Production of all-male non-transgenic zebrafish by conditional primordial germ cell ablation.

Many fish species exhibit remarkable sexual dimorphism, with males possessing numerous advantageous traits for commercial production by aquaculture such as faster growth rate, more efficient food energy utilization for muscle development, and better breeding performance. Several studies have shown that a decrease in the number of primordial germ cells (PGCs) during early development leads predominantly to male progeny. In this study, we developed a method to obtain all-male zebrafish (Danio rerio) by targeted PGC ablation using the nitroreductase/metronidazole (NTR/Mtz) system. Embryos generated by female heterozygous Tg(nanos3:nfsB-mCherry-nanos3 3'UTR) and male wild-types (WTs) were treated with vehicle or Mtz. Compared to vehicle-treated controls, 5.0 and 10.0 mM Mtz treatment for 24 h significantly reduced the number of PGCs and yielded an exclusively male phenotype in adulthood. The gonads of offspring treated with 5.0 mM Mtz exhibited relatively normal morphology and histological characteristics. Furthermore, these males were able to chase females, spawn, and produce viable offspring, while about 20.0% of males treated with 10.0 mM Mtz were unable to produce viable offspring. The 5.0 mM Mtz treatment protocol may thus be suitable for large-scale production of fertile male offspring. Moreover, about half of these males were WT as evidenced by the absence of nfsB gene expression. It may thus be possible to breed an all-male WT fish population by Mtz-mediated PGC ablation.

Molecular identification and expression of the Foxl2 gene during gonadal sex differentiation in northern snakehead Channa argus.

Channa argus is one of the most commercially important fish species in China. Studies show that males of C. argus grow faster than females at the same age. In order to explore the sex differentiation mechanism of C. argus, we isolated the full length of the sex-related gene Foxl2 cDNA and analysed its expression patterns during gonadal sex differentiation. Alignment of known Foxl2 amino acid sequences from vertebrates confirmed the conservation of the Foxl2 open reading frame, especially the forkhead domain and C-terminal region. Quantitative RT-PCR revealed that Foxl2 is predominantly expressed in brain, pituitary, gill and ovary, with its highest level in ovary but low levels in testis and other tissues, reflecting a potential role for Foxl2 in the brain-pituitary-gonad axis in C. argus. Our ontogenetic stage data showed that C. argus Foxl2 expression was significantly upregulated from 1 to 11 days posthatching (dph) and that the initiation of expression preceded the first anatomical ovarian differentiation (27 dph), suggesting that Foxl2 might play a potential role in early gonadal sex differentiation in C. argus. In addition, the Foxl2 protein was primarily located in granulosa cells surrounding the oocytes of mature C. argus, implying that Foxl2 may have a basic function in granulosa cell differentiation and the maintenance of oocytes.

Mercury biomagnification in the aquaculture pond ecosystem in the Pearl River Delta.

This is the first study to investigate the rate of mercury (Hg) biomagnification in the aquaculture pond ecosystem of the Pearl River Delta (PRD), China, by analyzing total mercury (THg) and methyl mercury (MeHg) concentrations in various species of fish at different trophic levels (TLs). Species representing a gradient of trophic positions in the aquaculture pond food chains were chosen for analyzing THg and MeHg concentrations. In this study, there were two kinds of the aquaculture pond food chains: (1) omnivorous (fish feeds, zooplankton, grass carp [Ctenopharyngodon idellus], and bighead carp [Aristichthys nobilis]) and (2) predatory (zooplankton, mud carp [Cirrhina molitorella], and mandarin fish [Siniperca kneri]). Bighead carp and mandarin fish had the highest MeHg and THg concentrations, i.e., an order of magnitude higher than other species, in their respective food chains. More than 90% of the THg concentrations detected in bighead carp, mandarin fish, and mud carp were in the methylated form. In this study, %MeHg increased with TLs and MeHg concentrations, reflecting that MeHg is the dominant chemical species of Hg accumulated in higher concentrations in biota, especially biota associated with higher TLs in the food chains. The trophic magnification factors were 2.32 and 2.60 for MeHg and 1.94 and 2.03 for THg in omnivorous and predatory food chains, respectively, in PRD. Hg concentrations in fish tissue correlated to Hg levels in the ambient environment, and sediment seemed to be the major source for Hg accumulated in fish. In addition, feeding habit also affected Hg accumulation in different fish species. Four significant linear relationships were obtained between log-THg and δ(15)N and between log-MeHg and δ(15)N. The slope of the regression equations, as biomagnification power, was smaller in magnitude compared with those reported for temperate and arctic marine and freshwater ecosystems, indicating that THg and MeHg biomagnifications were lower in this PRD subtropical aquaculture pond ecosystem. This was probably due to low Hg bioavailability at lower TLs as well as individual feeding behavior of fish.

An NGS-based approach for the identification of sex-specific markers in snakehead (Channa argus).

We described a next generation sequencing (NGS)-based approach to identify sex-specific markers and subsequently determine whether a species has male or female heterogamety. To test the accuracy of this technique, we examined the snakehead (Channa argus), which is economically important freshwater fish in China. Males grow faster than females, and there is significant interest in developing methods to skew breeding towards all-males to increase biomass yields. NGS was conducted on DNAs of individual female and male, the male reads were spitted into 60 bp K-mers and aligned to the female reference genome assembled by female reads, unaligned male K-mers-60 were kept in next filter process. Meanwhile, DNA sample of 48 females was pooled and sequenced, this data was further used to filter out the previous unaligned male K-mers-60. Hence, numbers of candidate Y chromosome-specific sequences were screened out, their sex-specificity were validated in wild snakeheads through PCR amplification. Finally, three Y chromosome-specific fragments (Contig-275834, Contig-359642, and Contig-418354) were identified, and specific primers were obtained to distinguish the sex of snakehead. Additionally, a pair of primers of Contig-275834 (275834X/Y-F and 275834X/Y-R) was exploited to distinguish XX females, XY males, and YY super-males, whose amplification products of different lengths were produced for different sexes. Therefore, our work demonstrated the ability of NGS data in identification of sex-specific markers, and the pipeline adopted in our study could be applied in any species of sex differentiation. Furthermore, the sex-specific markers have tremendous potential for improving the efficiency of all-male breeding practices in snakehead.

Arsenic contamination in the freshwater fish ponds of Pearl River Delta: bioaccumulation and health risk assessment.

This study investigated the extent of arsenic (As) contamination in five common species of freshwater fish (northern snakehead [Channa argus], mandrarin fish [Siniperca chuatsi], largemouth bass [Lepomis macrochirous], bighead carp [Aristichthys nobilis] and grass carp [Ctenopharyngodon idellus]) and their associated fish pond sediments collected from 18 freshwater fish ponds around the Pearl River Delta (PRD). The total As concentrations detected in fish muscle and sediment in freshwater ponds around the PRD were 0.05-3.01 mg kg(-1) wet weight (w. wt) and 8.41-22.76 mg kg(-1) dry weight (d. wt), respectively. In addition, the As content was positively correlated (p < 0.05) to total organic carbon (TOC) contents in sediments. Biota sediment accumulation factor (BSAF) showed that omnivorous fish and zooplankton accumulated higher concentrations of heavy metals from the sediment than carnivorous fish. In addition, feeding habits of fish also influence As accumulation in different fish species. In this study, two typical food chains of the aquaculture ponds were selected for investigation: (1) omnivorous food chain (zooplankton, grass carp and bighead carp) and (2) predatory food chain (zooplankton, mud carp and mandarin fish). Significant linear relationships were obtained between log As and δ (15)N. The slope of the regression (-0.066 and -0.078) of the log transformed As concentrations and δ (15)N values, as biomagnifications power, indicated there was no magnification or diminution of As from lower trophic levels (zooplankton) to fish in the aquaculture ponds. Consumption of largemouth bass, northern snakehead and bighead carp might impose health risks of Hong Kong residents consuming these fish to the local population, due to the fact that its cancer risk (CR) value exceeded the upper limit of the acceptable risk levels (10(-4)) stipulated by the USEPA.

[Residues and potential ecological risk assessment of metal in sediments from lower reaches and estuary of Pearl River].

In order to investigate the heavy metal concentrations and their potential ecological risks in surface sediments of lower reaches and estuary of Pearl River, 21 bottom sediment samples were collected from lower reaches and estuary of Pearl River. Total contents of Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Cd, Sb, Pb and Hg in these samples were measured by the inductively coupled plasma mass spectrometry (ICP-MS) and the atomic fluorescence spectrometry (AFS) and using the index of geoaccumulation and the potential ecological risk index to evaluate the pollution degree of heavy metals in the sediments. Results indicated that the concentration of total Fe and total Mn were 41658.73 and 1104.73 mg x kg(-1) respectively and toxic trace metals, such as Cr, Co, Ni, Cu, Zn, As, Se, Cd, Sb, Pb and Hg were 86.62, 18.18, 54.10, 80.20, 543.60, 119.55, 4.28, 10.60, 20.26, 104.58 and 0.520 mg x kg(-1). The descending order of pollution degree of various metals is: Cd > As approximately Zn > Hg > Pb approximately Cu approximately Cr, while the single potential ecological risk followed the order: Cd > Hg > As > Cu > Pb > Zn > Cr. The pollution extent and potential ecological risk of Cd were the most serious among all heavy metals. The distribution pattern of Cd individual potential ecological risk indices is exactly the same as that of general potential ecological risk indices for all heavy metals. Clustering analysis indicates that the sampling stations may be classified into five groups which basically reflected the characteristics of the heavy metal contamination and sedimentation environments along the different river reaches in lower reaches and estuary of Pearl Rive. In general, the serious heavy metal pollution and the high potential ecological risk existed in three river reaches: Chengcun-Shawan, Chengcun-Shundegang and Waihai-Hutiaomen. The pollution degree and potential ecological risk are higher in related river reaches of Beijiang than that in other lower reaches and estuary of Pearl River.

Interferon regulatory factor-2 in orange-spotted grouper (Epinephelus coioides): gene, inductive expression pattern and subcellular localization.

Interferon regulatory factor 2 (IRF-2) is a multifunctional transcription factor which exhibits both transcriptional activating and repressing activities in the IRF family. In this study, we report an IRF-2 gene isolated from orange-spotted grouper (Epinephelus coioides). The 1854bp full-length cDNA sequence of EcIRF-2 has been cloned, encoding a putative peptide of 336 amino acids which is highly consistent with the feature of IRF family members. The genomic fragment of EcIRF-2 contains nine exons and eight introns, spanning over approximate 8.8kb. The expression of EcIRF-2 gene was detected in various tissues of healthy orange-spotted grouper and in four tissues after being challenged with poly I:C or LPS. EcIRF-2 gene is ubiquitously expressed in various healthy fish tissues and is up-regulated in vivo in response to poly I:C or LPS. Subcellular localization analysis of EcIRF-2 suggests it is an intranuclear protein in the fish cells. We believe this research is the first report of fish IRF-2 protein localization. The results in this research establish the base for further study of function mechanism of IRF family members in orange-spotted grouper.

[Investigation of quinolones in aquaculture environment of the Pearl River Delta].

The concentrations and distributions of three sorts of quinolones (norfloxacin, ciprofloxacin and enorfloxacin) in water, sediments and the tissues of 8 kinds of fishes from 7 sites in Pearl River Delta aquaculture regions (freshwater and marine) were determined by high performance liquid chromatography (HPLC) with fluorescence detector. Results showed that no quinolones were found in either fresh waterborne or marine water. Norfloxacin, ciprofloxacin and enorfloxacin in sediments collected from freshwater aquaculture region ranged from 5.03-13.28, 3.64-9.32 and 0-7.13 ng x g(-1), respectively, ranged from 1.88-8.81, 0-1.09 ng x g(-1) in marine aquaculture area, respectively. Enorfloxacin were not found in sediments from marine aquaculture. Three kinds of quinolones in liver tissues were higher than that in muscle tissues. The residues of norfloxacin, ciprofloxacin and enorfloxacin in fish muscle tissues ranged from 1.95-100.54, 0.48-33.26 and 1.18-51.89 ng x g(-1), respectively. The concentration of pharmaceuticals in fish tissues were ranked by size as following: norfloxacin, ciprofloxacin and enofloxacin. Higher concentration of quinolones was found in fish from freshwater aquaculture than marine aquaculture.