cDNA cloning and characterization of the warm-temperature-acclimation-associated protein Wap65 from carp, Cyprinus carpio

We determined full-length cDNA of carp warm-temperature-acclimation-associated 65-kDa protein (Wap65). It encoded 439 amino acid residues with a signal peptide of 22 residues and showed an amino acid sequence identity of 88% to that of goldfish reported before (J. Biol. Chem. 1995. 270: 17087–17092). The number of potential N-linked glycosylation sites of carp Wap65 was two in contrast to three for goldfish. In addition, molecular mass determined by SDS-PAGE was apparently different from that of goldfish. These results suggest that the amount of oligosaccharide is different between the carp and goldfish protein. As in goldfish, carp Wap65 mRNA showed marked accumulation in hepatopancreas of the 30 °C- acclimated fish, which was 8-fold higher than that of the 10 °C-acclimated fish. Carp Wap65 showed 30% amino acid identity to mammalian hemopexins, which appeared to be considerably low in comparison with those among mammalian hemopexins (72 to 80%), or among carp Wap65 and rainbow trout hemopexin-like protein (70%). However, although mammalian hemopexins contain residues comprising the heme binding pocket, carp Wap65 lacked one of the two histidine residues to serve as heme axial ligands in hemopexins. Our data on carp protein substantiates the previous observation for goldfish and indicates that Wap65 might have some important functions in warm-temperature-acclimation of fish.     

Identification of Wap65, a human homologue of hemopexin as a copper-inducible gene in swordtail fish, <Emphasis Type="Italic">Xiphophorus helleri</Emphasis>

Copper is one of the major heavy metal pollutants found in the aquatic environment. Therefore, it is important for determining the genes that play a key role in copper metabolism in aquatic organisms. This study, thus, aimed to identify a new copper-inducible gene in swordtail fish, Xiphophorus helleri. Using ACP-based RT-PCR coupled with RLM-RACE, we cloned Wap65, a mammalian homologue of hemopexin gene. The gene exhibits high identity at amino acid levels with the Wap65 gene of other fish species (42–68%) and mammalian hemopexin gene (35–37%). In addition, ten cysteine and two histidine residues are conserved in the swordtail fish Wap65 gene. These cysteine residues are vital for structural integrity, and histidine residues provide high binding affinity towards heme. As revealed by RT-PCR, the gene was upregulated in swordtail fish that were exposed to copper in a dose- and time-dependent manner. Therefore, the identification of Wap65, a mammalian homologue of hemopexin, as a new copper-inducible gene will provide greater insight into the role of this gene in copper metabolism.     

Effects of temperature and dietary protein on gene expression of Hsp70 and Wap65 and immunity of juvenile mirror carp (Cyprinus carpio)

Juvenile mirror carp were fed diets containing 303.4, 321.7, 341.2, 361.0 and 379.1 g kg^?1 proteins, respectively, and reared at different water temperatures (18, 23 and 28°C) for 60 days. Gene expression of heat shock protein gene (Hsp70) and the warm temperature acclimation‐related 65 kDa protein gene (Wap65), immunity and antioxidant status in the carp were investigated. Results indicated that the contents of serum complement 3 (C3), complement 4 (C4) and immunoglobulin M (IgM), as well as activities of liver superoxide dismutase (SOD) and lysozyme (LSZ) were significantly enhanced with increasing dietary protein (P < 0.05), while content of malondiadehyde (MDA) decreased. Gene expression level of Wap65 in the liver significantly increased with dietary protein, while gene expression of Hsp70 decreased. The contents of C3, C4 and IgM, the activities of SOD and LSZ and gene expression level of Wap65 in the liver significantly increased with temperature. These results suggest that: Serum immune parameter, antioxidant enzymes and Hsp70 and Wap65 expression interact in fish to improve ability to adapt to the environment; and the optimal conditions for the immunity of carp are 348.1?354.5 g kg^?1 protein at 18°C, 352.3?364.9 g kg^?1 at 23°C and 360.2?364.3 g kg^?1 at 28°C, and the optimum temperature for carp is 23°C.     

Reinvestigation of positional distribution of tetracosahexaenoic acid in triacyl-sn-glycerols of flathead flounder flesh

Positional distribution of fatty acids in triacyl-sn-glycerols (TAG) of the flathead flounder Hippoglossoides dubius has been reinvestigated in order to accurately determine the contents of tetracosahexaenoic acid (24:6n-3) in the sn-1, sn-2, and sn-3 positions. Flesh TAG obtained from three flounders were subjected to stereospecific analysis using a suitable procedure for fish TAG analysis. The 24:6n-3 acid was found in the three positions at the concentrations of 0.3–5.5 mole% (the sn-1 position), 1.6–23.3 mole% (the sn-2 position), and 0.6–8.9 mole% (the sn-3 position). In contrast to a previous analysis, the present analysis revealed that 24:6n-3 is preferentially esterified in the sn-2 position followed by the sn-3 and sn-1 positions. Other polyunsaturated fatty acids, docosahexaenoic acid (22:6n-3; DHA) and docosapentaenoic acid (22:5n-3; DPA), showed a similar distribution pattern. These results indicate that the general tendency observed for long-chain polyunsaturated fatty acids in marine fish TAG can be extended to the distribution of 24:6n-3 in flathead flounder TAG. Because the use of flathead flounders is entirely for human food, we thus intake 24:6n-3 concentrated in the sn-2 position of their TAG.     

Diet and fish choice of Eurasian otters (<Emphasis Type="Italic">Lutra lutra</Emphasis> L.) in fish wintering ponds in Hungary

The diet composition and fish preference of piscivorous Eurasian otters (Lutra lutra) were studied in two fish farm systems in Hungary using spraint (otter faeces) analysis during two wintering periods. The primary food source of otters in both fish farms was fish (97–99% of biomass). The main fish prey was small-sized, below 100 g in weight (96% in both areas), while fish prey above 500 g comprised only 0.1–0.4% of the diet. The bulk of the otters’ diet consisted of less-valued species, especially non-native Prussian carp (Carassius auratus gibelio). Consumption of commercial fish species ranged between 15 and 31% of the total diet. Otters preferred fish below 100 g in weight (Ivlev’s electivity index, E _i = 0.65–0.70), and showed a lesser preference for (or avoided) fish above 100 g in weight (E _i = −0.37–1.00). With regard to species distribution, otters preferred small (below 100 g) grass carp (Ctenopharyngodon idella), zander (Sander lucioperca), pike (Esox lucius), Prussian carp, topmouth gudgeon (Pseudorasbora parva), while they consumed common carp (Cyprinus carpio), the most important commercial species, proportionally to its abundance in the environment (E _i = −0.18–0.29).     

Molecular cloning,genomic structure and expression analysis of major histocompatibility complex class Iα gene of half-smooth tongue sole (<Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis>)

Major histocompatibility complex (MHC) has a central role in the adaptive immune system by presenting foreign peptide to the T-cell receptor. The full length of MHC class Iα cDNA was cloned from half-smooth tongue sole by homology cloning and rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR), genomic organization and expression of MHC Iα were examined to study the function of MHC gene in fish. The domain structure feature and antigen-binding motifs of other teleost and mammals MHC are conserved in the half-smooth tongue sole MHC Iα gene. The deduced amino acid sequence of half-smooth tongue sole MHC Iα (GenBank accession no. FJ372720) had 12.1–61.8% identity with those of human and other fish. Eight exons and seven introns were identified in MHC Iα gene. Real-time quantitative PCR demonstrated that MHC Iα gene was ubiquitously expressed in normal tissues, while that in Vibrio anguillarum infected fish was significantly increased in intestines and decreased in spleen and liver from 24 to 72 h after infection, followed by a recovery to normal level after 96 h.     

Cloning of two Atlantic salmon growth hormone receptor isoforms and <Emphasis Type="Italic">in vitro</Emphasis> ligand-binding response

Two isoforms of the full-length cDNA of the growth hormone receptor (GHR) of the Atlantic salmon (Salmo salar; ss) were cloned by a PCR approach using RACE. Respectively, the cDNA sequences of ssGHR isoforms 1 and 2 are 2654 and 2608 nucleotides long, with 1782 and 1773 nucleotide ORFs. The resulting coded proteins are 594 and 590 aa long, with 19 and 20 aa signal peptides. The two isoforms share 86% protein and 87% cDNA sequence similarity. Isoform 1 is most similar to other salmonid GHR isoforms 1 while isoform 2 is most similar to salmonid GHR isoforms 2 (93–95%). Similarity with other teleost species was lower (37–44%). The bioactivity of the cloned ssGHR was tested by transfecting the ssGHR isoform 1 cDNA into CHO-K1 hamster cells, incubating with recombinant salmon GH (sGH) or native ovine prolactin (oPRL), and measuring cell proliferation by the MTT assay. The ssGHR-transfected cells significantly increased proliferation when stimulated by sGH at all concentrations. oPRL stimulated ssGHR-transfected cells at higher concentrations due to receptor cross reaction. ssGHR isoforms 1 and 2 contain a single transmembrane domain and the typical conserved motifs found in other teleost GHRs, including four paired cysteine residues and five potential N-glycosylation sites in the extracellular domain, Box I and Box II, as well as seven potential tyrosine phosphorylation sites in the intracellular domain. However, in salmonids, these motifs differ from those of other teleosts, and could be responsible for differentiated hormone binding, signal transduction and response.     

Effect of ration size on growth,body composition,and energy and protein maintenance requirement of fingerling Indian major carp, <Emphasis Type="Italic">Labeo rohita</Emphasis> (Hamilton)

An eight-week feeding trial has been conducted to determine the optimum ration for Indian major carp, Labeo rohita, fingerling (4.10 ± 0.30 cm, 0.55 ± 0.16 g) by feeding a purified diet (40% CP; 3.61 kcal g⁻¹ GE) at six levels, 2, 4, 6, 8, 10, and 12% of body weight per day, at 0800 and 1600 h, in triplicate, to 20 fish per trough fitted with a water flow-through system. Highest weight gain, best feed conversion ratio (FCR), best specific growth rate (SGR%), and highest protein efficiency ratio (PER) were evident for rations of 6–8% body weight. Second-degree polynomial regression analysis for FCR, PER, protein, and energy retention data indicated the break-points occurred at 6.55, 6.75, 6.80, and 6.95% bw per day, respectively. Significant (P < 0.05) differences between body composition were observed for fish fed different rations. Maximum body protein content was recorded for 6% and 8% rations. A linear increase in body fat content was evident with increasing ration. Body moisture and ash content remained non-significantly (P > 0.05) low for higher rations, however. On the basis of these results it is recommended that feeding in the range 6.5–7.0% bw per day corresponding to 2.6–2.8 g protein and 23.49–25.31 kcal energy per 100 g of the diet per day is optimum for growth and efficient feed utilization of Labeo rohita. Results for 2–4% rations (0.8–1.6 g protein and 7.23–14.46 kcal energy) suggest these amounts approximate to the maintenance requirement of this fish.     

Molecular cloning and sequencing of hemoglobin-β gene of channel catfish, Ictalurus punctatus Rafinesque

The hemoglobin-β gene of channel catfish, Ictalurus punctatus, was cloned and sequenced. Total RNA from head kidneys was isolated, reverse transcribed and amplified. The sequence of the channel catfish hemoglobin-β gene consists of 600 nucleotides. Analysis of the nucleotide sequence reveals one open reading frame and 5′- as well as 3′-untranslated regions. The open reading frame of the sequence potentially encodes 148 amino acids with a calculated molecular mass of 16.3 kDa. The pI and charge at pH 7.0 of the deduced hemoglobin-β protein were 7.28 and 0.47, respectively. Overall, 22 amino acid residues were conserved throughout the sequences, including His64 and His93, the sites for heme-binding. Unlike the counterpart of other common cultured fish such as Salmo salar, Oncorhynchus nerka, Oncorhynchus mykiss, Cyprinus carpio and Ctenopharyngodon idella, the hemoglobin-β of channel catfish did not have cysteine. The amino acid sequence of channel catfish hemoglobin-β shows 84% homology with that of Silurus asotus (both are in the order Siluriformes). However, comparison with those of other fish species shows homology ranging from 53 to 68%. Structural analysis by the 3D-PSSM program displays that channel catfish hemoglobin-β has eight α-helices, A–H.     

Computer-assisted image analysis in the evaluation of fish wounding by cormorant [Phalacrocorax carbo sinensis (L.)] attacks

Fish that have escaped from a cormorant’s (Phalacrocorax carbo sinensis (L.)) grasp and/or which could not be swallowed due to their size suffer from various injuries resulting in consecutive infections and subsequent increased mortality. Computer-assisted image analysis was applied to describe the extent of such injuries. Two-year-old mirror carp, Cyprinus carpio L. (TL 200–300 mm, W 200–300 g) showed injuries ranging over approximately 10% of the total body surface. Immediately after wounding, the damaged epithelium (scars) cover 5–35%, and deeper subdermal wounds caused by the beak tip pervading into muscle tissue cover an area of 1–2% of the total body surface. On the side impacted by the cormorant’s lower mandible, extensive areas of epidermal contusions (scars) occur. As time progresses, these ratios change—deeper necroses represent up to 10% of the total body surface and healing epithelial scars comprise just 1–2%. In wounded silver carp, Hypophthalmichthys molitrix Val. (300–400 mm, W 500–700 g), the share of subdermal wounds usually does not exceed 0.5% due to their compact scaly cover. During pond draining due to fish harvesting, the size spectrum of wounded fish increases and may also often include bigger fish (e.g., European catfish, Silurus glanis L.) up to 2.2 kg.     

Chromosome set manipulations in the black carp

The mollusc-eating black carp (Mylopharyngodon piceus) has economic and health-care potential for biological control of nuisance aquatic molluscs. The present study investigates the production of gynogenetic-monosex and triploid-sterile populations of black carp. The goal was to provide a method which would eliminate unwanted biological and environmental impacts of introducing this exotic species into areas with nuisance mollusc infestation. Meiotic gynogenesis was induced by inseminating black carp eggs with UV-irradiated (800 Jm⁻²) sperm of common carp (Cyprinus carpio) or Japanese ornamental (koi) carp. Diploidy was restored through retention of the second polar body (2PB), by shocking activated eggs at 1–8 min post-fertilization (embryological age of 0.07–0.57τ₀, a parameter defined by the cell cycle duration) at 1 min intervals, with heat-shocks (41.0±1.0 °C for 1 min) or pressure-shocks (7500–7600 psi for 1.5 min). Highest survival was found when embryos were heat-shocked 1.5–4.5 min post-fertilization (0.10–0.25τ₀). The highest survival of free-swimming larvae from pressure-shocked eggs, was achieved at 7500 psi at 1–2 min post-fertilization (0.08–0.16τ₀). Triploidy was induced by retention of 2PB following normal fertilization. Batches of 30 000 eggs were fertilized with intact sperm and pressure-shocked (6000–8500 psi for 1.5 min) 2 min post-fertilization (0.15–0.16τ₀). The highest survival of triploid swim-up larvae was 5.1% in eggs shocked with 7500 psi. In random samples of individual larvae taken from each treatment, triploidy was analysed by cytofluorometry of the cellular DNA content. In DNA analysis performed in fingerlings (N≥15), 50% of the fish were triploids.     

Extractive components and taste aspects of fermented fish pastes and bean pastes prepared using different koji molds as starters

We have evaluated four commercially available mold starters (SP-01, NY, M1, and “for kome miso”) for fermented fish pastes with the aim of determining the effect of different koji molds on the nutritional and sensory characteristics of the fermented product. Comparative studies among different fermented fish and bean pastes were also conducted. Protein contents were considerably higher in the fermented fish pastes (22.7–24.1%) than the fermented bean pastes (8.2–12.8%). Free amino acids, oligopeptides, organic acids, mineral content, and sensory scores of the fermented fish pastes were superior to those of the bean pastes in terms of nutritional aspects. Sensory evaluation of fermented fish and bean pastes clearly indicated that different koji molds used as starters had little effect on the taste of the fermented fish pastes. However, in terms of nutritional and sensory characteristics, fermented fish pastes from trash fishes were found to be both commercially valuable and a potential gain to food processing firms among the many new generic fermented food products becoming available.     

Molecular characterization and expression pattern of three zona pellucida 3 genes in the Chinese sturgeon, <Emphasis Type="Italic">Acipenser sinensis</Emphasis>

Chinese sturgeon (Acipenser sinensis) is a rare and endangered species and also an important resource for the sturgeon aquaculture industry. SMART cDNAs were synthesized from the ovary of A. sinensis, and the full-length cDNAs of three zona pellucida glycoprotein 3 genes (the new gene named AsZP3) were cloned and sequenced. AsZP3.1, AsZP3.2, and AsZP3.3 were 1,388 base pairs (bp), 1,288, and 1,290 bp in length, respectively, and they could be translated into proteins with 440, 394, and 398 amino acids, respectively. High level of amino acids sequence identity was seen between AsZP3.2 and AsZP3.3 (about 82%), but they share low identity with AsZP3.1 (26 and 23%, respectively). The AsZP3.1 has 42–50% amino acids sequence identity values with other fish and lower values with higher vertebrates (38%); AsZP3.2 and AsZP3.3 shared about 30–44% sequence identity with higher vertebrates and other fish. RT–PCR analysis indicated that AsZP3.1 displayed a wide tissue distribution at the mRNA levels including liver, kidney, spleen, heart, and ovary, but AsZP3.2 and AsZP3.3 mRNAs were expressed exclusively in the gonad. All three AsZP3 mRNAs were not detected during embryogenesis and early larval development; furthermore, they were not detected in the gonads of 1- and 2-year-old Chinese sturgeons. All three AsZP3 mRNAs were detected in the testes of 3-year-old males and in the ovaries of 4- and 5-year-old female Chinese sturgeons.     

Effect of varying dietary protein levels on growth and production of Chanos chanos (Forsskal) in inland saline groundwater: laboratory and field studies

Two experiments were conducted to investigate the effects of varying dietary protein (35–45%) and energy levels (17.34–19.44 kJ g⁻¹) on the growth performance of milkfish (Chanos chanos) maintained under laboratory (experiment 1) and field conditions (Experiment 2) in inland saline groundwater. The results of experiment 1 (initial weight of fish: 0.25 g) revealed that, irrespective of the protein source (fish meal or processed full fat soybean), fish fed a diet containing 40% protein showed significantly (p<0.05) higher growth in terms of live weight gain and specific growth rate, low feed conversion ratio [1.71 (fish meal) and 1.58 (soybean)], high nutrient retention (gross protein retention: 28.59 and 31.05%; gross energy retention: 24.23 and 26.04%), apparent protein digestibility (81.74 and 85.91%) and digestive enzyme activity (specific protease and amylase). An observation on the postprandial excretion of metabolites (N–NH₄ and o–PO₄) indicated significantly (p<0.05) low levels of these metabolites in aquaria where the fish were fed 40% dietary protein irrespective of the protein source. The results of experiment 2 also indicate (initial weight of fish: 4.43 g) a significantly (p<0.05) higher growth performance (weight gain, biomass, specific growth rate, growth day⁻¹ and fish production) in ponds where the fish were fed 40% dietary protein. Irrespective of the experimental conditions (laboratory/field), feeding the fish higher dietary protein levels (beyond 40%) not only repressed growth performance but also affected proximate composition by lowering protein accumulation and energy assimilation. An investigation on the effects of feeds on water quality parameters revealed that values for total alkalinity, NH₄–N, NO₃–N, turbidity, total dissolved solids and parameters indicative of productivity (chlorophyll a, net primary productivity and plankton population) increased significantly (p<0.05) from treatments 1–3 (35–40% protein) and declined thereafter, in treatment 4 (42% dietary protein), indicating that water quality characteristics correlated well with fish growth. The results suggest that in order to obtain a high-yield in milkfish culture system the fish should be fed supplementary diets containing appropriate (40%) protein levels, especially when the stocking rates are high.     

The content of total mercury and methylmercury in common carp from selected Czech ponds

The aim of the present study was to investigate concentrations of total mercury (THg) and methylmercury (MeHG) in the common carp from Czech ponds of Rožmberk, Spolsky, Nezmar, and Velky Bědny. Seven common carps (Cyprinus caprio) from each of the ponds were caught. Muscle tissue, the liver, and the soft and hard roe were used for the tests. Total mercury and methylmercury were found in all the muscle tissue samples examined. Detection limits for total mercury and methylmercury determination methods were 0.001 mg kg^–1 and 0.013 mg kg⁻¹ respectively. Methylmercury levels in the liver and gonads were below the method’s limits of detection. THg and MeHg concentrations in muscle tissues were 0.018–0.063 mg kg^–1 w.w. and 0.019–0.063 mg kg^–1 w.w. respectively. MeHg made up 90–100% of THg in muscle tissues. Total mercury and methylmercury concentrations were significantly (P < 0.05) higher in fish from the Spolsky pond than from the Nezmar pond. MeHg/THg ratios were significantly (P < 0.05) higher in fish from the Rožmberk, Spolsky and Nezmar ponds compared with fish from the Velky Bědny pond.