Unveiling the dynamics of embryogenesis and immune genes expression pattern in the amur common carp (Cyprinus carpio haematopterus).

Amur common carp (Cyprinus carpio haematopterus), is a commercially important fish species that has been genetically improved over the years through selective breeding. Despite its significance in aquaculture, limited knowledge exists regarding its embryogenesis and immune genes associated with its early stages of life. This article represents a detailed study of the embryogenesis and innate immune gene expression analysis of the Amur common carp during its ontogenic developments. The entire embryonic developmental process of ∼44 h could be divided into eight periods, beginning with the formation of the zygote, followed by cleavage, morula, blastula, segmentation, pharyngula, and hatching. The segmentation period, which lasted for ∼ 6 h, exhibited the most significant changes, such as muscle contraction, rudimentary heart formation, increased somites number, and the initiation of blood circulation throughout the yolk. The expression of immune-related genes, namely toll-like receptor (TLR)4, nucleotide-binding oligomerization domain (NOD)1, NOD2 and interleukin (IL)-8 showed stage-specific patterns with varying levels of expression across the developmental stages. The TLR4 gene exhibited the highest expression during the neurella stage, while NOD1 and NOD2 peaked during hatching and IL-8 reached its maximum level during the gastrula stage. This is the first report of the innate immune gene expression during the embryogenesis of Amur common carp.

The stage-specific long non-coding RNAs and mRNAs identification and analysis during early development of common carp, Cyprinus carpio.

Cyprinus carpio is considered an alternative vertebrate fish model to zebrafish. However, systemic times-series research on the lncRNAs and mRNAs during early development of C. carpio has not been reported yet. This study provides the first long non-coding RNA (lncRNA)-mRNA expression profiles during six main early development stages (2 h post-fertilization hpf, 6 hpf, 12 hpf, 20 hpf, 64 hpf and 1 day post-hatching). A total of 51,979 lncRNAs were identified. We screened the top 10 abundance lncRNAs and mRNAs and stage-specific lncRNAs and mRNAs (specificity measure SPM > 0.9). We identified significant differentially expressed lncRNAs and mRNAs (|log2 (fold change)| ≥ 1 and false discovery rate FDR of <0.05). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis identified numerous signaling pathways. Additionally, the lncRNA-mRNA co-regulated network analysis of two lncRNAs (lncrps25 and malat1) and two mRNAs (mitf and troponin T) were investigated. Our results provide new insight into the role of lncRNAs and mRNAs, and would advance the understanding of lncRNA-mediated mechanisms in early development of fish.

Geno-cytotoxicity and congenital malformations produced by relevant environmental concentrations of aluminum, diclofenac and their mixture on Cyprinus carpio. An interactions study.

Several studies highlight the presence of aluminum and diclofenac in water bodies around the world and their ability to induce oxidative stress and a negative effect on biomolecules in several aquatic species. However, studies evaluating the toxic effect of mixtures of these contaminants are scarce. The objective of this work was to determine the genotoxic, cytotoxic and embryotoxic effect of the mixture of aluminum and diclofenac at environmentally relevant concentrations on Cyprinus carpio. Juveniles of Cyprinus carpio were exposed to 0.31 µg L-1 of diclofenac, 24.45 mg L-1 of aluminum, and a mixture of both contaminants at the same concentrations for 12, 24, 48, 72 and 96 h. After the exposure time the liver, gills and blood were extracted and the following biomarkers were evaluated: micronucleus frequency, comet assay, caspase activity and TUNEL test. On the other hand, Cyprinus carpio embryos were exposed to diclofenac (0.31 µg L-1), aluminum (0.06 mg L-1) and their mixture at the same concentrations and exposure time. Microscopic observation was performed to evaluate embryonic development at 12, 24, 48, 72 and 96 h. Diclofenac (0.31 µg L-1) induces significant increases in micronucleus frequency with respect to control (p < 0.05), in all tissues. Aluminum (24.45 mg L-1) significantly increases DNA damage index in liver and blood cells with respect to control (p < 0.05). All treatments increase caspases activity in all tissues with respect to control (p < 0.05). Diclofenac increases the percentage of TUNEL-positive cells in liver and blood; while aluminum and the mixture increases it significantly in gills and blood with respect to the control (p < 0.05). The mixture significantly delays embryonic development, while aluminum and the mixture significantly increase teratogenic index with respect to control (p < 0.05). In conclusion, exposure to environmental concentrations of aluminium, diclofenac and their mixture induces genotoxic damage, cell death by apoptosis and negative effects on the development of Cyprinus carpio and the toxic response is modified by the interaction of the xenobiotics.

Comparative microRNAs expression profiles analysis during embryonic development of common carp, Cyprinus carpio.

MicroRNAs (miRNAs) play important roles in biological processes by regulating specific gene expression. Limited miRNAs information is available on embryonic development in common carp (Cyprinus carpio) so far. In this study, six important embryonic development stages of C.carpio were collected to perform a times-series of small RNA-seq experiments from cleavage, blastocyst, gastrulation, organ formation, hatching stage to 1 day post-hatching larva. The expression profiles of miRNAs were identified and differentially expressed miRNAs (DEMs) were screened out based on pairwise comparison. A mean of 12,744,989 raw reads and 9,888,123 clean reads were obtained from each library. A total of 2565 miRNAs were identified. 68 of 204 DEMs were overlapped with stage-specific miRNAs, in which 15 were known miRNAs and seemed to play a key role in embryogenesis. Additionally, time-course expression reveals several intriguing fluctuations during embryogenesis. Numerous signaling pathways were identified in embryonic development, including the phototransduction, hippo signaling pathway, Wnt, melanogenesis, histidine metabolism and fatty acid biosynthesis. The results would provide new insight into the roles of miRNAs in embryonic development, and would help us to advance the understanding of miRNA-mediated mechanisms in embryonic development of fish.

Experimental evidence of dispersal of invasive cyprinid eggs inside migratory waterfowl.

Fish have somehow colonized isolated water bodies all over the world without human assistance. It has long been speculated that these colonization events are assisted by waterbirds, transporting fish eggs attached to their feet and feathers, yet empirical support for this is lacking. Recently, it was suggested that endozoochory (i.e., internal transport within the gut) might play a more important role, but only highly resistant diapause eggs of killifish have been found to survive passage through waterbird guts. Here, we performed a controlled feeding experiment, where developing eggs of two cosmopolitan, invasive cyprinids (common carp, Prussian carp) were fed to captive mallards. Live embryos of both species were retrieved from fresh feces and survived beyond hatching. Our study identifies an overlooked dispersal mechanism in fish, providing evidence for bird-mediated dispersal ability of soft-membraned eggs undergoing active development. Only 0.2% of ingested eggs survived gut passage, yet, given the abundance, diet, and movements of ducks in nature, our results have major implications for biodiversity conservation and invasion dynamics in freshwater ecosystems.

Ontogenetic and tissue-specific expression of gonadotropin-inhibitory hormone (GnIH) and its receptors in Catla catla.

Gonadotropin-inhibitory hormone (GnIH) is an RFamide peptide, and its role in reproduction is well studied from fish to mammals, but very few reports are available about the function of GnIH during larval development. In this study, we examined the GnIH and GnIH receptors (GnIHRs) expression from embryogenesis to adult stage and tissue-specific expression in adult Catla catla using quantitative real-time (qRT) PCR. The qRT PCR analysis of GnIH mRNA during ontogenetic development showed the increasing trend from early developmental stages to the adult stage with the highest expression in 24 months fish. However, the expression of two GnIH receptors, GnIHR1 and GnIHR2 also increased from larval stages to the adults with a peak at 17 days post-hatching, while GnIHR3 showed the higher mRNA expression during embryogenesis and then decreasing gradually. Tissue distribution analysis of GnIH showed the highest mRNA expression of GnIH in the brain, followed by gonads of both the sexes. GnIHR1 and GnIHR2 were also highly expressed in the brain and gonads of both the sexes, while GnIHR3 showed the highest expression in gonads of both the sexes without any expression in the brain. These results suggest that the brain is the primary site of action for GnIH, GnIHR1 and GnIHR2, while gonads for GnIHR3.

Embryotoxic and teratogenic profile of tretracycline at environmentally relevant concentrations on Cyprinus carpio.

The objective of this work was to evaluate whether tetracycline (TC) in environmentally relevant concentrations was able to induce alterations to embryonic development and teratogenic effects in oocytes and embryos of Cyprinus carpio. For this purpose, an embryolethality study was conducted and the lethal concentration 50 (LC50) and effective concentration 50 of malformations (EC50) were calculated, and with these data the teratogenic index (TI) was determined. The main alterations to embryonic development and the teratogenic effects produced by TC on embryos of C. carpio were determined using the Kimmel and Hersem scale adapted for Cyprinus carpio. LC50 and EC50 were respectively 500.08 and 145.3 µg L-1.TC was shown to be teratogenic with teratogenic index of 3.44, and the main malformations identified in concentrations of 90-900 µg L-1 were malformation in tail, modified chorda structure, pericardical edema, scoliosis and malformations of the heart. A significant decrease in concentration-dependent in Kimmel and Hersem score was observed. The results allow us to conclude that TC at environmentally relevant concentrations is capable of inducing embryotoxic and teratogenic effects, generating risk in the integrity of the common carp C. Carpio.

Characterization and expression analysis of FGF6 (fibroblast growth factor 6) genes of grass carp (Ctenopharyngodon idellus) reveal their regulation on muscle growth.

The present study was conducted to investigate the regulative function of FGF6 in the muscle growth of grass carp (Ctenopharyngodon idellus) by the bioinformatics analysis and expression pattern analyses of FGF6 genes in different developmental stages and tissues, as well as the correlation analysis between muscle growth and FGF6 expression after fish were fed with different levels of dietary lotus leaf flavonoids (LLF) (0, 0.03%, 0.06%, 0.09%). Results showed that the FGF6a and FGF6b genes are two homologs of the FGF6 family, encoding 205 and 209 amino acids, respectively. Alignment of amino acid sequences and phylogenetic analysis demonstrated that FGF6a and FGF6b are highly conserved with other vertebrates. Quantitative RT-PCR analysis showed both FGF6a and FGF6b expressions were high in brain and muscle but low in other examined tissues. During embryonic development, FGF6a and FGF6b mRNA expressions could be detected as early as at fertilized egg stage and displayed the highest value at cleavage stage. Dietary LLF affected the gene expression of FGF6 in white muscle. The relative expression of FGF6a of 0.06% LLF group was significantly higher than that of 0.09% LLF group, while FGF6b expression of 0.06% LLF group was higher than those of other groups (P < 0.05). The muscle fiber diameter was significantly higher in 0.06% LLF group in comparison with other groups, while the fiber density in this group was lower (P < 0.05). Both FGF6a and FGF6b expressions were positively correlated with fiber diameter but negatively correlated with fiber density. These results collectively suggest that FGF6a and FGF6b play an important role in muscle growth regulation in grass carp.

Cloning and characterization of wnt4a gene in a natural triploid teleost, Qi river crucian carp (Carassius auratus).

WNT4 (wingless-type MMTV integration site family, member 4) plays a key role in the ovarian differentiation and development in mammals. However, the possible roles of Wnt4 during gonadal differentiation and development need further clarification in teleosts. In this study, we cloned and characterized the full-length cDNA of Qi river crucian carp (Carassius auratus) wnt4a gene (CA-wnt4a). The cDNA of CA-wnt4a is 2337 bp, including the ORF of 1059 bp, encoding a putative protein with a transmembrane domain and a WNT family domain. Sequence and phylogenetic analyses revealed that the CA-Wnt4a identified is a genuine Wnt4a. Tissue distribution analysis showed that CA-wnt4a is expressed in all the tissues examined, including ovary. CA-wnt4a undergoes a stepwise increase in the embryonic stages, suggesting that CA-wnt4a might be involved in the early developmental stage. Ontogenic analysis demonstrated that CA-wnt4a expression is upregulated in the ovaries at 30-50 days after hatching (dah), the critical period of sex determination/differentiation in Qi river crucian carp. From 90 dah, the expression of CA-wnt4a was gradually downregulated in the developing ovaries. Immunohistochemistry demonstrated that CA-Wnt4a was expressed in the somatic and germ cells of the ovary by 30 dah, thereafter, positive signals of Wnt4a were detected in the somatic cells, oogonia and primary growth oocytes from 60 dah. In the sex-reversed testis induced by letrozole treatment, the expression level of CA-wnt4a was significantly downregulated. When CA-wnt4a expression was inhibited by injection of FH535 (an inhibitor of canonical Wnt/ß-catenin signal pathway) in the ovaries, levels of cyp19a1a, foxl2 mRNA were significantly downregulated, while sox9b and cyp11c1 were upregulated, which suggested that together with Foxl2-leading estrogen pathway, CA-wnt4a signaling pathway might be involved in ovarian differentiation and repression of the male pathway gene expression in Qi river crucian carp.

Alterations to embryonic development and teratogenic effects induced by a hospital effluent on Cyprinus carpio oocytes.

Hospital functioning generates a great quantity of contaminants, among which organic materials, heavy metals, and diverse pharmaceuticals are noteworthy that can affect organisms if they are not properly removed from the effluents. The hospital effluent evaluated in the present study came from IMSS (Instituto Mexicano del Seguro Social) Clinic 221 in downtown Toluca, State of Mexico, a secondary care facility. The contaminants identified in hospitals have been associated with deleterious effects on aquatic organisms; however, it is necessary to continue with more studies in order to be able to regulate the production of said contaminants which are generally dumped into the city sewage system. The present study had the purpose of evaluating the alterations to embryonic development and teratogenic effects on oocytes Cyprinus carpio after exposure to different proportions of hospital effluent. For said purpose, the physicochemical properties of the effluent were determined. Concentrations of the main microcontaminants were also determined. An embryolethality study out and the determination of the main alterations to embryonic development and teratogenic effects produced, due to exposure of C. carpio at different proportions of the effluent, were carried out. The results showed that the physicochemical properties were within the values permitted by Mexican regulation; however, the presence of contaminants such as NaClO, metals, anti-biotics, anti-diabetics, non-steroidal anti-inflammatory drugs, hormones and beta-blockers, was detected. Lethal concentration 50 was 5.65% and the effective concentration for malformations was 3.85%, with a teratogenic index of 1.46. The main teratogenic alterations were yolk deformation, scoliosis, modified chorda structure, tail malformation, fin deformity and mouth hyperplasia. A high rate of hatching delay was observed. The results suggest that the hospital effluent under study is capable of inducing embryotoxicity and teratogenicity in oocytes of C. carpio.

Toxic effects of cyhalofop-butyl on embryos of the Yellow River carp (Cyprinus carpio var.): alters embryos hatching, development failure, mortality of embryos, and apoptosis.

As a universal environmental contaminant, the herbicide cyhalofop-butyl is considered to have infested effects on the embryonic development of aquatic species. The present study focused on an assessment of the impacts of cyhalofop-butyl on Yellow River carp embryos. It was found that cyhalofop-butyl inhibited the hatching of the embryos, and the hatching rate decreased with higher concentrations of the herbicide. The mortality rate was increased on exposure to cyhalofop-butyl and was significantly higher in the 1.6 and 2 mg/L treatment groups over 48 h. All of the embryos of the 2 mg/L treatment group died within the 48 h post-hatching stage. And the transcription of several embryos related to apoptosis was also influenced by cyhalofop-butyl exposure. Further, cyhalofop-butyl exposure leads to a series of morphological changes (pericardial edema, tail deformation, and spine deformation) in embryos, which were consistent with significant modifications in the associated genes. These results provided a scientific basis for further studies into the effects of cyhalofop-butyl on aquatic organisms.

Molecular characterization and expression patterns of a non-mammalian toll-like receptor gene (TLR21) in larvae ontogeny of common carp (Cyprinus carpio L.) and upon immune stimulation.

BACKGROUND: In the host innate immune system, various pattern recognition receptors (PRRs) recognize conserved pathogen-associated molecular patterns (PAMPs) and represent an efficient first line of defense against invading pathogens. Toll-like receptors (TLRs) are a major class of PRRs, which are able to recognize a wide range of PAMPs and play a central role in initiating innate immune responses. TLR21 is one of the non-mammalian TLRs identified in some bird and fish species. RESULTS: In the present study, we reported the cloning and identification of a TLR21 cDNA from the head kidney of common carp (Cyprinus carpio L.), named CcTLR21. The full-length CcTLR21 cDNA was 3557 bp long, including an open reading frame (ORF) of 2895 bp, which encoded a putative protein of 964 amino acids. The putative CcTLR21 protein was found to comprise a signal peptide, 14 LRR domains in the extracellular region and a TIR domain in the cytoplasmic region, which fits with the characteristic TLR domain architecture. The phylogenetic analysis showed that CcTLR21 possessed high amino acid identities with the TLR21s in other freshwater teleosts. A Real-time PCR assay showed that CcTLR21 mRNA was expressed in almost all tissues examined in healthy common carp, while the levels obviously varied among different tissues. During the embryonic and early larval developmental stages of common carp, the CcTLR21 showed two peaks of expression, with the first at 1 dpf and the second at 10 dpf. When challenged with poly(I:C) (a viral model) or Aeromonas hydrophila, the expression level of CcTLR21 was up-regulated in a variety of common carp tissues. CONCLUSIONS: Our findings indicate that CcTLR21 plays a significant role in innate immune defense during larvae ontogeny and in responses to viral or bacterial pathogens.

Identification, molecular characterization and analysis of the expression pattern of SoxF subgroup genes the Yellow River carp, Cyprinus carpio.

Sox7, Sox17 and Sox18 are the members of the Sry-related high-mobility group box family (SoxF) of transcription factors. SoxF factors regulate endothelial cell fate as well as development and differentiation of blood cells and lymphatic vessels. There is very less information about the functions of these genes in fish. We obtained the full-length cDNA sequence of SoxF genes including Sox7, Sox17 and Sox18 in Cyprinus carpio, where Sox7 and Sox18 had two copies. The construction of a phylogenetic tree showed that these genes were homologous to the genes in other species. Chromosome synteny analysis indicated that the gene order of Sox7 and Sox18 was highly conserved in fish. However, immense change in genomic sequences around Sox17 had taken place. Numerous putative transcription factor binding sites were identified in the 5_ flanking regions of SoxF genes which may be involved in the regulation of the nervous system, vascular epidermal differentiation and embryonic development. The expression levels of SoxF genes were highest in gastrula, and was abundantly expressed in the adult brain.We investigated the expression levels of SoxF genes in five specific parts of the brain. The expression levels of Sox7 and Sox18 were highest in the mesencephalon, while the expression level of Sox17 was highest in the epencephalon. In carp, the expression patterns of SoxF genes indicated a potential function of these genes in neurogenesis and in vascular development. These results provide new information for further studies on the potential functions of SoxF genes in carp.

The effects of Roundup on gametes and early development of common carp (Cyprinus carpio L).

To determine the effects of Roundup, a commercial formulation of glyphosate, gametes, and embryos of common carp (Cyprinus carpio L) was exposed to wide range of herbicide concentrations (0.0, 0.1, 0.5, 2.0, 5.0, 10.0, 20.0, and 50.0 mg/l). The obtained results showed different effects of Roundup on common carp gametes. Herbicide reduced swelling of eggs (but the effect was not concentration-related), while sperm showed low sensitivity to Roundup (time of spermatozoa motility was reduced in a significant way only at 20 mg/l, and at remaining concentrations, only a slight tendency was observed). During the embryonic development, Roundup caused a decrease of common carp embryonic survival (and the effect was concentration-related); however, it had no effect on development rate. During the embryogenesis, three types of embryo body malformation were observed: yolk sac edema, spine curvature, and shortening of body, but their frequencies were not associated with the presence or concentration of herbicide. However, Roundup affected quality of newly hatched larvae of common carp by increasing their mortality. No effect of herbicide on percentage of deformed larvae was observed but larvae hatched in water with Roundup tended to show more complex anomalies compared to those from the control. Obtained data showed that even low concentrations of this herbicide in waters can significantly reduce egg swelling, survival of embryos, and quality of fish larvae.

Tissue distribution and early developmental expression patterns of aldolase A, B, and C in grass carp Ctenopharyngodon idellus.

Aldolase is a key enzyme involved in glycolysis, gluconeogenesis, and the pentose phosphate pathway. To establish the expression patterns of all three aldolase isozyme genes in different tissues and during early embryogenesis in lower vertebrates, as well as to explore the functional differences between these three isozymes, the grass carp was selected as a model owing to its relatively high glucose-metabolizing capability. Based on the cDNA sequences of the aldolase A, B, and C genes, the expression patterns of these three isozymes were analyzed in different tissues and during early embryogenesis using quantitative real-time polymerase chain reaction (qRT-PCR). Sequence analysis of cDNAs indicated that aldolase A, B, and C (GenBank accession numbers: KM192250, KM192251, and KM192252) consist of 364, 364, and 363 amino acids, respectively. The qRT-PCR results showed that the expression levels of aldolase A, B, and C were highest in the muscle, liver, and brain, respectively. Aldolase A and C exhibited similar expression patterns during embryogenesis, with high levels observed in unfertilized and fertilized eggs and at the blastocyst stage, followed by a decline and then increase after organogenesis. In contrast, aldolase B transcript was not detected during the unfertilized egg stage, and appeared only from gastrulation; the expression increased markedly during the feeding period (72 h after hatching), at which point the level was higher than those of aldolase A and C. These data suggest that the glucose content of grass carp starter feed should be adjusted according to the metabolic activity of aldolase B.

Cloning, expression, and nutritional regulation of the glutamine synthetase gene in Ctenopharyngodon idellus.

Glutamine synthetase (GS) is considered a master enzyme that catalyzes ATP-dependent biosynthesis of glutamine from glutamate. In the present study, the GS gene was cloned from the intestine of grass carp (Ctenopharyngodon idellus). The full-length cDNA sequence of GS encodes a 371-amino-acid polypetide. Phylogenetic analysis of the C. idellus GS sequence reveals common carp (Cyprinus carpio) as its closest neighbor. GS mRNA was differentially expressed in different tissues, with high to low gradient expression the intestine, brain, muscle, heart, gill, liver, pituitary gland, and spleen. Additionally, GS exhibited a dynamic pattern of expression during embryonic development, reaching maximal and minimal levels in the organ and hatching stages, respectively, and constant low levels from 7 to 28days post-hatching. We also assessed dietary protein levels and feed sources in diet-regulated fish, and the results suggested that low crude protein (CP) and fish meal stimulate GS gene expression. Furthermore, intestinal GS mRNA expression was significantly increased by 0.2, 0.4, 0.6, 0.8mM concentrations of glutamine dipeptide in vitro. This study provides valuable knowledge about the regulation of GS expression in teleosts.

Two grass carp (Ctenopharyngodon idella) insulin-like growth factor-binding protein 5 genes exhibit different yet conserved functions in development and growth.

Insulin-like growth factor binding-protein 5 (igfbp5), the most conserved member of the IGFBP family in vertebrates, plays a critical role in controlling cell survival, growth, differentiation, and apoptosis. Here, we characterized the expression patterns of igfbp5a and igfbp5b in grass carp (Ctenopharyngodon idella), which are retained in many fish species, likely from the teleost-specific whole-genome duplication. Both igfbp5a and igfbp5b encode 268- and 263-aa peptides, respectively, which share a sequence identity of 71%. Their mRNAs are not detected in zygotes. At 14hpf, grass carp igfbp5b mRNA was detected in the somites, while igfbp5a mRNA has some possible signal around the eye and head region. At 24hpf, both igfbp5a and igfbp5b mRNA appear to be limited to the presomitic mesoderm. At 36hpf, igfbp5a mRNA was only detected in the midbrain, while igfbp5b mRNA was detected in both the midbrain and notochord. Overall, both mRNAs were expressed in most adult tissues. igfbp5a and igfbp5b were significantly upregulated in the muscle and liver after injection of 10µg per kilogram body weight of zebrafish growth hormone (zGH), while their hepatic expression was downregulated by 50µg zGH. During fasting, both igfbp5a and igfbp5b mRNAs were significantly downregulated in the muscle but upregulated in the liver. Collectively, the results suggest that the two igfbp5 genes play important but different roles in the regulation of growth and development in grass carp.

Effect of single and combination of three triazine metabolites at environmental concentrations on early life stages of common carp (Cyprinus carpio L.).

The sensitivity of early life stages of common carp (Cyprinus carpio L.) to chronic exposure to single and combined environmental concentrations of the triazine metabolites terbuthylazine 2-hydroxy, terbuthylazine-desethyl and atrazine 2-hydroxy was evaluated under laboratory conditions. Their effects were assessed on lipid peroxidation, antioxidant enzymes (total superoxide dismutase, glutathione reductase, catalase, glutathione S-transferase, reduced glutathione), mortality, growth, development and histology. Single metabolites (terbuthylazine 2-hydroxy-0.73 µg/L; terbuthylazine-desethyl-1.80 µg/L; atrazine 2-hydroxy-0.66 µg/L) and combinations were not associated with negative effects on hatching, behaviour, embryo viability, growth or early ontogeny. Carp exposed to terbuthylazine-desethyl at 1.80 µg/L showed significantly lower total superoxide dismutase and glutathione reductase activity compared with the control group. Liver histology revealed diffused steatosis associated with the presence of lipid inclusions in hepatic cells in groups exposed to terbuthylazine-desethyl, atrazine 2-hydroxy and the tested combination of metabolites.

Open and closed evolutionary paths for drastic morphological changes, involving serial gene duplication, sub-functionalization, and selection.

Twin-tail goldfish strains are examples of drastic morphological alterations that emerged through domestication. Although this mutation is known to be caused by deficiency of one of two duplicated chordin genes, it is unknown why equivalent mutations have not been observed in other domesticated fish species. Here, we compared the chordin gene morphant phenotypes of single-tail goldfish and common carp (close relatives, both of which underwent chordin gene duplication and domestication). Morpholino-induced knockdown depleted chordin gene expression in both species; however, while knockdown reproduced twin-tail morphology in single-tail goldfish, it had no effect on common carp morphology. This difference can be explained by the observation that expression patterns of the duplicated chordin genes overlap completely in common carp, but are sub-functionalized in goldfish. Our finding implies that goldfish drastic morphological changes might be enhanced by the subsequent occurrence of three different types of evolutionary event (duplication, sub-functionalization, and selection) in a certain order.

The effects of ciprofloxacin on early life stages of common carp (Cyprinus carpio).

The authors performed a toxicity test with ciprofloxacin in fertilized eggs of common carp according to guideline 210 of the Organisation for Economic Co-operation and Development. The tested concentrations were 1 µg L(-1) , 100 µg L(-1) , 500 µg L(-1) , 1000 µg L(-1) , and 3000 µg L(-1) . Accelerated hatching was found in all groups exposed to ciprofloxacin, but significant growth reduction was found only in the group exposed to the highest concentration (3000 µg L(-1) ). Increased numbers of macroscopic morphological anomalies were observed on day 6 of the test (after hatching). The highest numbers of macroscopic morphological anomalies were observed in the groups of free embryos and larvae exposed to ciprofloxacin concentrations of 100 µg L(-1) , 500 µg L(-1) , 1000 µg L(-1) , and 3000 µg L(-1) (20-23% of tested samples). A gradual decrease in glutathione S-transferase activity was detected in all experimental groups exposed to ciprofloxacin, but significant differences (p < 0.01) were found only in groups treated with 500 µg L(-1) and 3000 µg L(-1) . Glutathione peroxidase and catalase exhibited increased activity in most of the tested concentrations (p < 0.01 and <0.05, respectively), whereas decreased glutathione reductase activity was found in the groups exposed to ciprofloxacin concentrations of 500 µg L(-1) and 3000 µg L(-1) (p < 0.05). The concentration of thiobarbituric acid-reactive substances was significantly lower (p < 0.01) in all experimental groups exposed to ciprofloxacin. The lowest-observed-effect concentration of ciprofloxacin was 1 µg L(-1) . These results suggest that hatching, early ontogeny, occurrence of morphological anomalies, antioxidant and biotransformation enzyme activity, and lipid peroxidation in fish can be affected by ciprofloxacin. Environ Toxicol Chem 2016;35:1733-1740. © 2015 SETAC.