Metabolite features and oxidative response in kidney of red crucian carp (Carassius auratus red var) after Aeromonas hydrophila challenge.

Aeromonas hydrophila can threaten the survival of freshwater fish. In this study, A. hydrophila challenge could induce tissue damage, promote antioxidant imbalance as well as alter the transcript levels of oxidative stress indicators, apoptotic genes and metabolic enzyme genes in kidney of red crucian carp (RCC). Metabolomics analysis revealed that A. hydrophila challenge had a profound effect on amino acid metabolism and lipid metabolism. In addition, we further identified dipeptides, fatty acid derivatives, cortisol, choline and tetrahydrocortisone as crucial biomarkers in kidney of RCC subjected to A. hydrophila infection. These results highlighted the importance of metabolic strategy against bacterial infection.

A novel ferritin L (FerL) in hybrid crucian carp could participate in host defense against Aeromonas hydrophila infection and diminish inflammatory signals.

FerL, a multifunctional iron-storage polypeptide, not only exhibited a regulatory role in iron metabolism, but also participated in the regulation of fish immunity. In this study, ORF sequence of WR-FerL was 522 bp, encoding 173 amino acid residues. Tissue-specific analysis revealed that the highest expression of WR-FerL was detected in spleen. A. hydrophila challenge and LPS stimulation could sharply enhance WR-FerL mRNA expression in tissues and fish cells, respectively. Purified WR-FerL fusion peptide exhibited in vitro binding activity to A. hydrophila and endotoxin, limited bacterial dissemination to tissues as well as attenuated A. hydrophila-induced production of pro-inflammatory cytokines. Moreover, WR-FerL overexpression could abrogate NF-κB and TNFα promoter activity in fish cells. These results indicated that WR-FerL could play an important role in host defense against A. hydrophila infection.

Blood cell characterization and transcriptome analysis reveal distinct immune response and host resistance of different ploidy cyprinid fish following Aeromonas hydrophila infection.

Aeromonas hydrophila can pose a great threat to survival of freshwater fish. In this study, A. hydrophila infection could decrease blood cell numbers, promote blood cell damage as well as alter the levels of alkaline phosphatase (ALP), lysozyme (LZM), aspartate aminotransferase (AST), total antioxidant capacity (T-AOC), total superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) in immune-related tissues of red crucian carp (RCC, 2 N = 100) and triploid cyprinid fish (3 N fish, 3 N = 150). In addition, the significant alternation of antioxidant status was observed in PBMCs isolated from RCC and 3 N following LPS stimulation. The core differential expression genes (DEGs) involved in apoptosis, immunity, inflammation and cellular signals were co-expressed differentially in RCC and 3 N following A. hydrophila challenge. NOD-like receptor (NLR) signals appeared to play a critical role in A. hydrophila-infected fish. DEGs of NLR signals in RCCah vs RCCctl were enriched in caspase-1-dependent Interleukin-1ß (IL-1ß) secretion, interferon (IFN) signals as well as cytokine activation, while DEGs of NLR signals in 3Nah vs 3Nctl were enriched in caspase-1-dependent IL-1ß secretion and antibacterial autophagy. These results highlighted the differential signal regulation of different ploidy cyprinid fish to cope with bacterial infection.

Comparative analysis of erythrocyte hemolysis, plasma parameters and metabolic features in red crucian carp (Carassius auratus red var) and triploid hybrid fish following Aeromonas hydrophila challenge.

Aeromonas hydrophila can pose a great threat to survival of freshwater fish. In this study, A. hydrophila challenge could promote the erythrocyte hemolysis, increase free hemoglobin (FHB) level and generate malondialdehyde (MDA) production in plasma but decrease the levels of total antioxidant capacity (T-AOC), total superoxide dismutase (SOD), catalase (CAT), alkaline phosphatase (ALP) and lysozyme (LZM) of red crucian carp (RCC, 2 N = 100) and triploid hybrid fish (3 N fish, 3 N = 150) following A. hydrophila challenge. Elevated expression levels of heat shock protein 90 alpha (HSP90α), matrix metalloproteinase 9 (MMP-9), free fatty acid receptor 3 (FFAR3), paraoxonase 2 (PON2) and cytosolic phospholipase A2 (cPLA2) were observed in A. hydrophila-infected fish. In addition, A. hydrophila challenge could significantly increase expressions of cortisol, leucine, isoleucine, glutamate and polyunsaturated fatty acids (PUFAs) in RCC and 3 N, while glycolysis and tricarboxylic acid cycle appeared to be inactive. We identified differential fatty acid derivatives and their metabolic networks as crucial biomarkers from metabolic profiles of different ploidy cyprinid fish subjected to A. hydrophila infection. These results highlighted the comparative metabolic strategy of different ploidy cyprinid fish against bacterial infection.

Ferritin H can counteract inflammatory response in hybrid fish and its parental species after Aeromonas hydrophila infection.

Ferritin H can participate in the regulation of fish immunity. Tissue-specific analysis revealed that the highest expressions of Ferritin H in parental species were observed in spleen, while peaked level of Ferritin H mRNA in hybrid fish was observed in liver. In addition, A. hydrophila challenge could sharply enhance their Ferritin H mRNA expression in liver, kidney and spleen. To further investigate their roles in immune regulation, their Ferritin H fusion proteins were produced in vitro. Ferritin H fusion proteins could exhibit a direct binding activity to A. hydrophila and endotoxin in a dose-dependent manner, restrict dissemination of A. hydrophila to tissues and abrogate inflammatory cascades. Moreover, treatment with Ferritin H fusion proteins could reduce A. hydrophila-induced lipid peroxidation. These results indicated that Ferritin H in hybrid fish elicited a similar immune regulation of A. hydrophila-induced inflammatory signals in comparison with those of its parents.

A novel NK-lysin in hybrid crucian carp can exhibit cytotoxic activity in fish cells and confer protection against Aeromonas hydrophila infection in comparison with Carassius cuvieri and Carassius auratus red var.

NK-lysin, an effector of natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), not only exhibits cytotoxic effect in fish cells, but also participates in the immune defense against pathogenic infection. In this study, ORF sequences of RCC-NK-lysin, WCC-NK-lysin and WR-NK-lysin were 369 bp. Tissue-specific analysis revealed that the highest expressions of RCC-NK-lysin and WCC-NK-lysin were observed in gill, while the peaked level of WR-NK-lysin mRNA was observed in spleen. A. hydrophila infection sharply increased RCC-NK-lysin, WCC-NK-lysin and WR-NK-lysin mRNA expression in liver, trunk kidney and spleen. In addition, elevated levels of NK-lysin mRNA were observed in cultured fin cell lines of red crucian carp (RCC), white crucian carp (WCC) and their hybrid offspring (WR) after Lipopolysaccharide (LPS) challenge. RCC-NK-lysin, WCC-NK-lysin and WR-NK-lysin exerted regulatory roles in inducing ROS generation, modulating mitochondrial membrane potential, decreasing fish cell viability and antagonizing survival signalings, respectively. RCC/WCC/WR-NK-lysin-overexpressing fish could up-regulate expressions of inflammatory cytokines and decrease bacterial loads in spleen. These results indicated that NK-lysin in hybrid fish contained close sequence similarity to those of its parents, possessing the capacities of cytotoxicity and immune defense against bacterial infection.

Effect of Lipopolysaccharide (LPS) stimulation on apoptotic process and oxidative stress in fibroblast cell of hybrid crucian carp compared with those of Carassius cuvieri and Carassius auratus red var.

Bacterial LPS is a heat-stable endotoxin and wall components of gram negative bacteria, which can exhibit a toxicological effect on physiology and biochemical activities of fish. In this study, we investigated the effect of LPS exposure on cell viability, oxidative stress, caspase activity and immune-related gene expressions in cultured fin cell lines of red crucian carp, white crucian carp and their hybrid offspring. LPS stimulation could reduce fish cell viability, whereas gene expression levels and promoter activities in inflammatory signals increased dramatically. Moreover, enhanced levels of intracellular oxidative stress and decreased levels of mitochondrial membrane potential (MMP) were observed in LPS-induced fish cells. N-Acetyl-L-cysteine (NAC) could alleviate LPS-stimulated reactive oxygen species (ROS) generation and caspase-3 activity in fish cells. These results suggested that ROS-mediated cytotoxic stress was involved in LPS-induced inflammation and mitochondrial damage in cultured fish cells.

Chimeric ferritin H in hybrid crucian carp exhibits a similar down-regulation in lipopolysaccharide-induced NF-κB inflammatory signal in comparison with Carassius cuvieri and Carassius auratus red var.

Ferritin H can participate in the regulation of teleostean immunity. ORF sequences of RCC/WCC/WR-ferritin H were 609 bp, while WR-ferritin H gene possessed chimeric fragments or offspring-specific mutations. In order to elucidate regulation of immune-related signal transduction, three fibroblast-like cell lines derived from caudal fin of red crucian carp (RCC), white crucian carp (WCC) and their hybrid offspring (WR) were characterized and designated as RCCFCs, WCCFCs and WRFCs. A sharp increase of ferritin H mRNA was observed in RCCFCs, WCCFCs and WRFCs following lipopolysaccharide (LPS) challenge. Overexpression of RCC/WCC/WR-ferritin H can decrease MyD88-IRAK4 signal and antagonize NF-κB, TNFα promoter activity in RCCFCs, WCCFCs and WRFCs, respectively. These results indicated that ferritin H in hybrid offspring harbors highly-conserved domains with a close sequence similarity to those of its parents, playing a regulatory role in inflammatory signals.

Antibacterial and immunoregulatory activity of a novel hepcidin homologue in diploid hybrid fish (Carassius auratus cuvieri ♀ × Carassius auratus red var ♂).

Hepcidin, a multifunctional hormone oligopeptide, not only exhibits a regulatory role in iron metabolism, but also participates in the regulation of teleostean immunity. In this study, ORF sequence of WR-hepcidin was 258 bp and encoded 85 amino acid residues. Tissue-specific analysis revealed that the highest expression of WR-hepcidin was observed in liver. Aeromonas hydrophila challenge can sharply increased WR-hepcidin mRNA expression in liver, trunk kidney and spleen. The purified WR-hepcidin fusion peptide can directly bind to A. hydrophila and Streptococcus agalactiae, reduce the relative bacterial activity, limit bacterial growth and attenuate their dissemination to tissues in vivo. In addition, the treatment of WR-hepcidin fusion protein can diminish the production of pro-inflammatory cytokines. These results indicated that WR-hepcidin can play a negative regulatory role in bacteria-stimulated pro-inflammatory cytokines production and MyD88-IRAK4 activation.

ITLN in diploid hybrid fish (Carassius auratus cuvieri ♀ × Carassius auratus red var ♂) is involved in host defense against bacterial infection.

Hybrid genotypes in fish may be less susceptible to pathogenic infection. ITLN, a novel lectin, not only exhibits a regulatory role in pathogenic infection, but also participates in the regulation of teleostean immunity. In this study, ORF sequence of WR-ITLN was 945 bp and encoded 314 amino acid residues. Tissue-specific analysis revealed that the highest expression of WR-ITLN was observed in liver. Aeromonas hydrophila challenge can sharply increased WR-ITLN mRNA expression in liver, kidney and spleen. The purified WR-ITLN protein can directly bind to A. hydrophila and S. agalactiae, reduce their relative bacterial activity and limit bacterial growth in vitro in the presence of Ca2+. In addition, the treatment of WR-ITLN + Ca2+ can restrict bacterial dissemination in vivo and attenuate production of pro-inflammatory cytokines. These results indicated that WR-ITLN can confer protection against bacteria-stimulated MyD88-dependent pro-inflammatory cytokines activation in a Ca2+-dependent manner.

A novel LEAP-2 in diploid hybrid fish (Carassius auratus cuvieri ♀ × Carassius auratus red var. ♂) confers protection against bacteria-stimulated inflammatory response.

LEAP-2, a multifunctional peptide, not only exhibits a regulatory role in pathogenic infection, but also participates in the regulation of teleostean immunity. In this study, ORF sequence of WR-LEAP-2 was 240 bp and encoded 79 amino acid residues. Tissue-specific analysis revealed that the highest expression of WR-LEAP-2 was observed in liver. Aeromonas hydrophila challenge can sharply increase WR-LEAP-2 mRNA expression in liver, kidney and spleen. The purified WR-LEAP-2 peptide can directly bind to A. hydrophila and S. agalactiae, reduce the relative bacterial activity and limit bacterial growth in vitro. In addition, the treatment of WR-LEAP-2 can restrict bacterial dissemination in vivo and reduce production of pro-inflammatory cytokines. These results indicated that WR-LEAP-2 can confer protection against A. hydrophila- or S. agalactiae-stimulated MyD88-dependent pro-inflammatory cytokines activation.

[Cytogenetic and molecular genetic analysis of gynogenesis in Megalobrama amblycephala using spermatozoa of Erythroculter ilishaeformis].

In the present study we used both cytogenetics (measurement of DNA content, detection of chromosome number, observation of gonadal development)and molecular genetics(microsatellite analysis)to analyze the biological characteristics of gynogenetic M. amblycephala, which werecreatedthrough gynogenesis induced via UV-irradiated E. ilishaeformis spermatozoa to fertilize M. amblycephala eggs. The maternal genome was duplicated by cold shock in 0~4℃ cold water to form a population of M. amblycephala with 48 chromosomes whose DNA content was identical to the diploid maternal parent. Morphologically, this group of gynogenetic M. amblycephala was similar to the control group. All gynogenetic M. amblycephala were female, and no males were found in any of the examined gynogenetic M. amblycephala, providing cytogenetic evidence that our gynogenetic M. amblycephala are type XY. At the same time, microsatellite analysis showed that 63 alleles were amplified in the three test groups of gynogenetic M. amblycephala. Overall, the population of gynogenetic M. amblycephala observed heterozygosity average, and the expected average was significantly lower than the parental averages, demonstrating that after generation gynogenesis the gene homozygosity of M. amblycephala was significantly higher than the ordinary bream and E. ilishaeformis, making it a pure line. The genetic proximity of gynogenetic M. amblycephala to M. amblycephala demonstrates that gynogenesis passes on maternal DNA. Gynogenetic groups developed in this study may provide good genetic material for future breeding projects of M. amblycephala.

Evidence of different ploidy eggs produced by diploid F2 hybrids of Carassius auratus (female) x Cyprinus carpio (male).

Based on the presence of three types of eggs with different diameters 0.13, 0.17 and 0.2 cm, we made two crosses: F2 (female) x diploid red crucian carp (male), and F2 (female) x F10 tetraploid (male). The ploidy levels of the progeny of the two crosses were examined by chromosome counting and DNA content measurement by flow cytometer. In the offspring of the former cross, tetraploids, triploids, and diploid were obtained. In the progeny of the latter cross, tetraploids and triploids were observed. The production of the different ploidy level fish in the progeny of the two crosses provided a further evidence that F2 might generate triploid, diploid and haploid eggs. The presence of the male tetraploid found in F2 (female) x diploid red crucian carp (male) suggested that the genotype of XXXY probably existed in the tetraploid progeny. The gonadal structures of the tetraploids and triploids indicated that both female and male tetraploids were fertile and the triploids were sterile. We concluded that the formations of different ploidy level eggs from F2 were contributed by endoreduplication and fusion of germ cells.

[Triploid crucain carp-allotetraploid hybrids (male) x goldfish( hermaphrodite)].

A new type of triploid crucian carp was generated by mating the males of the allotetraploid hybrids of red crucian carp (Carassius auratus red var.) (female) x common carp (Cyprinus carpio L.) (male), with the females the goldfish( Carassius auratus red var.) which was red in color and possessed the biforked tails. The biological characteristics of one-year old triploid crucian carp including the number and karotype of chromosome, gonadosomatic indices and gonadal structures, appearance and growth rate were investigated. The results indicated that the triploid crucian carp possessed 150 chromosomes with the karyotype: 33m + 51sm + 33st + 33t. In the breeding season, the gonadosomatic indices (GSI) of the ovaries and testes in the triplpoid crcuian carp were, respectively, lower than those in the diploid red crucian carp, and the tissue sections showed that both the ovaries and the testes in the triploid cruican carp were not able to produce the mature ova and sperm, respectively, indicating that they were sterile during one year. The triploid crucian carp was gray in color and had a single fork tail. They had other morphological traits intermediate to the paternal allotetraploid hybrids and the maternal goldfish, for example, one pair of very short and not readily visible barbells, the formula of lateral line scales: 31-6/7, 18 dorsal fin spines, and 0.5 in ratio of the width to the length of the body which was evidently higher than that (0.41) in another kind of triploid crucian carp called Xiangyun crucian carp, generated by crossing the males of the allotetraploid hybrids with the females of the Japanese crucain carp (Carassius auratus cuvieri T. et S.). Following 8-month culture, the triploid crucian carp averagely reached 350 gram in which the maximum individual weighed 550 gram. Among one- year old triploid crucian carp, the females grew faster than the males. Compared with Xiangyun crucian carp, the new type of triploid crucian carp not only presented some advantages the Xiangyun crucian carp had, for example, fast growth rate and potential sterility, but also got some new good performances like the high ratio of the width to the length of the body and the sweet flesh, suggesting they will be very potential in the commercial interest.

[The chromosome number and gonadal structure of F9-F11 allotetraploid crucian-carp].

In this paper, the chromosome number of F9-F11 hybridis of Red crucian (Carassius auratus) x common carp(Cryprinus carpio) was examined by means of kidney cell-PHA culture. The results showed that F9-F11 allotetraploids possessed 200 chromosomes, with the karyotype formula 44 m + 68 sm + 44 st + 44 t, which were the same as the data indicated in F3-F8 allotetraploids. Both female and male of F9-F11 allotetraploids had the normal ovaries and testes that were able to produce the normal dipoid ova and sperm. In nature conditions, without the injection of the extraneous hormone, the females and males of F9-F11 allotetraploids mated each other to produce new generation of tetraploids. With the stable genetic tetraploidy and the fertility in the nature environments, this allotetraploid population presented the key factors to form a new species with 200 chromosomes.