An integrin alpha 4 (ChIntα 4) from oyster Crassostrea hongkongensis mediates the hemocytes phagocytosis towards Vibrio alginolyticus.

Integrins, a family of cell adhesion transmembrane receptors, mediate cell adhesion, migration, proliferation, apoptosis, and phagocytosis. In the present study, an integrin ChIntα 4 from Crassostrea hongkongensis was characterized to investigate its role in defensing against pathogenic bacterium Vibrio alginolyticus. The full-length cDNA sequence of ChIntα 4 was 3572 bp with an open reading frame (ORF) of 3168 bp, which encoded a polypeptide with 1055 amino acids. The mRNA expression of ChIntα 4 in the hemocytes was significantly up-regulated at 6 h and 24 h post V. alginolyticus stimulation (p < 0.01). The recombinant ChIntα 4 protein could agglutinate the rabbit red blood cells and Gram-negative bacteria V. alginolyticus and Escherichia coli. Moreover, the phagocytic activity of the hemocytes was significantly down-regulated from 46.9% to 32.7% when blocked with anti-ChIntα 4 antibody, and it was significantly up-regulated from 42.7% to 59.5% post transfection with pCI-neo-ChIntα 4 plasmid (p < 0.05). In conclusion, these findings demonstrated that ChIntα 4 might be involved in resisting V. alginolyticus infection and regulating phagocytosis as a cell adhesion receptor in C. hongkongensis.

Application of potential probiotic strain Streptomyces sp. SH5 on anti-Aeromonas infection in zebrafish larvae.

Pre-treatment of Streptomyces sp. SH5 on zebrafish lead to a significant enhancement of larvae survival upon Aeromonas hydrophila challenging. SH5 was able to colonize in zebrafish approximately at 1 × 102.6 cells per fish for at least seven days. The presence of SH5 strongly repelled the A. hydrophila colonization in zebrafish, and maximally, a 67.53% reduction rate was achieved. A more diversified flora was discovered in the SH5-treated zebrafish larvae at both phylum and genus levels. The expression of immune response genes of SH5-treated zebrafish, including TLR3, lysozyme and NOS2α, were enhanced at initial stage, while, that of various inflammatory stimuli genes including 1L-1ß, 1L-6 and MyD88 were decreased at all tested timepoints. SH5 was shown to inhibit virulence factors production and the expression of corresponding virulence genes in A. hydrophila, suggesting its quorum sensing inhibitory potential. These results indicated favorable application perspectives of SH5 in resisting pathogenic infection in aquaculture.

Establishment and application of a TaqMan probe-based qPCR for the detection of Enterocytozoon hepatopenaei in shrimp Litopenaeus vannamei.

Enterocytozoon hepatopenaei (EHP) is a common parasite that invades the epithelial cytoplasm in the hepatopancreas of shrimp Litopenaeus vannamei and results in slow growth of the host shrimps to cause significant economic loss in shrimp aquaculture. In this study, a TaqMan probe-based qPCR for quantitative detection of EHP was established. A pair of specific primers and a TaqMan probe were designed based on the sequence of cysteine desulfurase gene (NFS1) of EHP. The standard curve between cycle threshold (Ct) and the logarithmic starting quantity (SQ) of the template was determined as Ct = - 3.231 lg (SQ) + 40.638, with a correlation coefficient (R2) of 0.998 and an amplification efficiency of 103.9%. The lower limit of quantification was 1.67 × 101 copies/µL for this TaqMan probe-based qPCR and 1.67 × 103 copies/µL for the conventional PCR. The TaqMan probe-based qPCR established in the research was 100 times more sensitive than the conventional PCR method. In addition, the results of clinical sample detection indicated that the present technique was efficient in detecting EHP in the hepatopancreas, feces, water, and pond bottom mud samples. Therefore, the established TaqMan probe-based qPCR is a suitable technique for detecting EHP in both shrimp and aquatic environment samples.

Expression analysis and tissue localization of IgZ in the grouper Epinephelus coioides after Vibrio alginolyticus infection and vaccination.

The orange-spotted grouper (Epinephelus coioides) is an important marine farmed fish in China. It is affected by the bacterial pathogen Vibrio alginolyticus, which causes high mortality and substantial economic losses. We studied the transcriptional changes of the IgZ gene in E. coioides following V. alginolyticus stimulation and investigated the distribution of IgZ in different tissues. The highest expression level of IgZ occurred in the head kidney. When fish were stimulated with live and inactivated V. alginolyticus, the expression levels of IgZ in the head kidney, spleen, intestine, gills and blood cells were significantly upregulated. In an in situ hybridization study, IgZ mRNA-positive cells were detected in the head kidney, spleen and gill, but positive signals were not detected in the liver and intestine. IgZ-labelled cells increased in the head kidney, spleen and gills post-infection with V. alginolyticus for 21 days. The present study provides additional evidence that IgZ is involved in mucosal immune responses and helps explain the role of IgZ in E. coioides defence against V. alginolyticus infection.

Antiparasitic Efficacy of Crude Plant Extracts and Compounds Purified from Plants against the Fish Monogenean Neobenedenia girellae.

Neobenedenia girellae is a pathogenic ectoparasite of many marine fishes, and it causes major epidemics in marine aquaculture. In this study, the efficacy of ethanol extracts of huangqi Astragalus membranaceus (known as milkvetch in North America), guanzhong Dryopteris setosa (known as beaded wood fern in North America), gancao Glycyrrhiza uralensis (known as Chinese licorice in North America), danshen Salvia miltiorrhiza (known as red sage in North America), and pomegranate Punica granatum, as well as seven phytochemicals (10-gingerol, curcumin, cynatratoside-C, emodin, kuwanon-G, kuwanon-O, and sophoraflavanone-G), against adult N. girellae was investigated. In vitro results indicated that pomegranate extract killed all adult N. girellae at a 62.5-mg/L concentration with an 8-h exposure, but gancao extract did not cause 100% mortality until a 1,000-mg/L concentration was used. Additionally, all adult N. girellae died after an 8-h exposure to cynatratoside-C, kuwanon-G, kuwanon-O, or sophoraflavanone-G at a concentration of 125 mg/L. Curcumin, emodin, and 10-gingerol at a concentration of 1,000 mg/L did not kill all parasites after an 8-h exposure. These findings demonstrate that plant extracts and active phytochemicals are potential sources of botanical drugs for controlling N. girellae infection in aquaculture.

The transcriptional factor GATA-4 negatively regulates Hsp70 transcription in Crassostrea hongkongensis.

To better explore the application potential of heat shock protein Hsp70s in diverse areas including biomonitoring, a further investigation of the details of the regulatory mechanism governing Hsp70 transcription is required. A transcriptional factor ChGATA-4 that displayed affinity to the ChHsp70 promoter of Crassostrea hongkongensis was isolated and identified by DNA affinity purification as well as mass spectrometry analysis. The ChGATA-4 cDNA is 2162 bp in length and the open reading frame encodes a polypeptide containing 482 amino acids with a conserved zinc finger domain. The over-expression of ChGATA-4 significantly inhibited the expression of ChHsp70 promoter in heterologous HEK293T cells. However, the depletion of ChGATA-4 mRNA by RNAi technique resulted in significant increase of ChHsp70 transcription in oyster hemocytes. The RT-PCR results demonstrated that the transcription of both ChHsp70 and ChGATA-4 were induced by heat, Cd, or NP (Nonyl phenol) stress. This suggested a potential correlation between ChHsp70 and ChGATA-4 in the stress-mediated genetic regulatory cascade. This study demonstrated that ChGATA-4 acts in a negative manner in controlling ChHsp70 transcription in C. hongkongensis and promotes to further understand the mechanisms leading Hsp70 transcription.

Molecular characterization and expression analysis of complement component C3 in southern catfish (Silurus meridionalis) and a whole mount in situ hybridization study on its ontogeny.

The complement system plays an important role in protecting fish against attack by pathogens early in life. Complement component C3 is a central component in the complement system. The present work aimed to clone the full length C3 cDNA sequence of southern catfish (Silurus meridionalis), detect the tissue expression patterns of C3, investigate the ontogeny of C3 in embryo and larva, and assess the expression of C3 in response to pathogen infection. The full length C3 cDNA sequence of 5157 bp with an open reading frame (ORF) of 4938 bp was cloned from southern catfish. The deduced amino acid sequence showed similarity with other teleost fish. The mRNA expression of C3 was detected in liver, spleen, stomach, intestine, and head kidney with RT-PCR and in situ hybridization. Whole mount in situ hybridization results revealed that C3 was first expressed in the yolk syncytial layer at 34 h post fertilization (hpf), followed by the liver at 36 h post hatching (hph). When challenged with Aeromonas hydrophila, the transcripts of C3 showed a significant up-regulation in liver and spleen at 24 h. The results suggested that complement C3 played a key role in defense against invading pathogens in the early development stages of southern catfish. Therefore, these results provide important information to understand the functions of C3 during fish early development in Siluriformes.

Molecular characterization, expression analysis, and ontogeny of complement component C9 in southern catfish (Silurus meridionalis).

The complement system plays an important role in host defense against invading microorganisms. Complement component C9 is the last component that is involved in the formation of the membrane attack complex (MAC) on the surface of target cells. In the present study, the full length C9 cDNA sequence of 1984 bp with an open reading frame (ORF) of 1809 bp was cloned from southern catfish (Silurus meridionalis). The deduced amino acid sequence showed similarity with other teleost fish. The mRNA expression of C9 was detected in the liver, spleen, stomach, intestine, and head kidney, with highest levels detected in the liver. The mRNA of C9 was first detected in the yolk syncytial layer at 34 h post fertilization (hpf) with whole mount in situ hybridization, followed by the liver at 36 h post hatching (hph). The mRNA expression of C9 was upregulated significantly in the liver, spleen, and intestine following the injection with Aeromonas hydrophila, suggesting that C9 played an important role in defense against invading pathogens in southern catfish. Therefore, these results provide important information to understand the functions of C9 during fish early development in fish.

The transcriptional factor YB-1 positively regulates Hsc70 transcription in Crassostrea hongkongensis.

A Y-box binding protein ChYB-1 was discovered as a ChHsc70 promoter-associated protein in Crassostrea hongkongensis by DNA-affinity purification and mass spectrometry analysis. The overexpression of ChYB-1 in heterologous HEK293T cells led to clear enhancement of ChHsc70 promoter expression, while ChYB-1 depletion correlated with significant reduction of ChHsc70 transcription in the hemocytes of C. hongkongensis. Quantitative Real-time PCR analysis revealed that both ChHsc70 and ChYB-1 were transcriptionally responsive to external chemical or physical stressors. This indicates a plausible correlation between ChHsc70 and ChYB-1 in the genetic regulatory pathway triggered by external stresses. This study presents the first evidence of positive regulator for Hsc70 transcription and contributes to a better understanding of the regulatory mechanisms governing Hsc70 expression.

The guanine nucleotide-binding protein α subunit protein ChGnaq positively regulates Hsc70 transcription in Crassostrea hongkongensis.

Gnaq, one of Guanine nucleotide-binding protein α subunits, was isolated from cellular nucleus extracts of oyster Crassostrea hongkongensis gills with biotin-labeled ChHsc70 promoter by means of DNA-affinity purification, and preliminarily identified with mass spectrometry analysis. ChGnaq mRNA depletion by RNAi technique led to clear reduction in ChHsc70 mRNA expression of C. hongkongensis hemocytes. Correspondently, ChGnaq over-expression in heterologous HEK293T cells correlated with elevated expression activation of ChHsc70 promoter. Quantitative real time PCR analysis showed that both ChHsc70 and ChGnaq transcriptions were responsive to external physical/chemical stresses by heat, CdCl2 and NP. This suggested a plausible association between ChHsc70 and ChGnaq in the stress-induced genetic regulatory pathway. This study discovered a positively regulatory role of ChGnaq in controlling ChHsc70 transcription of C. hongkongensis, and conduced to a better understanding of the regulatory mechanisms in control of Hsc70 transcription.

Grass carp which survive Dactylogyrus ctenopharyngodonid infection also gain partial immunity against Ichthyophthirius multifiliis.

Dactylogyrus ctenopharyngodonid and Ichthyophthirius multifiliis are 2 important ectoparasites of fish. Both parasites can induce an immune response in fish that leads to a decrease in parasitic infection intensity and the development of resistance against parasitic reinfection. The present study evaluated whether grass carp Ctenopharyngodon idella that survived a D. ctenopharyngodonid infection could develop immunity against infection by D. ctenopharyngodonid and I. multifiliis. The results demonstrated that when grass carp were infected with D. ctenopharyngodonid, the number of red blood cells and the percentages of thrombocytes, monocytes, and neutrophils in the white blood cells increased significantly in the early stage of infection. The percentage of lymphocytes increased over time following parasitic infection. The mean infection intensity of D. ctenopharyngodonid decreased to 0 on Day 28. The activities of serum acid phosphatase, alkaline phosphatase, lysozyme, and superoxide dismutase increased significantly after D. ctenopharyngodonid infection. In addition, the grass carp that survived a previous D. ctenopharyngodonid infection could completely resist D. ctenopharyngodonid reinfection and partially resist I. multifiliis infection.

Combined effects of Chinese medicine feed and ginger extract bath on co-infection of Ichthyophthirius multifiliis and Dactylogyrus ctenopharyngodonid in grass carp.

Dactylogyrus ctenopharyngodonid and Ichthyophthirius multifiliis are two important ectoparasites of freshwater fish. Co-infection by the two parasites leads to high fish mortality and results in heavy economic losses. This study aimed to evaluate the efficacy of medicated feed and a ginger extract bath against D. ctenopharyngodonid and I. multifiliis on grass carp and investigate the hematological response of grass carp co-infected by the two parasites. These results demonstrated that red blood cell (RBC) and thrombocyte percentage among leucocytes significantly decreased after grass carp were co-infected by D. ctenopharyngodonid and I. multifiliis. The monocyte and neutrophil percentages significantly increased with the increment of parasite mean intensities, while the lymphocyte percentage decreased. The activities of serum acid phosphatase (ACP), alkaline phosphatase (AKP), lysozyme (LZM), and superoxide dismutase (SOD) significantly increased after co-infection. When grass carp treated with medicated feed containing 4% of Astragalus membranaceus, Allium sativum, Morus alba, and Glycyrrhiza uralensis, the activities of ACP, AKP, LZM, and SOD were significantly enhanced, and the mean intensities of D. ctenopharyngodonid and I. multifiliis were significantly decreased. When grass carp was treated with medicated feed and a 4-mg/L ginger extract bath, all parasites were eliminated during 28 days. The bath of ginger extract at a concentration of 4 mg/L kept a low mean intensity of I. multifiliis and D. ctenopharyngodonid, then the two parasites were eliminated by oral administration of the medicated feed with an immunostimulant (Chinese medicine compound).

High mobility group protein DSP1 negatively regulates HSP70 transcription in Crassostrea hongkongensis.

HSP70 acts mostly as a molecular chaperone and plays important roles in facilitating the folding of nascent peptides as well as the refolding or degradation of the denatured proteins. Under stressed conditions, the expression level of HSP70 is upregulated significantly and rapidly, as is known to be achieved by various regulatory factors controlling the transcriptional level. In this study, a high mobility group protein DSP1 was identified by DNA-affinity purification from the nuclear extracts of Crassostrea hongkongensis using the ChHSP70 promoter as a bait. The specific interaction between the prokaryotically expressed ChDSP1 and the FITC-labeled ChHSP70 promoter was confirmed by EMSA analysis. ChDSP1 was shown to negatively regulate ChHSP70 promoter expression by Luciferase Reporter Assay in the heterologous HEK293T cells. Both ChHSP70 and ChDSP1 transcriptions were induced by either thermal or CdCl2 stress, while the accumulated expression peaks of ChDSP1 were always slightly delayed when compared with that of ChHSP70. This indicates that ChDSP1 is involved, very likely to exert its suppressive role, in the recovery of the ChHSP70 expression from the induced level to its original state. This study is the first to report negative regulator of HSP70 gene transcription, and provides novel insights into the mechanisms controlling heat shock protein expression.

Molecular immune response of channel catfish immunized with live theronts of Ichthyophthirius multifiliis.

The parasite Ichthyophthirius multifiliis (Ich) has been reported in various freshwater fishes worldwide and results in severe losses to both food and aquarium fish production. The fish surviving natural infections or immunized with live theronts develop strong specific and non-specific immune responses. Little is known about how these immune genes are induced or how they interact and lead to specific immunity against Ichthyophthirius multifiliis in channel catfish Ictalurus punctatus. This study evaluated the differential expression of immune-related genes, including immunoglobulin, immune cell receptor, cytokine, complement factor and toll-like receptors in head kidney from channel catfish at different time points after immunization with live theronts of I. multifiliis. The immunized fish showed significantly higher anti-Ich antibody expressed as immobilization titer and ELISA titer than those of control fish. The vast majority of immunized fish (95%) survived theront challenge. Expression of IgM and IgD heavy chain genes exhibited a rapid increase from 4 hour (h4) to 2 days (d2) post immunization. Expression of immune cell receptor genes (CD4, CD8-α, MHC I, MHC II ß, TcR-α, and TcR-ß) showed up-regulation from h4 to d6 post immunization, indicating that different immune cells were actively involved in cellular immune response. Cytokine gene expression (IL-1ßa, IL-1ßb, IFN-γ and TNF-α) increased rapidly at h4 post immunization and were at an up-regulated level until d2 compared to the bovine serum albumin control. Expression of complement factor and toll-like receptor genes exhibited a rapid increase from h4 to d2 post immunization. Results of this study demonstrated differential expression of genes involved in the specific or non-specific immune response post immunization and that the vaccination against Ich resulted in protection against infection by I. multifiliis.

Evaluation of medicated feeds with antiparasitical and immune-enhanced Chinese herbal medicines against Ichthyophthirius multifiliis in grass carp (Ctenopharyngodon idellus).

Since malachite green was banned for using in food fish due to its carcinogenic and teratogenic effects on human, the search of alternative drug to treat Ichthyophthirius multifiliis becomes urgent. This study aimed to (1) evaluate the ethanol extracts of medicinal plants Cynanchum atratum, Zingiber officinale, Cynanchum paniculatum, immunostimulant (A), and immunostimulant (B) for their efficacy against I. multifiliis, and (2) determine effects of medicated feeds with C. atratum, Z. officinale, C. paniculatum, and immunostimulant (A) to treat I. multifiliis in grass carp. The results in this study showed that the minimum concentrations of C. atratum, Z. officinale, and C. paniculatum extracts for killing all theronts were 16, 8, and 16 mg/L, respectively. In vivo experiments, fish fed with medicated feeds of C. atratum for 10 days, or Z. officinale for 3 days, or combination of three plants for 10 days resulted in a significant reduction in the I. multifiliis infective intensity on grass carp after theronts exposure. Grass carp fed with medicated feeds of immunostimulant (A) for 21 days showed no infection and 100 % of survival 15 days post theronts exposure. Therefore, immunostimulant (A) is a promising feed supplement to treated I. multifiliis with good antiparasitic efficacy.

Cynatratoside-C efficacy against theronts of Ichthyophthirius multifiliis, and toxicity tests on grass carp and mammal blood cells.

Infection by Ichthyophthirius multifiliis, a ciliated protozoan parasite, results in high fish mortality and causes severe economic losses in aquaculture. To find new, efficient anti-I. multifiliis agents, cynatratoside-C was isolated from Cynanchum atratum by bioassay-guided fractionation in a previous study. The present study investigated the anti-theront activity, determined the toxicity of cynatratoside-C to grass carp Ctenopharyngodon idellus and mammalian blood cells, and evaluated the protection of cynatratoside-C against I. multifiliis theront infection in grass carp. Results showed that all theronts were killed by 0.25 mg l-1 of cynatratoside-C in 186.7 ± 5.8 min. Cynatratoside-C at 0.25 mg l-1 was effective in treating infected grass carp and protecting naive fish from I. multifiliis infestation. The 96 h median lethal concentration (LC50) of cynatratoside-C to grass carp and 4 h median effective concentration (EC50) of cynatratoside-C to theront were 46.8 and 0.088 mg l-1, respectively. In addition, the hemolysis assay demonstrated that cynatratoside-C had no cytotoxicity to rabbit red blood cells. Therefore, cynatratoside-C could be a safe and effective potential parasiticide for controlling I. multifiliis.

Structure and regulatory targets of SCO3201, a highly promiscuous TetR-like regulator of Streptomyces coelicolor M145.

SCO3201, a regulator of the TetR family, is a strong repressor of both morphological differentiation and antibiotic production when overexpressed in Streptomyces coelicolor. Here, we report the identification of 14 novel putative regulatory targets of this regulator using in vitro formaldehyde cross-linking. Direct binding of purified His6-SCO3201 was demonstrated for the promoter regions of 5 regulators (SCO1716, SCO1950, SCO3367, SCO4009 and SCO5046), a putative histidine phosphatase (SCO1809), an acetyltransferase (SCO0988) and the polyketide synthase RedX (SCO5878), using EMSA. Reverse transcriptional analysis demonstrated that the expression of the transcriptional regulators SCO1950, SCO4009, SCO5046, as well as of SCO0988 and RedX was down regulated, upon SCO3201 overexpression, whereas the expression of SCO1809 and SCO3367 was up regulated. A consensus binding motif was derived via alignment of the promoter regions of the genes negatively regulated. The positions of the predicted operator sites were consistent with a direct repressive effect of SCO3201 on 5 out of 7 of these promoters. Furthermore, the 2.1Å crystal structure of SCO3201 was solved, which provides a possible explanation for the high promiscuity of this regulator that might account for its dramatic effect on the differentiation process of S. coelicolor.

Anthelmintic activity of saikosaponins a and d from radix bupleuri against Dactylogyrus spp. infecting goldfish.

Disease caused by the parasitic helminths Dactylogyrus spp. results in significant economic damage to the aquaculture industry. Treatment using common chemicals (e.g. formalin) is usually dissatisfactory due to environmental problems, risk of residues, toxicity to fish, and the possibility of anthelmintic resistance. The search for an alternative drug is thus becoming more urgent. This study was designed to evaluate in vivo the anthelmintic efficacy of total saponin (TS), saikosaponin a (SSa), and saikosaponin d (SSd) from radix bupleuri (i.e. the dried root of Bupleurum sp.) based on our previous screening works, with the aim of determining which has commercial potential. Results showed that median effective concentration (EC50) values for TS, SSa, and SSd were 2.01, 1.46, and 0.74 mg l⁻¹, respectively. The acute toxicities against goldfish Carassius auratus for TS, SSa, and SSd were also determined, with median lethal concentration (LC50) of 8.99, 11.20, and 1.54 mg l-1, respectively. The resulting therapeutic indices (TIs) indicated that SSa (TI = 7.67) is a potential therapeutic agent for treating Dactylogyrus infection.

Parasiticidal effects of Morus alba root bark extracts against Ichthyophthirius multifiliis infecting grass carp.

Ichthyophthirius multifiliis (Ich), an important fish parasite, can cause significant losses in aquaculture. To find efficacious drugs to control Ich, the root bark of white mulberry Morus alba was evaluated for its antiprotozoal activity. Bark was powdered and extracted with 1 of 5 organic solvents: petroleum ether, chloroform, ethyl acetate, acetone, or methanol. The extracts were concentrated, dissolved in 0.1% (v/v) DMSO, and used for anti-Ich trials. Acetone and ethyl acetate extracts significantly reduced the survival of Ich tomonts and theronts. In vitro, acetone extract at 25 mg l-1 killed all non-encysted tomonts, at 50 mg l-1 eradicated all encysted tomonts, and at 8 mg l-1 caused mortality of all theronts. Ethyl acetate extract at 50 mg l-1 eliminated all non-encysted tomonts, at 100 mg l-1 killed all encysted tomonts and terminated tomont reproduction, and at 8 mg l-1 killed all theronts. Low concentrations (2 and 4 mg l-1) of acetone and ethyl acetate extracts could not kill all theronts after 4 h exposure, but a significant decrease in theront infectivity was observed following 30 min of pretreatment with the extracts. The 96 h LC(50) values of acetone and ethyl acetate extracts to grass carp were 79.46 and 361.05 mg l-1, i.e. much higher than effective doses for killing Ich theronts (8 mg l-1 for both extracts) and non-encysted tomonts (12.5 and 25 mg l-1, respectively). Thus M. alba extract may be a potential new, safe, and efficacious drug to control Ich.