Evolutionary Conservation of Dazl Genomic Organization and its Continuous and Dynamic Distribution Throughout Germline Development in Gynogenetic Gibel Carp

To investigate germline development and germ cell specification, we identified a Dazl homolog (CagDazl) from gynogenetic gibel carp (Carassius auratus gibelio). Its cDNA sequence and BAC clone sequence analyses revealed the genomic organization conservation and conserved synteny of the Dazl family members and their neighborhood genes among vertebrates, especially in fish. Moreover, a polyclonal antibody specific to CagDazl was produced and used to examine its expression and distribution throughout germline development at protein level. Firstly, ovary-specific expression pattern of CagDazl was confirmed in adult tissues by RT-PCR and Western blot. In addition, in situ hybridization and immunofluorescence localization demonstrated its specific expression in germ cells, and both its transcript and protein were localized to germ plasm. Then, co-localization of CagDazl and mitochondrial cloud was found, confirming that CagDazl transcript and its protein are germ plasm component and move via METRO pathway during oogenesis. Furthermore, the CagDazl is abundant and continuous throughout germline development and germ cell specification including primordial germ cell (PGC) formation, oogonium differentiation, oocyte development, and embryogenesis, and the dynamic distribution occurs at different development stages. The data suggest that maternal CagDazl might play an important role in gibel carp PGC formation. Therefore, CagDazl is a useful and specific marker for tracing germ plasm and germ cell development in the gynogenetic gibel carp. In addition, in comparison with previous studies in sexual reproduction species, the continuous and dynamic distribution of CagDazl protein in the germ plasm throughout the life cycle seems to have significant implication in sex evolution of vertebrates. J. Exp. Zool. (Mol. Deu. Euol.) 312B:855-871, 2009. (C) 2009 Wiley-Liss, Inc.

Waterborne exposure to PFOS causes disruption of the hypothalamus-pituitary-thyroid axis in zebrafish larvae

Thyroid hormones (THs) play an important role in the normal development and physiological functions in fish. Environmental chemicals may adversely affect thyroid function by disturbing gene transcription. Perfluorooctane sulfonate (PFOS), a persistent compound, is widely distributed in the aquatic environment and wildlife. In the present study, we investigated whether PFOS could disrupt the hypothalamic-pituitary-thyroid (HPT) axis. Zebrafish embryos were exposed to various concentrations of PFOS (0, 100, 200 and 400 mu g L-1) and gene expression patterns were examined 15 d post-fertilization. The expression of several genes in the HIPT system, i.e., corticotropin-releasing factor (CRF), thyroid-stimulating hormone (TSH), sodium/iodide symporter (NIS), thyroglobulin (TG), thyroid peroxidase (TPO), transthyretin (TTR), ioclothyronine deiodinases (Dio1 and Dio2) and thyroid receptor (TR alpha and TR beta), was quantitatively measured using real-time PCR. The gene expression levels of CRF and TSH were significantly up-regulated and down-regulated, respectively, upon exposure to 200 and 400 mu g L-1 PFOS. A significant increase in NIS and Diol gene expression was observed at 200 mu g L-1 PFOS exposure, while TG gene expression was down-regulated at 200 and 400 mu g L-1 PFOS exposure. TTR gene expression was down-regulated in a concentration-dependent manner. Up-regulation and down-regulation of TR alpha and TR beta gene expression, respectively, was observed upon exposure to PFOS. The whole body thyroxine (T-4) content remained unchanged, whereas triiodothyronine (T-3) levels were significantly increased, which could directly reflect disrupted thyroid hormone status after PFOS exposure. The overall results indicated that PFOS exposure could alter gene expression in the HPT axis and that mechanisms of disruption of thyroid status by PFOS could occur at several steps in the synthesis, regulation, and action of thyroid hormones. (C) 2009 Elsevier Ltd. All rights reserved.

Isolation of Y- and X-linked SCAR markers in yellow catfish and application in the production of all-male populations

P>Sex controls have been performed in some farmed fish species because of significant growth differences between females and males. In yellow catfish (Pelteobagrus fulvidraco), adult males are three times larger than female adults. In this study, six Y- and X-linked amplified fragment length polymorphism fragments were screened by sex-genotype pool bulked segregant analysis and individual screening. Interestingly, sequence analysis identified two pairs of allelic genes, Pf33 and Pf62. Furthermore, the cloned flanking sequences revealed several Y- and X-specific polymorphisms, and four Y-linked or X-linked sequence characterized amplified region (SCAR) primer pairs were designed and converted into Y- and X-linked SCAR markers. Consequently, these markers were successfully used to identify genetic sex and YY super-males, and applied to all-male population production. Thus, we developed a novel and simple technique to help commercial production of YY super-males and all-male populations in the yellow catfish.

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.

Histone H2A Has a Novel Variant in Fish Oocytes

Histone variants and their modification have significant roles in many cellular processes. In this study, we identified and characterized the histone H2A variant h2af1o in fish and revealed its oocyte-specific expression pattern during oogenesis and embryogenesis. Moreover, posttranslational modification of H2af1o was observed that results from phosphorylation during oocyte maturation. To understand the binding dynamics of the novel core histone variant H2af1o in nucleosomes, we cloned ubiquitous gibel carp h2afx as a conventional histone control and investigated the dynamic exchange difference in chromatin by fluorescence recovery after photobleaching. H2af1o has significantly higher mobility in nucleosomes than ubiquitous H2afx. Compared with ubiquitous H2afx, H2af1o has a tightly binding C-terminal and a weakly binding N-terminal. These data indicate that fish oocytes have a novel H2A variant that destabilizes nucleosomes by protruding its N-terminal tail and stabilizes core particles by contracting its C-terminal tail. Our findings suggest that H2af1o may have intrinsic ability to modify chromatin properties during fish oogenesis, oocyte maturation, and early cleavage.

Waterborne exposure to fluorotelomer alcohol 6:2 FTOH alters plasma sex hormone and gene transcription in the hypothalamic-pituitary-gonadal (HPG) axis of zebrafish

Fluorotelomer alcohols (FTOHs) have shown estrogenic activity in vitro and in vivo, but the mechanism of this activity is not known. In this study, 18-week-old zebrafish (Danio rerio) were exposed to 0, 0.03, 0.3 and 3.0 mg/l 1H, 1H, 2H, 2H-perfluorooctan-1-ol (6:2 ETCH) for 7 days, and the effects on plasma sex hormone levels were measured followed by use of real-time PCR to examine selected gene expression in hypothalamic-pituitary-gonadal (HPG) axis and liver. Exposure to 6:2 FTOH significantly increased plasma estradiol (E2) and testosterone (T) levels in both males and females. Furthermore, the ratio of T/E2 was reduced in females while increased in males. In females, the increase of E2 was accompanied by up-regulated hepatic estrogenic receptor alpha (ER alpha) and vitellogenin (VTG1 and VTG3) expression. In males, the elevation of the T level is consistent with the up-regulation of cytochrome P450 c17 alpha-hydroxylase, 17, 20-lase (CYP17) and the down-regulation of cytochrome P450 aromatase A (CYP19A). The present study demonstrated that waterborne exposure to 6:2 FTOH alter plasma sex hormone levels and the ratio of T/E2, as well as the transcriptional profiles of some genes in the HPG axis and liver. The results suggested that FTOHs may disturb fish reproduction through endocrine disrupted activity. (C) 2009 Elsevier B.V. All rights reserved.

Effect of high dietary starch levels on the growth performance, blood chemistry and body composition of gibel carp (Carassius auratus var. gibelio)

An 8-week growth trial was carried out in a semi-recirculation system to investigate the effect of high dietary starch levels on the growth performance, blood chemistry, starch utilization and body composition of gibel carp (Carassius auratus var. gibelio). Five isonitrogenous and isocarloric experimental diets were formulated to contain different starch levels (24%, 28%, 32%, 36% and 40% respectively). Triplicate groups of fish (24 fish per tank with an average body weight, of 8.5 g) were assigned to each diet. The results showed that dietary carbohydrate levels significantly affected the growth performance, hepatopancreatic lipid content, pyruvate kinase (PK) activity and whole-body lipid content. Growth performance, body crude lipid and plasma glucose concentrations showed a decreasing trend with an increase in dietary starch from 24% to 40%. Pyruvate kinase activities and hepatopancreatic lipid content showed an increasing trend with the dietary starch increasing from 24% to 32%, and then a decreasing trend with the dietary starch increasing from 32% to 40%. No significant difference in the hepatopancreatic hexokinase (HK) activity, plasma triglyceride contents, body crude protein, ash and calcium (Ca) and phosphorus (P) contents was observed between different treatments. In conclusion, higher dietary starch levels (32-40%) significantly (P < 0.05) decreased the growth of gibel carp in the present study.

Identification of Prorocentrum minimum and Takayama pulchella by fluorescence in situ hybridization through epifluorescence microscopy and flow cytometry

Partial rDNA sequences of Prorocentrum minimum and Takayama pulchella were amplified, cloned and sequenced. and these sequence data were deposited in the GenBank. Eight oligonucleotide probes (DNA probes) were designed based on the sequence analysis. The probes were employed to detect and identify P. minimum and T. pulchella in unialgal and mixed algal samples with a fluorescence in situ hybridization method using flow cytometry. Epifluorescence micrographs showed that these specific probes labeled with fluorescein isothiocyanate entered the algal cells and bound to target sequences, and the fluorescence signal resulting from whole-cell hybridization varied from probe to probe. These DNA probes and the hybridization protocol we developed were specific and effective for P. minimum and T. pulchella, without any specific binding to other algal species. The hybridization efficiency of different probes specific to P. minimum was in the order: PM18S02 > PM28S02 > PM28S01 > PM18S01, and that of the probes specific to T. pulchella was TP18S02 > TP28S01 > TP28S02 > TP18S01. The different hybridization efficiency of the DNA probes could also be shown in the fluorescent signals between the labeled and unlabeled cells demonstrated using flow cytometry. The DNA probes PM18S02, PM28S02; TP18S02 and TP28S01, and the protocol, were also useful for the detection of algae in natural samples.

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

Natural killer (NK) cell enhancing factor (NKEF) belongs to the newly defined peroxiredoxin (Prx) family. Its functions are to enhance NK cell cytotoxicity and to protect DNA and proteins from oxidative damage. In this study, a partial cDNA sequence of carp NKEF-B was isolated from thymus cDNA library. Subsequently, the full-length cDNA of carp NKEF-B was obtained by means of 3' and 5' RACE, respectively. The full-length cDNA of carp NKEF-B was 1022 bp, consisting of a 73 bp 5'-terminal untranslated region (UTR), a 355 bp T-terminal UTR, and a 594 bp open reading frame coding for a protein of 197 amino acids. Carp NKEF-B contained two consensus Val-Cys-Pro (VCP) motifs and three consensus cysteine (Cys-51, Cys-70 and Cys-172) residues. Sequence comparison showed that the deduced amino acid sequence of carp NKEF-B had an overall similarity of 74-96% to that of other species homologues. Phylogenetic analysis revealed that carp NKEF-B forms a cluster with other known teleost NKEF-Bs. Then, by PCR we obtained a 5.1 -k long genomic DNA of carp NKEF-B containing six exons and five introns. Realtime RT-PCR results showed that carp NKEF-B gene was predominantly detected in kidney and head kidney under un-infected conditions. Whereas under SVCV-infection condition, the expression of NKEF-B gene was significantly increased in blood cells, gill, intestine and spleen, but maintained in liver, and decreased significantly in kidney and head kidney. Finally, the rNKEF-B was constructed and expressed in Escherichia coli. By using an antibody against carp rNKEF-B, immunohistochemical study further indicated that NKEF-B positive cells are mainly some RBCs and a few epithelial cells in gill and intestine, and that under SVCV-infection condition, these positive cells or positive products in their cytoplasm were mainly increased in gill and spleen sections of carp. The results obtained in the present study will help to understand the function of NKEF-B in teleost innate immunity. (C) 2008 Elsevier Ltd. All rights reserved.

Distribution and depuration of the potentially carcinogenic malachite green in tissues of three freshwater farmed Chinese fish with different food habits

The use of malachite green (MG) in fish farming is prohibited in China due to its potentially toxicological and carcinogenic nature, but it is still illegally used in some places. Uptake, accumulation and deputation of MG in various tissues were studied under laboratory conditions in three common freshwater fish, Parabramis pekinensis (plant-eating fish), Carassius auratus (omnivorous fish) and Ophiocephalus argus (carnivorous fish). The concentrations of MG and its primary metabolite, the reduced and colorless leucomalachite green (LMG), were analyzed by liquid chromatography-mass spectrometry (LC-MS2). Absorption of MG occurred during the waterborne exposure and the MG concentrations in gills of the three fish species all showed a maximum at 0 h after an acute water exposure (6 mg l(-1) MG for 20 min). Afterwards, both MG and LMG declined very rapidly in the blood of the fish. Levels of MG and LMG were still above 0.002 mu g g(-1) in fresh weight muscle at 240 h and may persist for as long as 10 days. Most MG was converted rapidly to LMG in the fish and deputation of LMG was very slow in fat tissue. skin and gonads of the fish. Distribution of LMG was strongly dependent on the fat content in the tissues of the fish, but not related to their different feeding habits. Therefore, it appears that fat tissue, skin and gonads of the fish contaminated by MG and LMG pose the greatest risk for human consumption. (C) 2008 Published by Elsevier B.V.

Chronic effects of water-borne PFOS exposure on growth, survival and hepatotoxicity in zebrafish: A partial life-cycle test

Perfluorooctane sulfonate (PFOS) is widely distributed and persistent in the environment and wildlife. The main aim of this study was to investigate the impact of long-term exposure to low concentrations of PFOS in zebrafish. Zebrafish fry (F-0, 14d post-fertilization, dpf) were exposed via the water for 70d to 0 (control), 10, 50 and 250 mu g L-1 PFOS, followed by a further 30d to assess recovery in clean water. The effects on survival and growth parameters and liver histopathology were assessed. Although growth suppression (weight and length) was observed in fish treated with high concentrations PFOS during the exposure period, no mortality was observed throughout the 70d experiment. Embryos and larvae (F-1) derived from maternal exposure suffered malformation and mortality. Exposure to 50 and 250 mu g L-1 PFOS could inhibit the growth of the gonads (GSI) in the female zebrafish. Histopathological alterations, primary with lipid droplets accumulation, were most prominently seen in the liver of males and the changes were not reversible, even after the fish were allowed to recover for 30d in clean water. The triiodothyronine (T-3)) levels were not significantly changed in any of the exposure groups. Hepatic vitellogenin (VTG) gene expression was significantly up-regulated in both male and female zebrafish, but the sex ratio was not altered. The overall results suggested that lower concentrations of PFOS in maternal exposure could result in offspring deformation and mortality. (c) 2008 Elsevier Ltd. All rights reserved.

Analysis of heavy metals of muscle and intestine tissue in fish - in Banan section of Chongqing from Three Gorges Reservoir, China

The present study was carried out to investigate contamination of heavy metals in 19 fish species from the Banan section of Chongqing in the Three Gorges, Yangtze River. The results showed that the mean concentrations of heavy metals were higher in intestine than muscle, except zinc in upper strata. In the fish inhabiting the upper strata, there were significant differences between mean concentrations of As, Cr, Cu and Hg in muscle and intestine (P <0.05). There were also significant differences between mean concentrations of Cr and Cu in muscle and intestine in the fish inhabiting middle strata. However, significant differences between mean concentrations of As, Cd, Hg, Pb and Zn were measured in fish inhabiting bottom strata in both intestine and muscle tissues (P <0.05). For the fish inhabiting different strata, the concentrations of As, Cd, Cr, Cu, Hg and Ph in muscle and intestine of the fish from bottom strata (BS) were higher than those in both upper strata (US) and middle strata (MS); whereas a higher concentration of Zn was measured in muscle and intestine from fish inhabiting upper strata. Mean metal concentrations were found to be higher in age 11 than those in age I in Coreius heterodon (2- and 1-year odl fish respectively). The overall results indicated that fish muscle in the Banan section were slightly contaminated by heavy metals, but did not exceed Chinese food standards.

Occurrence of Camallanus cotti in greatly diverse fish species from Danjiangkou Reservoir in central China

Two thousand four hundred fifty-eight fish comprised of 53 species were captured in the Danjiangkou Reservoir, in the northwestern part of Hubei Province, central China during 2004, to examine Camallanus cotti infections. We found that 19 cypriniform, 3 siluriforme, and 4 perciforme fishes were infected by the nematode. Our study revealed the species, Hemiculter bleekeri bleekeri, Culter oxycephaloide, Pseudolaubuca sinensis, Acanthobrama simony, Mylopharyngodon piceus, Ctenopharyngodon idella, Gnathopogon imberbis, G. argentatus, Saurogobio dabryi, S. dumerili, Gobiobotia ichangensis, Liobagrus marginatoides, and Ctenogobius shennongensis as new hosts of the worm. The number and range of fish host species found in this survey were much greater than any of the previous investigations. The mean prevalence, prevalence, mean abundance, and intensity of infection varied in different fish species, indicating a possible host preference. Moreover, we suggest that this nematode is a native parasite of cypriniform fishes in China, perhaps initially in the reaches of the Yangtze River.

Differential expression and dynamic changes of SOX3 during gametogenesis and sex reversal in protogynous hermaphroditic fish

SOX3 has been suggested to play significant roles in gametogenesis and gonad differentiation of vertebrates, but the exact cellular localization evidence is insufficient and controversial. In this study, a protogynous hermaphrodite fish Epinephelus coioides is selected to analyze EcSox3 differential expression and the expression pattern in both processes of oogenesis and spermatogenesis by utilizing the advantages that gonad development undergoes transition from ovary to intersexual gonad and then to testis, and primordial germ cells and different stage cells during oogenesis and spermatogenesis are synchronously observed in the transitional gonads. The detailed and clear immunofluoresence localization indicates that significantly differential expression and dynamic changes of Sox3 occur in the progresses of gametogenesis and sex reversal, and EcSOX3 protein exists in the differentiating primordial germ cells, oogonia, and different stage oocytes of ovaries, and also in the differentiating primordial germ cells and the Sertoli cells of testis. One important finding is that the EcSox3 expression is a significant time point for enterable gametogenesis of primordial germ cells because EcSOX3 is obviously expressed and localized in primordial germ cells. As EcSox3 continues to express, the EcSOX3-positive primordial germ cells develop toward oogonia and then oocytes, whereas when EcSox3 expression is ceased, the EcSOX3-positive primordial germ cells develop toward spermatogonia. Therefore, the current finding of EcSOX3 in the differentiating primordial germ cells again confirms the potential regulatory role in oogenesis and germ cell differentiation. The data further suggest that SOX3, as a transcription factor, might have more important roles in oogenesis than in spermatogenesis.

First description of the male and redescription of the female of Philometra clavaeceps Dogiel and Akhmerov, 1959 (Nematoda : Philometridae), a parasite of east Asian cyprinid fish

The first description of the male and a redescription of the female of the nematode Philometra clavaeceps Dogiel and Akhmerov, 1959, a parasite of east Asian cyprinids, are presented on the basis of specimens collected from Culter erythropterus Basilewsky and Culler dabryi Bleeker from Liangzi Lake (the Yangtze River basin), Hubei Province, central China. Gravid females from the fish abdominal cavity, penetrating often into ovaries, occurred in May-June, whereas conspecific males and young mature females on the swimbladder were recorded in January. Philometra clavaeceps seems to have a pronounced annual maturation cycle in the locality. The finding of P. clavaeceps in C. dabryi represents a new host record.