Molecular cytogenetic study of genome ploidy in the German mirror carp Cyprinus carpio

We examined the polyploidy of Cyprinus carpio, the German mirror carp. Specimens were collected in the field in Hulan, China, and treated with phytohemagglutinin and colchicine before the chromosome spreads and the karyotype of kidney cells were examined using silver staining, chromomycin A₃ (CMA₃)/distamycin (DA)/4,6-diamidino-2-phenylindole (DAPI) staining, and fluorescence in situ hybridization (FISH) with a 5.8S + 28S rDNA probe. One to twosilver stained (Ag) nucleolus organizing regions (NORs) were detected during metaphase and interphase events, with 80 % of the metaphase spreads and 69 % of the interphase nuclei showing two Ag-NORs signals. One CMA₃ signal was detected on the terminus of the short arm of each submetacentric chromosome 8 (SMC8) homolog (n = 2). The 5.8S + 28S rDNA probe hybridized at the terminus of the short arm of each SMC8 homolog (n = 2). The results of the Ag-NORs, CMA₃/DA/DAPI, and 5.8S + 28S rDNA FISH analyses were consistent with regard to the total number and location of the SMC8 NORs in the chromosome spreads and karyotype, indicating that, at the molecular cytogenetic level, the German mirror carp is an evolutionary tetraploid with two sets of chromosomes after diploidization.     

The role of Ca2+ and Na+ membrane transport in brook trout (Salvelinus fontinalis) spermatozoa motility

The role of environmental ion composition and osmolality in Ca²⁺ signaled activation was assessed in spermatozoa of brook trout Salvelinus fontinalis. Milt from ten mature males was obtained by abdominal massage. Spermatozoa motility was evaluated in 0, 100, and 300 mOsm/kg NaCl or sucrose solutions, buffered by 10 mM Tris–HCl pH 8.5. For investigation of spermatozoa reaction to external Ca²⁺ concentration, 2 mM ethylene glycol tetraacetic acid (EGTA) was added to the activation media as a calcium ions chelator. For investigation of the effect of external Na⁺ concentration in conditions of low external Ca²⁺, 100 µM amiloride was added to the EGTA-containing solutions as a Na⁺ transport blocker. Low motility was observed in sucrose (Na⁺ free) solutions containing 2 mM EGTA but not in Na⁺ solutions containing 2 mM EGTA. Addition of amiloride led to significantly increased motility (P < 0.05) compared with sucrose (Na⁺ free) solutions containing 2 mM EGTA. We conclude that Na⁺ transport in Ca²⁺-free solutions plays a regulatory role in brook trout spermatozoa activation. The influence of competitive Na⁺ and Ca²⁺ transport on the control of spermatozoa activation requires further study with respect to its application for improvement of artificial activation and storage media.     

Heritability and phenotypic correlations of gonad sweetness in the sea urchin Strongylocentrotus intermedius

Gonad is the only edible part of sea urchins. Thus, a number of studies have been focusing on how to improve both quantity and quality of their gonads. However, as far as our knowledge, the genetic basis of gonad flavor remains totally unknown in sea urchins. In the present study, we found that the heritability of gonad sweetness was at a high level of 0.56, clearly indicating that it is, to a large extent, under genetic control. Gonad sweetness was significantly positively correlated with gonad weight (P < 0.05), a* (P < 0.01) and b* (P < 0.01), while significantly negative correlated with L* (P < 0.01), ΔE ₁ (P < 0.01) and ΔE ₂ (P < 0.01). The present study provides valuable information into the genetic basis of gonad sweetness and evidences that gonad sweetness is potential to be improved in sea urchin genetic breeding programs.     

Effects of heating conditions on fatty acids and volatile compounds in foot muscle of abalone Haliotis discus hannai Ino

Abalone Haliotis discus hannai Ino is a popular delicacy consumed as a luxury food owing to its unique flavor and texture. In this study, we investigated the effects of heating conditions on the fatty acids and volatile compounds in its foot muscle based on gas chromatography–mass spectrometry (GC–MS) and solid-phase microextraction (SPME)–GC–MS. The contents of fatty acids significantly decreased after heating at 80 °C for 2 h. In total, 52 volatile compounds, including aldehydes, aromatic compounds, alkanes, alcohols, ketones, and furans, were detected in the heated samples. Principal component analysis revealed an interaction between heating temperature and time. Heating at 80 °C for 0.5–2 h generated higher contents of volatile compounds. In particular, the contents of hexanal, heptanal, octanal, nonanal, and undecanal mainly derived from autoxidation of fatty acids during heating increased at least fourfold.     

Effects of dietary cholesterol supplementation on growth and cholesterol metabolism of rainbow trout (Oncorhynchus mykiss) fed diets with cottonseed meal or rapeseed meal

This study was conducted to evaluate the effects of cholesterol on growth and cholesterol metabolism of rainbow trout (Oncorhynchus mykiss) fed diets with cottonseed meal (CSM) or rapeseed meal (RSM). Four experimental diets were formulated to contain 550 g kg^?1 CSM or 450 g kg^?1 RSM with or without 9 g kg^?1 supplemental cholesterol. Growth rate and feed utilization efficiency of fish fed diets with 450 g kg^?1 RSM were inferior to fish fed diets with 550 g kg^?1 CSM regardless of cholesterol level. Dietary cholesterol supplementation increased the growth rate of fish fed diets with RSM, and growth rate and feed utilization efficiency of fish fed diets with CSM. Similarly, dietary cholesterol supplementation increased the plasma total cholesterol (TC), high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and triiodothyronine levels, but decreased the plasma triglycerides and cortisol levels of fish fed diets with RSM or CSM. In addition, supplemental cholesterol increased the free cholesterol and TC levels in intestinal contents, but decreased the hepatic 3-hydroxy-3-methyl-glutaryl-CoA reductase activity of fish fed diets with RSM or CSM. These results indicate that 9 g kg^?1 cholesterol supplementation seems to improve the growth of rainbow trout fed diets with CSM or RSM, and the growth-promoting action may be related to the alleviation of the negative effects caused by antinutritional factors and/or make up for the deficiency of endogenous cholesterol in rainbow trout.     

Impact of season and grafter skill on nucleus retention and pearl oyster mortality rate in Pinctada margaritifera aquaculture

The mollusc Pinctada margaritifera is the top economic aquaculture species in French Polynesia (export value) and forms the basis of the black pearl industry. Mass production of this unique gem, produced by a living organism, relies on a surgical operation requiring tissue from a donor pearl oyster to be grafted, together with a nucleus made of shell, into the gonad of a receiving pearl oyster. This technique is performed by expert grafters, whose work constitutes the first step influencing pearl farm production yield. This study makes the first report of effects mediated by individual grafters and by the season of grafting on three rates scored at 45 days post operation: nucleus retention, nucleus rejection and pearl oyster mortality. These results were obtained in a very large scale grafting experiment, designed and conducted in a single culture site in the atoll of Arutua (Tuamotu Archipelago) and involving a total of 52,910 grafts performed by ten professional grafters, during two contrasting seasons: autumn (four grafting experiments) and spring (three grafting experiments). Statistical analysis using linear mixed-effect model for both univariate and multivariate analysis was performed. The results show a very highly significant season effect (p < 0.001), with a higher rate of nucleus retention in autumn than spring: 91.1 versus 79.4 %, respectively. A very highly significant season effect was also recorded for nucleus rejection rate (p < 0.001), which was greater in spring than in autumn: 12.6 versus 7.7 %. Oyster mortality rate was up to six times higher in spring than in autumn (p < 0.001). Within the two seasons, there was no significant difference between the grafting experiments for any of the three variables, adding robustness to the inter-season results. Taking into account this seasonal variation effect, grafter skill had a significant impact on the three variables (p < 0.001), making it possible to rank grafters from most to least efficient. Aside from skill differences between grafters, grafting in autumn may significantly increase graft success. Water temperature and its indirect consequences could be one of the most important environmental factors implicated in overall efficiency. These findings may help the potential development of an annual grafting schedule according to season/ lagoon temperature as a way to maximize production yield for the black-lipped pearl oyster industry in French Polynesia.     

Oxidative damage repair by glutamine in fish enterocytes

Fish intestine is very sensitive to oxidative damage. Repair of damaged enterocytes may be involved to restore normal function of fish intestine. However, studies of fish enterocyte repair are scarce. The present study aimed to investigate the potential repair role of glutamine after a H₂O₂ challenge. In this study, fish enterocytes were post-treated with graded levels of glutamine (0, 4, 8, 12 and 20 mM of glutamine) after expose to 100 μM H₂O₂. The basal control cells were kept in the glutamine-free minimum essential medium only. Results showed that the H₂O₂-induced decreases in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide optical density, alkaline phosphatase and Na⁺, K⁺-ATPase activities were completely restored by subsequent glutamine treatments. In addition, cellular injury (lactate dehydrogenase), lipid peroxidation (malondialdehyde) and protein oxidation (protein carbonyls) caused by H₂O₂ were reversed by subsequent glutamine treatments. Furthermore, the H₂O₂-induced decreases in glutathione contents, glutathione reductase, superoxide dismutase and glutathione peroxidase activities were completely restored by subsequent glutamine treatments. In summary, the present study indicated that glutamine improved the repair activity in fish enterocytes after challenge with H₂O₂.     

Observations on the increase of wild gilthead seabream,Sparus aurata abundance,in the eastern Adriatic Sea: problems and opportunities

The recent increase of the local population of gilthead seabream, Sparus aurata, in three areas along the southeastern Adriatic Sea: Malostonski Bay (Croatia and Bosnia and Herzegovina), Neretva Estuary (Croatia) and Boka Kotorska Bay (Montenegro) and its adverse effects on shellfish culture by preying on Mediterranean mussels, Mytilus galloprovincialis, and the European flat oyster, Ostrea edulis, are studied. The results from the analysis of the existing information show that the main reason for the recent increase is the escapes from local fish farm which enrich the local population constantly with new gilthead sea bream. The existence of practically endless food in the area of the shellfish farms allows the concentration of the population in the region instead of its dispersion along the Adriatic coast. Moreover, ecological analysis indicates that the gilthead seabream is facing a very low competition from other local species which enhances its capacity to further populate the region. While the impact on the ecosystem is not yet known, the socio-economic impact of the increase of the gilthead seabream population is evident today. Many shellfish farms are closing today in the region since the damages may reach over 90 % of the production.     

Cloning,molecular characterization and expression analysis of insulin-like growth factor binding protein-2 (IGFBP-2) cDNA in goldfish,Carassius auratus

In the present study, a full-length cDNA encoding the insulin-like growth factor binding protein-2 (IGFBP-2) was cloned from the liver of goldfish (Carassius auratus) by rapid amplification of cDNA ends technique. The goldfish IGFBP-2 cDNA sequence was 1,513 bp long and had an open reading frame of 825 bp encoding a predicted polypeptide of 274 amino acid residues. Semi-quantitative RT-PCR results revealed that goldfish IGFBP-2 mRNA was expressed in all detected tissues. In liver, central nervous system and pituitary gland, goldfish IGFBP-2 expressed at high levels, followed by anterior intestine, middle intestine and kidney. In posterior intestine, ovary, skin, fat, spleen, muscle and gill, the goldfish IGFBP-2 expression levels were very low. Fasting and refeeding experiment showed that the mRNA expression of goldfish IGFBP-2 was up-regulated significantly in liver compared to the fed group and restored rapidly to normal level after refed. However, the mRNA expressions of IGFBP-2 in hypothalamus and pituitary of goldfish were insensitive to fasting. Furthermore, the mRNA expressions of IGFBP-2 in hypothalamus, pituitary and liver were varied in periprandial changes and significantly down-regulated at 2 and 4 h after meal. These results imply that the IGFBP-2 mRNA expression may be associated with anabolic and catabolic metabolism and regulated by metabolic factors in goldfish.     

Oocyte maturation and active motility of spermatozoa are triggered by retinoic acid in pen shell <Emphasis Type="Italic">Atrina pectinata</Emphasis>

For recovery of the declining population of pen shells in the wild, the production of pen shell juveniles for transplantation or aquaculture is underway in Japan. For more stable juvenile production, artificial fertilization methods for pen shells are needed, but methods to induce oocyte maturation (meiosis resumption) used in other bivalves, which make oocytes fertilizable, were ineffective for pen shells. Here, we report evidence showing that retinoic acid (RA) has strong activity in inducing oocyte maturation and activating sperm motility in pen shells. Treatment of fully developed oocytes with 1.0 μM all-trans-RA (at-RA) induced germinal vesicle breakdown, a typical morphological sign of oocyte maturation, but 1.0 μM at-retinol and at-retinal, 2 mM ammonia, and 1.0 μM serotonin were ineffective. Treatment with at-RA for 30 min was sufficient for oocyte maturation and was more potent than its isomers, 9-cis- and 13-cis-RA. Parallel results were obtained for sperm motility activation. Oocyte responsiveness to at-RA increased during the final stage of ovary development. Artificial fertilization was successful only with the oocytes treated with at-RA, and fertilized eggs developed to D-shaped (veliger) larvae without apparent morphological abnormalities. These results indicate the possible application of RA for the artificial fertilization of pen shells.     

Effects of dietary selenium on the pathological changes and oxidative stress in loach (Paramisgurnus dabryanus)

In this study, loach (Paramisgurnus dabryanus) were fed artificial diets containing 0.31 (control), 0.39, 0.48, 0.50 and 0.62 mg kg^?1 of selenium (Se) for 60 days, respectively. Liver histopathology, hepatocyte ultrastructure, blood indices, biochemical parameters of liver functions and oxidative stress in the Se-treated loach were then assayed. The results showed the following: histopathological and ultrastructural lesions in liver were only observed in loach fed the 0.62 mg Se kg^?1 diet; Haemoglobin and total protein were significantly increased in the 0.50 mg Se kg^?1 group; albumin and high-density lipoprotein were increased significantly in the 0.48–0.50 mg Se kg^?1 groups. However, white blood cell count was significantly decreased in the 0.48 mg Se kg^?1 group; alanine aminotransferase, aspartate aminotransferase and lactate dehydrogenase were decreased in the 0.39–0.50 mg Se kg^?1 groups. In liver tissue, the content of hydrogen peroxide was lower than that of controls in the 0.48–0.50 mg Se kg^?1 groups, and the malondialdehyde level was lowest in the 0.48 mg Se kg^?1 group. The activities of superoxide dismutase and glutathione peroxidase were significantly increased in the 0.50 mg Se kg^?1 group; catalase and total antioxidant capacity were markedly increased in the 0.48–0.50 mg Se kg^?1 group. These present results indicated that the dietary Se requirement for loach is 0.48–0.50 mg Se kg^?1 diet.     

Dynamic expression pattern of corticotropin-releasing hormone,urotensin I and II genes under acute salinity and temperature challenge during early development of zebrafish

Corticotropin-releasing hormone (CRH), urotensin I (UI) and urotensin II (UII) are found throughout vertebrate species from fish to human. To further understand the role of crh, uI and uII in teleosts during development, we investigated the expression pattern of crh, uI, uIIα and uIIβ genes, and their response to acute salinity and temperature challenge during early development of zebrafish, Danio rerio. The results reveal that crh, uI, uIIα and uIIβ mRNA are detected from 0hpf, and the expression levels increase to a maximum at 6 days post fertilization (dpf), with the exception of uIIα that peak at 5dpf. Exposure of zebrafish embryos and larvae to acute osmotic (30ppt) stress for 15 min failed to modify expression levels of crh, uI, uIIα and uIIβ mRNA from levels in control fish except at 6dpf when uIIα and uIIβ were significantly (P < 0.05) modified. Exposure of embryos and larvae to a cold (18 °C) or hot stress (38 °C) generally down-regulated mRNA levels of crh, uI, uIIα and uIIβ apart from at 3dpf. The results indicate that the contribution of crh, uI, uIIα and uIIβ genes to the stress response in zebrafish may be stressor-specific during early development. Overall, the results from this study provide a basis for further research into the developmental and stressor-specific function of crh, uI, uIIα and uIIβ in zebrafish.     

The bioactivity of gonadotropin releasing hormones and its regulation in the gilthead seabream,Sparus aurata: in vivo andin vitro studies

Thein vivo andin vitro potency of native and modified forms of gonadotropin releasing hormone (GnRH) to release gonadotropin (GtH) was studied inSparus aurata and correlated with their relative susceptibility to degradation by cytosolic-bound enzymes of the pituitary, kidney, and liver. Salmon (s) GnRH and luteinizing hormone-releasing hormone (LHRH) are equipotent whereas analogs of these peptides ((D-Arg⁶-Pro⁹NET)-sGnRH, (D-Ala⁶-Pro⁹NET)-LHRH, (D-Trp⁶)-LHRH) are superactive in inducingin vivo GtH release (at 10 μg/kg body weight). In anin vitro superfusion system of pituitary fragments all analogs are equipotent to the native peptides (at 10⁻¹⁰ to 2.5 × 10⁻⁷M). sGnRH and LHRH are rapidly degraded by cytosolic peptidases of the pituitary, liver, and kidney. The preferred site of cleavage is the Tyr⁵-Gly⁶ bond. Substitution of the position 6 glycine by D-amino acids renders the 5–6 bond resistant to degradation and shifts the main site of cleavage to the Pro⁹-Gly¹⁰NH₂ bond. Substitution at position 6 (as above) and at position 10 with Pro⁹NET results in analogs that are resistant to degradation. We propose that enzymatic cleavage terminates GnRH bioactivityin vivo and thus increased resistance to degradation is a major determinant of GnRH analog superactivity.     

Responses to ROS inducer agents in zebrafish cell line: differences between copper and UV-B radiation

Fish are commonly exposed to environmental pollutants, which in turns could induce an oxidative stress. So, it is important to understand the effects and the responses elicited by these toxicants in fish species, being fish cell lines important tools for this purpose. Thus, the aim of the present study was to compare the effects of copper and UV-B radiation exposure on zebrafish hepatocytes (ZFL lineage) in terms of reactive oxygen species (ROS) levels, sulfhydril groups content and mRNA levels of important genes related to cellular response to toxic agents. Exposure of ZFL cells to UV-B radiation (23.3 mJ/cm²) significantly increased levels of intracellular ROS and mRNA of both superoxide dismutase isoforms (sod1 and sod2), three glutathione S-transferase isoforms (gstα, gstµ and gstπ) and a heat shock protein (hsp70). However, no changes in nonprotein sulfhydryl groups (NP-SH) content, as well as in the mRNA levels of genes related to glutathione (GSH) synthesis and recycling, were observed. Contrary to this, copper exposure (20 mg/L) diminished NP-SH content and increased the levels of mRNA of genes related to GSH synthesis (gclc and gs). Moreover, copper exposure increases the mRNA levels of some genes related to antioxidant defenses (gpx and gstπ), biotransformation reactions (cyp1a1) and protein repair (hsp70). In conclusion, these results demonstrated that both toxicants could increase ROS levels in ZFL cell line, but the responses are different, which could be related to activation of different signaling pathways.     

Molecular cloning,characterization, and expression analysis of luteinizing hormone receptor gene in turbot (Scophthalmus maximus)

The luteinizing hormone receptor (LHR) plays a crucial role in female reproduction. In the present study, full-length sequence coding for the LHR was obtained from female turbot (Scophthalmus maximus) by homology cloning and a strategy based on rapid amplification of cDNA end-polymerase chain reaction. The full-length LHR cDNA was 3,184 bp long and contained a 2,058-bp open reading frame which encoded a protein of 685 amino acids. Multiple sequence alignments of the turbot LHR manifested high homologies with the corresponding sequences of available teleosts and representative vertebrates, and significant homology with that of Hippoglossus hippoglossus. In addition, the turbot LHR showed typical characteristics of glycoprotein receptors, including a long N-terminal extracellular domain, seven transmembrane domains, and a short C-terminal intracellular domain. LHR mRNA was abundant in the ovary, but was deficient in extra-ovarian tissues. Furthermore, LHR mRNA gradually developed from previtellogenesis to migratory nucleus stage, with the highest values observed in migratory nucleus stage during reproductive cycle. However, LHR mRNA sharply decreased in atresia stage. These results suggested that LHR is a typical G protein-coupled receptor that is involved in the promotion of turbot ovarian development and may be related to the final maturation and ovulation of oocyte. These findings contribute to the understanding of the potential roles of LHR in controlling the fish reproductive cycle.     

Effects of larval cryopreservation on subsequent development of the blue mussels,Mytilus galloprovincialis Lamarck

In this study, the effects of three commonly used chemicals, dimethyl sulphoxide (DMSO), ethylene glycol (EG), propylene glycol (PG) and their combinations with trehalose, were evaluated on the cryopreservation of D‐larvae of the blue mussel Mytilus galloprovincialis. The larvae were harvested 30 h post‐fertilization at 21 °C and cryopreserved using a standard protocol in 5%, 10% or 15% of DSMO, EG and PG either as single chemical solutions or in combination with 0.2 M trehalose. Among these cryoprotectants, 5% DMSO resulted in the highest post‐thaw survival rate of 55.3±7.8%, although it did not significantly differ from those with 10% and 15% EG. The addition of 0.2 M trehalose did not improve the post‐thaw larval survival rates in all the combinations assessed. The cryo‐effects on subsequent development were evaluated using the D‐larvae frozen with 5% DMSO. The results showed that cryopreservation affected both larval survival and growth in this species. The relative daily mortality rate was significantly higher in treated than control groups over the period from 3 h post‐thaw to day 11 post‐fertilization. On day 6 post‐fertilization, the average larval length in the treated group was significantly smaller than that in the control. From day 11 post‐fertilization, and onwards, differences in these two traits were not significant between treated and control groups. On day 21 post‐fertilization, about 80% of the larvae in both treated and control groups developed eyes and the normalized survival rate in the treated group was 12.5%.     

Cryopreservation of male and female gonial cells by vitrification in the critically endangered cyprinid honmoroko <Emphasis Type="Italic">Gnathopogon caerulescens</Emphasis>

We investigated the feasibility of cryopreservation of spermatogonia and oogonia in the critically endangered cyprinid honmoroko Gnathopogon caerulescens using slow-cooling (freezing) and rapid-cooling (vitrification) methods. Initially, we examined the testicular cell toxicities and glass-forming properties of the five cryoprotectants: ethylene glycol (EG), glycerol (GC), dimethyl sulfoxide (DMSO), propylene glycol (PG), and 1,3-butylene glycol (BG), and we determined cryoprotectant concentrations that are suitable for freezing and vitrification solutions, respectively. Subsequently, we prepared the freezing solutions of EG, GC, DMSO, PG, and BG at 3, 2, 3, 2, and 2 M and vitrification solutions at 7, 6, 5, 5, and 4 M, respectively. Following the cryopreservation of the testicular cells mainly containing early-stage spermatogenic cells (e.g., spermatogonia and primary spermatocytes), cells were cultured for 7 days and immunochemically stained against germ cell marker protein Vasa. Areas occupied by Vasa-positive cells indicated that vitrification led to better survival of germ cells than the freezing method, and the best result was obtained with 5 M PG, about 50% recovery of germ cells following vitrification. In the case of ovarian cells containing oogonia and stage I, II, and IIIa oocytes, vitrification with 5 M DMSO resulted the best survival of oogonia, with equivalent cell numbers to those cultured without vitrification. The present data suggest that male and female gonial cells of the endangered species G. caerulescens can be efficiently cryopreserved using suitable cryoprotectants for spermatogonia and oogonia, respectively.