Plasma biochemical responses of the omnivorous crucian carp (Carassius auratus) to crude cyanobacterial extracts

Healthy crucian carp (Carassius auratus) were treated by intraperitoneal (i.p.) injection of crude cyanobacterial extracts at two doses, 50 and 200 mu g MC-LR equiv kg(-1) BW. High mortality (100%) was observed within 60 h post injection in the high-dose group. In the treated fish, activities of four plasma enzymes, alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH), all showed substantial increases, with both dose and time-dependent effects. These increases of enzyme activity indicate severe impairment occurred in the liver of crucian carp over time. Plasma concentrations of energy-related biomolecules including glucose (GLU), cholesterol (CHO), triglyceride (TG), and total protein (TP) showed marked changes in the high-dose group, possibly a nutritional imbalance correlated with the liver injury caused by intraperitoneal exposure to crude cyanobacterial extracts.

Determination of the surface area of common carp, Cyprinus carpio L.

A wrap method adaptation combined with AutoCAD2005 and Scion Image for Windows were used to determine the surface area of a fish. Compared with the corresponding r(2) and F of many models, the most accurate formula: S = 752.15W(0.675) (r(2) = 0.999, F = 18362.94, P < 0.0001) for estimating the surface area of common carp was obtained. Similarly, the fin formula: S = 1834.12W(0.708) (r(2) = 0.992, F = 2690.47, P < 0.0001) was also obtained for the same purpose. It was proven that these two formulae gave good estimates of surface and fin areas of four strains of common carp: Yellow-river carp, fancy carp, mirror carp and Xingguo red carp.

Molecular characterization and expression pattern of fetuin-B in gibel carp (Carassius auratus gibelio)

Fetuin-B has recently been cloned and identified from rats, mice, and humans; their expression patterns, however, have not been elucidated yet. In this study, Cagfetuin-B has been cloned in gibel carp. RT-PCR and Western blot detection revealed that Cagfetuin-B is first transcribed from the blastula stage and at a relatively stable level afterward during embryogenesis and the larval stage. Cagfetuin-B transcripts are predominantly distributed over the yolk syncytial layer in the early embryos and later restricted to the cells of liver and brain in newly hatched larvae. Moreover, a dynamic distribution of Cagfetuin-B protein was observed in brain, kidney, liver, and skin during morphogenesis. In adult fish, Cagfetuin-B transcripts are restricted in liver and ovary. Our work, for the first time, revealed the extra-hepatic transcription and a dynamic distribution of fetuin-B during embryogenesis and in adults, which indicates the potential roles of fetuin-B in fish organogenesis.

Effects of dietary manganese on growth and tissue manganese concentrations of juvenile gibel carp, Carassius auratus gibelio

A 68-day growth trial was conducted in a flow-through system to determine the effect of dietary manganese levels on growth and tissue manganese concentration of juvenile gibel carp (Carassius auratus gibelio). Seven purified diets containing 7.21, 8.46, 9.50, 10.50, 13.03, 19.72 and 22.17 mg manganese (as manganic sulfate) per kilogram diet were fed to triplicate groups of fish (initial weight 3.21 +/- 0.01 g). The results showed that dietary manganese levels did not significantly affect feed intake of the fish. Specific growth rate, feed efficiency, total hepatic superoxide dismutase activity, carcass and skeletal manganese concentration increased significantly with increased dietary manganese(P < 0.05) while condition factor decreased significantly(P < 0.05). It was concluded that dietary requirement of manganese was 13.77 mg Mn per kilogram diet. Carcass and skeletal manganese concentration could also be used to evaluate the manganese requirement. Total hepatic superoxide dismulase activity was not a sensitive indicator for dietary requirement.

Ultrastructural alteration of lymphocytes in spleen and pronephros of grass carp (Ctenopharyngodon idella) experimentally exposed to microcystin-LR

Relatively little is known in relation to pathological changes of immune organs in fish when exposed to MC-LR. The ultrastructural alteration of lymphocytes was examined in the spleen and pronephros of grass carp Ctenopharyngodon idella injected experimentally with microcystin-LR. The fish were intraperitoneally injected with MC-LR at a dose of 50 mu g/kg body weight, and the spleen and pronephros were dissected out at 1, 2, 7, 14 and 21 days post intraperitoneal injection (dpi). Pathological changes were then examined by transmission electron microscopy. Apoptosis was detected only in lymphocytes in the spleen, with obvious apoptotic features observed at 2 dpi; pathological changes of lymphocytes in the pronephros were also serious with mitochondria being highly edematous. However, damaged lymphocytes were almost un-observed in the spleen and pronephros at 21 dpi. These findings suggest that MC-LR can induce toxic effect on immune organs in grass carp, and the spleen may be much more sensitive to MC-LR stimulation. (C) 2008 Elsevier B.V. All rights reserved.

Tissue distribution and depuration of the extracted hepatotoxic cyanotoxin microcystins in crucian carp (Carassius carassius) intraperitoneally injected at a sublethal dose

An acute toxicity experiment was conducted by intraperitoneal injection with a sublethal dose of extracted microcystins (MCs), 50 mu g MC-LR (where L = leucine and R = arginine) equivalent/kg body weight (BW), to examine tissue distribution and depuration of MCs in crucian carp (Carassius carassius). Liver to body weight ratio increased at 3, 12, 24, and 48 h postinjection compared with that at 0 h (p < 0.05). MC concentrations in various tissues and aquaria water were analyzed at 1, 3, 12, 24, 48, and 168 h postinjection using liquid chromatography coupled with mass spectrometry (LC-MS). The highest concentration of MCs (MC-RR + MC-LR) was found in blood, 2 -270 ng/g dry weight (DW), followed by heart (3 -100 ng/g DW) and kidney (13 -88 ng/g DW). MC levels were relatively low in liver, gonad, intestine, spleen, and brain. MC contents in gills, gallbladder, and muscle were below the limit of detection. Significant negative correlation was present between MC-RR concentration in blood and that in kidney, confirming that blood was important in the transportation of MC-RR to kidney for excretion. Rapid accumulation and slow degradation of MCs were observed in gonad, liver, intestine, spleen, and brain. Only 0.07% of injected MCs were detected in liver. The recovery of MCs in liver of crucian carp seemed to be dose dependent.

Detection of cutaneous antibodies in excised skin explants from grass carp, Ctenopharyngodon idella (Valenciennes), immune to Scophthalmus maximus rhabdovirus

This study determined whether cutaneous antibodies were present in excised skin explants of grass carp, Ctenopharyngodon idella, immune to Scophthalmus maximus rhabdovirus (SMRV). Culture fluid from immune skin explants were assayed by indirect enzyme-linked immunosorbent assay (iELISA), Western blot, indirect immunofluorescent assay (IFA) and flow cytometry (FCM). iELISA showed that cutaneous antibody titres were much lower (1:12) than antiserum titres (1:1458) from intraperitoneally immunized grass carp. The phosphoprotein and matrix protein antigens of purified SMRV proteins were recognized by cutaneous antibodies from skin culture fluid using Western blot. The skin culture fluid produced staining signals in viral assembly sites and cytoplasm of SMRV-infected epithelioma papulosum cyprini (EPC) cells by IFA. FCM showed that 4.39% SMRV-infected EPC cells were detected, while non-specific reaction was seen in 2% of control cells. This is the first description of cutaneous antibodies against SMRV in grass carp.

Development and characterization of monoclonal antibodies to spring viraemia of carp virus

Five monoclonal antibodies (mAbs) against spring viraemia of carp (SVCV0504, isolated from common carp in China) were produced from mice immunized with purified virus preparations. The virion of SVCV contains five structural proteins, representing the nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase (Q. Western blotting analysis revealed that three mAbs (1145, IE10, and 11-17) recognized specifically to a single protein of 47 kDa (N), the mAb 3G4 reacted with, two SVCV0504 proteins of 69 kDa (G) and 47 kDa (N), while the mAb 1A9 reacted with three SVCV0504 proteins of 69 kDa (G), 50 kDa (P), and 47 kDa (N). By indirect ELISA, two mAbs (1H5 and 11-17) showed cross-reactivity with pike fry rhabdovirus (PFRV), but no cross-reactions with the Siniperca chuatsi rhabdovirus (SCRV), Scophthalmus maximus rhabdovirus (SMRV), Paralichthys olivaceus rhabdovirus (PoRV) were demonstrated with the five mAbs. Indirect immunofluorescence showed intense fluorescence in the cytoplasm of the SVCV0504-infected epithelioma papulosum cyprini (EPC) cells in areas corresponding to the location of granular structures. The sucrose gradient-purified SVCV0504 particles could be detected successfully by these mAbs using immunodot blotting. mAb 1A9 could completely neutralize 100 TCID50 (50% tissue culture infective dose) of SVCV0504 at a dilution of 1:8. This is the first report of development of the neutralizing mAbs against SVCV. The mAb 1A9 was analyzed further and could be used to successfully detect viral antigens in the infected-EPC cell cultures or in cryosections from experimentally infected crucian carp (Carassius auratus) by immunohistochemistry assay. Furthermore, a flow cytometry procedure for the detection and quantification of cytoplasmic SVCV0504 in cell cultures was developed with mAb 1A9. At 28 h after inoculation with the virus (0.01 PFU/cell), 10.12% of infected cells could be distinguished from the uninfected cells. These mAbs will be useful in diagnostic test development and pathogenesis studies for fish rhabdovirus. (c) 2008 Elsevier B.V. All rights reserved.

Cloning, characterization and expression analysis of SIMP (source of immunodominant MHC-associated peptides) in grass carp Ctenopharyngodon idella

SIMP (source of immunodominant MHC-associated peptides) plays a key rote in N-linked glycosylation with the active site of oligosaccharyltransferase, being the source of MHC-peptides in the MHC I presentation pathway. In the present study, the SIMP gene has been cloned from grass carp Ctenopharyngodon idella by rapid amplification of cDNA ends (RACE). The full length of the cDNA sequence is 4384 bp, including a 1117 bp 5' UTR (untranslated region), a 2418 bp open reading frame, and a 849 bp 3' UTR. The deduced amino acids of the grass carp SIMP (gcSIMP) are a highly conserved protein with a STT3 domain and 11 transmembrane regions. The gcSIMP spans over more than 24,212 bp in length, containing 16 exons and 15 introns. Most encoding exons, except the first and the 15th, have the same length as those in human and mouse. The gcSIMP promoter contains many putative transcription factor binding sites, such as Oct-1, GCN4, YY1, Sp1, Palpha, TBP, GATA-1, C/EBP beta, and five C/EBP alpha binding sites. The mRNA expression of gcSIMP in different organs was examined by real-time PCR. The gcSIMP was distributed in all the organs examined, with the highest level in brain, followed by the level in the heart, liver, gill, trunk kidney, muscle, head kidney, thymus, and the lowest level in spleen. Furthermore, the recombinant gcSIMP has been constructed successfully and expressed in Escherichia coli by using pQE-40 vector, and the polyclonal antibody for rabbit has been successfully obtained, which was verified to be specific. Identification of gcSIMP will help to explore the function in fish innate immunity. (c) 2007 Elsevier Ltd. All rights reserved.

Structure, organization and expression of common carp (Cyprinus carpio L.) SLP-76 gene

SLP-76 is an important member of the SLP-76 family of adapters, and it plays a key role in TCR signaling and T cell function. Partial cDNA sequence of SLP-76 of common carp (Cyprinus carpio L.) was isolated from thymus cDNA Library by the method of suppression subtractive hybridization (SSH). Subsequently, the full length cDNA of carp SLP-76 was obtained by means of 3' RACE and 5' RACE, respectively. The full Length cDNA of carp SLP-76 was 2007 bp, consisting of a T-terminal untranslated region (UTR) of 285 bp, a T-terminal. UTR of 240 bp, and an open reading frame of 1482 bp. Sequence comparison showed that the deduced amino acid sequence of carp SLP-76 had an overall similarity of 34-73% to that of other species homotogues, and it was composed of an NH2-terminal domain, a central proline-rich domain, and a C-terminal SH2 domain. Amino acid sequence analysis indicated the existence of a Gads binding site R-X-X-K, a 10-aa-long sequence which binds to the SH3 domain of LCK in vitro, and three conserved tyrosine-containing sequence in the NH2-terminal domain. Then we used PCR to obtain a genomic DNA which covers the entire coding region of carp SLP-76. In the 9.2 k-long genomic sequence, twenty one exons and twenty introns were identified. RT-PCR results showed that carp SLP-76 was expressed predominantly in hematopoietic tissues, and was upregulated in thymus tissue of four-month carp compared to one-year old carp. RT-PCR and virtual northern hybridization results showed that carp SLP-76 was also upregulated in thymus tissue of GH transgenic carp at the age of four-months. These results suggest that the expression level of SLP-76 gene may be related to thymocyte development in teleosts. (c) 2007 Published by Elsevier Ltd.

Phenotypic plasticity in gut length in the planktivorous filter-feeding silver carp (Hypophthalmichthys molitrix)

Phenotypic plasticity widely exists in the external morphology of animals as well as the internal traits of organs. In the present study, we studied the gut length plasticity of planktivorous filter-feeding silver carp under different food resources in large-net cage experiments in Meiliang Bay of Lake Taihu in 2004 and 2005. There was a significant difference in stocking density between these 2 years. Under a low stocking density and abundant food resources, silver carp increased their energy intake by feeding on more zooplankton. Meanwhile, silver carp adjusted their gut length to match the digestive requirements of food when exposed to different food resources. In the main growth seasons (from April to October), silver carp significantly increased their relative gut length when feeding on more phytoplankton in 2005 (p < 0.01, 9.23 +/- 1.80 in 2004 and 10.77 +/- 2.05 in 2005, respectively). There was a nearly significant negative correlation between zooplankton proportion in the diet and the relative gut length when silver carp were stocked in a high density (p = 0.112). It appears that silver carp might have evolved plasticity to change their gut length rapidly to facilitate efficient utilization of food resources. Such resource polymorphisms in the gut may be a good indication of temporal adaptation to resource conditions. Our work provided field evidence for understanding the functional basis of resource polymorphisms and the evolution of phenotypic plasticity in planktivorous filter-feeding fish.

Distribution of toxins in various tissues of crucian carp intraperitoneally injected with hepatotoxic microcystins

An acute toxicity experiment was conducted to examine the distribution and depuration of microcystins (MCS) in crucian carp (Carassius aurutus) tissues. Fish were injected intraperitoneally with extracted MCs at a dose of 200 mu g MC-LR (where L = leucine and R = arginine) equivalent/kg body weight. Microcystin concentrations in various tissues and aquaria water were analyzed at 1, 3, 12, 24, and 48 h postinjection using liquid chromatography coupled with mass spectrometry. Microcystins were detected mainly in blood (3.99% of injected dose at 1 h), liver (1.60% at I h), gonad (1.49% at 3 h), and kidney (0.14% at 48 h). Other tissues, such as the heart, gill, gallbladder, intestine, spleen, brain, and muscle, contained less than 0.1% of the injected MCs. The highest concentration of MCs was found in blood (526-3,753 ng/g dry wt), followed by liver (103-1,656 ng/g dry wt) and kidney (279-1,592 ng/g dry wt). No MC-LR was detectable in intestine, spleen, kidney, brain, and muscle, whereas MC-RR was found in all examined fish tissues, which might result from organ specificity of different MCs. Clearance of MC-RR in brain tissue was slow. In kidney, the MC-RR content was negatively correlated with that in blood, suggesting that blood was important in the transportation of MC-RR to kidney for excretion.

Sixteen polymorphic microsatellites in bighead carp (Aristichthys nobilis) and cross-amplification in silver carp (Hypophthalmichthys molitrix)

A (GT)(n) enriched partial genomic library of bighead carp (Aristichthys nobilis) was constructed by employing the (fast isolation by AFLP of sequences containing repeats) FIASCO protocol. Sixteen loci exhibited polymorphism with two to seven alleles/locus (mean 3.263) in a test population and the observed heterozygosity ranging from 0.100 to 0.690 (mean 0.392). Eleven of the 16 bighead carp microsatellites were found to be also polymorphic in silver carp. These polymorphic loci should provide sufficient level of genetic diversity to evaluate population structure of bighead carp.

Molecular cloning and characterization of common carp (Cyprinus carpio L.) TCR gamma and CD3 gamma/delta chains

Partial cDNA sequences of TCR gamma and CD3 gamma/delta were isolated from the thymus of common carp (Cyprinus carpio L.) by the method of suppression subtractive hybridization (SSH). Subsequently the full length cDNAs of carp TCR gamma and CD3 gamma/delta were obtained by means of 3' RACE and 5' RACE, respectively. The full length of carp TCR gamma chain is 1368 bp and encodes 326 amino acids including a signal peptide region of 19 amino acids and a transmembrane region of 23 amino acids at the C-terminal region from aa 291 to 313. The V region of carp TCR gamma contains 109 amino acids, the core motif FGXG in J segment was also found in carp TCR gamma. The C region of carp TCR gamma contains the characteristic CX6PX6WX45C motif. The CP region of carp TCR C gamma contains 37 amino acids. The full length of carp CD3 gamma/delta is 790 bp and encodes 175 amino acids including a signal peptide region of 17 amino acids and a transmembrane region of 23 amino acids from aa 93 to 115. Similar to other known CD3 gamma/delta s, four cysteine residues in the extracellular domain and an immunoreceptor tyrosine-based activation motif ITAM (YxxL/Ix6-8YxxL/I) in the intracellular domain are also included in carp CD3 gamma/delta. Differing from other known CD3 gamma/delta s, carp CD3 gamma/delta tacks the CXXCXE motif in the extracellular domain. RTPCR analysis demonstrated that the expression of TCR gamma gene was mainly in the thymus and gill of 6-month carp, but in 18-month carp, TCR gamma gene was detected in all the examined tissues. The expression of CD3 gamma/delta gene was detected in all examined tissues of 6 and 18-month carp; among them, the highest expression level was in the thymus of 6-month carp. In situ hybridization showed that CD3 gamma/delta-expressing cells were widely distributed in the head kidney, spleen and kidney of carp, whereas in the thymus, they were densely distributed in the lymphoid outer zone and scattered in the epithelioid inner zone. (c) 2007 Published by Etsevier Ltd.

Acute effects of microcystins on the transcription of antioxidant enzyme genes in crucian carp Carassius auratus

Recent evidences suggested that oxidative stress may play a significant role in the pathogenesis of MCs toxicity. In the present study, the acute effects of microcystins on the transcription of antioxidant enzyme genes were investigated in liver of crucian carp i.p.-injected with 50 mu g MC-LReq per kg body weight (BW). We reported the cDNA sequences for four kinds of antioxidant enzyme (GSH-PX, CAT, Cu/Zn SOD, and GR) genes, and evaluated the oxidant stress induced by MCs through analyzing the transcription abundance of antioxidant enzyme genes using real-time PCR method. The time-dependent change of relative transcription abundance and expression of the antioxiclant enzyme genes were determined at 1, 3, 12, 24, and 48 h. The transcription abundance varied among antioxiclant enzymes, with GSH-PX and GR down-regulation, and CAT and SOD significantly upregulation. Based on these data, we tentatively concluded that the oxidant stress was induced by MCs, and caused the different response of the antioxiclant enzyme genes. (c) 2008 Wiley Periodicals, Inc.