Integration of physiological, miRNA-mRNA interaction and functional analysis reveals the molecular mechanism underlying hypoxia stress tolerance in crucian carp (Carassius auratus).

Hypoxia has become one of the most critical factors limiting the development of aquaculture. Crucian carp (Carassius auratus) is widely consumed fish in China, with excellent tolerance to hypoxic environment. However, the molecular mechanisms underlying hypoxia adaptation and tolerance in crucian carp remain unclear. Compared with the control, increased T-SOD, CAT, GSH-Px, T-AOC, ALT, and AST activities and MDA, TCHO, and TG contents, and decreased TP and ATP contents were observed after hypoxia stress. Based on RNA-seq, 2479 differentially expressed (DE) mRNAs and 60 DE miRNAs were identified, and numerous DE mRNAs involved in HIF signaling pathway (hif-1α, epo, vegfa, and ho), anaerobic metabolism (hk1/hk2, pfk, gapdh, pk, and ldh) and immune response (nlrp12, cxcr1, cxcr4, ccr9, and cxcl12) were significantly upregulated after hypoxia exposure. Integrated analysis found that ho, igfbp1, hsp70, and hk2 were predicted to be regulated by novel_867, dre-miR-125c-3p/novel_173, dre-miR-181b-5p, and dre-miR-338-5p/dre-miR-17a-3p, respectively, and targets of DE miRNAs were significantly enriched in MAPK signaling pathway, FoxO signaling pathway, and glycolysis/gluconeogenesis. Expression analysis showed that the mRNA levels of vegfa, epo, ho, hsp70, hsp90aa.1, igfbp1, ldh, hk1, pfk, pk, and gapdh exhibited a remarkable increase, whereas sdh and mdh were downregulated in the H3h, H12h, and H24h groups compared with the control. Furthermore, research found that hk2 is a target of dre-miR-17a-3p, overexpression of dre-miR-17a-3p significantly decreased the expression level of hk2, while the opposite results were obtained after dre-miR-17a-3p silencing. These results contribute to our understanding of the molecular mechanisms of hypoxia tolerance in crucian carp.

GCN2-eIF2α signaling pathway negatively regulates the growth of triploid crucian carp.

GCN2-eIF2α signaling pathway plays crucial roles in cell growth,development, and protein synthesis. However, in polyploid fish, the function of this pathway is rarely understood. In this study, genes associated with the GCN2-eIF2α pathway (pkr, pek, gcn2, eif2α) are founded lower expression levels in the triploid crucian carp (3nCC) muscle compared to that of the red crucian carp (RCC). In muscle effect stage embryos of the 3nCC, the mRNA levels of this pathway genes are generally lower than those of RCC, excluding hri and fgf21. Inhibiting gcn2 in 3nCC embryos downregulates downstream gene expression (eif2α, atf4, fgf21), accelerating embryonic development. In contrast, overexpressing of eif2α can alter the expression levels of downstream genes (atf4 and fgf21), and decelerates the embryonic development. These results demonstrate the GCN2-eIF2α pathway's regulatory impact on 3nCC growth, advancing understanding of fish rapid growth genetics and offering useful molecular markers for breeding of excellent strains.

Finotypic plasticity: Predator-induced plasticity in fin size, darkness and display behaviour in a teleost fish.

Fish fins are remarkable devices of propulsion. Fin morphology is intimately linked to locomotor performance, and hence to behaviours that influence fitness, such as foraging and predator avoidance. This foreshadows a connection between fin morphology and variation in predation risk. Yet, whether prey can adjust fin morphology according to changes in perceived risk within their lifetime (a.k.a. predator-induced plasticity) remains elusive. Here, we quantify the structural size of five focal fins in crucian carp (Carassius carassius) following controlled manipulations to perceived predation risk (presence/absence of pike Esox lucius). We also assess if crucian carp respond to increased predation risk by shifts in dorsal fin colouration, and test for differences in how fish actively use their dorsal fins by quantifying the area of the fin displayed in behavioural trials. We find that crucian carp show phenotypic plasticity with regards to fin size as predator-exposed fish consistently have larger fins. Individuals exposed to perceived predation risk also increased dorsal fin darkness and actively displayed a larger area of the fin to potential predators. Our results thus provide compelling evidence for predator-induced fin enlargement, which should result in enhanced escape swimming performance. Moreover, fin-size plasticity may evolve synergistically with fin colouration and display behaviour, and we suggest that the adaptive value of this synergy is to enhance the silhouette of deep-bodied and hard-to-capture prey to deter gape-limited predators prior to attack. Together, our results provide new perspectives on the role of predation risk in development and evolution of fins.

Cellular evidence and spatial distribution of endosomal biosynthesis and autophagy in intestinal immune barrier cells of crucian carp (Carassiuscarassius).

Crucian carp (Carassius carassius) is an important aquatic economic animal, and the immune barrier function of its intestine has been a focus of research into oral vaccines and drugs. However, the histological structures of the intestinal barrier and its adjacent areas have not been clearly established, and little subcellular evidence is available to elucidate the spatial distribution of intracellular biological processes. In this study, the spatial distribution of autophagy and endosome formation in the intestinal epithelial cells (IECs) of crucian carp were analyzed. These two biological activities are closely related to intestinal homeostasis, immunity, and cell communication. Periodic acid-Schiff (PAS) and Masson's trichrome staining were employed to elucidate the distinctive histological framework of the Crucian carp's myoid cell network, which resides within the subepithelial layer and is characterized by gap junctions. Transmission electron microscopy (TEM), immunohistochemistry (IHC), and immunofluorescence (IF) were used to detect the structural and functional aspects of the IEC in different intestinal segments. TEM and immunohistochemical analyses captured the biogenesis and maturation of early and late endosomes as well as multivesicular bodies (MVBs), as well as the initiation and progression of autophagy, including macroautophagy and mitophagy. The endosome and MVBs-specific marker CD63 and autophagy-related protein LC3 were highly expressed in IECs and were correlated with autophagy and endosome biosynthesis in the apical and basal regions of individual cells, and differed between different intestinal segments. In summary, this study elucidated the ubiquity and morphological characteristics of autophagy and endosome formation across different intestinal segments of crucian carp. A unique myoid cell network beneath the intestinal epithelium in crucian carp was also identified, expanding the histological understanding of this animal's intestinal tract.

Streptomyces enissocaesilis L-82 has broad-spectrum antibacterial activity and promotes growth for Carassius auratus.

Aeromonas is the main pathogen causing bacterial diseases in fish. The disadvantages of chemical drugs to control fish diseases have been highlighted, and it is urgent to find an eco-friendly control method. In this study, an actinomycete strain with antibacterial activity against fish pathogenic bacteria was screened from soil samples. Combined with morphological characteristics, physiological and biochemical characteristics, and gyrB gene and whole genome comparison analysis, it was identified as a new strain of Streptomyces enissocaesilis, named Streptomyces enissocaesilis L-82. The strain has broad-spectrum antibacterial activity against fish pathogens. A substance with a mass-to-charge ratio of 227.20 [M + H] + was isolated and purified by high-performance liquid chromatography and mass spectrometry. It was presumed to be a derivative of 5-dimethylallylindole-3-acetonitrile. The strain is safe and non-toxic to crucian carp, and can stably colonize crucian carp and inhibit the proliferation of A. hydrophila. After feeding the feed containing 1 × 108 CFU/mL strain concentration, the weight growth rate and specific growth rate of crucian carp increased, the activity of ACP and SOD in serum increased, and the survival rate of crucian carp increased after challenge. Genome-wide analysis showed that the strain had strong ability to metabolize and tolerate extreme environments. And has a strong potential for disease resistance. Therefore, the strain is expected to be developed as a feed additive for fish farming. KEY POINTS: • The new Streptomyces enissocaesilis L-82 has a broad spectrum and stable antibacterial activity and meets the safety standards of feed additives. • Strain L-82 can colonize crucian carp, improve the growth, antioxidant, and immune performance of the host, and improve the survival rate after being infected with A. hydrophila. • Genome-wide analysis suggests that the strain has great disease resistance potential and is expected to be developed as a feed additive for fish culture.

Immersion immunization with recombinant Saccharomyces cerevisiae displaying ORF25 induced protective immunity against cyprinid herpesvirus 2.

Displaying antigens on yeast surface as an oral vaccine has been widely explored, while its potential as an immersion vaccine has not been evaluated. Here, an immersion vaccine was prepared by displaying ORF25 of Cyprinid herpesvirus 2 (CyHV-2) on the surface of Saccharomyces cerevisiae. Carassius auratus gibelio was immersion immunized by 2 × 107 CFU/mL yeast for 2 h, and reinforce the immunity using the same method 14 days after the first immunization. The results showed that ORF25 specific antibody in immunized crucian carp serum was detected at a high level, and the mRNA expression level of IgM, IgT, IL-1ß, and IFN-1 in vaccinated head-kidney and spleen tissues were higher than the control group, indicating that innate and adaptive immunity were induced. Moreover, the immersion vaccination provided effective protection for fish against CyHV-2, leading to a relative percent survival of 50.2%. Meanwhile, immersion vaccination restrained virus replication and histological damage in CyHV-2 infected crucian carp. Our data suggested that immersion immunization of S. cerevisiae-displayed ORF25 could be served as a candidate vaccine to prevent CyHV-2 infection.

Effect of chronic deltamethrin exposure on brain transcriptome and metabolome of juvenile crucian carp.

Deltamethrin (Del), a widely administered pyrethroid insecticide, has been established as a common contaminant of the freshwater environment and detected in many freshwater ecosystems. In this study, we investigated the changes in brain transcriptome and metabolome of crucian carp after exposure to 0.6 µg/L Del for 28 days. Elevated MDA levels and inhibition of SOD activity indicate damage to the antioxidant system. Moreover, a total of 70 differential metabolites (DMs) were identified using the liquid chromatography-mass spectrometry, including 32 upregulated and 38 downregulated DMs in the Del-exposed group. The DMs associated with chronic Del exposure were enriched in steroid hormone biosynthesis, fatty acid metabolism, and glycerophospholipid metabolism for prostaglandin G2, 5-oxoeicosatetraenoic acid, progesterone, androsterone, etiocholanolone, and hydrocortisone. Transcriptomics analysis revealed that chronic Del exposure caused lipid metabolism disorder, endocrine disruption, and proinflammatory immune response by upregulating the pla2g4, cox2, log5, ptgis, lcn, and cbr expression. Importantly, the integrative analysis of transcriptomics and metabolomics indicated that the arachidonic acid metabolism pathway and steroid hormone biosynthesis were decisive processes in the brain tissue of crucian carp after Del exposure. Furthermore, Del exposure perturbed the tight junction, HIF-1 signaling pathway, and thyroid hormone signaling pathway. Overall, transcriptome and metabolome data of our study offer a new insight to assess the risk of chronic Del exposure in fish brains.

Tumor necrosis factor α1 decreases mucosal immune and antioxidant response in the midgut of hybrid fish (white crucian carp ♀ × red crucian carp ♂).

Tumor necrosis factor α1 (TNFα) is a pleiotropic cytokine involved in immune regulation and cellular homeostasis, but the crucial role of TNFα in fish gut remained unclear. The current study aimed to evaluate the immunoregulatory function of TNFα1 on gut barrier in a novel hybrid fish (WR), which was produced by crossing white crucian carp (Carassius cuvieri, ♀) with red crucian carp (Carassius auratus red var, ♂). In this study, WR-tnfα1 sequence was identified, and a high-level expression was detected in the intestine. Elevated levels of WR-tnfα1 expressions were detected in immune-related tissues and cultured fish cells on stimulation. The appearance of vacuolization and submucosal rupture was observed in TNFα1-treated midgut of WR, along with elevated levels of goblet cell atrophy, whereas no significant changes were detected in most expressions of tight-junction genes and mucin genes. In contrast, WR receiving gut perfusion with WR-TNFα1 showed a remarkable decrease in antioxidant status in midgut, whereas the expression levels of apoptotic genes and redox responsive genes increased sharply. These results suggested that TNFα1 could exhibit a detrimental effect on antioxidant defense and immune regulation in the midgut of WR.

microRNA regulation of skin pigmentation in golden-back mutant of crucian carp from a rice-fish integrated farming system.

BACKGROUND: MicroRNAs (miRNAs) are endogenous small non-coding RNAs (21-25 nucleotides) that act as essential components of several biological processes. Golden-back crucian carp (GBCrC, Carassius auratus) is a naturally mutant species of carp that has two distinct body skin color types (golden and greenish-grey), making it an excellent model for research on the genetic basis of pigmentation. Here, we performed small RNA (sRNA) analysis on the two different skin colors via Illumina sequencing. RESULTS: A total of 679 known miRNAs and 254 novel miRNAs were identified, of which 32 were detected as miRNAs with significant differential expression (DEMs). 23,577 genes were projected to be the targets of 32 DEMs, primarily those involved in melanogenesis, adrenergic signaling in cardiomyocytes, MAPK signaling pathway and wnt signaling pathway by functional enrichment. Furthermore, we built an interaction module of mRNAs, proteins and miRNAs based on 10 up-regulated and 13 down-regulated miRNAs in golden skin. In addition to transcriptional destabilization and translational suppression, we discovered that miRNAs and their target genes were expressed in the same trend at both the transcriptional and translational levels. Finally, we discovered that miR-196d could be indirectly implicated in regulating melanocyte synthesis and motility in the skin by targeting to myh7 (myosin-7) gene through the luciferase reporter assay, antagomir silencing in vivo and qRT-PCR techniques. CONCLUSIONS: Our study gives a systematic examination of the miRNA profiles expressed in the skin of GBCrC, assisting in the comprehension of the intricate molecular regulation of body color polymorphism and providing insights for C. auratus breeding research.

Cellular evidence of mucus cell immunological and differentiation characteristics in allogeneic crucian carp intestinal lamina propria.

The allogeneic crucian carp is an important fish farm animal with a very different digestive system structure from that of mammals. The lamina propria of the fish intestine is also considered to be an important site of intestinal immunity in fish, but functional histological studies of the lamina propria of the allogeneic crucian carp intestine are still lacking. In this study, Identification of the ubiquitous lamina propria mucus cells in the lamina propria of the intestine by hematoxylin-eosin staining, and determination of the mucocytic properties, class, and distribution of these cells in each intestinal segment by Alcian Blue-Periodic Acid-Schiff (AB-PAS) staining. The results show that type III mucus cells were abundant in the lamina propria of the foregut and midgut, while type II and type IV mucus cells predominate in the hindgut, possibly reflecting the distinct functions of these intestinal segments. Transmission electron microscopy dissected the differentiation of mucus cells in the lamina propria of the intestine at the ultrastructural level and investigated their morphology and distribution patterns in different intestinal segments, the findings revealed that lamina propria mucus cells perform rudimentary functions such as mucous secretion, phagocytosis, and degradation functions. Moreover, immunohistochemistry labeling with CD68 and LAMP1 revealed that numerous cells in the anterior, middle, and posterior intestines were positive for both proteins. Immunofluorescence double-labeling demonstrated that these cells highly co-expressed CD68 and LAMP1. Besides, the distribution and morphology of CD68+ and LAMP1+ cells were similar to those of AB-PAS positive cells and they accounted for the majority of parenchyma cells. Considering the above results, there were abundant cells with both mucous secretion and phagocytosis in the intestinal lamina propria of allogeneic crucian carp, which are a essential component of the intestinal immune process of allogeneic crucian carp.

Adjuvant effect of allogeneic blood in vaccines against edwardsiellosis in ginbuna crucian carp Carassius auratus langsdorfii.

Edwardsiella tarda (E. tarda), an intracellular pathogen, has caused severe economic losses in aquaculture. Effective vaccine development for E. tarda prevention is urgently needed. A previous study indicates that cell-mediated immunity (CMI) might play an important role in E. tarda infection. We believe that the involvement of allograft rejection and CMI has now been well documented in mammals and some fishes. However, there is still little research on the application of blood allograft rejection in vaccine development. In the current study, we investigate the immune response and vaccine effect in fish vaccinated with allogeneic blood + formalin-killed cells vaccine (FKC), allogeneic blood + phosphate-buffered saline (PBS), PBS + FKC and PBS + PBS. In the challenge test, the relative percentage survival (RPS) of the allogeneic + FKC, the allogeneic blood + PBS and the PBS + FKC group was 61.46, 35.41, and 30.63 % respectively. The up-regulated expression of Th1-related genes IFN-γ 1, IFN-γ 1rel2, IL-12p35 and T-bet suggests the protection is via CMI induction. Only in the allogeneic + FKC group, gene expression of IFN-γ 1, IL-12p35 and T-bet is significantly higher, indicating synergy between the two substances. Furthermore, among the fish injected with the allogeneic blood cells, syngeneic blood cells and PBS group, only in the fish of the allogenic blood cells injection group, did expression of IFN-γ 1, IFN-γ 2 and IFN-γ rel2 gene expression significantly increased. The results indicate that the rejection was induced by allogeneic components. Thus, our findings might provide essential information and insights into vaccine development in aquaculture.

The collectin subfamily member 11 (Ca-Colec11) from Qihe crucian carp (Carassius auratus) agglutinates and inhibits Aeromonas hydrophila and Staphylococcus aureus.

The collectin subfamily member 11 (Colec11), plays an important role in innate immunity as a pattern recognition molecule. In the present study, a colec11 homolog was identified and characterised from Qihe crucian carp, namely, Ca-colec11. The full-length cDNA of Ca-colec11 was composed of 1129 bp, with a 99 bp 5'-untranslated region (UTR), 816 bp open reading frame (ORF) encoding a 271-aa protein and 214 bp 3'-UTR with a polyadenylation signal sequence (aataaa) and a poly(A) tail. The deduced amino acid sequence of Ca-Colec11 contained a si gnal peptide, collagen domain, neck region and carbohydrate-recognition domain (CRD), which had four conserved cysteine residues (Cys170-Cys256 and Cys242-Cys264) and an EPN/WND motif required for carbohydrate-binding specificity. Tissue expression profile analysis by quantitative real-time polymerase chain reaction (RT-qPCR) showed that Ca-colec11 was ubiquitously distributed in the tested tissues and highly expressed in the liver. The gene expression levels of Ca-colec11 were evidently up-regulated in the liver, spleen, kidney and head kidney after infection with A. hydrophila and S. aureus. The recombinant Ca-Colec11 (rCa-Colec11) purified from Escherichia coli BL21 (DE3) could agglutinate A. hydrophila and S. aureus, and it possessed haemagglutination activity against rabbit erythrocytes, which was inhibited by various carbohydrates, including d-Mannose, N-Acetyl-d-mannosamine, l-Fucose, d-Glucose, N-Acetyl-d-glucosamine, d-Galactose, LPS and PGN. Furthermore, rCa-Colec11 could inhibit the growth of A. hydrophila and S. aureus. These findings collectively demonstrated that Ca-Colec11, as a PRR, could play a role in the immune defence of Qihe crucian carp.

CD4-1 and CD4-2 single positive are two major CD4 lymphocyte subpopulations in ginbuna crucian carp Carassius auratus langsdorfii.

In this study, we established a murine cell line that expresses ginbuna crucian carp (ginbuna) CD4-2 and used it to develop an anti-CD4-2 monoclonal antibody (mAb). An established mAb, named D5, showed good reactivities to BALB/c 3T3 cells expressing CD4-2 and a lymphocyte population in the ginbuna leukocytes. Gene expression analysis showed that D5+ cells express CD4-2 and TCRß genes but not CD4-1 and IgM genes, meanwhile May Grunwald-Giemsa staining of sorted D5+ cells had the typical morphology of lymphocytes. Two-color immunofluorescence analysis with anti-CD4-1 mAb (6D1) and anti-CD4-2 mAb (D5) by flow cytometry revealed that the percentages of CD4-1 single positive (SP) and CD4-2 SP lymphocytes were comparatively higher than CD4-1/CD4-2 double positive (CD4 DP) lymphocytes in all tissues examined in ginbuna. The highest percentage of CD4-2 SP cells (∼40%) was found in the thymus, while the head-kidney exhibited the highest percentages of CD4-1 SP (∼30%) and CD4 DP (∼5%) cells. These findings indicated that ginbuna CD4+ lymphocyte population consists of two major subpopulations (CD4-1 SP and CD4-2 SP) and a minor subset (CD4 DP).

Efficacy of a recombinant Lactobacillus plantarum Lp-pPG-Malt as an oral vaccine candidate against Aeromonas hydrophila infection in crucian carp.

Aeromonas hydrophila (A. hydrophila), a gram-negative bacterium, causes serious diseases with various clinical symptoms in farm raised fish. Thus, different ways to prevent and control A. hydrophila infection need to be explored, including a vaccine. In this study, we evaluated the protective efficacy of an oral vaccine prepared from the A. hydrophila TPS maltoporin (Malt) with Lactobacillus plantarum (L. plantarum) against A. hydrophila infection in crucian carp (Carassius auratus). For the in vivo experiment, the oral vaccine was administered to crucian carp by feeding them fish diets containing Lp-pPG-Malt, Lp-pPG and PBS for 28 days. The enzyme-linked immunosorbent assay (ELISA), leukocyte phagocytosis assay and real-time quantitative polymerase chain reaction (RT-qPCR) were performed to measure the protective efficacy of the Lp-pPG-Malt. ELISA and leukocyte phagocytosis assay confirmed that Lp-pPG-Malt significantly enhanced the IgM level and nonspecific immune response of crucian carp compared with the control groups (Lp-pPG and PBS). The RT-qPCR results showed that the Lp-pPG-Malt increased the relative expression of immune-related genes (IL-10, IL-1ß, TNF-α, IFN-γ) of crucian carp in various tissues (liver, spleen, head kidney and hind intestine). Moreover, Lp-pPG-Malt significantly increased the relative percent survival of fish after intraperitoneal injection with A. hydrophila (55%) compared with the Lp-pPG and PBS groups (0%). These findings suggest that Lp-pPG-Malt can serve as an oral vaccine candidate for A. hydrophila infection and that Malt can be used as an effective antigen in crucian carp farming.

Alteration of synergistic immune response in gut-liver axis of white crucian carp (Carassius cuvieri) after gut infection with Aeromonas hydrophila.

Aeromonas hydrophila can pose a great threat to the survival of farmed fish. In current study, we investigated the pathological characteristics and immune response in gut-liver axis of white crucian carp (WCC) upon gut infection. WCC anally intubated with A. hydrophila exerted a tissue deformation in damaged midgut with elevated levels of goblet cells along with a significant decrease in tight junction proteins and villi length-to-width ratios. In addition, immune-related gene expressions and antioxidant properties increased dramatically in gut-liver axis of WCC following gut infection with A. hydrophila. These results highlighted the immune modulation and redox alteration in gut-liver axis of WCC in response to gut infection.

Microplastic polyamide toxicity: Neurotoxicity, stress indicators and immune responses in crucian carp, Carassius carassius.

This study aimed to determine the toxicity standard and potential risks and effects of polyamide (PA) exposure on neurotoxicity, stress indicators, and immune responses in juvenile crucian carp Carassius carassius. Numerous microplastics (MPs) exists within aquatic environments, leading to diverse detrimental impacts on aquatic organisms. The C. carassius (mean weight, 23.7 ± 1.6 g; mean length, 13.9 ± 1.4 cm) were exposed to PA concentrations of 0, 4, 8, 16, 32 and 64 mg/L for 2 weeks. Among the neurotransmitters, the acetylcholinesterase (AChE) activity in the liver, gill, and intestine of C. carassius was significantly inhibited by PA exposure. Stress indicators such as cortisol and heat shock protein 70 (HSP70) in the liver, gill, and intestine of C. carassius were significantly increased, while immune responses to lysozyme and immunoglobulin M (IgM) were significantly decreased. Our study demonstrates the toxic effects of MP exposure on crucian carp's neurotoxicity, stress indicators, and immune responses.

Effects of chlorantraniliprole-based pesticide on transcriptional response and gut microbiota of the crucian carp, Carassius carassius.

Chlorantraniliprole (CAP) is a presentative diamide pesticide utilized in agricultural area and as well as rice-fish co-culture system for pest control. However, the understanding of toxic effects of CAP on fish species is still incomplete. In the present study, we performed an integrated study of the acute toxicity and bioaccumulation of CAP on the crucian carp, Carassius carassius, a fish species widely distributed in freshwater area in China and commonly farmed in the rice-fish co-culture systems. Besides, biochemical changes, transcriptional responses and gut microbiota of fish were investigated upon sub-chronic CAP exposure. The results showed that CAP is low toxic to crucian carp with a 96 h LC50 of 74.824 mg/L, but has considerable accumulation in the fish muscles when exposed to 3 mg/L of CAP for 14 d and still detectable after 18 d recovery in fresh water. For sub-chronic test, fish were exposed to CAP at 0, 0.3, 3 and 30 mg/L respectively for 14 d. CAP induced oxidative stress and detoxification inhibition in the liver of fish by decreasing antioxidative and detoxicated enzymes activities and downregulating relevant genes expression. In addition, disrupted gut flora composition was found in all experimental groups by the 16 S rRNA sequencing data, indicating the gut microbiota dysbiosis in crucian carp and potential adverse host effect. All the results suggest that CAP at sublethal concentrations has prominent toxic effect on crucian carp and more attentions should be paid especially using directly in an integrated aquaculture system.

Metabolism and residue differences of Enrofloxacin between the brain and peripheral tissues and the resulting brain damages in crucian carp (Carassius auratus var. Pengze).

This study aimed to explore the metabolism and residue differences of Enrofloxacin (ENR) at two doses between the brain and peripheral tissues (liver, kidney, and muscle) along with the brain damages caused by ENR in crucian carp (Carassius auratus var. Pengze). The concentrations of ENR in tissues were determined using a validated high-performance liquid chromatography (HPLC) analysis. Relying on the hematoxylin-eosin (HE) staining method, brain damages caused by the drug were evaluated by the section of pathological tissue. Metabolism and residue results showed that ENR could be detected in the brain throughout the experiment both at median lethal dose (LD50 at 96 h, 1949.84 mg/kg) and safe dose (SD, 194.98 mg/kg), as well as in the three peripheral tissues. The maximum residue at LD50 followed the decreasing order of liver >kidney > brain > muscle. Although the Cmax of ENR at SD in the brain was significantly lower than that in other peripheral tissues (p < .05), it still reached 41.91 µg/g. The T1/2 of ENR in brain tissue at the same dose was both shorter than that in peripheral tissues. At LD50 , the amount of ENR residues in brain was lower than that in peripheral tissues on the whole, except that it had been higher than in the muscle for the first 3 h. At SD, the drug residue in brain tissue was lower than that in peripheral tissues from 12 h to 960 h, but it exceeded the muscle and kidney at 1 h and 6 h, respectively. At 960 h, the residual amount of ENR at SD in the brain was 0.09 µg/g, while it was up to 0.15 µg/g following the oral administration at LD50 . Demonstrated by the HE staining, there were pathological lesions caused by ENR in the brain at LD50 , which were characterized by sparse neural network and increased staining of glial cells. The present results indicated that metabolism and residue of ENR in crucian carp were affected by the tissue type and drug dosage, and the ENR could also bring about histopathological changes in the brain.

UPLC-MS/MS Method for Simultaneous Determination of Valnemulin and Its Metabolites in Crucian Carp: In Vivo Metabolism and Tissue Distribution Analyses.

Valnemulin (VML) is a semi-synthetic pleuromutilin derivative widely used to treat animal bacterial diseases. However, no study has comprehensively evaluated VML metabolism in aquatic animals, including crucian carp. This study aimed to investigate VML metabolism in crucian carp. VML metabolites in crucian carp were quantified via intraperitoneal injection and analyzed via ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Three VML metabolites were detected in crucian carp via ultra-performance liquid chromatography-tandem quadrupole and time-of-flight mass spectrometry (UPLC-Q-TOF/MS) structural analysis. The enrichment and metabolism rules of the metabolites were summarized based on tissue distribution and concentration changes of the three metabolites. The metabolites were mainly found in the liver at 0.1 h after VML injection. The levels of the metabolites were abundant in the bile from 4 h to 12 h and in the skin after 72 h. The levels of the metabolites in the bile first increased, then decreased. The metabolism in the liver was completed at 72 h. The metabolites were detected in the skin following a 72 h period, which increased with time.

Dietary supplementation with Weissella cibaria C-10 and Bacillus amyloliquefaciens T-5 enhance immunity against Aeromonas veronii infection in crucian carp (Carassiu auratus).

With the aim to discover novel lactic acid bacteria and Bacillus strains from fish as potential probiotics to replace antibiotics in aquaculture, the present study was conducted to isolate lactic acid bacteria and Bacillus from intestinal tract of healthy crucian carp (Carassiu auratus) and largemouth bass (Micropterus salmoides) and evaluate their resistance against Aeromonas veronii. Based on the evaluation of antibacterial activity and tolerance test, one strain of lactic acid bacteria (Weissella cibaria C-10) and one strain of Bacillus (Bacillus amyloliquefaciens T-5) with strong environmental stability were screened out. The safety evaluation showed that these two strains were non-toxic to crucian carp and were sensitive to most antibiotics. In vivo study, the crucian carps were fed a basal diet supplemented with W. cibaria C-10 (C-10), B. amyloliquefaciens T-5 (T-5) and W. cibaria C-10 + B. amyloliquefaciens T-5 (C-10+T-5), respectively, for 5 weeks. Then, various immune parameters were measured at 35 days of post-feeding. Results showed both probiotics could improve the activities of related immune enzymes, immune factors and non-specific immune antibodies in blood and organs (gill, gut, kidney, liver, and spleen) of crucian carp in varying degrees. Moreover, after 7 days of challenge experiment, the survival rates after challenged with A. veronii of W. cibaria C-10 (C-10), B. amyloliquefaciens T-5 (T-5) and W. cibaria C-10 + B. amyloliquefaciens T-5 (C-10+T-5) supplemented groups to the crucian carps were 20%, 33% and 22%, respectively. Overall, W. cibaria C-10 and B. amyloliquefaciens T-5 could be considered to be developed into microecological preparations for the alternatives of antibiotics in aquaculture.