Combined administration routes of marine yeasts enhanced immune-related genes and protection of white shrimp (Penaeus vannamei) against Vibrio parahaemolyticus.

Antibiotic usage to control infectious diseases in shrimp aquaculture has led to serious problems on antimicrobial resistance. An alternative to mitigate this issue is the use of probiotics, which can be easily administered by feed and water. This study examines immunomodulatory and protective effects of the marine yeasts Debaryomyces hansenii CBS8339 (Dh) and Yarrowia lipolytica Yl-N6 (Yl) -alone and mixed-in white shrimp Penaeus vannamei post-larvae. Administration routes (fed and water alone or in combination), supplementation frequency and time elapsed after the last dietary supplement were tested on growth and gene expression of penaeidin, lectin, lysozyme, superoxide dismutase, catalase, and peroxidase, as well as survival upon Vibrio parahaemolyticus IPNGS16 challenge. Penaeidin and lectin genes were upregulated in post-larvae fed orally with Yl or combined Dh + Yl. Higher growth and survival for yeast supplementation treatments were observed compared to the control group, mainly when yeasts (Dh + Yl) and administration routes (feed and water) were combined. In conclusion, mixed yeast and combined administration routes improved growth and immunity against V. parahaemolyticus.

Physiological and antioxidant response of Litopenaeus vannamei against Vibrio parahaemolyticus infection after feeding supplemented diets containing Dunaliella sp. flour and ß-glucans.

The presence of pathogen agents in shrimp farming is the main obstacle for successful aquaculture. Vibrio species are naturally part of water because they play an important role as opportunistic bacteria. Vibrio parahaemolyticus was identified as the causative agent of the Early Mortality Syndrome in 2009, causing the loss of shrimp farming worldwide. Dunaliella sp. flour has been tested against Vibrio infection proving to be an effective prophylactic method that decreases mortality and improves physiological and immune response in Litopenaeus vannamei. Juvenile shrimp were exposed to 2% Dunaliella sp. flour and commercial 1.1% ß -glucan diet provided every other day for 15 days and a posterior infection with V. parahaemolyticus (1 × 106 CFU/mL). To evaluate shrimp stress status, some parameters as glucose, lactate, cholesterol, triglycerides, relative superoxide dismutase (SOD) gene expression and circulating hemocytes were analyzed in hemolymph at zero and seven days before infection and at 0, 24, and 48 h post-infection. L. vannamei fed with Dunaliella sp. showed 93% and ß -glucan 87% survival, compared with 79% in the infected control group. Additionally, Dunaliella sp. improved hemocyte and lipid concentrations compared to ß -glucan while both immunostimulants showed an increase in SOD response against bacteria. The addition of 2% Dunaliella sp. every other day in L. vannamei diet enhanced stress response against V. parahaemolyticus infection.

Dietary supplementation of marine yeast Yarrowia lipolytica modulates immune response in Litopenaeus vannamei.

The immunostimulatory potential of the marine yeast Yarrowia lipolytica (D1 and N6 strains) administered orally was evaluated in the white shrimp Litopenaeus vannamei. Yeasts and commercial glucans were mixed with a commercial feed to formulate diets with a 1.1% concentration of immunostimulants. The shrimp were fed daily for a period of 21 days. Weekly determinations were performed for immunological parameters in hemolymph, such as total hemocyte count (THC), lysozyme activity (LYZ), prophenoloxidase activity, antioxidant enzymatic activities (superoxide dismutase [SOD], catalase [CAT], and peroxidases), and bactericidal activity against Vibrio parahaemolyticus. Expression profiles of penaeidin (PEN), lysozyme (LYZ), and prophenoloxidase (proPO) immune genes were evaluated in hemocytes. In general, an increase in the immune parameters was observed in shrimp fed yeast diet compared to glucan and the control diets. Yarrowia lipolytica, especially strain N6, provided maximum immunostimulatory effects evidenced by the increase of immune parameters (THC, LYZ, SOD, CAT) and gene expression profile. In conclusion, this study demonstrated that Y. lipolytica had immunostimulatory effects and increased bactericidal activity in L. vannamei hemocytes against V. parahaemolyticus. These findings open the path for the potential application of Y. lipolytica-based immunostimulant for shrimp aquaculture.

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.

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.

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.

Antioxidant screening and phenolic content of ethanol extracts of selected Baja California Peninsula macroalgae.

The Baja California Peninsula in México has about 670 species of macroalgae along its coast. Species richness increases the probability of finding native macroalgae with potential as sources of bioactive compounds suitable for health, pharmacological, and cosmetic ingredients. To understand the biotechnological value of macroalgae from the peninsula, ethanol extracts from 17 macroalgae (four Chlorophyta, six Rhodophyta, seven Ochrophyta) were screened for antioxidant potential. To determine the antioxidant capacity of macroalgal extracts, 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing antioxidant power, and nitric oxide radical scavenging as well as total phenolic content (TPC) were measured. Extracts of the brown macroalgae were most active. Among these, Eisenia arborea, Padina concrecens, and Cystoseira osmundacea had the highest TPC and exhibited the strongest radical scavenging activities. Correlations were found between TPC macroalgal and scavenging capacity, indicating an important role of polyphenols as antioxidants. This suggests that some brown macroalgae from Baja California Peninsula may be a good source of natural bioactive compounds.

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.

Antioxidant Activity and Oxidative Damage Associated with Seeding Surgery for Pearl Culture in the Winged Pearl Oyster Pteria sterna.

To evaluate the antioxidant activity and oxidative damage by relaxing, wounding, and seeding of a saibo of different origin on Pteria sterna hosts, five oyster treatments were included: (1) relaxed (REL) but neither wounded nor seeded; (2) relaxed and wounded (WOU) but not seeded; (3) relaxed, wounded, and seeded with an allograft (ALL); (4) relaxed, wounded, and seeded with an autograft (AUT); and (5) unrelaxed, unwounded, and unseeded as control (CTR). Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and thiobarbituric acid (TBARS) activity were quantified between 3 and 24 h post-seeding. Compared to the CTR oysters, which did not suffer oxidative stress, SOD activity significantly decreased in the gonad and digestive gland in all treatments and decreased in mantle tissue in AUT oysters; this indicates that the entire process of preparing oysters for pearl culture (relaxing, wounding, and seeding) generates oxidative stress in the host. CAT was not a sensitive enzyme for measuring the short-term response of oysters to the wounding-seeding processes but rather a more prolonged or chronic stress. Similar to SOD, the lowest GPx and TBARS activity in seeded oysters evidenced their susceptibility to oxidative stress and damage, particularly in the WOU treatment. Evidence from this study indicates that SOD is a more sensitive enzyme for measuring the short-term response of the host oyster to the wounding and seeding of a saibo. It is also clear that the host undergoes stress at all stages of the pearl culture process, mostly during gonad wounding and regardless of the origin of saibo.

Probiotics in the intestinal tract of juvenile whiteleg shrimp Litopenaeus vannamei: modulation of the bacterial community.

Molecular analysis of the 16S rDNA of the intestinal microbiota of whiteleg shrimp Litopenaeus vannamei was examined to investigate the effect of a Bacillus mix (Bacillus endophyticus YC3-b, Bacillus endophyticus C2-2, Bacillus tequilensisYC5-2) and the commercial probiotic (Alibio(®)) on intestinal bacterial communities and resistance to Vibrio infection. PCR and single strain conformation polymorphism (SSCP) analyses were then performed on DNA extracted directly from guts. Injection of shrimp with V. parahaemolyticus at 2.5 × 10(5) CFU g(-1) per shrimp followed 168 h after inoculation with Bacillus mix or the Alibio probiotic or the positive control. Diversity analyses showed that the bacterial community resulting from the Bacillus mix had the highest diversity and evenness and the bacterial community of the control had the lowest diversity. The bacterial community treated with probiotics mainly consisted of α- and γ-proteobacteria, fusobacteria, sphingobacteria, and flavobacteria, while the control mainly consisted of α-proteobacteria and flavobacteria. Differences were grouped using principal component analyses of PCR-SSCP of the microbiota, according to the time of inoculation. In Vibrio parahaemolyticus-infected shrimp, the Bacillus mix (~33 %) induced a significant increase in survival compared to Alibio (~21 %) and the control (~9 %). We conclude that administration of the Bacillus mix induced modulation of the intestinal microbiota of L. vannamei and increased its resistance to V. parahaemolyticus.

Toxicity of cultured bullseye puffer fish Sphoeroides annulatus.

The toxin content in various life cycle stages of tank-cultivated bullseye puffer (Sphoeroides annulatus) were analyzed by mouse bioassay and ESI-MS spectrometry analysis. The presence of toxin content was determined in extracts of sperm, eggs, embryo, larvae, post-larvae, juvenile, pre-adult, and adult fish, as well as in food items used during the cultivation of the species. Our findings show that only the muscle of juveniles, the viscera of pre-adults, and muscle, liver, and gonad of adult specimens were slightly toxic (<1 mouse unit). Thus, cultivated S. annulatus, as occurs with other cultivated puffer fish species, does not represent a food safety risk to consumers. This is the first report of toxin analysis covering the complete life stages of a puffer fish under controlled conditions.

[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.

Acute mercury toxicity and bioconcentration in shrimp Litopenaeus vannamei juveniles: Effect of low salinity and chemical speciation.

This study evaluated low salinity effect on acute Hg toxicity and bioconcentration capacity in L. vannamei juveniles (8.4 ± 0.7 g), because this species is frequently exposed to hypo-osmotic environments in its natural habitat, and in farming ponds. Hg LC50 values were 1723, 1272, 983 and 536 µg L-1 at salinity of 5 ppt (parts per thousand); 2203, 1740, 1340 and 873 µg L-1 at 10 ppt; and 7013, 5693, 1759 and 1534 µg L-1 at 25 ppt for 24, 48, 72 and 96 h, respectively. After 96 h, acute Hg toxicity in shrimp was significantly higher in salinity of 5 ppt than in 25 ppt; likewise, Hg bioconcentration in shrimp exposed to different Hg treatments was statistically greater in salinity of 5 ppt than in 25 ppt. The chemical speciation calculated in experimental waters suggested that neutral chemical Hg species (HgCl2 and HgClOH) were the most bioavailable because their fractions (51-62%) increased when salinity decreased. Therefore, the inverse relationship between Hg toxicity and salinity was due to osmotic stress and the neutral chemical Hg species fractions that increased at lower salinities. Hg bioconcentration factors indicated that the higher Hg waterborne concentrations were the most saturated uptake and storage mechanisms in shrimp. Thus, Hg concentrations in organisms did not increase in proportion to waterborne Hg concentrations in the three salinities. These results support the hypothesis of an effect of low salinity on Hg toxicity and bioconcentration capacity in L. vannamei. The safe Hg concentrations 5.4, 8.7 and 15.3 µg L-1 were proposed for shrimp exposed to salinity of 5, 10 and 25 ppt, respectively. This information allows recognizing risky environments for both wild and cultured healthy growth of these shrimp, which can help decision makers on coastal management and shrimp pond managers to have better water quality.

Genetically-engineered plants yield an orally immunogenic PirA-like toxin from Vibrio parahaemolyticus.

Vibrio parahaemolyticus is the main etiological agent of human gastroenteritis by seafood consumption and some strains from this species causing the Acute Hepatopancreatic Necrosis Disease in shrimp have been recently reported. The PirA-like toxin from V. parahaemolyticus (ToxA) has been recently reported as an attractive antigen implicated in subunit vaccine development. Since plants are attractive hosts for the production and delivery of vaccines in the present study plants expressing ToxA were developed to account with a low cost platform for the production and oral delivery of ToxA. Tobacco plants were genetically engineered by Agrobacterium-mediated transformation to stably integrate the ToxA-coding gene into the nuclear genome. Transgenic lines were rescued in kanamycin-containing medium and analyzed by ELISA to determine ToxA yields observing levels up to 9 µg g-1 FW leaf tissues. Western blot analysis confirmed the presence of the ToxA protein in plant extracts. Immunogenicity assessment of the plant-made ToxA was performed in mice, comprising a 4-dose oral immunization scheme; revealing the induction of anti-ToxA humoral responses (IgG in serum and IgA in feces). This study opens the path for the development of low cost plant-based vaccines against Vibrio parahaemolyticus.

Effect of low salinity on acute arsenic toxicity and bioconcentration in shrimp Litopenaeus vannamei juveniles.

This study investigated acute arsenic toxicity and bioconcentration capacity in Litopenaeus vannamei because it has been frequently exposed to lower salinities than its isosmotic point (25 g L-1). Juveniles (9.9 ±â€¯0.4 g) were exposed to low (5-10 g L-1) and isosmotic salinity (25 g L-1) levels; As values were 30.8, 20.2, 16.8 and 13.9 mg L-1 at 5 g L-1; 30.4, 19.1, 16.8 and 14.8 mg L-1 at 10 g L-1; 31.5, 19.0, 15.0 and 11.9 mg L-1 at 25 g L-1 at 24, 48, 72 and 96 h LC50, respectively. No significant differences were found among As LC50 values calculated for different salinity levels and same exposure times, concluding that low salinity did not affect shrimp sensitivity to As. Likewise, no significant differences were observed in As bioconcentration in shrimp exposed to the same waterborne As and distinct salinity, supporting the results of acute toxicity. Bioconcentration factors of As maintained a relatively stable tendency where all values (0.8 ±â€¯0.2 to 1.7 ±â€¯0.4) were statistically comparable to 1, indicating that As was accumulated in a similar proportion to waterborne As concentration at three salinity levels. This study proposed 135.3 ±â€¯12.1 µg L-1 for salinities from 5 to 25 g L-1 as provisional safe As concentration. According to these results, the hypothesis that sustains an effect of low salinity on As acute toxicity and its bioconcentration capacity cannot be acceptable. Therefore, the information provided allows knowing the threshold levels of As in water to avoid ecological and economic losses.

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.

Immunostimulant effects and potential application of ß-glucans derived from marine yeast Debaryomyces hansenii in goat peripheral blood leucocytes.

Debaryomyces hansenii has been described to be effective probiotic and immunostimulatory marine yeast in fish. Nonetheless, to the best of our knowledge, it has been not assayed in ruminants. This study attempts to describe the immunostimulatory effects of its ß-glucan content through in vitro assays using goat peripheral blood leukocytes at 24 h of stimulation. The structural characterization of yeast glucans by proton nuclear magnetic resonance indicated structures containing (1-6)-branched (1-3)-ß-D-glucan. In vitro assays using peripheral blood leukocytes stimulated with ß-glucans derived from three D. hansenii strains and zymosan revealed that ß-glucans significantly increased cell immune parameters, such as phagocytic ability, reactive oxygen species production (respiratory burst), peroxidase activity and nitric oxide production. Antioxidant enzymes revealed an increase in superoxide dismutase and catalase activities in leukocytes stimulated with yeast ß-glucans. This study revealed that yeast ß-glucans were able to activate dectin-1 mRNA gene expression in leukocytes. The TLR4 gene expression was up-regulated in leukocytes after stimulation with yeast ß-glucans. In conclusion, ß-glucans were able to modulate the immune system by promoting cell viability, phagocytic activity, antioxidant immune response and immune-related gene expression in leukocytes. Therefore, ß-glucans derived from Debaryomyces hansenii should be considered a potential immunostimulant for goat production systems.

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.

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.