Felipe Ascencio-Valle

Oral delivery of live yeast Debaryomyces hansenii modulates the main innate immune parameters and the expression of immune-relevant genes in the gilthead seabream (Sparus aurata L.)

Microorganisms isolated from fish can be used as prophylactic tools for aquaculture in the form of probiotic preparations. The purpose of this study was to evaluate the effects of dietary administration of the live yeast Debaryomyces hansenii CBS 8339 on the gilthead seabream (Sparus aurata L.) innate immune responses. Seabream were fed control or D. hansenii-supplemented diets (10(6) colony forming units, CFU g(-1)) for 4 weeks. Humoral (seric alternative complement and peroxidase activities), and cellular (peroxidase, phagocytic, respiratory burst and cytotoxic activities) innate immune parameters and antioxidant enzymes (superoxide dismutase (SOD) and catalase (CAT)) were measured from serum, head-kidney leucocytes and liver, respectively, after 2 and 4 weeks of feeding. Expression levels of immune-associated genes, Hep, IgM, TCR-beta, NCCRP-1, MHC-II alpha, CSF-1R, C3, TNF-alpha and IL-1 beta, were also evaluated by real-time PCR in head-kidney, liver and intestine. Humoral immune parameters were not significantly affected by the dietary supplementation of yeast at any time of the experiment. On the other hand, D. hansenii administration significantly enhanced leucocyte peroxidase and respiratory burst activity at week 4. Phagocytic and cytotoxic activities had significantly increased by week 2 of feeding yeast but unchanged by week 4. A significant increase in liver SOD activity was observed at week 2 of feeding with the supplemented diet; however CAT activity was not affected by the dietary yeast supplement at any time of the experiment. Finally, the yeast supplemented diet down-regulated the expression of most seabream genes, except C3, in liver and intestine and up-regulated all of them in the head-kidney. These results strongly support the idea that live yeast Debaryomyces hansenii strain CBS 8339 can stimulate the innate immune parameters in seabream, especially at cellular level.

Phenoloxidase activity in larval and juvenile homogenates and adult plasma and haemocytes of bivalve molluscs.

Phenoloxidase (PO) activity was studied in larval and juvenile homogenates and in the plasma and haemocytes of adult Crassostrea gigas, Argopecten ventricosus, Nodipecten subnodosus, and Atrina maura. Samples were tested for the presence of PO activity by incubation with the substrate L-3, 4-dihydroxyphenylalanine using trypsin, alpha-chymotrypsin, laminarin, lipopolysaccharides (LPS), and sodium dodecyl sulphate (SDS) to elicit activation of prophenoloxidase (proPO) system. PO activity was not detected in larval homogenate. In juvenile homogenate, PO activity was found only in C. gigas and N. subnodosus. PO activity was present in adult samples and was enhanced by elicitors in the plasma of all species tested, but in haemocyte lysate supernatant (HLS) of only N. subnodosus. Activation of proPO by laminarin was suppressed by a protease inhibitor cocktail (P-2714) in plasma and HLS of all species tested.

Effects of Dietary Arginine on Hematological Parameters and Innate Immune Function of Channel Catfish

The effects of elevated dietary arginine on the hematology and immune function of juvenile channel catfish Ictalurus punctatus were evaluated by means of in vivo and in vitro experiments. Healthy juvenile channel catfish (average weight, 34.8 g) were fed casein-gelatin-based diets containing 28% crude protein and supplemented with crystalline L-arginine (ARG) at 0.5, 1, 2, or 4% of diet. An intact-protein diet containing 1.3% arginine also was included to investigate the effects of amino acid form (crystalline-free amino acids versus intact protein). Each purified diet was fed to apparent satiation to triplicate groups of fish for 6 weeks. At the end of the experimental feeding period, the fish were injected intraperitoneally with two doses (3 d apart) of 2 mg lipopolysaccharide/kg body weight. Six days after the initial injection, the fish were anesthetized and tissue samples were obtained to evaluate hematological and humoral and cellular immune parameters, including phagocytic activity of peritoneal macrophages, hemoglobin, hematocrit, mean corpuscular volume (MCV), blood cell counts, plasma protein, and hepatic superoxide dismutase activity. High dietary levels (4% ARG) resulted in significantly higher levels of hemoglobin, hematocrit, and circulating erythrocytes. Dietary ARG did not significantly affect MCV and the number of circulating leukocytes, lymphocytes, eosinophils, and monocytes. In vitro, a moderate level (2 mM) of ARG in the culture media was found to be ideal in significantly enhancing phagocytosis. This study demonstrates that some aspects of the immune system of channel catfish are sensitive to changes in dietary ARG.

Dietary administration of β-1,3/1,6-glucan and probiotic strain Shewanella putrefaciens, single or combined, on gilthead seabream growth, immune responses and gene expression

It is widely known that β-glucans and probiotic bacteria are good immunostimulants for fish. In the present work we have evaluated the dietary effect of β-1,3/1,6-glucan (isolated from Laminarina digitata) and Pdp 11 (Shewanella putrefaciens, probiotic isolated from gilthead seabream skin), single or combined, on growth, humoural (seric level of total IgM antibodies and peroxidase and antiprotease activities) and cellular innate immune response (peroxidase and phagocytic activities of head-kidney leucocytes), as well as the expression of immune-related genes in gilthead seabream (Sparus aurata). Four treatment groups were established: control (non-supplemented diet), Pdp 11 (10(9) cfu g(-1)), β-1,3/1,6-glucan (0.1%) and β-1,3/1,6-glucan + Pdp 11 (0.1% and 10(9) cfu g(-1), respectively). Fish were sampled after 1, 2 and 4 weeks of feeding. Interestingly, all supplemented diets produced increments in the seabream growth rates, mainly the Pdp 11-suplemented diet. Overall, Pdp 11 dietary administration resulted in decreased serum IgM levels and peroxidase activity. However, the seric antiprotease activity was increased in fish fed with both supplements together. Furthermore, β-1,3/1,6-glucan and combined diet increased phagocytic activity after 2 or 4 weeks. At gene level, IL-1β and INFγ transcripts were always up-regulated in HK but only the interleukin reached significance after 4 weeks in the group fed with β-glucan. On the contrary, IgM gene expression tended to be down-regulated being significant after 1 week in seabream specimens fed with β-glucan or β-glucan plus Pdp 11. These results suggest that β-1,3/1,6-glucan and Pdp 11 modulate the immune response and stimulates growth of the gilthead seabream, one of the species with the highest rate of production in Mediterranean aquaculture.

Immune response of gilthead seabream (Sparus aurata) following experimental infection with Aeromonas hydrophila.

The Gram-negative bacteria Aeromonas hydrophila is a heterogeneous organism that causes the disease known as motile aeromonad septicaemia, which is responsible for serious economic loss in seabream culture due to bacterial infections. However, the immune mechanisms involved in this disease in fish are still poorly understood. For the purpose of this study, gilthead seabream (Sparus aurata L.) specimens received a double intraperitoneal injection of bacterial inoculums: a primary infection with 1 × 10(7) cell ml(-1) A. hydrophila, followed by a secondary infection with 1 × 10(8) cell ml(-1) fourteen days later. Changes in cellular innate immune parameters - phagocytosis, respiratory burst activity and peroxidase leucocyte content - were evaluated 24 and 48 h after each injection. Simultaneously, the expression levels of nine immune-relevant genes (TLR, NCCRP-1, HEP, TCR, IgM, MHC-IIα, IL-1β, C3 and CSF-1R) were measured in the head-kidney, spleen, intestine and liver, by using q-PCR. Generally, the results showed a significant decrease in cellular immune responses during the primary infection and a significant enhanced during the second infection, principally in respiratory burst and peroxidase activity, thus indicating a recovery of the immune system against this bacterial pathogen. Finally, transcript levels of immune genes were down-regulated during the first infection, except for the IL-1β gene. In contrast, mRNA expression levels during the re-infection were significantly up-regulated. The results seem to suggest a relatively fast elimination of the bacteria and recovery of fish during the secondary infection.

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.

Ontogenetic variations of hydrolytic enzymes in the Pacific oyster Crassostrea gigas.

Occurrence and level of hydrolytic enzymatic activity (proteases, glycosidases, phosphatases, lipases, and esterases) were studied in oocytes, larvae, juveniles, and adult haemolymph of the Pacific oyster Crassostrea gigas. Samples were obtained as oocyte lysate supernatant, larval homogenate supernatant, juvenile homogenate supernatant, haemocyte lysate supernatant, and plasma. The presence of enzymes was demonstrated by colorimetric and lysoplate assay techniques. Between stages, significant differences in enzymatic activity determined by the colorimetric technique were found. Higher levels of enzymatic activity were found in the adult stage. Lysozyme-like activity was not found in oocytes, but was present in larvae, juveniles, and adults. In larvae, the highest lysozyme-like activity was in 3-d larvae. Juveniles had a 48-fold higher level of lysozyme-like activity, compared with 20-h larvae and was six-fold higher compared with 3-d larvae. In adults, lysozyme-like activity had a five-fold higher level in haemocyte lysate supernatant compared with plasma and was 98-fold higher compared with 20-h larvae. As determined with the API ZYM kit, 19 hydrolytic enzymatic activities were present, in oocytes, larvae, juveniles, and adult haemolymph of C. gigas. The presence of important lysozyme-like activity was confirmed from trochophora larvae (20 h) to adult stages.

Dietary administration of microalgae Navicula sp. affects immune status and gene expression of gilthead seabream (Sparus aurata)

Effects of silage microalgae enriched with a probiotic and lyophilized microalgae were evaluated on main immune parameters and different gene expression of gilthead seabream (Sparus aurata L.). A total of 60 seabream were grouped into 3 treatment diets which were a control diet (commercial diet) without microalgae (C), commercial diet supplemented with silage microalgae Navicula sp. plus Lactobacillus sakei 5-4 (10(6) CFU g(-1)) (SM), and commercial diet supplemented with lyophilized microalgae (LM) for 4 weeks. Generally, the results showed a significant increase in the immune parameters, principally in leucocyte peroxidase, phagocytosis and complement activities in fish fed with SM diet compared to control group. About the gene expression in head-kidney, transcript levels (Interleukin-8, Interleukin-1β and β-defensin) were upregulated in fish fed with SM after 4 weeks of treatments. However, the gene expression was upregulated in intestine from fish fed with LM with significant difference in transferrin and cyclooxygenase 2 gene at 2 weeks, and in occludin, transferrin, interleukin-8 and interleukin-1β at 4 weeks. Finally, about the digestive enzymes, LM diet caused an upregulated of α-amylase and alkaline phosphatase genes at 2 weeks; however SM diet caused an upregulated trypsin gene at 4 weeks. SM diet a higher enhancing effect on gilthead seabream immune parameters than that observed when using LM. Furthermore, dietary administration of microalgae Navicula sp. provokes upregulation of several genes in the gut that correlates with slight inflammation. Further studies are needed to know if this diatom could be useful for administering as diet supplement for farmed fish.

Modulation of the Intestinal Microbiota and Immune System of Farmed Sparus aurata by the Administration of the Yeast Debaryomyces hansenii L2 in Conjunction with Inulin

Fish gastrointestinal tract is one of the most important sites of interaction with the external world. Interactions between the intestinal microbiota and the host modulate the functionality of the intestinal mucosa and gene expression. Application of probiotics, prebiotics and synbiotics for aquatic animals is increasing to improve welfare and promote growth. However, data about the use of probiotics in conjunction with prebiotics, in farmed marine organisms are scarce. The objective of this work is to study the modulation ability of the intestinal microbiota and the immune system of gilthead seabream exerted by the probiotic yeast Debaromycess hansonii L2 in conjunction with the prebiotic inulin. Fish were fed a commercial diet (control diet I), and with the same diet supplemented with 1.1% D. hansenii strain L2 (106 CFU g-1) plus inulin (3%) (diet II) for 4 weeks. The whole intestines of healthy fish from each group were aseptically removed at 2 and 4 weeks after starting the experiment and they were analysed by PCR-Denaturing Gradient Gel Electrophoresis (DGGE). Samples of blood and head kidney were obtained for humoral and cellular immune parameters determination. The expression of 12 selected genes related to the immune response (IgM, MHCI?, MHCII?, C3, IL-1?,TLR, TNF?, CSF-1R, NCCRP-1, Hep, TCR? y CD8) were analyzed by real-time PCR from skin, intestine, liver and head-kidney tissue. In this study, relevant changes in the intestinal microbiota of gilthead seabream specimens fed the diets assayed has been demonstrated. An important effect on the intestinal microbiota by the dietary administration of a synbiotic mixture has also been detected, especially in fish receiving the supplemented diet for 4 weeks. These changes coincided in the same time with an up-regulation of the expression of immunological genes in skin and head kidney of gilthead seabream at 2 and 4 weeks, respectively.

Effects of polyamines on cellular innate immune response and the expression of immune-relevant genes in gilthead seabream leucocytes.

It is well known that the polyamines spermidine and spermine, along with the diamine putrescine, are involved in many cellular processes and they are known to play an important role in the control of the innate immune response in higher vertebrates. However, to the best of our knowledge, no studies have focused on their immunological implications in other vertebrates, such as fish. For this reason, the effects of polyamines on the cellular innate immune response and immune-related gene expression were evaluated in vitro, using seabream head-kidney leucocytes (HKL). For this study, head-kidney leucocytes were incubated with the polyamines putrescine, spermine or spermidine (0.005 and 0.0025%) for 0.50, 1, 2 or 4 h. No significant effect was observed on either leucocyte viability or the innate cellular immune responses (peroxidase content and phagocytic and respiratory burst activities). The polyamines produced an increase in respiratory burst and phagocytic ability when leucocytes were incubated principally with putrescine (0.005 and 0.0025%) after 2 and 4 h of the experiment. Finally, the expression levels of immune-associated genes (IgM, MHCIα, MHCIIα, C3, IL-1β, CD8, Hep, NCCRP-1, CSF-1 and TLR) were quantified by real-time PCR and some of them (C3, MHCI, CD8, IgM and Hep) were up-regulated by the higher polyamine concentration. Further studies are needed to ascertain how polyamines control the immune system of seabream as well as which mechanisms are involved.