Shrimp that have received carrageenan via immersion and diet exhibit immunocompetence in phagocytosis despite a post-plateau in immune parameters

The effect of carrageenan on the immune response of white shrimp Litopenaeus vannamei, was studied in vitro and in vivo. Shrimp haemocytes receiving carrageenan at 1 mg ml⁻¹ experienced change in cell size, reduction in cell viability, increase in PO activity, serine proteinase activity, and RB in vitro. Shrimp received carrageenan via immersion at 200, 400 and 600 mg L⁻¹ after 3 h and orally at 0.5, 1.0 and 2.0 g kg⁻¹ after 3 weeks showed higher proliferation of haematopoietic tissues (HPTs) together with increases in haemocyte count and other immune parameters. Shrimp that fed a diet containing carrageenan at 0.5 g kg⁻¹ after 3 weeks significantly up-regulated gene expressions of several immune-related proteins. The immune parameters of shrimp that received carrageenan via immersion and orally increased to a plateau after 3 h and after 3 weeks, but decreased after 5 h and 6 weeks, respectively. Phagocytosis and clearance of Vibrio alginolyticus remained high in shrimp that had received carrageenan via immersion after 5 h and orally after 6 weeks, respectively. Resistances of shrimp against V. alginolyticus and white spot syndrome virus were higher over 24–144 h and 72–144 h, respectively in shrimp that received carrageenan at 600 mg L⁻¹ via immersion after 3 and 5 h. It was concluded that carrageenan effectively triggers an innate immunity in vitro, and increases mitotic index of HPT, immune parameters, gene expressions and resistance against pathogens in vivo. Shrimp received carrageenan via immersion and orally exhibited immunocompetence in phagocytosis and clearance of V. alginolyticus, and resistance to pathogen despite the trend in immune parameters to recover to background values.     

Determination of aloenin,barbaloin and isobarbaloin in Aloe species by micellar electrokinetic chromatography

Aloenin, barbaloin and isobarbaloin in JP Aloe¹, Aloe barbadensis (Aloe vera) and Aloe arborescens Miller var. natalensis Berger (Aloe arborescens Miller) were determined by micellar electrokinetic chromatography (MEKC) with 50 mM sodium dodecyl sulfate. Aloenin, barbaloin and isobarbaloin were well separated by MEKC and as little as 5.5 pg/11 nl of the three compounds could be detected. The determination took around 14 min.     

Termiticidal activity of lectins from Myracrodruon urundeuva against Nasutitermes corniger and its mechanisms

The isolation of lectins from Myracrodruon urundeuva bark (MuBL) and heartwood (MuHL) as well as the termiticidal activity of MuHL against Nasutitermes corniger has already been described. This work reports on the purification of a leaf lectin (MuLL) and the characterization of MuBL, MuHL, and MuLL; also described are the resistance of hemagglutinating activity of the three lectins to trypsin activity from N. corniger gut and the termiticidal activity on N. corniger of MuBL (LC₅₀ of 0.974 mg ml⁻¹ on workers and 0.787 mg ml⁻¹ on soldiers) and MuLL (LC₅₀ of 0.374 mg ml⁻¹ on workers and 0.432 mg ml⁻¹ on soldiers). The antibacterial effect of MuBL, MuHL, and MuLL on bacteria from gut of N. corniger was also investigated and lectins showed similar bacteriostatic activity (MIC of 62.5 ??g ml⁻¹ for workers and 125 ??g ml⁻¹ for soldiers). MuBL and MuHL were more efficient bactericidal agents on bacteria in the workers’ gut (MBC of 125 ??g ml⁻¹) than MuLL (MBC of 250 ??g ml⁻¹) and similar bactericidal activity was detected on bacteria in the gut of soldiers (MBC of 250 ??g ml⁻¹). The termiticidal activity of M. urundeuva lectins can be explained by the chitin-binding property, resistance to termite digestive enzyme, and the antibacterial effect on symbiotic bacteria of N. corniger gut.     

Administration of the hot-water extract of Spirulina platensis enhanced the immune response of white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus

White shrimp Litopenaeus vannamei which had been injected with the hot-water extract of Spirulina platensis at 6, 10, and 20 μg g^?1, or immersed in aerated seawater containing extract at 200, 400, and 600 mg L^?1 were challenged with Vibrio alginolyticus at 1.5 × 10⁶ or 1.4 × 10⁶ colony-forming units (cfu) shrimp^?1, and then placed in seawater. Survival rates of shrimp that received the extract of S. platensis at 6–20 μg g^?1, and those of shrimp immersed in seawater containing the extract at 400 and 600 mg L^?1 were significantly higher than those of control shrimp after 24–96 and 48–96 h, respectively. In a separate experiment, the hyaline cell (HC) count, granular cell (GC, including semi-granular cell) count, total haemocyte count (THC), phenoloxidase (PO) activity, respiratory burst (RB), superoxide dismutase (SOD) activity, glutathione peroxidase (GPx) activity, and lysozyme activity were measured when shrimp were injected with the extract at 6, 10, and 20 μg g^?1, and immersed in seawater containing the extract at 200, 400, and 600 mg L^?1. These parameters directly increased with the concentration, and significantly increased when shrimp were immersed in the seawater containing the extract at 0.5–4 h. L. vannamei that received all doses of the extract via injection or via immersion all had increased phagocytic activity and clearance efficiency to V. alginolyticus at 12–72 h and 3–4 h, respectively. It was concluded that L. vannamei that received the hot-water extract of S. platensis had enhanced innate immunity and increased resistance against V. alginolyticus infection.     

White shrimp Litopenaeus vannamei that received the hot-water extract of Gracilaria tenuistipitata showed earlier recovery in immunity after a Vibrio alginolyticus injection

White shrimp Litopenaeus vannamei which had been immersed in seawater containing the hot-water extract of Gracilaria tenuistipitata at 0 (control), 200, 400, and 600 mg L^?1 for 3 h, were challenged with Vibrio alginolyticus at 4.6 × 10⁶ colony-forming units (CFU) shrimp^?1 and then placed in normal seawater (34‰). The survival rates of shrimp immersed in 200, 400, and 600 mg L^?1 of the hot-water extract were significantly higher than those of control shrimp over 48–120 h. In another experiment, L. vannamei which had been immersed in the hot-water extract at 0, 200, 400, and 600 mg L^?1 for 3 h, were challenged with V. alginolyticus at 4.0 × 10⁶ CFU shrimp^?1, and the immune parameters examined included the haemocyte count, phenoloxidase (PO) activity, respiratory burst (RB), and superoxide dismutase (SOD) activity at 12–120 h post-challenge after shrimp had been released into normal seawater. Shrimp not exposed to the hot-water extract or V. alginolyticus served as the background control. Results indicated that the haemocyte count, PO activity, RB, and SOD activity of shrimp immersed in 600 mg L^?1 were significantly higher than those of control shrimp at 12–72 h post-challenge. Results also indicated that total haemocyte count (THC), PO activity, RB and SOD activity of shrimp immersed in 400 and 600 mg L^?1 of the hot-water extract returned to the background values at 96, 48, 48, and 72 h, whereas these parameters of control shrimp returned to the original values at >120, >120, 96, and 96 h post-challenge, respectively. It was therefore concluded that L. vannamei that had been immersed in seawater containing the hot-water extract of G. tenuistipitata exhibited protection against V. alginolyticus as evidenced by the earlier recovery of immune parameters.     

The effects of bee (Apis mellifera) venom phospholipase A2 on Trypanosoma brucei brucei and enterobacteria

The potential role of phospholipases in trypanosomiasis was investigated using bee venom phospholipase A2 (bvPLA2) as a model. The effects of bvPLA2 on the survival of Trypanosoma brucei brucei, 2 h and 12 h cultures of Enterobacter cloacae, Escherichia coli, Citrobacter freundii were studied. About 1 mg ml⁻¹ bvPLA2 was trypanocidal after 30 min. Some growth occurred at lower concentrations up to 2 h after treatment but viability decreased up to 8 h. Even very low concentrations of bvPLA2 (10⁻¹² mg ml⁻¹) had some trypanocidal activity. Bee venom PLA2 was bactericidal to 2 h bacterial cultures but bacteriostatic to 12 h ones. Minimum bactericidal concentrations were 10⁻⁵-10⁻⁶ mg ml⁻¹. The results showed that bvPLA2 had significant trypanocidal and antibacterial effects on Gram-negative bacteria. The relationship to events occurring during infection is discussed. Phospholipases may play a role in increased endotoxin levels in trypanosomiasis.     

Role of extracellular protease in nitrogen substrate management during antibiotic fermentation: a process model and experimental validation

The production of compound K and aglycon protopanaxadiol (APPD) from ginsenoside Rd and ginseng root extract was performed using a recombinant β-glycosidase from Pyrococcus furiosus. The activity for Rd was optimal at pH 5.5 and 95°C with a half-life of 68 h at 95°C. β-Glycosidase converted Rb₁, Rb₂, Rc, and Rd to APPD via compound K. With increases in the enzyme activity, the productivities of compound K and APPD increased. The substrate concentration was optimal at 4.0 mM Rd or 10% (w/v) ginseng root extract; 4 mM of Rd was converted to 3.3 mM compound K with a yield of 82.5% (mol/mol) and a productivity of 2,010 mg l⁻¹ h⁻¹ at 1 h and was hydrolyzed completely to APPD with 364 mg l⁻¹ h⁻¹ after 5 h. Rb₁, Rb₂, Rc, and Rd at 3.9 mM in 10% ginseng root extract were converted to 3.1 mM compound K with 79.5% and 1,610 mg l⁻¹ h⁻¹ at 1.2 h and were hydrolyzed completely to APPD with 300 mg l⁻¹ h⁻¹ after 6 h. The concentrations and productivities of compound K and APPD in the present study are the highest ever reported.     

Development of cell culture system from the giant freshwater prawn <Emphasis Type="Italic">Macrobrachium rosenbergii</Emphasis> (de Man)

A new cell culture system (MRH) was developed for the first time from 2 months old freshwater prawn, Macrobrachium rosenbergii. Primary cultures were developed from heart tissues by explant culture technique. Cell outgrowth was obtained from the heart explant after 14 days of explant culture. The culture medium used was Leibovitz-15 supplemented with 20% Fetal Bovine Serum along with 1% prawn hemolymph serum, 0.1% glucose, 0.5% NaCl and antibiotics (Penicillin 10,000 Units ml⁻¹, Streptomycin 10,000 μg ml⁻¹, Amphotericin B 500 mg ml⁻¹) with a final osmomolality of 470–550 mmol kg⁻¹. The pH of the growth medium found suitable for the growth of the cells was 7.20. The viability of cells was found to be 60% when revived after a month of storage in liquid nitrogen.     

Two new guaiane sesquiterpenes from Datura metel L. with anti-inflammatory activity

Chemical investigation of an acidic methanol extract of the whole plants of Datura metel resulted in the isolation of two new guainane sesquiterpenes, 1β,5α,7β-guaiane-4β,10α,11-triol (1) and 1α,5α,7α-11-guaiene-2α,3β,4α,10α,13-pentaol (2), along with eight known compounds: pterodontriol B (3), disciferitriol (4), scopolamine (5), kaempferol 3-O-β-d-glucosyl(1 → 2)-β-d-galactoside 7-O-β-d-glucoside (6), kaempferol 3-O-β-glucopyranosyl(1 → 2)-β-glucopyranoside-7-O-α-rhamnopyranoside (7), pinoresinol 4′′-O-β-d-glucopyranoside (8), (7R,8S,7′S,8′R)-4,9,4′,7′-tetrahydroxy-3,3′-dimethoxy-7,9′-epoxy-lignan-4-O-β-d-glucopyranoside (9), and (7S,8R,7′S,8′S)-4,9,4′,7′-tetrahydroxy-3,3′-dimethoxy-7,9′-epoxylignan-4-O-β-d-glucopyranoside (10). Their structures were elucidated by extensive spectroscopic methods, including 1D and 2D NMR and MS spectra. Compounds 2-4 and 6-10 were shown to have modest anti-inflammatory effects through inhibition of NO production in LPS-stimulated BV cells.     

The effects of algae species and densities on the population growth of the marine rotifer, Colurella dicentra

Colurella dicentra clones isolated from bay water in the Mississippi Gulf Coast were cultured with artificial seawater. Experiments were conducted to determine the effects of six algae species (Nannochloropsis oculata, Tetraselmis chuii, Chaetoceros gracilis, Rhodomonas salina, Isochrysis galbana, and Prorocentrum micans), six C. gracilis densities, and six N. oculata densities (25,000, 50,000, 100,000, 250,000, 500,000, and 1,000,000 cells ml^− 1) on C. dicentra population growth. Algae type influenced rotifer production (p < 0.0001). C. gracilis treatment (9120 ± 3351SD) produced the highest number of rotifers followed by N. oculata (5760 ±2232SD). P. micans had the lowest number of rotifers, although not significantly different from numbers in T. chuii, R. salina, and I. galbana treatments (p > 0.05).The population growth rate (r) varied with algae species treatment. The highest values were recorded for C. gracilis treatment (0.22 to 0.26 d^− 1), followed by N. oculata (0.21 to 0.24 d^− 1), and the lowest for P. micans (− 0.19 to 0.14 d^− 1). C. gracilis and N. oculata densities had significant effects (p < 0.0001) on C. dicentra population growth. The highest rotifer production was recorded at a C. gracilis density of 100,000 cells ml^− 1, followed by 250,000 cells ml^− 1 and 50,000 cells ml^− 1. Algae densities of 500,000 cells ml^− 1 and above produced the lowest rotifer numbers. Population growth rate (r) varied with C. gracilis densities. The highest values were observed for C. gracilis concentrations of 100,000 cells ml^− 1 (0.17 to 0.19 d^− 1), and the lowest for concentrations of 500,000 cells ml^− 1 and above (− 0.19 to 0.09 d^− 1). The 100,000 cells ml^− 1N. oculata density gave the highest rotifer production followed by 50,000, 250,000, 25,000, and 500,000 cells ml^− 1. Algae densities of 1,000,000 cells ml^− 1 produced the lowest rotifer numbers. Population growth rate (r) varied with N. oculata densities, with the highest values obtained for algae densities of 100,000 cells ml^− 1 (0.35 to 0.40 d^− 1), and the lowest for concentrations of 1,000,000 cells ml^− 1 (0.05 to 0.012 d^− 1). This is the first report of C. dicentra in Mississippi Coastal waters, and perhaps the smallest marine rotifer species (93 by 49 μm) ever cultured successfully.     

New cytotoxic dihydrochalcone and steroidal saponins from the aerial parts of Sansevieria cylindrica Bojer ex Hook

A new dihydrochalcone, 2‘,4‘-dihydroxy-3‘-methoxy-3,4-methylenedioxy-8-hydroxymethylene dihydrochalcone 1 and two new steroidal saponins, (25S)-ruscogenin-1-O-α-l-rhamnopyranosyl-(1 → 2)-β-d-glucopyranoside 2, (25S)-ruscogenin-3-O-α-l-rhamnopyranosyl-(1 → 4)-β-d-glucopyranoside 3, together with three known steroidal saponins (25S)-ruscogenin-3-O-β-d-glucopyranoside 4, (25S)-ruscogenin-1-O-α-l-rhamnopyranosyl-(1 → 2)-[β-d-xylopyranosyl-(1 → 3)]-α-l-arabinopyranoside 5 and (25R)-26-O-β-d-glucopyranosyl-furost-5-ene-1β,3β,22α,26-tetrol-1-O-α-L-rhamnopyranosyl-(1 → 2)-[β-d-xylopyranosyl-(1 → 3)]-α-l-arabinopyranoside 6 were isolated from the aerial parts of Sansevieria cylindrica. The structures of the new compounds were established by UV, IR, EI-MS, HR-ESI–MS as well as 1D (¹H,¹³C and DEPT-135) and 2D (HSQC, HMBC and TOCSY) NMR spectral analysis. The isolated compounds 1-6 were assayed for in vitro cytotoxicities against the three human tumor cell lines HT116, MCF7 and HepG2. Compound 1 showed a moderate cytotoxicity against MCF7. Compounds 2, 3 and 6 exhibited moderate cytotoxicities against the three used cell lines and compound 5 showed marked cytotoxicities against all used cell lines.     

Enhanced regeneration of haploid plantlets from microspores of <Emphasis Type="Italic">Brassica napus</Emphasis> L. using bleomycin,PCIB, and phytohormones

The effects of three periods of incubation (10, 20 and 30 min) at different levels of bleomycin (0, 0.1, 0.2, 0.3, 0.4 and 0.5 μg ml⁻¹), as well as three periods of exposure (12, 24 and 48 h) to different levels of the anti-auxin p-chlorophenoxyisobutyric acid (PCIB), including 1, 2, 3, 4 and 5 mg l⁻¹, on microspore embryogenesis of rapeseed cv. ‘Amica’ were investigated. Microspore embryogenesis was significantly enhanced following 20 min treatment with 0.2 μg ml⁻¹ bleomycin compared with untreated cultures. Highest embryo yield (163 embryos Petri dish⁻¹) was observed with 24 h treatment of 4 mg l⁻¹ PCIB. The highest percentage of secondary embryogenesis was observed on B₅ medium containing 0.15 mg l⁻¹ of gibberellic acid (GA₃) and 0.2 mg l⁻¹ 6-benzyladenine (BA) in 4–6 mm microspore-derived embryos (MDEs). Most callus formed on B₅ medium containing 0.15 mg l⁻¹ GA₃, 0.1 mg l⁻¹ BA and 0.1 mg l⁻¹ indole-3-acetic acid (IAA) when 4–6 mm embryos were used. Regeneration was highest on B₅ medium containing 0.05 mg l⁻¹ GA₃ or 0.1 mg l⁻¹ BA and 0.2 mg l⁻¹ IAA with 2–4 mm embryos. Microspore embryogenesis and plant regeneration could be improved by both bleomycin and PCIB when the appropriate MDE length and phytohormone level were selected.     

A new complex triterpenoid saponin from Samanea saman with haemolytic activity and adjuvant effect

A new complex triterpenoid saponin was isolated from the stem bark of Samanea saman by using chromatographic methods. Its structure was established as 3-[(2-O-β-d-glucopyranosyl-β-d-glucopyranosyl)oxy]-2,23-dihydroxy-(2β,3β,4α)-olean-12-en-28-oic acid O-β-d-glucopyranosyl-(1 → 3)-O-[O-β-d-glucopyranosyl-(1 → 4)]-O-6-deoxy-α-l-mannopyranosyl-(1 → 2)-6-O-[4-O-[(2E,6S)-2,6-dimethyl-1-oxo-2,7-octadienyl]-6-deoxy-α-l-mannopyranosyl)oxy]-β-d-glucopyranosyl ester (1). Structural elucidation was performed using detailed analyses of ¹H and ¹³C NMR spectra including 2D NMR spectroscopic techniques and chemical conversions. The haemolytic activity of the saponin was evaluated using in vitro assays, and its adjuvant potential on the cellular immune response against ovalbumin antigen was investigated using in vivo models.     

Pyrogenic effects of cytokines (IL-1β, IL- 6, TNF-α) and their mode of action on thermoregulatory centers and functions

The objective of this study was to compare the thermoregulatory responses of rabbits to fever-inducing doses of various cytokines (IL-1β, IL-6, TNF-α) injected peripherally (i.v.), or centrally (i.h.).TNF-α (1 μg kg⁻¹), when applied i.v. at thermoneutral conditions, induced a long lasting increase in hypothalamic temperature, which reached maximal values within 50 min and then persisted for at least 3 h. This monophasic fever-like response was due to extensive and long lasting attenuation of panting and due to transient vasoconstriction. Metabolic rate tended to increase during the early phase of the fever, only. On the other hand, IL-6 when applied i.v. in the same dose (1 μg kg⁻¹) and IL-1β, in a much lower dose (60 ng kg⁻¹), induced a short lasting monophasic hyperthermia due to transient attenuation of panting and due to transient vasoconstriction. No significant increase in metabolic rate was observed. The immediate attenuation of panting and induction of vasoconstriction rather resembled reflex (shock) responses than specific thermoregulatory reactions. Thirty minutes after i.v. administration of TNF-α, temperature thresholds for induction of cold thermogenesis, panting and vasomotion were shifted to higher body temperatures and remained elevated for at least 3 h. In case of IL-1β the increased temperature thresholds were observed 30 min after i.v. administration, only. I.v. applied IL-6 did not influence the thresholds neither 30 or 180 min after injection. Thus, peripheral IL-6 rather induced hyperthermia than fever.When injected i.h. all cytokines studied induced a long lasting increase in body temperature similar to that after i.v. injection of LPS. Temperature thresholds for induction of all thermoregulatory outputs were increased and remained increased for at least 3 h. No changes in hypothalamic thermosensitivity were observed. Data indicate a nonspecific effect of central cytokines on body temperature control.IL-1β appeared to be the most potent fever inducer. Nanogram doses of IL-1β injected i.v. induced a similar febrile response as microgram doses of other cytokines. On the other hand, TNF-α induced a longer lasting fever than other cytokines.     

Two new antimicrobial steroids and other constituents from the whole plant of Ludwigia abyssinica

The genus Ludwigia belongs to the Onagraceae family and it encompasses seventy-five species of aquatic plants. The chemistry of this genus is scarcely investigated, although some studies have demonstrated the potential of Ludwigia leptocarpa to produce important bioactive compounds. Herein, we describe the phytochemical investigation of Ludwigia abyssinica A. Rich. Two new steroids named 3β-formyloxy-5α,6α-dihydroxysitostane (Ludwigiaformyl A, 1) and 3β,6α-diformyloxy-5α-hydroxysitostane (Ludwigiaformyl B, 2), along with six known compounds, 3β-formyloxysitost-5-en (3), 5α,6β-dihydroxysitosterol (4), maslinic acid (5), oleanolic acid (6) and a mixture of two iridoids: linearin (7) and 1-epilinearin (8) were obtained from whole plant of L. abyssinica. The structures of the isolated compounds were established by extensive analysis of their spectroscopic and spectrometric data, which included HR-TOF-ESIMS, ¹D NMR (¹H, ¹³C) and ²D NMR (¹H–¹H COSY, HSQC, HMBC and ROESY) and by comparison with data reported in the literature. The antimicrobial activities of extracts, fractions, and new compounds (1) and (2) were evaluated using broth microdilution method against fungi and bacteria strains. The MeOH extract and the ethyl acetate fraction displayed different degrees of antibacterial and antifungal activities (MIC = 32 – 512 µg/mL; MMC = 64 – 512 µg/mL) whereas compounds 1 and 2 showed moderate antimicrobial activities against Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Shigella flexneri and Cryptococcus neoformans (MIC = 8 – 32 µg/mL; MMC = 8 – 64 µg/mL).     

Bactericidal activity of ethanolic extracts of propolis against Staphylococcus aureus isolated from mastitic cows

Staphylococcus aureus is an important pathogen for both humans and animals, and it has been an ubiquitous etiological agent of bovine mastitis in dairy farms worldwide. Elimination of S. aureus with classic antibiotics is difficult, and the current study aimed to evaluate the efficacy of ethanolic extracts of propolis (EEP) against S. aureus cultivated in complex media or milk. EEP (0–0.5 mg ml⁻¹) decreased growth of S. aureus in BHI media and 1 mg ml⁻¹ was bactericidal against washed cell suspensions (10⁷ CFU ml⁻¹). Propolis extracts also killed S. aureus cells resuspended in milk, but the bactericidal dose was at least 20-fold greater. Cultures that were transferred for at least 60 generations with sub-lethal doses of propolis did not change much their sensibility to EEP. Atomic force microscopy images revealed changes in morphology and cell size of S. aureus cells exposed to EEP (0.5 mg ml⁻¹). Our results indicate that propolis extracts might be effective against mastitis-causing S. aureus strains in vivo, but milk constituents affect the inhibitory activity of propolis. Considering that propolis-resistance appears to be a phenotype not easily selected, the use of EEP combined or not with other antimicrobial agents might be useful for mastitis control in vivo.     

Herbal supplementation diets on hematology and innate immunity in goldfish against Aeromonas hydrophila

Goldfish, Carassius auratus (47 ± 3 g, n = 300) were inoculated intramuscularly (50 μl) with Aeromonas hydrophila (1.8 × 10⁶ cells ml^?1). On the 6th day of post-infection the fishes were divided into i) control, without infection fed with normal diet (C), ii) infected fish, fed with normal diet (IU), and infected fishes treated with different doses of iii) 100 mg kg^?1, iv) 200 mg kg^?1, iv) 400 mg kg^?1 and vi) 800 mg kg^?1 mixed herbal extracts supplementation diets. Hematological and immunological parameters were determined on week 1, 2 and 4. In infected goldfish were fed diets containing 100 and 200 mg kg^?1 of mixed herbal extracts supplementation feeds, the white blood cell (WBC) levels significantly increased (P < 0.05) throughout the experimental trial compared to the control. During the experimental period, the red blood cell (RBC) and haemoglobin (Hb) level in goldfish significantly decreased (P < 0.05) when fish fed with 100 and 200 mg kg^?1 of mixed herbal extracts supplementation feeds while it was restored near control when infected fish fed with 400 or 800 mg kg^?1 of herbal extracts supplementation feeds. On the other hand, the haematocrit (Ht) values decline significantly (P < 0.05) in 100, 200 and 400 mg kg^?1 of mixed herbal supplementation feeding groups on weeks 2 and 4 when compared to control group. The mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) values almost significantly differ from the control values. The infected goldfish and treated with 100 or 200 mg kg^?1 of herbal supplementation feeds exhibited significantly decline (P < 0.05) in total protein (TP), glucose (GLU) and cholesterol (CHO) levels on week 1–4 whereas it was restored when infected fish fed with 400 or 800 mg kg^?1 of herbal supplementation feeds on week 4. In comparison to untreated control goldfish, the respiratory burst activity and phagocytic activity of blood cells was significantly enhanced in infected fish feeding with 200, 400 and 800 mg kg^?1 of herbal supplementation feeds compared to the control. On the other hand, infected fish fed with all the doses of mixed herbal supplementation feeds, the lysozyme activity was significantly enhanced throughout the experimental period. This study shows that the infected goldfish treated with 400 and 800 mg kg^?1 of herbal supplementation feeds preceding the challenge with live A. hydrophila had 30% and 25% mortality. However, 100 and 200 mg kg^?1 of herbal supplementation feeds treated groups were found the percentage mortality 50% and 45%, respectively. Our results indicate that 400 or 800 mg kg^?1 of mixed herbal supplementation feeds were restored the altered hematological parameters and triggering the innate immune system of goldfish against A. hydrophila.     

Cystathionine gamma-synthase is essential for methionine biosynthesis in Fusarium graminearum

Methionine (Met) plays an important role in various cellular processes in both eukaryotes and prokaryotes. Cystathionine gamma-synthase encoded by STR2 gene is a key enzyme in Met biosynthesis in Saccharomyces cerevisiae. In this study, we identified FgMETB, a homologue of S. cerevisiae STR2, from Fusarium graminearum using the Protein Basic Local Alignment Search Tool (BLASTP) program. The FgMETB deletion mutants were unable to grow on fructose gelatin agar (FGA) medium containing SO₄²⁻ as sole sulphur source. In addition, more than 90 % conidia of the mutants were not able to germinate in 2 % sucrose solution within 6 or 12 h of incubation. Supplementation of 1 mM Met or 0.5 mg ml⁻¹ homocysteine, but not 1 mM cysteine or 0.5 mg ml⁻¹ glutathione, rescued the defect of mycelial growth and spore germination of FgMETB deletion mutants. These results indicated that the enzyme encoded by FgMETB is involved in conversion of cysteine into homocysteine. Inoculation tests showed that the FgMETB deletion mutant exhibited decreased virulence significantly on wheat heads, which is consistent with a low level of deoxynivalenol (DON) production of the mutant in wheat kernels. Fungicide sensitivity assays revealed FgMETB deletion mutants showed increased sensitivity to the sterol demethylation inhibitor tebuconazole, but did not change their sensitivities to other fungicides. Taken together, results of this study indicated that FgMETB plays a critical role in the regulation of various cellular processes in F. graminearum.