide, plays an important role in the regulation of food intake and energy homeostasis in teleost and mammals. In the present study, we obtained the full-length preproghrelin cDNA in Chinese perch. The preproghrelin messenger RNA (mRNA) tissue expression showed that level was much higher in stomach and pituitary than in other tissues. The fasting study showed, after gastric emptying (3–6 h), short-term fasting (6–12 h) increased preproghrelin expression in the stomach. While in the pituitary, fasting reduced preproghrelin expression at 1, 3, 12, and 48 h, presenting state fluctuation of self-adjustment. The temperature study showed that the mRNA expression of preproghrelin was the highest in the brain at 26 °C and highest in the stomach at 32 °C, respectively, with different optimum temperature in these two tissues, reflecting spatiotemporal differences of regulation by central nervous system and peripheral organs. The photoperiod study showed that normal light (11 h of lightness and 13 h of darkness) led to highest preproghrelin expression, both in the brain and in the stomach, than continuous light or continuous dark, proving food intake is adapted to natural photoperiod or normal light in this study. These results all indicated that tissue-specific preproghrelin expression of Chinese perch could be significantly affected by environmental factors. Short-term fasting of 6 h after gastric emptying, 26 °C, and normal light led to higher preproghrelin expression, which indicated potential appetite increase in Chinese perch.

Seasonal expression of arginine vasotocin mRNA and its correlations to gonadal steroidogenic enzymes and sexually dimorphic coloration during sex reversal in the gilthead seabream ( Sparus aurata )

Published on 17/3/2017

Abstract

Arginine vasotocin is a hormone produced in the hypothalamus of teleost fish that has been shown to regulate gonad development and sexual behavior. To study the role of arginine vasotocin in the gonadal cycle of the hermaphrodite gilthead seabream, Sparus aurata, we cloned the seabream arginine vasotocin (avt) complementary DNA (cDNA). We investigated the expression of brain avt throughout the gonad cycle using real-time quantitative PCR and compared its expression levels to the expression levels of two key gonadal steroidogenic enzymes, cyp19a1a and cyp11b2. In July, when the process of sex reversal is thought to begin, avt expression was elevated over the previous 2 months. Avt in the brain remained at or above the level of July until November then peaked again in December. There was no difference between males and females in the expression levels of brain avt throughout the year. However, only in ambisexual fish was the expression of the cyp19a1a gonadal aromatase correlated to the expression of avt in the brain. Cyp11b2 did not show any correlation to brain avt expression. We also found that females had more intense body coloration than males and that this intensity peaked prior to spawning. Avt expression and female coloration were positively correlated. The fact that brain avt expression was lowest during gonad quiescence, together with the observation of a correlation between brain avt with gonadal cyp19a1a and body coloration during that time suggests that avt may play a role during the process of sex reversal and spawning of the gilthead seabream.

Activity of metabolic enzymes and muscle-specific gene expression in parr and smolts Atlantic salmon Salmo salar L. of different age groups

Published on 17/3/2017

Abstract

This study was conducted to characterize the energy metabolism level and the features of muscle growth regulation during the development of Atlantic salmon (Salmo salar) inhabiting the Indera River (Kola Peninsula, Russia). The activities of aerobic and anaerobic enzymes (cytochrome c oxidase and lactate dehydrogenase) and carbohydrate metabolism enzymes (glucose-6-phosphate dehydrogenase, glycerol-3-phosphate dehydrogenase, and aldolase) were measured in muscle and liver tissue. Gene expression levels of myosin heavy chain (MyHC), myostatin (MSTN-1a), and myogenic regulatory factors (MRFs—MyoD1a, MyoD1b, MyoD1c, Myf5, myogenin) were measured in the white muscles of salmon parr of ages 0+, 1+, 2+, and 3+ and smolts of ages 2+ and 3+. Multidirectional changes in the activity of enzymes involved in aerobic and anaerobic energy metabolism with age were shown in the white muscles of the parr. The cytochrome c oxidase activity was higher in muscles of underyearlings (0+) and yearlings (1+) and decreased in 2+ and 3+ age groups. The activity of lactate dehydrogenase, in contrast, increased with age. The patterns of changes in expression levels of MyoD1a, MyoD1b, myogenin, MyHC, and MSTN-1a at different ages of the parr were similar. Particularly, the expression of these genes peaked in the yearling parr (1+) and then decreased in elder groups. The differences were revealed in parameters studied between the parr and smolts. The level of aerobic and anaerobic metabolism enzyme activities was higher in the white muscles of smolts than in parr. The activity of carbohydrate metabolism enzymes was decreased in the smolts’ livers. The expression levels of MyHC, MyoD1a, MyoD1b, and myogenin were lower in smolts at age 2+ compared to parr. These findings expand our knowledge of age-related and stage-related features of energy metabolism and muscle development regulation in young Atlantic salmon in their natural habitat. The results might be used for monitoring of the salmon population during restoration and rearing.

Dietary supplementation of curcumin augments heat stress tolerance through upregulation of nrf-2 -mediated antioxidative enzymes and hsps in Puntius sophore

Published on 16/3/2017

Abstract

Heat stress is one of the major environmental concerns in global warming regime and rising temperature has resulted in mass mortalities of animals including fishes. Therefore, strategies for high temperature stress tolerance and ameliorating the effects of heat stress are being looked for. In an earlier study, we reported that Nrf-2 (nuclear factor E2-related factor 2) mediated upregulation of antioxidative enzymes and heat shock proteins (Hsps) provide survivability to fish under heat stress. In this study, we have evaluated the ameliorative potential of dietary curcumin, a potential Nrf-2 inducer in heat stressed cyprinid Puntius sophore. Fishes were fed with diet supplemented with 0.5, 1.0, and 1.5% curcumin at the rate 2% of body weight daily in three separate groups (n = 40 in each group) for 60 days. Fishes fed with basal diet (without curcumin) served as the control (n = 40). Critical thermal maxima (CTmax) was determined for all the groups (n = 10, in duplicates) after the feeding trial. Significant increase in the CTmax was observed in the group fed with 1.5% curcumin- supplemented fishes whereas it remained similar in groups fed with 0.5%, and 1% curcumin-supplemented diet, as compared to control. To understand the molecular mechanism of elevated thermotolerance in the 1.5% curcumin supplemented group, fishes were given a sub-lethal heat shock treatment (36 °C) for 6 h and expression analysis of nrf-2, keap-1, sod, catalase, gpx, and hsp27, hsp60, hsp70, hsp90, and hsp110 was carried out using RT-PCR. In the gill, expression of nrf-2, sod, catalase, gpx, and hsp60, hsp70, hsp90, and hsp110 was found to be elevated in the 1.5% curcumin-fed heat-shocked group compared to control and the basal diet-fed, heat-shocked fishes. Similarly, in the liver, upregulation in expression of nrf-2, sod, catalase, and hsp70 and hsp110 was observed in 1.5% curcumin supplemented and heat shocked group. Thus, this study showed that supplementation of curcumin augments tolerance to high temperature stress in P. sophore that could be attributed to nrf-2-induced upregulation of antioxidative enzymes sod, catalase, gpx, and the hsps.

The effect of live feeds bathed with the red seaweed Asparagopsis armata on the survival, growth and physiology status of Sparus aurata larvae

Published on 16/3/2017

Abstract

Larval rearing is affected by a wide range of microorganisms that thrive in larviculture systems. Some seaweed species have metabolites capable of reducing the bacterial load. However, no studies have yet tested whether including seaweed metabolites on larval rearing systems has any effects on the larvae development. This work assessed the development of Sparus aurata larvae fed preys treated with an Asparagopsis armata product. Live prey, Brachionus spp. and Artemia sp., were immersed in a solution containing 0.5% of a commercial extract of A. armata (Ysaline 100, YSA) for 30 min, before being fed to seabream larvae (n = 4 each). In the control, the live feed was immersed in clear water. Larval parameters such as growth, survival, digestive capacity (structural-histology and functional-enzymatic activity), stress level (cortisol content), non-specific immune response (lysozyme activity), anti-bacterial activity (disc-diffusion assay) and microbiota quantification (fish larvae gut and rearing water) were monitored. Fish larvae digestive capacity, stress level and non-specific immune response were not affected by the use of YSA. The number of Vibrionaceae was significantly reduced both in water and larval gut when using YSA. Growth was enhanced for YSA treatment, but higher mortality was also observed, especially until 10 days after hatching (DAH). The mortality peak observed at 8 DAH for both treatments, but higher for YSA, indicates larval higher susceptibility at this development stage, suggesting that lower concentrations of YSA should be used until 10 DAH. The application of YSA after 10 DAH onwards promotes a safer rearing environment.

Modulatory role of L-carnitine against microcystin-LR-induced immunotoxicity and oxidative stress in common carp

Published on 16/3/2017

Abstract

Microcystin-LR (MCLR), one of the most popular microcystins (MCs) found in many field water bodies around the world, poses great health risks to animals and humans. In the present study, healthy common carp (initial weight 24.8 ± 2.3 g) were randomly assigned to five groups. Group I was fed on normal diet as control. Group II was maintained on normal diet and received MCLR intraperitoneal injection (150 μg kg−1 BW). Common carp in groups III, IV, and V were daily pretreated with L-carnitine (LC) at doses of 0.5, 1.0, and 2.0 g kg−1 of the diet for 4 weeks prior to MCLR intraperitoneal injection. The results showed that MCLR alone led to a significant downregulation in immune response, including serum complement C3, lysozyme, and bactericidal activity. However, oxidative stress response: catalase (CAT), superoxide dismutase (SOD), glutathione (GSH), glutathione peroxidase (GPx), and lipid peroxidation (LPO) levels were significantly increased. Similarly, gene expressions of inflammatory IL-1β, TNF-α, IFN I, and heat shock proteins (HSP70 and HSP90) were also upregulated after challenged with MCLR. However, LC pretreated group caused a significant elevation in immune response (C3, lysozyme, and bactericidal activity) and gene expressions of inflammatory IL-1β, TNF-α, IFN I, and heat shock proteins (HSP70 and HSP90) after MCLR stress. Antioxidant activities (CAT, SOD, GSH, GPx, and LPO) were returned to background levels at 96 h after MCLR challenge. Strikingly, LC supplementation at 2.0 g kg−1 has been considered the optimum for common carp since it exhibited enhancement of immune response and antioxidant activity over the level 0.5 and 1.0 g kg−1, and even better than that of control level. It was concluded that LC as a functional feed additive significantly inhibited the progression of MCLR-induced immunotoxicity and oxidative stress in common carp.

Tissue-specific daily variation in the oxidative status of sturgeon ( Acipenser naccarii ) and rainbow trout ( Oncorhynchus mykiss ): a comparative study

Published on 13/3/2017

Abstract

The oxidative status is associated with animal lifespan, metabolism, activity and circadian rhythms. The objective of this work is to study the time course of the oxidative status over a daily cycle in the plasma, liver and brain, and the changes in the plasma cortisol levels of sturgeon and trout. The knowledge of daily oxidative status will provide a better understanding of the trout and sturgeon physiology and adequate maintenance and food supply of farmed fish in relation to photoperiod. Superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione transferase, DT-diaphorase activities, lipid peroxidation and cortisol were measured. Our results showed that the antioxidative enzyme activities and lipid peroxidation in the liver of trout and sturgeon changed through the day, with increased levels in lipid peroxidation of liver in the dark period for sturgeon. This could be related to the different activity time in both species, an issue to be taken into account when designing the guidelines of the maintenance of these species in fish farms. On the contrary, there was not clear influence of the daily rhythms on brain oxidative status. The higher efficiency of the antioxidant defences in the brain of sturgeon, which displays less lipid peroxidation and higher antioxidative activity, could be related to its longer life expectancy. The absence of any apparent daily rhythm in the plasma cortisol levels in sturgeon could indicate a lower susceptibility to stress, and that mechanisms involved in cortisol secretion in chondrosteans could be different from that described for teleosteans.

Identification and characterization of microRNAs in the muscle of Schizothorax prenanti

Published on 13/3/2017

Abstract

MicroRNAs (miRNAs) regulate gene expression by fully or partially binding to complementary sequences and play important roles in skeletal muscle development. However, the roles of miRNAs in the skeletal muscle of Schizothorax prenanti remain unknown. In this study, we analyzed the miRNA profiling in the skeletal muscle of S. prenanti at 30 days post-hatching (dph), 1 year, and 3 years by high-throughput sequencing. Two hundred twenty-nine unique miRNA types aligned to 201 independent pre-miRNA loci according to sequence similarity and 28 novel miRNAs were obtained. The nucleotide bias analysis of identified miRNAs showed that the miRNAs in S. prenanti were highly conserved. Eight identified miRNAs validated using stem-loop qRT-PCR were differentially expressed in the process of skeletal muscle development of S. prenanti. This study provide not only an overall insight into the miRNA landscape in the skeletal muscle of S. prenanti but also a basis for further investigation of miRNA roles in skeletal muscle development of S. prenanti.

Hemolysis interference in measuring fish plasma biochemical indicators

Published on 13/3/2017

Abstract

The present study aimed to determine hemolysis interference in measurement of plasma biochemical parameters in fish specimens. For this purpose, blood samples were harvested from 24 Huso huso juveniles. After centrifugation, each plasma sample was divided into seven portions to make seven levels of hemolysis. Hemolysis was induced by addition of different percentages of the corresponding whole blood [0 (non-hemolyzed control group), 1, 3, 5, 10, 15, and 20% of whole blood]. Albumin, total protein, calcium, phosphorus, glucose, sodium, potassium, chloride, alkaline phosphatase (ALP), aspartate transaminase (AST), and alanine transaminase (ALT) were measured in different samples. Results showed that plasma albumin, ALP, potassium, and AST significantly increased (more than 200% at the highest hemolysis level) in the hemolyzed samples. Also, plasma total protein and phosphorus showed significant elevation in the hemolyzed samples (more than 70% at the highest hemolysis level). Plasma glucose, calcium, chloride, and ALT showed narrow but significant increase in hemolyzed samples (11.8–35.2% at the highest hemolysis level). Plasma sodium showed no significant changes in the hemolyzed samples. In conclusion, the present results show that hemolysis markedly affects plasma parameters levels, which interferes with plasma results interpretation. Therefore, analysis of hemolyzed samples should be avoided or the results should be interpreted with caution. This study encourages further investigations to develop methods for omitting the effects of hemolysis by sample blanking and/or presenting correction coefficients for measurement of plasma parameters in samples with different levels of hemolysis.

Endocrine disruption, oxidative stress, and testicular damage induced by 4-nonylphenol in Clarias gariepinus : the protective role of Cydonia oblonga

Published on 10/3/2017

Abstract

Exposure to xenoestrogens like 4-nonylphenol (NP) is recognized by disrupting endocrine functions and causes reproductive dysfunction in male fish. The present study aimed at investigating the 4-nonylphenol propensity to induce oxidative stress and hormonal disturbances in male catfish and at studying the protective role of quince (Cydonia oblonga). To fulfill this aim, catfish Clarias gariepinus were exposed to pure 100 μg/L 4-NP and to quince the leaf extract added to 4-NP, both for 15 days. The 4-NP exposure induced a marked increase in 17ß-estradiol (E2), LH, and cortisol, while thyroid hormone (TSH, T3), testosterone (T), and FSH levels noticeably decreased; however, 4-NP had no effect on T4 level. Moreover, 4-NP exposure was accompanied by histological impairments in testes. Existence of 4-NP was associated with oxidative damage as evidenced by the significant increase (p < 0.05) of the enzymes, superoxidase dismutase (SOD), catalase (CAT), acetylcholinesterase (AchE), glutathione s-transferase, total antioxidant capacity (TAC), and malondialdehyde (MDA). Adding quince was effective to neutralize hormonal levels and to repair the testicular histological alterations. In response to quince remedy, the enzymes AchE and MDA reduced significantly (p < 0.05), while limited or no response was detected for other tested enzymes. Our results concluded that quince can antagonize 4-NP toxicity in catfish, confirming that quince leaf extract displayed antioxidant activities against the toxicity of hazardous chemicals.

Involvement of the miR-462/731 cluster in hypoxia response in Megalobrama amblycephala

Published on 8/3/2017

Abstract

MicroRNAs (miRNAs) are non-coding small RNAs showing both evolutionarily conserved and unique features and are involved in nearly all biological processes. In the present study, the role played by miR-462/731 cluster miRNAs in hypoxia response in Megalobrama amblycephala, an important freshwater fish, was investigated. The M. amblycephala miR-462/731 cluster locus and their 5′ flanking sequences were sequenced and analyzed. In M. amblycephala and other teleost fish species, the mature sequences of miR-462 and miR-731 were identical and hypoxia-responsive elements (HREs) were identified upstream of the miR-462/731 loci. The two miRNAs were significantly induced in the liver, spleen, gill, muscle, and brain after hypoxia treatment. The expression of both miRNAs was also upregulated in cells that received treatment which mimicked hypoxia. Furthermore, reporter assay revealed that M. amblycephala HREs can be activated by hypoxia. Taken together, the 462/731 cluster may play a role in the regulation of the hypoxia response in M. amblycephala.

Effects of dietary n -3 fatty acids on Toll-like receptor activation in primary leucocytes from Atlantic salmon ( Salmo salar )

Published on 8/3/2017

Abstract

The shortage of the n-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on the international markets has led to increasing substitution of fish oil by plant oils in Atlantic salmon (Salmo salar) feed and thereby reducing the EPA and DHA content in salmon. However, the minimum required levels of these fatty acids in fish diets for securing fish health are unknown. Fish were fed with 0, 1 or 2% EPA or DHA alone or in combination of both over a period, growing from 50 to 400 g. Primary head kidney leucocytes were isolated and stimulated with Toll-like receptor (TLR) ligands to determine if EPA and DHA deficiency can affect expression of important immune genes and eicosanoid production. Several genes related to viral immune response did not vary between groups. However, there was a tendency that the high-level EPA and DHA groups expressed lower levels of IL-1β in non-stimulated leucocytes. These leucocytes were also more responsive to the TLR ligands, inducing higher expression levels of IL-1β and Mx1 after stimulation. The levels of prostaglandin E2 and leukotriene B4 in serum and media from stimulated leucocytes were lower in both low and high EPA and DHA groups. In conclusion, leucocytes from low EPA and DHA groups seemed to be less responsive towards immunostimulants, like TLR ligands, indicating that low levels or absence of dietary EPA and DHA may have immunosuppressive effects.

Potential contributions of heat shock proteins and related genes in sexual differentiation in yellow catfish ( Pelteobagrus fulvidraco )

Published on 27/2/2017

Abstract

Sex determination and differentiation in ectotherms are very complicated affairs and usually affected by both genetic and environmental factors. Because of their temperature-sensitive expression, heat shock proteins (HSPs) are good candidates for temperature-dependent sex determination (TSD). Similar to most thermosensitive fish species, the male to female ratio increases with temperature in yellow catfish (Pelteobagrus fulvidraco). Yellow catfish is also a type of sexual size dimorphic fish, and the male individuals grow much faster than females of the same age. Therefore, research of sex differentiation in yellow catfish is important in aquiculture. In this attempt, a total of seven HSPs and related genes were identified from transcriptomes of yellow catfish by 454 pyrosequencing and Solexa sequencing that we did previously, including five genes with complete open reading frame (ORF). Phylogenetically, all these genes were compared with their counterparts from other vertebrates. All these genes were sex-biased expressed in gonads. Hspa5, Hip, and Cdc37 were expressed more highly in ovary than in testis, whereas Hsp90α, Hspb2, Hspb8, and Hspbp1 were expressed more highly in testis than in ovary. Additionally, the expression of these genes was assessed after 17α-methyltestosterone (MT) and 17α-ethinylestradiol (EE2) treatment, respectively. Our result showed that working as co-chaperones, these HSPs and related genes may regulate sex steroid receptor activities to influence gonad development in yellow catfish. Our work would help in the understanding of the mechanism of sexual differentiation in teleosts.

Purification and development of ELISAs for two forms of vitellogenin in Indian walking catfish, Clarias batrachus (L.)

Published on 27/2/2017

Abstract

Two forms of vitellogenin (Vg: Vg1 and Vg2) were purified from the plasma of estradiol-17β (E2)-treated Indian walking catfish, Clarias batrachus, by gel filtration and adsorption chromatography. Native Vg1 and Vg2 had apparent molecular masses of 375 and 450 kDa, respectively, and both Vgs resolved into two similar major bands (95 and 67 kDa) in SDS-PAGE under reducing condition. Polyclonal antisera raised against each form of Vg were absorbed with a combination of hypophysectomized male catfish serum proteins and alternate Vg to ensure specificity. Immunological analyses verified the presence of Vg1 and Vg2 in the plasma of female catfish. Homologous ELISAs were developed for Vg1 and Vg2 using their respective harvested antisera, which exhibited the detection limit of 100 ng ml−1 for Vg1 and 40 ng ml−1 for Vg2, and low level of cross-reactivity (not parallel to the standard) was found with alternate Vg in each assay. Treatment of male catfish with E2 induced both Vgs showing a proportionate ratio of Vg1 to Vg2 at 5.6:1. Plasma concentrations of both Vgs measured by ELISAs at different reproductive phases of field collected female catfish increased in accordance with the ovarian development, keeping the proportionate ratio of Vg1 to Vg2 at about 2:1 in fish undergoing vitellogenesis during prespawning period and 1:20 during spawning period, suggesting that Vg1 may be the major Vg to contribute in yolk formation, whereas Vg2, besides its role in yolk formation, may facilitate other physiological functions. The present study, thus, demonstrates the occurrence of two unequally synthesized Vgs in the catfish.

Molecular cloning and expression analysis of Megalobrama amblycephala transferrin gene and effects of exposure to iron and infection with Aeromonas hydrophila

Published on 23/2/2017

Abstract

Transferrin (Tf) plays an important function in iron homeostasis and metabolism of organisms. In this study, we identified and characterized the Tf gene in Megalobrama amblycephala and evaluated its expression in basal conditions as well as after iron overload and experimental infection with Aeromonas hydrophila. Furthermore, we studied the iron binding properties of recombinant Tf. The full-length M. amblycephala Tf complementary DNA (cDNA) (GenBank accession no.: KX698308) of 2245 bp was cloned and contained a 1953 bp open reading frame (ORF) encoding 650 amino acid residues and flanked by a 68 bp 5′ and a 204 bp 3′ untranslated regions (UTR). Predicted conservative structure illustrated that M. amblycephala Tf consisted of two conservative Tf domains. Amino acid sequence alignment revealed that M. amblycephala Tf had high similarity with that of cyprinids deposited in Genbank, and phylogenetic analysis showed that M. amblycephala Tf clustered with Ctenopharyngodon idella and Hypophthalmichthys molitrix. Tissue expression pattern analyses demonstrated that the liver was the main Tf mRNA expressing organ, being significantly higher than other tissues (p < 0.05). In the liver, Tf mRNA expression in fish artificially injected with the pathogenic bacteria A. hydrophila was significantly upregulated, reaching a peak at 12 h post injection (hpi) and then decreasing afterward. The expression in FeCl3-injected fish showed a similar tendency, but reached a peak at 8 hpi. Meanwhile, fish serum iron significantly decreased following A. hydrophila injection, but increased to peak at 4 hpi and then decreased in FeCl3-injected fish. The recombinant M. amblycephala Tf showed iron binding capacity using CAS analysis. These results are helpful to understand the structure and regulation of expression of Tf, as well as the specific function of Tf for both immune responses and iron homeostasis.

Ultramorphological changes in gill rakers of Astyanax altiparanae (Characidae) kept in contaminated environments

Published on 13/2/2017

Abstract

Most water bodies in Brazil, and in the world, are contaminated by some types of pollutants, ranging from sewage to metal/chemicals, carcinogenic products, and biodegradable detergents. Despite the extensive knowledge on their effects on fish biology and especially on gill morphology, research that concerns their impacts on gill rakers and implications in parameters such as food consumption cannot be found in the literature. Gill rakers are vital because, together with gills, they are responsible for the defense and protection of the organism and for selecting appropriate food for survival. When detergents, which can act as toxic chemical agents, get in contact with the body of the fish, they can cause severe effects that must be understood. Therefore, our study investigated ultramorphological changes in gill rakers of Astyanax altiparanae (Lambeth) caused by the exposure to biodegradable detergents. Fish were exposed to a 1 mg/L dilution of a mixture of detergents and pure water from an artesian well for 5 months. Results revealed that the first month of exposure to detergent caused dilation of chemical receptors in taste buds and the rise of a large number of orifices for mucus release among pavement cells in gill rakers, although only a small amount of mucus was found in fish exposed both to pure water and the detergent dilution. After 5 months, there was an increase in the dilation of these chemoreceptors, excess mucus on gill rakers of detergent groups, and the emergence of microbridges between microridges in pavement cells.

Plasma levels of immune factors and sex steroids in the male seahorse Hippocampus erectus during a breeding cycle

Published on 13/2/2017

Abstract

To better understand the endocrine- and immune-response pattern during reproduction in a fish species having parental care behaviors and also to accumulate the endocrine- and immune-related data for future explanations of the low reproductive efficiency in seahorse species, the variations of immune factors and sex steroids in the plasma of the male lined seahorse Hippocampus erectus at different breeding stages, i.e., pre-pregnancy, pregnancy (early, middle, and late periods), and post-pregnancy, were investigated in the present study. The immune factors included monocytes/leucocytes (M/L), leucocyte phagocytic rate (LPR), immunoglobulin M (Ig M), interleukin-2 (IL-2), interferon-α (IFN-α), and lysozyme (LZM). The sex steroids included testosterone (T), 11-ketotestosterone (11-KT), 11β-hydroxytestosterone (11β-OHT), 17α-methyltestosterone (17α-MT), 17β-estradiol (E2), and 17α-hydroxy-20β-dihydroprogesterone (17α-20β-P). Moreover, the immune metabolic activity of epithelium cells in the brood pouch at different breeding stages was also analyzed through ultrastructural observations of the abundance of cytoplasmic granules, mitochondria, endoplasmic reticulum, lysosomes, and exocytosis. The results show that a higher immune level was observed during pregnancy, particularly in the early and middle periods, and a lower immune level was noted during pre-pregnancy. Correspondingly, the epithelium cells in the brood pouch also showed a stronger immune metabolic activity during pregnancy and weaker activity during pre-pregnancy. Four sex steroids of T, 11β-OHT, 17α-MT, and E2 were higher during pre-pregnancy and lower during post-pregnancy, whereas 11-KT and 17α-20β-P, which were positively correlated with part immune factors, were higher during pregnancy. No negative correlations between sex steroids and immune factors were observed. In conclusion, the higher immune competence during pregnancy may indicate that parental care could improve immunity, which may be the major factor for no immunosuppressive effect of sex steroids during reproduction in the seahorse H. erectus, unlike noncaregiving fishes in which inhibitions of sex steroids on immunity are frequently observed. Moreover, higher 11-KT and 17α-20β-P during pregnancy than during pre-pregnancy and post-pregnancy may suggest that these two steroids are also involved in parental care regulation.

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