Dosage and frequency effects of the microalgae Dunaliella sp. on the diet of Litopenaeus vannamei challenged with Vibrio parahaemolyticus.

Carotenoid sources in shrimp diets have shown to be effective for improving survival, growth, reproductive capacity, stress resistance, and also for diminishing disease. Dunaliella sp. is known to have high levels of ß-carotenes, which works as pro-vitamin A, enhancing the immune response in shrimp. However, the administration of Dunaliella sp. in shrimp diet needs to be evaluated to determine the appropriate dose and frequency of administration needed to optimize performance in cultured white shrimp. Diets with three different concentrations of Dunaliella sp. flour (1.5, 2 and 3%) were tested, and each one was administered at three different time frequencies: daily, and at 3- and 7-days intervals. Shrimp fed for 20 days were then infected with Vibrio parahaemolyticus (1 × 106 CFU/mL). Hemolymph parameters including protein, glucose, lactate, cholesterol and triglycerides were analyzed to evaluate shrimp stress status. Additionally, L. vannamei innate non-specific immune response was examined by evaluating the activity of prophenoloxidase (proPO), phenoloxidase (PO) and superoxide dismutase (SOD) in hemolymph; shrimp survival was also recorded. Survival after infection with V. parahaemolyticus was higher for shrimp fed with diets consisting of 2% Dunaliella sp. administered every 3 and 7 days. Shrimp fed a diet consisting of 2% or 3% Dunaliella sp. administered every third day showed positive physiological and immune responses to infection. A decrease in lipid oxidation in plasma triglycerides was observed at 48 h post inoculation in shrimp fed at all diets regimes due to Dunaliella sp. antioxidant action. Experimental results suggest the importance of Dunaliella sp. dosage and feeding frequency in L. vannamei diet to improve the survival and immune response.

Survival of Litopenaeus vannamei shrimp fed on diets supplemented with Dunaliella sp. is improved after challenges by Vibrio parahaemolyticus.

Survival of Litopenaeus vannamei was evaluated during a Vibrio parahaemolyticus infection. This bacteria has been causing significant economic losses in the shrimp industry due to the appearance of early mortality syndrome (EMS), also known as acute hepatopancreatic necrosis disease (AHPND). Control of bacteria in ponds is difficult to achieve with antibiotics due to environmental infections and antibiotic resistance. New methods have been proposed to control and prevent the impact of bacterial infections. The physiological response indicated by plasma biochemical parameters in shrimp can determine their health and stress status. Meanwhile, shrimp immunology is the key factor in establishing strategies to control diseases. Immunostimulants are the best alternative to antibiotics to prevent or minimize disease damage, and at the same time, these stimulants improve the immune system in shrimp. Four diets containing 1.5, 2, 2.5 and 3% of Dunaliella sp. with high ß-carotene content were tested in the present study. After 20days of feeding, organisms were infected with V. parahaemolyticus. Protein, glucose, lactate, triglyceride and cholesterol levels, as well as activity of prophenoloxidase and phenoloxidase, were determined 48 h post-infection (hpi). Shrimp fed a diet with 3% Dunaliella sp. showed the highest survival. Glucose, cholesterol, and triglyceride levels, as well as prophenoloxidase and phenoloxidase activity, were not observed to be suitable indicators during this bacterial infection. The results indicated that the inclusion of Dunaliella sp. in diet increases survival in L. vannamei infected with V. parahaemolyticus.

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.

[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.

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.

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.

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.