Resveratrol Reduces ROS by Increasing GSH in Vitrified Sheep Embryos.

The in vitro production and cryopreservation of mammalian embryos generates reactive oxygen species (ROS) due to conditions of the system that can overcome their antioxidant protection. Resveratrol is an antioxidant used in in vitro systems to improve blastocyst rates, but its effect on antioxidant enzymes such as glutathione (GSH) in embryos produced by in vitro fertilization (IVF) after vitrification has not been reported. The objective of this study was to evaluate the effects of resveratrol on the in vitro maturation medium (IVM) of sheep oocytes (Ovis aries) on the levels of ROS and GSH in embryos produced by IVF subjected to vitrification. Resveratrol was added at 0 µM, 0.25 µM, 0.5 µM, and 1 µM during oocyte in vitro maturation (IVM). Matured oocytes were fertilized with thawed ram sperm. Embryos were cultured in sequential media until blastocysts, were then vitrified for 24 h, and, after heating, they were stained with DCFH-DA (2',7'-dichlorodihydrofluorescein diacetate) to determine the presence of ROS and with Cell Tracker Blue® for the presence of GSH. The quantitative values of ROS and GSH were obtained through the Image J image processor. The results showed that resveratrol increased GSH and decreased ROS production (p < 0.05) in a dose-dependent manner. It is concluded that its use in sheep oocytes during IVM has a beneficial effect on embryos produced by IVF subjected to vitrification by decreasing reactive oxygen species levels and facilitating the generation of embryo antioxidant enzymes like glutathione.

Structure and predictive metabolic contribution of intestinal microbiota of Longfin yellowtail (Seriola rivoliana) juveniles in aquaculture systems.

Seriola rivoliana intestinal microbiota (IM) was characterised under aquaculture conditions through 16S rRNA amplicon sequencing. Specimens of 30 days after hatching (DAH) were maintained in three tanks and fed under the same environmental conditions for characterisation 15 days prior to sampling. Three fish were randomly taken from each tank; total DNA extraction of the gut microbiota was performed to characterise microbial composition and its metabolic prediction. The V3 hypervariable region of the 16S rRNA was amplified and sequenced with Illumina pair-end technology. The prokaryotic components in the S. rivoliana intestine were dominated mainly by the phyla Proteobacteria, Firmicutes, Bacteroidetes, Cyanobacteria and Actinobacteria. No significant differences in beta diversity were detected in the three samples (tanks). However in alpha diversity, they were detected in juveniles of the same cohort within the same group, as exemplified by enrichment of certain bacterial groups, mainly of the Clostridia class, which were specific in each fish within the same tank. The metabolic prediction analyses suggested that S. rivoliana IM contribute to the metabolism of amino acids, carbohydrates, lipids, and immune system. This study provides the first IM characterisation under rearing conditions of S. rivoliana-a species with broad economic potential-and contributes to novel information for potential use of probiotics in future trials.

Probiotic Bacterium and Microalga Interaction on Rearing Kumamoto Oyster Crassostrea sikamea Spat.

This study assessed in vitro interaction between Bacillus bacteria and microalgae and their posterior in vivo effect on rearing Kumamoto oyster Crassostrea sikamea. The probiotic strains Bacillus licheniformis (MAt32), B. subtilis (MAt43) and B. subtilis (GAtB1) were individually inoculated in triplicate into 250 mL flasks containing 1 × 104 colony forming units (CFU) mL-1 of bacteria and 4.5 × 104 cell mL-1 of microalgae (Isochrysis galbana or Chaetoceros calcitrans) to evaluate their growth during a 7-day culture. Single cultures of microalgae or bacilli served as control. Additionally, C. sikamea spat was treated for 28 days with four single/combined bacillus treatments in triplicate at a concentration of 1 × 106 CFU mL-1 as follows: (a) control, without treatments; (b) combination of two antibiotics (10 mg L-1); (c) B. licheniformis; (d) B. subtilis; (e) B. subtilis subtilis and (f) mixed bacilli. The results showed a significantly (P < 0.05) increased growth of Bacillus strains co-cultured with microalgae, while the growth of I. galbana co-cultured with bacteria was not reduced significantly (P > 0.05) compared with the control group. C. sikamea spat treated with Bacillus showed significantly (P < 0.05) higher growth and survival than the control group. In this study, C. calcitrans microalgae were susceptible to the presence of probiotic bacteria. Nonetheless, this reduction in microalgal growth observed in vitro increased growth and survival of C. sikamea spat exposed to probiotic bacteria when compared to spat without probiotics.

Transcriptome analysis of Catarina scallop (Argopecten ventricosus) juveniles treated with highly-diluted immunomodulatory compounds reveals activation of non-self-recognition system.

Marine bivalve hatchery productivity is continuously challenged by apparition and propagation of new diseases, mainly those related to vibriosis. Disinfectants and antibiotics are frequently overused to prevent pathogen presence, generating a potential negative impact on the environment. Recently, the use of highly diluted compounds with immunostimulant properties in marine organisms has been trailed successfully to activate the self-protection mechanisms of marine bivalves. Despite their potential as immunostimulants, little is known about their way of action. To understand their effect, a comparative transcriptomic analysis was performed with Argopecten ventricosus juveniles. The experimental design consisted of four treatments formulated from pathogenic Vibrio lysates at two dilutions: [(T1) Vibrio parahaemolyticus and Vibrio alginolyticus 1D; (T2) V. parahaemolyticus and V. alginolyticus 7C]; minerals [(T3) PhA+SiT 7C], scorpion venom [(T4) ViT 31C]; and one control (C1) hydro-alcoholic solution (ethanol 1%). The RNA sequencing (RNAseq) analysis showed a higher modulation of differentially expressed genes (DEG) in mantle tissue compared to gill tissue. The scallops that showed a higher number of DEG related to immune response in mantle tissue corresponded to T1 (V. parahaemolyticus and V. alginolyticus lysate) and T3 (Silicea terra® - Phosphoric acid®). The transcriptome analysis allowed understanding some interactions between A. ventricosus juveniles and highly-diluted treatments.

Evaluation of Homeopathic Phosphoric Acid, Silica and Pathogenic Vibrio on Digestive Enzyme Activity of Longfin Yellowtail Fish (Seriola rivoliana).

BACKGROUND: This research aimed to observe the effect of homeopathically prepared Vibrio parahaemolyticus (ViP) and V. alginolyticus (ViA) and the commercial homeopathic compound Similia (Phosphoricum acidum and Silicea terra) on the digestive enzyme activities of Seriola rivoliana juveniles under usual culture conditions. MATERIALS AND METHODS: Biochemical analysis was used to study the effect of highly diluted substances (7C potency) prepared from ViP and ViA (Treatment 1: T1) and the homeopathic compound Phosphoricum acidum and Silicea terra (Treatment 2: T2) on changes in the main digestive enzymes on weaning-state fish (WS; 30 days post-hatching [DPH]) and early juveniles (EJ; 62 DPH) versus a reference control group that received no homeopathic medicines. RESULTS: Treatment T2 significantly increased the activity of trypsin and lipase and decreased the activity of amylase, whereas treatment T1 increased the activity of chymotrypsin and reduced the activity of aminopeptidase-N in WS fish. Except for alkaline phosphatase, which was significantly reduced in the intestine, no significant differences in enzymatic activity were found between treated EJ fish and controls. The fish of the WS group had a higher growth rate with the T2 treatment. CONCLUSIONS: T1 treatment stimulated chymotrypsin in EJ fish and T2 promoted intestinal maturation of WS fish. Higher growth rate with the T2 treatment may be associated with the stimulation of trypsin activity. Thus, T2 may be applied, under hatchery conditions, during larval stages with an aim to enhance digestion and assimilation of inert food.

Effect of Streptomyces probiotics on the gut microbiota of Litopenaeus vannamei challenged with Vibrio parahaemolyticus.

This study assessed the intestinal microbiota of juveniles of the White shrimp Litopenaus vannamei, whose feed was enriched with three probiotic formulations: Streptomyces sp. RL8 (RL8); a mix of Lactobacillus graminis and Streptomyces spp. RL8 and N7 (Lac-Strep); and a mix of Bacillus spp. and Streptomyces spp. RL8 and N7 (Bac-Strep). The analysis was performed by sequencing the V3 region of the 16S rRNA gene of treated animals and the control group before and after Vibrio parahaemolyticus challenge. After challenge, the highest Shannon diversity indexes corresponded to RL8 and Bac-Strep (3.94 ± 0.11 and 3.39 ± 0.3, respectively) and the lowest to the control group (2.58 ± 0.26). The most abundant phyla before and after challenge were Proteobacteria, Actinobacteria, and Bacteroidetes. The principal component analysis and Statistical Analysis of Metagenomic Profiles (STAMP) showed that the gut microbiota of the groups RL8 and Bac-Strep after challenge was different from the other experimental groups, which was characterized by a higher bacterial diversity, as well as a significant stimulation of the Bacteriovorax population and other antimicrobial producing genera that protected shrimp from infection.

Efecto de medicamentos homeopáticos sobre crecimiento, supervivencia y microbiota gastrointestinal, en juveniles del pectínido Argopecten ventricosus / Effect of homeopathic medicines on growth, survival and gastrointestinal microbiota of juvenile scallop Argopecten ventricosus

RESUMEN Objetivo. Estudiar el efecto de medicamentos homeopáticos sobre el crecimiento, supervivencia y microbiota del tracto gastrointestinal (TGI) de almeja Catarina Argopecten ventricosus. Materiales y Métodos. Se aplicaron cinco tratamientos homeopáticos derivados de bacterias [(T1) ViP-ViA 1D, (T2) ViP-ViA 7C], minerales [(T3) AcF-MsS 1D, (T4) PhA-SiT 7C], o venenos [(T5) ViT 31C] y tres controles: (C1) etanol diluido 1:99, (C2) etanol dinamizado 1C y (C3) agua destilada. La microbiota se determinó secuenciando la región V3-V5 del gen 16S rRNA. Resultados. El mayor crecimiento en longitud de la concha correspondió a T1 (117 µm d-1) y T2 (108 µm d-1), la mayor supervivencia a T3 y T5 y el mejor resultado global a T3. Las curvas de rarefacción de los grupos tratados y controles mostraron una clara separación. Se encontraron diferencias significativas (p≤0.05) entre filos (Proteobacteria > Actinobacteria > Firmicutes > Bacterloidetes> Chloroflexi y para los Géneros: Symbiobacterium > Microbacterium > Methylobacillus > Bacillus > Paenibacillus > Burkholderia > Nostoc > Methylobacterium > Leucobacter). El género Symbiobacterium fue dominante (p≤0.05) para T5, respecto a todos los tratamientos y grupos controles. La especie Microbacterium maritypicum (Actinobacteria) mostró la mayor abundancia relativa (p≤0.05) en T1 y T3 y Symbiobacterium toebii (Firmicutes) en T5 y T2 (p≤0.05), ambas con respecto al inicio del estudio T0. Conclusiones. Se presenta por primera vez la composición de la microbiota del TGI de A. ventricosus y la aplicabilidad potencial de la homeopatía para mejorar el rendimiento productivo y modular la microbiota gastrointestinal de la especie.

ABSTRACT Objective. To study the effect of homeopathic medicines on growth, survival and gastrointestinal (GIT) microbiota of Catarina scallop Argopecten ventricosus. Materials and methods. Five homeopathic (HOM) treatments derived from bacteria [(T1) ViP-ViA 1D, (T2) ViP-ViA 7C], minerals [(T3) AcF-MsS 1D, (T4) PhA-SiT 7C] or venoms [T5) ViT 31C] and three controls: [(C1) diluted ethanol 1:99, (C2) diluted/succussed ethanol 1C and (C3) distilled water] were evaluated (21 days) in triplicate. Microbiota was analysed by sequencing the V3-V5 region of the 16S rRNA genes. Results. The best growth in shell-length corresponded to T1 (117 µm d-1) and T2 (108 µm d-1) and the highest survival to T3 and T5, stating T3 as the best HOM-treatment. A clear separation was found in rarefaction curves of HOM-treated against un-treated control scallops. Significant differences (p≤0.05) were found for Phyla (Proteobacteria> Actinobacteria> Firmicutes> Bacterloidetes>Chloroflexi and for Genera: Symbiobacterium> Microbacterium> Methylobacillus> Bacillus> Paenibacillus> Burkholderia> Nostoc> Methylobacterium> Leucobacter). The genus Symbiobacterium was dominant in T5, finding significant differences (p≤0.05) with respect to all treatments. At species level, Microbacterium maritypicum (Actinobacteria) showed a greater relative abundance (p≤0.05) in T1 and T3 and Symbiobacterium toebii (Firmicutes) was also significantly higher (p≤0.05) in abundance in T5 and T2, both against initial T0. Conclusions. This study showed for a first time, the composition of GIT microbiota in A. ventricosus and focused on the potential applicability of homeopathy to improve overall performance and modulate the GIT microbiota of the species.

Effect of Homeopathic Medicines on Intestinal Coccidia and Immune Response Cells in Spotted Rose Snapper (Lutjanus guttatus).

BACKGROUND: Homeopathy has been widely applied in freshwater species but rarely in marine fish. Farm stress destabilises host-pathogen equilibrium, favouring parasites and disease. Coccidian endoparasites cause intestinal infections and cell degeneration. MATERIALS AND METHODS: Naturally parasite-infested juvenile snapper Lutjanus guttatus (n = 430; weight 1.9 ± 0.01 g; length 4.9 ± 0.03 cm) specimens were distributed in group sizes of 43, in 10 fiberglass tanks (100 L) with aeration and continuous water change. Five groups in duplicate were assessed: Passival (PaV); Passival and Phosphoricum acid Similia (PaV-PhA); Passival and Silicea terra Similia (PaV-SiT); Endecto and Infecçoes (End-Inf) and a control (Ethanol) for 45 days. Feed was sprinkled with treatment (5% v/w) and dried to avoid ethanol side-effects. Statistical results were expressed as mean ± standard error of the mean. RESULTS: Intestinal coccidia were recorded histopathologically, with the least incidence attained at T45 with End-Inf treatment (p < 0.001), coinciding with the highest lymphocyte (p = 0.015) count. Fish treated with PaV-PhA recorded a higher percentage of neutrophils (p = 0.015), and those treated with PaV-PhA, PaV-SiT and End-Inf revealed a decrease in lesions, degree of alteration and change in intestine and stomach tissues (p < 0.05). The number of mucous cells in gills was greater (p < 0.001) for End-Inf (43 ± 0.58), PaV-SiT (40 ± 2.89) and PaV-PhA (39 ± 3.46) as compared respectively with T0 and control (19 ± 0.58 and 28 ± 2.31). PaV and PaV-SiT increased carbohydrate reserves in liver (28.4 ± 1.39% and 22.4 ± 0.12%, respectively) compared with T0 and control (3.4 ± 0.32 and 5.6 ± 0.66%). The highest survival rate was 97.7 ± 0.16% in fish treated with PaV and End-Inf. CONCLUSION: Homeopathic treatments had a positive effect on fish health. Besides the accumulation of metabolic reserves in the liver, homeopathic treatment was associated with mucin increase in gills as well as raised production of lymphocytes and neutrophils in blood, which could act as macrophages against intestinal coccidia.

Immune and Antioxidant Enzyme Response of Longfin Yellowtail (Seriola rivoliana) Juveniles to Ultra-diluted Substances Derived from Phosphorus, Silica and Pathogenic Vibrio.

BACKGROUND: This research aimed to observe the effect of homeopathic treatments prepared from Vibrio parahaemolyticus and V. alginolyticus (H1) and commercial homeopathic medication Phosphoricum acidum and Silicea terra (H2) on the immune and antioxidant response in Seriola rivoliana juveniles under usual culture conditions and challenged with V. parahaemolyticus. MATERIALS AND METHODS: Quantitative polymerase chain reaction analysis was used to study changes in the expression of key genes related to immune response, cytokines (interleukin-1ß [IL-1ß]), adapter protein for cytokine release (MyD88) and piscidin and spectrophotometric techniques to analyze the activity of antioxidant superoxide dismutase (SOD) and catalase (CAT) enzymes in Seriola rivoliana juveniles at 30 (weaning stage [WS]) and 60 (early juveniles [EJ]) days post-hatching. RESULTS: The H1 treatment led to over-expression of the IL-1ß and MyD88 genes in fish at WS and EJ with respect to control, contrary to the H2 treatment that led to under-expression of the IL-1ß, MyD88 and piscidin genes at the EJ stage. In fish challenged with V. parahaemolyticus, both H1 and H2 led to over-expression of IL-1ß and MyD88; H2 caused an over-expression of piscidin. The SOD activity was higher in H1 with respect to H2 and the control group. CAT remained relatively stable with both H1 and H2 treatments. CONCLUSIONS: The results suggest that the overall effect of H1 was due to the presence of unknown antigens in low concentrations, while the response to H2-specifically during challenge-may have been due to a stimulating effect of nano-structures, prevailing from mother tincture after sequential dilution/succussion, in a pathway similar to that attributed to nano-vaccines.

Assessment of homeopathic medicines on survival and antioxidant response in white shrimp Litopenaeus vannamei / Evaluación de medicamentos homeopáticos en la supervivencia y respuesta antioxidante del camarón blanco Litopenaeus vannamei infectado con Vibrio parahaemolyticus

ABSTRACT Objective. Evaluating the effect of homeopathic medicines on survival and activity of the superoxide dismutase (SOD) enzyme in white shrimp Litopenaeus vannamei subjected to infection with the pathogenic Vibrio parahaemolyticus. Materials and methods . The average lethal dosage (LD50) was determined for the pathogen strain in L. vannamei juveniles under immersion (Imm), injection (Inj) and incision + immersion (Inc+Imm) methods. Four treatments were applied: (1) Mix CIB®-HOM Heel-Mix (TH1), constituted by equal v/v ratio, of Cyme-Heel®, Gal-Heel®, Hepa-Heel®, Mucs-Heel® and Chol-Heel®; (2) Mix CIB®-HOM Pav-Mix (TH2), constituted by the same v/v ratio of Passiflora incarnata, Valeriana officinalis, Zincum valerianicum and Ignatia amara (Similia®); (3) Heel-Mix/Pav-Mix (TH3) consisting of a 1:1 v/v combination of the TH1 and TH2 treatments, and (4) ViT-Mix (TH4), constituted by Vidatox®, and a control (not treated/infected). Results. While applying the methods Imm, Inj and Inc+Imm, LD50 was 0.9 x 106; 0.6 x 106 and 0.5 x 106 UFC.mL-1, respectively. At the end of the challenge, the groups treated with TH2, TH3 and TH4 had a greater survival rate to that of the control group (p<0.05). Moreover, these two last treatments showed a greater SOD activity with respect to the control group (p<0.05). Conclusions. The homeopathic treatments (TH3 and TH4) increased survival and SOD activity in L. vannamei juveniles challenged with V. parahaemolyticus, which suggests that the homeopathic treatments employed had the potential as an alternative for the control of V. parahaemolyticus and its associated diseases, including the early mortality syndrome in shrimp farming.

RESUMEN Objetivo. Evaluar el efecto de medicamentos homeopáticos sobre la supervivencia y actividad de la enzima superóxido dismutasa (SOD) del camarón Litopenaeus vannamei sometido a infección con Vibrio parahaemolyticus. Materiales y métodos. Se determinó la dosis letal media (DL50) para la cepa patógena en juveniles de L. vannamei, bajo los método de inmersión (Inm), inyección (Iny) e incisión + inmersión (Inc+Inm). Luego el efecto de cuatro medicamentos homeopáticos sobre juveniles de L. vannamei retados con Vibrio parahaemolyticus fue evaluado usando el índice la supervivencia y la actividad SOD. Se aplicaron cuatro tratamientos: (1) Mezcla CIB®-HOM Heel-Mix (TH1), constituido por igual proporción v/v, de Cyme-Heel®, Gal-Heel®, Hepa-Heel®, Mucs-Heel® y Chol-Heel®; (2) Mezcla CIB®-HOM Pav-Mix (TH2), constituido por igual proporción v/v de Passiflora incarnata, Valeriana officinalis, Zincum valerianicum e Ignatia amara (Similia®); (3) Heel-Mix/Pav-Mix (TH3) constituido por una combinación 1:1 v/v de los tratamientos TH1 y TH2, y (4) ViT-Mix (TH4), constituido por Vidatox®, y un control (no tratado/infectado). Resultados. Al aplicar los método Inm, Iny e Inc+Inm la DL50 fue de 0.9 x 106; 0.6 x 106 y 0.5 x 106 UFC.mL-1, respectivamente. Los camarones tratados con TH3 y TH4 presentaron una mayor actividad de SOD con respecto al grupo control (p<0.05). Al final del reto, los grupos TH2, TH3 y TH4 tuvieron una supervivencia mayor a la del grupo control (p<0.05). Conclusiones. Los tratamientos homeopáticos (TH3 y TH4), aumentaron la actividad de la enzima SOD y la supervivencia en juveniles de L. vannamei, retados con V. parahaemolyticus. Esto sugiere que los tratamientos homeopáticos empleados tienen potencial como alternativa para el control de V. parahaemolyticus y sus enfermedades asociadas, incluido el síndrome de mortalidad temprana en el cultivo del camarón.

Digestive enzymes of the Californian two-spot octopus, Octopus bimaculoides (Pickford and McConnaughey, 1949).

Octopus bimaculoides is an important commercially fished species in the California Peninsula with aquaculture potential; however, to date limited information is available regarding its digestive physiology. The objective of this study was focused on biochemically characterizing the main enzymes involved in the digestion of O. bimaculoides. Optimum pH, temperature and thermostability were determined for amylases, lipases, trypsin and chymotrypsin; optimum pH and protease inhibitor effect were assessed for acidic and alkaline proteases, and the effect of divalent ions on trypsin and chymotrypsin activity was evaluated in enzymatic extracts from the digestive (DG) and salivary glands (SG) and crop gastric juices (GJ). High amylase activity was detected in GD and GJ whereas this activity is very low in other cephalopods. Salivary glands had the greatest activity in most of the enzyme groups, showing the importance of this organ in digestion. Optimum pH was different depending on the organ and enzyme analyzed. The optimum pH in DG was 3 showing the predominance of acidic proteases in the digestion process. All enzymes were resistant and stable at high temperatures in contrast with other marine species. Trypsin and chymotrypsin activity were highly incremented with the presence of Mg2+, Co2+, Cu2+ and Zn2+ in some tissues. The inhibitor assay showed the importance of serine proteases, metalloproteases and aspartic proteases in the digestive process of this species. This study is the first in assessing the main digestive enzymes of O. bimaculoides and in remarking the importance of other digestive enzyme groups besides proteases in octopuses.

A Solar Position Sensor Based on Image Vision.

Solar collector technologies operate with better performance when the Sun beam direction is normal to the capturing surface, and for that to happen despite the relative movement of the Sun, solar tracking systems are used, therefore, there are rules and standards that need minimum accuracy for these tracking systems to be used in solar collectors' evaluation. Obtaining accuracy is not an easy job, hence in this document the design, construction and characterization of a sensor based on a visual system that finds the relative azimuth error and height of the solar surface of interest, is presented. With these characteristics, the sensor can be used as a reference in control systems and their evaluation. The proposed sensor is based on a microcontroller with a real-time clock, inertial measurement sensors, geolocation and a vision sensor, that obtains the angle of incidence from the sunrays' direction as well as the tilt and sensor position. The sensor's characterization proved how a measurement of a focus error or a Sun position can be made, with an accuracy of 0.0426° and an uncertainty of 0.986%, which can be modified to reach an accuracy under 0.01°. The validation of this sensor was determined showing the focus error on one of the best commercial solar tracking systems, a Kipp & Zonen SOLYS 2. To conclude, the solar tracking sensor based on a vision system meets the Sun detection requirements and components that meet the accuracy conditions to be used in solar tracking systems and their evaluation or, as a tracking and orientation tool, on photovoltaic installations and solar collectors.

Homeopathy outperforms antibiotics treatment in juvenile scallop Argopecten ventricosus: effects on growth, survival, and immune response.

BACKGROUND: Mortality from vibriosis in mollusk production is attributed to pathogenic bacteria, particularly Vibrio alginolyticus. Use of increasingly potent antibiotics has led to bacterial resistance and increased pathogenicity. Alternatives in sanitation, safety, and environmental sustainability are currently under analysis. To-date, homeopathy has been investigated in aquaculture of freshwater fish, but not in marine mollusks. The effect of the homeopathic complexes in the growth, survival, and immune response of the Catarina scallop Argopecten ventricosus were assessed. METHODS: A bioassay to assess the potential of homeopathy in improving cultivation of juvenile A. ventricosus was conducted for 21 days, with a final challenge of 120 h with V. alginolyticus. The experimental design included two homeopathic formulas The homeopathic complex Passival, consisting of Passiflora incarnata 30 CH, Valeriana officinalis 30 CH, Ignatia amara 30 CH and Zincum valerianicum 30 CH plus Phosphoricum acid 30 CH (treatment TH1) or Silicea terra 30 CH (TH2), two antibiotics (ampicillin = AMP, oxytetracycline = OXY), and two reference treatments (without homeopathic or antibiotic treatment = CTRL, ethanol 30° GL = ETH). Additionally, a negative control CTRL- (untreated/uninfected) is included in the challenge test. Juvenile scallops (4.14 ± 0.06 mm, 13.33 mg wet weight) were cultivated in 4 L tanks provided with aerated, filtered (1 µm), and UV-sterilized seawater that was changed every third day. They were fed a blend of the microalgae Isochrysis galbana and Chaetoceros calcitrans (150,000 cells mL-1 twice a day). All treatments were directly added to the tank water and then 500 mL challenge units were inoculated with 1 × 107 CFU/mL (LD50) of V. alginolyticus. RESULTS: Juveniles grew significantly larger and faster in height and weight with TH2 compared to the ETH and CTRL (P < 0.05, ANOVA). Higher concentrations of proteins occurred in scallops exposed to TH2 (160.57 ± 7.79 mg g-1), compared to other treatments and reference treatments. Higher survival rate during the challenge bioassay occurred with TH1 (85%), compared to AMP (53%), OXY (30%), and CTRL (0%), and superoxide dismutase (P < 0.05) was significantly higher in scallops treated with TH1, compared to other treatments and reference treatments. CONCLUSIONS: Homeopathic treatments improved growth and survival and enhanced survival against V. alginolyticus in juvenile A. ventricosus. This suggests that homeopathy is a viable treatment for this mollusk to reduce use of antibiotics in scallops and its progressive increase in pathogenicity in mollusk hatcheries.

Effect of mixed-Bacillus spp isolated from pustulose ark Anadara tuberculosa on growth, survival, viral prevalence and immune-related gene expression in shrimp Litopenaeus vannamei.

The widespread overuse of antibiotics in aquaculture has led to the emergence of antibiotic-resistance shrimp pathogens, the negative impact on shrimp gut microbiota, and the presence of antimicrobial residues in aquaculture products, with negative consequences on human health. Alternatively, probiotics have positive effects on immunological responses and productive performance of aquatic animals. In this study, three probiotic bacteria, (Bacillus licheniformis MAt32, B. subtilis MAt43 and B. subtilis subsp. subtilis GAtB1), isolated from the Anadara tuberculosa were included in diets for juvenile shrimp, Litopenaeus vannamei, to evaluate their effects on growth, survival, disease prevalence, and immune-related gene expression. Shrimp naturally infected with WSSV and IHHNV were fed with the basal diet (control, T1) and diets supplemented with four levels of bacilli probiotic mix (1:1:1) at final concentration of (T2) 1 × 106, (T3) 2 × 106, (T4) 4 × 106, and (T5) 6 × 106 CFU g-1 of feed. The specific growth rate of shrimp was significantly higher in T2 than in T1 (control) treatment, and the final growth as well as the survival were similar among treated groups. The prevalence of WSSV and IHHNV infected shrimp was reduced in T2 and T4 treatments, respectively, compared with control. The mRNA expression of proPO gene was higher in treatment T4 than control. The LvToll1 gene was significantly up-regulated in treatments T4 and T5 compared to control. The SOD gene was up-regulated in treatment T5 compared to control. In contrast, the mRNA expression of the Hsp70 gene was down-regulated in treatments T4 and T5 respect to control, and the TGase gene remained unaffected by the level of bacillus probiotic mix. As conclusion, the bacilli probiotic mix (Bacillus spp.) enhanced immune-related gene expression in WSSV and IHHNV naturally infected shrimp. This is the first report of probiotic potential of bacteria isolated from A. tuberculosa on the immune response and viral prevalence in shrimp Litopenaeus vannamei.

Isolation and in vitro selection of actinomycetes strains as potential probiotics for aquaculture.

AIM: This study was designed to describe a series of in vitro tests that may aid the discovery of probiotic strains from actinomycetes. MATERIALS AND METHODS: Actinomycetes were isolated from marine sediments using four different isolation media, followed by antimicrobial activity and toxicity assessment by the agar diffusion method and the hemolysis of human blood cells, respectively. Extracellular enzymatic production was monitored by the hydrolysis of proteins, lipids and carbohydrates. Tolerance to different pH values and salt concentrations was also determined, followed by hydrophobicity analysis and genetic identification of the most promising strains. RESULTS: Five out of 31 isolated strains showed antimicrobial activity against three Vibrio species. Three non-hemolytic strains (N7, RL8 and V4) among these active isolates yielded positive results in hydrophobicity tests and exhibited good growth at salt concentrations ranging from 0% to 10%, except strain RL8, which required a salt concentration >0.6%. Although these strains did not grow at pH<3, they showed different enzymatic activities. Phylogenetic analysis revealed that strains N7 and V4 have more than 99% identity with several Streptomyces species, whereas the closest matches to strain RL8 are Streptomyces panacagri and Streptomyces flocculus, with 98% and 98.2% similarity, respectively. CONCLUSION: Three actinomycetes strains showing probiotic-like properties were discovered using several in vitro tests that can be easily implemented in different institutions around the world.

Changes in the composition and diversity of the bacterial microbiota associated with oysters (Crassostrea corteziensis, Crassostrea gigas and Crassostrea sikamea) during commercial production.

The resident microbiota of three oyster species (Crassostrea corteziensis, Crassostrea gigas and Crassostrea sikamea) was characterised using a high-throughput sequencing approach (pyrosequencing) that was based on the V3-V5 regions of the 16S rRNA gene. We analysed the changes in the bacterial community beginning with the postlarvae produced in a hatchery, which were later planted at two grow-out cultivation sites until they reached the adult stage. DNA samples from the oysters were amplified, and 31 008 sequences belonging to 13 phyla (including Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes) and 243 genera were generated. Considering all life stages, Proteobacteria was the most abundant phylum, but it showed variations at the genus level between the postlarvae and the adult oysters. Bacteroidetes was the second most common phylum, but it was found in higher abundance in the postlarvae than in adults. The relative abundance showed that the microbiota that was associated with the postlarvae and adults differed substantially, and higher diversity and richness were evident in the postlarvae in comparison with adults of the same species. The site of rearing influenced the bacterial community composition of C. corteziensis and C. sikamea adults. The bacterial groups that were found in these oysters were complex and metabolically versatile, making it difficult to understand the host-bacteria symbiotic relationships; therefore, the physiological and ecological significances of the resident microbiota remain uncertain.

Molecular analysis of bacterial microbiota associated with oysters (Crassostrea gigas and Crassostrea corteziensis) in different growth phases at two cultivation sites.

Microbiota presumably plays an essential role in inhibiting pathogen colonization and in the maintenance of health in oysters, but limited data exist concerning their different growth phases and conditions. We analyzed the bacterial microbiota composition of two commercial oysters: Crassostrea gigas and Crassostrea corteziensis. Differences in microbiota were assayed in three growth phases: post-larvae at the hatchery, juvenile, and adult at two grow-out cultivation sites. Variations in the microbiota were assessed by PCR analysis of the 16S rRNA gene in DNA extracted from depurated oysters. Restriction fragment length polymorphism (RFLP) profiles were studied using Dice's similarity coefficient (Cs) and statistical principal component analysis (PCA). The microbiota composition was determined by sequencing temperature gradient gel electrophoresis (TGGE) bands. The RFLP analysis of post-larvae revealed homology in the microbiota of both oyster species (Cs > 88 %). Dice and PCA analyses of C. corteziensis but not C. gigas showed differences in the microbiota according to the cultivation sites. The sequencing analysis revealed low bacterial diversity (primarily ß-Proteobacteria, Firmicutes, and Spirochaetes), with Burkholderia cepacia being the most abundant bacteria in both oyster species. This study provides the first description of the microbiota in C. corteziensis, which was shown to be influenced by cultivation site conditions. During early growth, we observed that B. cepacia colonized and remained strongly associated with the two oysters, probably in a symbiotic host-bacteria relationship. This association was maintained in the three growth phases and was not altered by environmental conditions or the management of the oysters at the grow-out site.

[Effect of probiotic bacteria on survival and growth of Cortez oyster larvae, Crassostrea corteziensis (Bivalvia: Ostreidae)]. / Efecto de bacterias probióticas en el cultivo larvario del ostión de placer Crassostrea corteziensis (Bivalvia: Ostreidae).

Disease control problems have major constraints in aquaculture production, and the use of probiotics in larviculture is a valid alternative to antibiotics. This study analyzed the effect of probiotic bacteria on survival and final size of Cortez oyster larvae Crassostrea corteziensis. Two different probiotic concentrations were evaluated, 1 x 10(4) and 1 x 10(5) CFU/ml of Lactic acid bacteria (strain NS61) isolated from Nodipecten subnodosus, and bacilli isolated from the white leg shrimp, Litopenaeus vannamei (Pseudomonas aeruginosa, strain YC58) and C. corteziensis (Burkholderia cepacia, strain Y021). Bacteria were added directly into culture tanks, starting the bioassays from veliger to pediveliger stages as follows: (1) Control, without probiotics; (2) lactic acid bacteria (Lb); (3) bacilli mix (Mb) in a proportion 1:1. Results showed a higher larval survival with Lb and Mb at a dose of 1 x 10(4) CFU/ml compared to the control group. Larvae exposed to Mb at 1 x 10(5) CFU/ml showed higher survival than Lb and control. Larval final size was not significantly increased with the tested probiotics, but larvae treated with Lb at 1 x 10(5) CFU/ml showed less survival rate than those treated at 1 x 10(4) CFU/ml. This study showed the beneficial effect of these probiotics, added individually or mixed in C. corteziensis larvae culture.

Efecto de bacterias probióticas en el cultivo larvario del ostión de placer Crassostrea corteziensis (Bivalvia: Ostreidae)

Effect of probiotic bacteria on survival and growth of Cortez oyster larvae, Crassostrea corteziensis (Bivalvia: Ostreidae). Disease control problems have major constraints in aquaculture production, and the use of probiotics in larviculture is a valid alternative to antibiotics. This study analyzed the effect of probiotic bacteria on survival and final size of Cortez oyster larvae Crassostrea corteziensis. Two different probiotic concentrations were evaluated, 1x10(4) and 1x10(5)CFU/ml of Lactic acid bacteria (strain NS61) isolated from Nodipecten subnodosus, and bacilli isolated from the white leg shrimp, Litopenaeus vannamei (Pseudomonas aeruginosa, strain YC58) and C. corteziensis (Burkholderia cepacia, strain Y021). Bacteria were added directly into culture tanks, starting the bioassays from veliger to pediveliger stages as follows: (1) Control, without probiotics; (2) lactic acid bacteria (Lb); (3) bacilli mix (Mb) in a proportion 1:1. Results showed a higher larval survival with Lb and Mb at a dose of 1x10(4)CFU/ml compared to the control group. Larvae exposed to Mb at 1x10(5)CFU/ml showed higher survival than Lb and control. Larval final size was not significantly increased with the tested probiotics, but larvae treated with Lb at 1x105CFU/ml showed less survival rate than those treated at 1x10(4)CFU/ml. This study showed the beneficial effect of these probiotics, added individually or mixed in C. corteziensis larvae culture. Rev. Biol. Trop. 59 (1): 183-191. Epub 2011 March 01.

El ostión de placer u ostra del Cortés (Crassostrea corteziensis) se considera como una especie con potencial para ser cultivada en gran escala. Sin embargo, al igual que en otros bivalvos, la alta mortalidad que se presenta durante la etapa larvaria y juvenil, es el principal problema que limita el desarrollo del cultivo en el laboratorio. Un método que está ganando aceptación en la acuicultura es el uso de bacterias probióticas para controlar patógenos microbianos. Este estudio analiza el efecto de estas bacterias en la supervivencia y talla final de larvas de ostión de placer Crassostrea corteziensis. Se utilizó una cepa de bacterias ácido lácticas (cepa NS61) aisladas N. subnodosus, así como de bacilos aislados de L. vannamei (Pseudomonas aeruginosa, cepa YC58) y de C. corteziensis (Burkholderia cepacia, cepa Y021). Las cepas se evaluaron por inmersión en cultivos larvarios de C. corteziensis a dos concentraciones diferentes, hasta completar el estadio pediveliger. Los organismos se trataron con bacterias ácido lácticas (Lb), una mezcla de bacilos (Lb) en proporción 1:1 y un grupo control. La concentración de 1x10(4)UFC/ml registró una mayor supervivencia con Lb y Mb respecto al grupo control. La supervivencia con Mb a una concentración de 1x10(5)UFC/ml fue mayor que la del grupo control y del grupo tratado con Lb. Los resultados mostraron que las larvas de C. corteziensis tratadas con probióticos no incrementaron significativamente su talla respecto a las larvas del grupo control. Mientras que las tratadas con Lb a la concentración mayor, 1x10(5)UFC/ml, mostraron una disminución de la supervivencia respecto a las tratadas con 1x10(4)UFC/ml. Este estudio demostró el efecto benéfico de cepas probióticas utilizadas individualmente o en mezcla en el cultivo larvario de C. corteziensis.