Ontogeny of IgM-producing cells in the mandarin fish Siniperca chuatsi identified by in situ hybridisation

The ontogeny of IgM-producing cells was studied in juvenile mandarin fish Simperca chuatsi, an important fish in China's aquaculture sector. The IgM-producing cells were localised through in situ hybridisation with a probe complementary to the Ig mu-chain in lymphoid-related tissues, including head kidney, spleen, thymus, intestine and gills. In head kidney, transcripts of Ig mu were first detected at 20 days post-hatching (dph) with a few positive signals. and the number of IgM-producing cells increased obviously from 39 dph onwards. At 136 dph, a large amount of positive cells were observed in the entire organ with clusters of these cells located around the blood vessels. In spleen, IgM-producing cells were found from 26 dph onwards, followed by an increase until 67 dph: clusters of positive cells were also detected around blood vessels at 102 dph. In thymus, IgM-producing cells were first observed at 39 dph; thereafter, no obvious increase was detected until 78 dph. The positive cells in thymus were distributed mainly in the outer zone of thymus. A few IgM-producing cells were still observed in thymus of 1-year-old mandarin fish. IgM-producing cells were not detected in the intestine until 87 dph, with several discrete positively stained cells distributed in the lamina propria. IgM-producing cells, scattered mainly in primary gill filaments around blood vessels, were detected in gills from 90 dph. As in other teleosts, these results indicated that the head kidney appears to be the primary organ for IgM production in mandarin fish, and IgM-producing cells exist in all organs examined in the present study, implying their lymphoid role in fish. In addition, it is suggested that vaccination after 20 dph may be much more effective in mandarin fish. (C) 2009 Elsevier B.V. All rights reserved.

Molecular and expression characterization of two somatostatin genes in the Chinese sturgeon, Acipenser sinensis

Chinese sturgeon (Acipenser sinensis) is a rare and endangered species and also an important resource for the sturgeon aquaculture industry. SMART cDNA was synthesized from the hypothalamus of Chinese sturgeon, and the full-length cDNAs of two somatostatin (SS) genes were cloned and sequenced. The first cDNA (AsSS1) encodes a 116-amino acid protein that contains the SS14 sequence at its C-terminal extremity. AsSS1 shows high identity to that of human and other vertebrates. The second cDNA (AsSS2) encodes a 111-amino acid protein that contains the somatostatin variant [Pro(2)]-SS14 at its C-terminal extremity. Both the two SS mRNAs were expressed in brain and pituitary with different mRNA levels. But in peripheral tissues, AsSS2 was more widely distributed than AsSS1. High mRNA levels of AsSS2 were found in liver, kidney and heart, while low mRNA levels of AsSS2 were also detected in ovary. Throughout embryogenesis and early larval development only AsSS2 mRNAs were detected. Furthermore, in the hypothalamus of one to five year-old Chinese sturgeon, AsSS2 but not AsSS1 maintained stable expression. The mRNA distribution suggests that the Chinese sturgeon AsSS2 products play important physiological functions in adult fish as well as in cell growth and organ differentiation in embryo and larva development. (C) 2009 Elsevier Inc. All rights reserved.

Involvement of Fas/FasL system in apoptotic signaling in testicular germ cells of male Wistar rats injected i.v. with microcystins

Previous studies have shown that gonads were the second target organ of microcystins (MCs), and that MCs exposure exerted obvious toxic effects on male reproductive system of mammals. However, relevant molecular evidences are still lacking. Fas-signaling pathway plays a key role in toxicant-induced germ cell apoptosis. This study was to evaluate the responses of Fas/FasL system related genes and proteins in testes of rats injected intravenously with MCs. Enhanced apoptosis of germ cells in the testes of MCs-treated rats was detected by the terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling (TUNEL) associated with up-regulation of the Fas/FasL system. Both Fas and FasL protein expression were induced evidently from I h post-injection, and this high expression level maintained throughout the experiment. In addition, the activation of caspase-8 and caspase-3 protein was also observed, which were indicators of apoptosis. These results suggested the likely involvement of Fas/FasL system in the MCs-induced germ cell apoptosis. It is also suggested that MCs can cause damage to Sertoli cells directly. (C) 2009 Elsevier Ltd. All rights reserved.

Zebrafish eaf1 and eaf2/u19 Mediate Effective Convergence and Extension Movements through the Maintenance of wnt11 and wnt5 Expression

Studies have attributed several functions to the Eaf family, including tumor suppression and eye development. Given the potential association between cancer and development, we set forth to explore Eaf1 and Eaf2/U19 activity in vertebrate embryogenesis, using zebrafish. In situ hybridization revealed similar eaf1 and eaf2/u19 expression patterns. Morpholino-mediated knockdown of either eaf1 or eaf2/u19 expression produced similar morphological changes that could be reversed by ectopic expression of target or reciprocal-target mRNA. However, combination of Eaf1 and Eaf2/U19 (Eafs)-morpholinos increased the severity of defects, suggesting that Eaf1 and Eaf2/U19 only share some functional redundancy. The Eafs knockdown phenotype resembled that of embryos with defects in convergence and extension movements. Indeed, knockdown caused expression pattern changes for convergence and extension movement markers, whereas cell tracing experiments using kaeda mRNA showed a correlation between Eafs knockdown and cell migration defects. Cardiac and pancreatic differentiation markers revealed that Eafs knockdown also disrupted midline convergence of heart and pancreatic organ precursors. Noncanonical Wnt signaling plays a key role in both convergence and extension movements and midline convergence of organ precursors. We found that Eaf1 and Eaf2/U19 maintained expression levels of wnt11 and wnt5. Moreover, wnt11 or wnt5 mRNA partially rescued the convergence and extension movement defects occurring in eafs morphants. Wnt11 and Wnt5 converge on rhoA, so not surprisingly, rhoA mRNA more effectively rescued defects than either wnt11 or wnt5 mRNA alone. However, the ectopic expression of wnt11 and wnt5 did not affect eaf1 and eaf2/u19 expression. These data indicate that eaf1 and eaf2/u19 act upstream of noncanonical Wnt signaling to mediate convergence and extension movements.

Accumulation of hepatotoxic microcystins in freshwater mussels, aquatic insect larvae and oligochaetes in a large, shallow eutrophic lake (Lake Chaohu) of subtropical China

This paper studied the seasonal changes of two common microcystins (MCs), MC-RR and -LR, in the commercially important mussel Corbicula fluminea in Lake Chaohu, where there occurred dense cyanobacteria. Occasional measurements were also made for MC in the mussel Arconaia lanceolat, the oligochaete Limnodilus hoffineisteri and the insect larva Chironomus sp. Mean MC of C. fluminea was much higher in hepatopancreas than in intestine and foot. Our study is the first to report accumulation of MCs in oligochaetes and aquatic insect larvae. The hi-h contents of MCs in the insect larvae suggest a great possibility for the transfer of MCs to benthos-feeding omnivores like common carp. According to the provisional standard by the WHO, 28.6% of the collected C. fluminea were harmful for human consumption, assuming a daily consumption of 300 by a person. It is recommended that edible mussels should not be collected for human consumption during toxic cyanobacterial blooms in Lake Chaohu.

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.

Microcystin-induced variations in transcription of GSTs in an omnivorous freshwater fish, goldfish

The glutathione S-transferases are important enzymes in the microcystin-induced detoxication processes. In this experiment, we cloned the full-length cDNA of alpha, pi and theta-class-like glutathione S-transferase genes from goldfish (Carassius auratus Q. Their derived amino acid sequences were clustered with other vertebrate alpha, pi and theta-class GSTs in a phylogenetic tree and the goldfish GST sequences have the highest similarity with those from common carp and zebrafish. Goldfish were i.p. injected with microcystins extract at two doses (50 and 200 mu g kg(-1) BW MC-LReq) and the relative changes of the mRNA abundance in liver, kidney and intestine were analyzed by real-time PCR. The transcription of GST alpha was suppressed in both liver and intestine, but induced in the kidney. Decreased transcription of GST theta was detected in liver, kidney and intestine in the low-dose group. The transcription of GST pi was suppressed in liver and intestine post-injection in both dose groups. These results suggested that the transcription of GST isoforms varied in different ways within an organ and among organs of goldfish exposed to MCs. (C) 2008 Elsevier B.V. All rights reserved.

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.

Impact of environmental and innate factors on the food habit of Chinese perch Siniperca chuatsi (Basilewsky) (Percichthyidae)

Laboratory and field investigations were conducted to study the food habit of Chinese perch Siniperca chuatsi (Basilewsky) from first feeding through adult stage. Only fish larvae were consumed by Chinese perch larvae (2-21 days from hatching), and the presence of zooplankton did not have any significant effect on their survival rate. The ability of Chinese perch to feed on zooplankton is clearly limited by some innate factor. Instead of gill rakers, Chinese perch larvae have well-developed sharp teeth at the first feeding stage, and are well adapted to the piscivorous feeding habit unique to the larvae of Chinese perch, e.g. they bite and ingest the tails of other fish larvae. At the first feeding stage (2 days from hatching), daily rations were both very low, either in light or complete darkness. Although early-staged Chinese perch larvae (7-17 days from hatching) could feed in complete darkness, their daily rations were always significantly higher in light than in complete darkness. Late-staged Chinese perch larvae (21 days from hatching) were able to feed in complete darkness as well as in light, similar to the case of Chinese perch yearlings. Chinese perch yearlings (total length, 14-16 cm) consumed prey fish only and refused shrimp when visual cues were available (in light), but they consumed both prey when visual cues were not available (in complete darkness), suggesting that prey consumption by Chinese perch yearlings is affected by their sensory modality in predation. Both prey were found in the stomachs of similar-sized Chinese perch (total length, 14-32 cm) from their natural habitat, suggesting that shrimp are consumed by Chinese perch at night. Prey selection of Chinese perch with a length >38 cm, which consumed only fish in the field, appears to be based upon prey size instead of prey type. These results suggest that although environmental factors (e.g. light intensity) affect prey detection by Chinese perch, this fish is anatomically and behaviourally predisposed to prey on live fish from first feeding. This makes it a difficult fish to cultivate using conventional feeds.

Molecular identification and expression analysis of tumor necrosis factor receptor-associated factor 2 in grass carp Ctenopharyngodon idella

Tumor necrosis factor receptor-associated factor 2 (TRAF2) is a crucial component of almost the entire tumor necrosis factor receptor superfamily signaling pathway. In the present study, a TRAF2 gene has been cloned from grass carp (Ctenopharyngodon idella) by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends. The full-length cDNA is 3162 bp, including a 60 bp 5' untranslated region (UTR), a 1611 bp open reading frame, and a 1491 bp 3' UTR. The polyadenylation signal (AATAAA) and the mRNA instability motifs (ATTTTA, ATTTA) were followed by a poly(A) tail in the 3' UTR. No signal peptide or transmembrane region has been found in the putative amino acids of grass carp TRAF2 (gcTRAF2). Phylogenetic tree analysis clearly showed that gcTRAF2 is nearest to the TRAF2 gene of goldfish. The identity of gcTRAF2 with its homologs in other vertebrates ranges from 56% to 97%. It is characterized by one RING-type signature at the N-terminus, one zinc finger in the middle part, and one conserved TRAF domain consisting of a C-proximal (TRAF-C) subdomain and a N-proximal (TRAF-N) subdomain. The identity of TRAF-C among all TRAF2 homologs in vertebrates varies from 78% to 97%, whereas the identity of TRAF-N ranges from 56% to 100%. The recombinant gcTRAF2 has been expressed in Escherichia coli using pET-32a expression vector. The rabbit anti-gcTRAF2 polyclonal antibody was obtained. The expression of gcTRAF2 in different organs was examined by real-time quantitative polymerase chain reaction and Western blot analysis. It was widely distributed in heart, head kidney, thymus, brain, gill, liver, spleen, and trunk kidney. This is the first report of a TRAF2 homolog molecule in fish.

The diverse Grania fauna (Clitellata : Enchytraeidae) of the Esperance area, Western Australia, with descriptions of two new species

Seven species of the marine enchytraeid genus Grania Southern, 1913 are described from sediments sampled during the 2003 International Workshop on the Marine Flora and Fauna of Esperance Bay and the Recherche Archipelago, on the southern coast of Western Australia. Two species are new to science, the euryhaline Tasmanian G. dolichura Rota and Erseus, 2000 represents a new record for the state, and the remaining four species were known from other parts of Western Australia. Grania quaerens sp. n. is recognized by having a high chaetal index (= 5 short chaetal foot), small coelomocytes, penial apparati with long whip-like terminal stylets, conspicuous spermathecae with ectally bulbous ducts, and ectally granulated ampullae housing sperm rings in their ental region. Grania sperantia sp. n. is readily distinguishable by the complete lack of lateral chaetae, a multiple-banded pattern of the clitellum, extremely long sperm funnels, and the intrasegmental location of the spermathecal pores. The latter new species and four others in the collection (G. bykane Coates, 1990, G. crassiducta Coates, 1990, G. dolichura, and G. ersei Coates, 1990) are remarkable in possessing the head organ, a sensory structure unique to Grania that was not noted previously in Western Australian species. When considering the whole genus, the geographic pattern of the head organ appears southern-centred: of the 17 species of Grania reported to possess it, as many as 13 inhabit the southern latitudes. The seventh species of the Esperance collection, G. vacivasa Coates and Stacey, 1993, is notable for the kind of items found in its gut and the unusual appearance of its pygidium.

Intelectin gene from the grass carp Ctenopharyngodon idella: cDNA cloning, tissue expression, and immunohistochemical localization

The cDNA encoding grass carp intelectin was isolated from a head kidney cDNA library, and termed gcIntL. The deduced amino acid sequence of gcIntL consists of 318 amino acids, and about 55% identical and 74% similar to human intelectin, which is a new type of lectin recognizing galactofuranose, and plays a role in the recognition of bacteria-specific components in animal hosts. The gcIntL gene consists of seven exons and six introns, spacing over approximately 3 kb of genomic sequence. Phylogenetic analysis clearly demonstrated that the gcIntL formed a clade with Danio rerio intelectin and 35 kDa serum lectin. By real-time quantitative RT-PCR analysis, gcIntL transcripts were significantly induced in head kidney, trunk kidney, spleen, and intestine from LPS-stimulated fish. RT-PCR and Western blotting analysis demonstrated that the mRNA and protein of gcIntL gene have the same expression pattern, and both were detected in brain, gill, intestine, head kidney, trunk kidney, spleen, and heart. Furthermore, gcIntL protein could be detected in gill, intestine, trunk kidney, head kidney, spleen, heart, and brain including medulla oblongata and optic lobe, as determined by immunohistochemistry. This is the first report of intelectin expression pattern in fish, and of recombinant gcIntL and polyclonal antibody against gcIntL. (c) 2006 Elsevier Ltd. All rights reserved.

Early ontogeny of Ancherythroculter nigrocauda and effects of delayed first feeding on larvae

Development of embryos and larvae in Ancherythroculter nigrocauda Yih et Woo (1964) and effects of delayed first feeding on larvae were observed after artificial fertilization. The fertilized eggs were incubated at an average temperature of 26.5 degrees C (range: 25.7-27) and the larvae reared at temperatures ranging from 21.8 to 28 degrees C. First cleavage was at 50 min, epiboly began at 7 h 5 min, heartbeat reached 72 per min at 24 h 40 min and hatching occurred at 43 h 15 min after insemination. Mean total length of newly hatched larvae was 4.04 +/- 0.03 mm (n = 15). A one-chambered gas bladder was observed at 70 h 50 min, two chambers occurred at 15 days, and scales appeared approximately 30 days after hatching. Larvae began to feed exogenously at day 4 post-hatch at an average temperature of 24 degrees C. Food deprivation resulted in a progressive atrophy of skeletal muscle fibres, deterioration of the larval digestive system and cessation of organ differentiation. Larval growth under food deprivation was significantly affected by the time of first exogenous feeding. Starved larvae began to shrink, with negative growth from day 6 post-hatch. The point of no return (PNR) was reached at day 11 after hatching. Mortality of starved larvae increased sharply from day 12 after hatching.

Effects of hexachlorobenzene on antioxidant status of liver and brain of common carp (Cyprinus carpio)

Hexachlorobenzene (HCB)-induced oxidative damages have been published in rats while the effects have not yet been reported in fishes. Juvenile common carps (Cyprinus carpio) were exposed to waterborne HCB from 2 to 200 mu g l(-1) for 5, 10 or 20 days. Liver and brain were analyzed for various parameters of oxidative stress. There were no significant changes of glutathione (GSH) content and superoxide dismutase (SOD) activity in liver after 5 or 10 days exposure, whereas obvious drops were observed at higher concentrations after 20 days exposure. Significant decreases of GSH content and SOD activity in brain were found during all the exposure days. In brain, HCB also significantly elevated the contents of reactive oxygen species (ROS), thiobarbituric acid-reactive substances (TBARS, as an indicator of lipid peroxidation products), glutathione disulfide (GSSG), and activities of nitric oxide synthase (NOS), glutathione peroxidase (GPx), and glutathione reductase (GR), and inhibited activities of acetylcholinesterase (AchE) and glutathione S-transferase (GST). The results clearly demonstrated that environmentally possible level of HCB could result in oxidative stress in fish and brain was a sensitive target organ of HCB toxicity. (c) 2006 Elsevier Ltd. All rights reserved.

First report of aphantoxins in China - waterblooms of toxigenic Aphanizomenon flos-aquae in Lake Dianchi

The oligohaline cyanobacterium Aphanizomenon flos-aquae (L.) Ralfs (A. flos-aquae) has been reported in several countries to produce paralytic shellfish poisons (PSPs) or protracted toxic effects. In the past years, A. flos-aquae blooms have occurred annually in the eutrophic Lake Dianchi (300 km(2) in area, located in southwestern China). Material from natural blooms dominated by A. flosaquae was collected and lyophilized. Acute toxicity testing was performed by mouse bioassay using extracts from the lyophilized material. Clear symptoms of PSPs, intoxications were observed. To confirm the production of PSPs, a strain of A. flos-aquae (DC-1) was isolated and maintained in culture. Histopathological effects were studied by examining the organ damages using transmission electron microscopy (TEM). Slight hepatocytic damage with swollen mitochondria was found. The ultrastructural pulmonary lesions were characterized by distortied nuclei and indenting of karyotheca, together with degeneration and tumefaction of mitochondria and endoplasmic reticulum. Control animals injected with acetic acid did not exhibit histopathological damage in any organ. Toxic effects of cultured algal cells on enzymatic systems in the mouse were studied using sublethal doses of extracts. Significant glutathione-S-transferase (GST) and lactate dehydrogenase (LDH) increases, together with decrease of the glutathione (GSH) level, were measured. These results indicated a potential role of PSPs intoxicating and metabolizing in the test animals. HPLC-FLD and LC/MS analysis of extracts from cultured material demonstrated the PSP toxins produced by A. flos-aquae bloom. To the best of our knowledge, this is the first study reporting chemically and toxicologically confirmed PSP toxins related to A. flosaquae in China. (c) 2005 Elsevier Inc. All rights reserved.