Dietary Application of the Microalga Lobosphaera incisa P127 Reduces Severity of Intestinal Inflammation, Modulates Gut-Associated Gene Expression, and Microbiome in the Zebrafish Model of IBD.

SCOPE: Microalgae are an emerging nutritional resource of biomolecules with potential to alleviate gut inflammation. The study explores the anti-inflammatory and immunomodulatory potential of the microalga Lobosphaera incisa P127, which accumulates a rare omega-6 LC-PUFA dihomo-É£-linolenic acid (DGLA) under nitrogen starvation. The therapeutic potential of dietary supplementation with P127 is investigated in the zebrafish model of IBD (TNBS-induced colitis). METHODS AND RESULTS: Guts are sampled from zebrafish fed experimental diets for 4 weeks, before and 24 h after TNBS challenge. Diets containing 15% non-starved (Ns) and 7.5% and 15% N-starved (St) algal biomass significantly attenuate the severity of gut injury and goblet cell depletion. In contrast, diets containing 7.5% Ns and DGLA ethyl ester have no effect on gut condition. Fish fed 15% St, high-DGLA biomass, have the fewest individuals with pathological alterations in the gut. Dietary inclusion of Ns and St distinctly modulates gut-associated expression of the immune and inflammatory genes. Fish fed 15% Ns biomass display a coordinated boost in immune gene expression and show major changes in the gut microbiome prior challenge. CONCLUSION: Dietary inclusion of L. incisa biomass at two physiological states, ameliorates TNBS-induced gut inflammation, suggesting the synergistic beneficial effects of biomass components not limited to DGLA.

Dietary Supplementation with Omega-6 LC-PUFA-Rich Microalgae Regulates Mucosal Immune Response and Promotes Microbial Diversity in the Zebrafish Gut.

The effect of dietary omega-6 long-chain polyunsaturated fatty acid (LC-PUFA) on host microbiome and gut associated immune function in fish is unexplored. The effect of dietary supplementation with the omega-6 LC-PUFA-rich microalga Lobosphaera incisa wild type (WT) and its delta-5 desaturase mutant (MUT), rich in arachidonic-acid and dihomo-gamma-linolenic acid (DGLA), respectively, on intestinal gene expression and microbial diversity was analyzed in zebrafish. For 1 month, fish were fed diets supplemented with broken biomass at 7.5% and 15% (w/w) of the two L. incisa strains and a control nonsupplemented commercial diet. Dietary supplementation resulted in elevated expression of genes related to arachidonic acid metabolism - cyclooxygenase 2 (cox-2), lipoxygenase 1(lox-1), anti-inflammatory cytokine - interleukin 10 (il-10), immune defense - lysozyme (lys), intestinal alkaline phosphatase (iap), complement (c3b), and antioxidants - catalase (cat), glutathione peroxidase (gpx). Microbiome analysis of the gut showed higher diversity indices for microbial communities in fish that were fed the supplemented diets compared to controls. Different treatment groups shared 237 operational taxonomic units (OTUs) that corresponded to the core microbiome, and unique OTUs were evident in different dietary groups. Overall, the zebrafish gut microbiome was dominated by the phylum Fusobacteria and Proteobacteria (averaging 38.4% and 34.6%, respectively), followed by Bacteroidetes (12.9%), Tenericutes, Planctomycetes, and Actinobacteria (at 3.1%-1.3%). Significant interaction between some of the immune-related genes and microbial community was demonstrated.

Mapping of the Sex Determining Region on Linkage Group 12 of Guppy (Poecilia reticulata).

Poecilia reticulata is one of the most popular ornamental fish species with a higher demand for males due to their large colorful fins. The objectives of this study were mapping of the sex-determining (SD) region on linkage group 12 of guppy, and identification of a sex specific marker. We generated eight polymorphic microsatellite markers distributed along the distal 5.4 Mbp sequence of the previously identified SD region on linkage group (LG) 12. The markers were tested for association with sex in 156 individuals of the Red Blonde and Flame strains, and 126 progeny of four full-sibs Red Blonde families. A male-specific allele was found for microsatellite gu1066 at position of 25.3 Mbp on LG12 for both strains, and gu832 at position 24.4 Mbp for the Flame strain. Thus, a region of 0.9 Mbp between these markers, harboring 27 annotated genes, was selected for analysis. Based on association of copy number variation and sex determination we mapped a duplicated region between LGs 9 and 12, of 1.26 Mbp, containing 59 genes on LG12. The common interval between the segment bounded by gu1066 and gu832, and the duplicated region of 0.43 Mbp on LG12 harbors 11 genes of which 6 have multiple copies (54%). Growth arrest and DNA damage inducible gamma-like (GADD45G-like) is a plausible positional and functional candidate gene for its role in male fertility. We characterized the genomic structure of the gene in guppy, and found two isoforms; but no sex-biased differences were evident in genomic sequence and gene expression.

The effect of tertiary treated wastewater on fish growth and health: Laboratory-scale experiment with Poecilia reticulata (guppy).

Treated wastewater (TWW) constitutes a sustainable water resource and has been used for fish culture in some countries around the world, although there are no comprehensive data on the effect of TWW on fish growth and health in the context of aquaculture production. Our objectives were to examine how fish culture in TWW affected fish growth and fitness, as well as compliance with the international standards for safe consumption. Guppy (Poecilia reticulata) fingerlings were reared in 0%, 50% and 100% tertiary TWW (TTWW), from the age of five days, for a period of four months. In water analyses, 33 out of 67 tested organic micropollutants (OMPs) were detected in the TTWW samples at least once, at concentrations that are typically reported in domestic TTWW. Fish survival ranged between 77-80% and did not differ between treatment groups. Fish growth and mortality following challenge infection with Tetrahymena sp. (which ranged between 64-68%), were similar among treatment groups. Of tested immunological parameters, lysozyme and anti-protease was similar among treatments while complement activity was highest in the 50% TTWW-reared fish. No abnormalities were observed in the histopathological analysis. Levels of heavy metals, polychlorinated-biphenyls (PCBs) and organochlorines (OCs) in fish were below the detection limit and below the Food and Agriculture Organization of the United Nations (FAO) and the European Union EU maximal permitted levels in food fish. Results suggest that the yield of fish grown in TTWW is potentially similar to that in freshwater, and the produced fish comply with the standards of consumer safety. The results are in line with previous studies that examined the feasibility of TWW-fed aquaculture.

The gut mucosal barrier of zebrafish (Danio rerio) responds to the time-restricted delivery of Lobosphaera incisa-enriched diets.

Recent studies in mammalian models revealed compelling evidence that along with the intrinsic characteristics of diets, the time of their delivery could have a profound impact on their benefits. In this study, we explored a time-dependent modulation of the gut mucosal barrier by delivering diets enriched with the green microalga (Lobosphaera incisa) either in a time-restricted regime or randomly to zebrafish (Danio rerio). The basal diet was enriched with microalgal biomass through two inclusion levels (i.e., 10% and 15% w/w), and the feeding trial lasted for six weeks. The control group was fed with the basal diet. After collection of tissue samples at week 6, the remaining fish were challenged by intraperitoneal injection of Streptococcus inaie. A histological analysis of the gut structure revealed that the fish that received the microalgae randomly exhibited shorter villi length. Genes coding for immunity were modulated in the gut by dietary treatments. Notably, the transcript levels of lysozyme, ß-defensin and hepcidin were significantly higher in the group subjected to the time-restricted feeding regime. Dietary microalgae affected the fatty acid content in the gut, particularly the level of arachidonic acid (ARA), and the time-restricted feeding influenced its accumulation. Groups that received diets enriched with 15% microalgae, regardless of the feeding strategy, displayed a significantly higher resistance to S. inaie 16 days post-infection, though differences between the delivery strategies were pronounced during the early stage of infection. In conclusion, the dietary inclusion of L. incisa modulated some of the features of the gut mucosal barrier of zebrafish, and the time of delivery appeared to have a considerable influence on immunomodulatory functions.

Dietary Supplementation With ω6 LC-PUFA-Rich Algae Modulates Zebrafish Immune Function and Improves Resistance to Streptococcal Infection.

Arachidonic acid (ARA, 20:4n-6) and dihomo-γ-linolenic acid (DGLA, 20:3n-6) are omega-6 long-chain polyunsaturated fatty acids (LC-PUFA), which are key precursors for lipid mediators of the immune system and inflammatory response. The microalga Lobosphaera incisa (WT) and its Δ5-desaturase mutant P127 (MUT) are unique photosynthetic sources for ARA and DGLA, respectively. This study explores the effect of dietary supplementation with L. incisa and P127 biomass on tissue fatty acid composition, immune function, and disease resistance in zebrafish (Danio rerio). The broken microalgal biomass was added to commercial fish feed at 7.5 and 15% (w/w), providing 21.8 mg/g feed ARA for the WT-supplemented group and 13.6 mg/g feed DGLA for the MUT-supplemented group at the 15% inclusion levels. An unsupplemented group was used as the control. After 1 month of feeding, fish were challenged with Streptococcus iniae. Fish were sampled before the challenge and 1 week after the challenge for various analyses. Tissue ARA and DGLA levels significantly increased in the liver, corresponding to microalgal supplementation levels. The elevated expression of specific immune-related genes was evident in the kidneys in all treatment groups after 1 month of feeding, including genes related to eicosanoid synthesis, lysozyme, and NF-κB. In the liver, microalgal supplementation led to the upregulation of genes related to immune function and antioxidant defense while the expression of examined genes involved in ARA metabolism was downregulated. Importantly, fish fed with 15% of both WT- and MUT-supplemented feed showed significantly (p < 0.05) higher survival percentages (78 and 68%, respectively, as compared to only 46% in the control group). The elevated expression of genes related to inflammatory and immune responses was evident post-challenge. Collectively, the results of the current study demonstrate the potential of microalgae-derived dietary ARA and DGLA in improving immune competence and resistance to bacterial infection in zebrafish as a model organism.

Analyzing complement activity in the serum and body homogenates of different fish species, using rabbit and sheep red blood cells.

Alternative complement activity was determined in whole body homogenates (WBHs) and serum samples of different fish species, by measuring the amount of sample that induces 50% hemolysis of red blood cells using the ACH50 assay (Alternative Complement pathway Hemolytic activity). Values of ACH50 obtained for serum samples were about two-fold higher when using rabbit red blood cells (RRBC), as compared to sheep red blood cells (SRBC). The increase in ACH50 when using RRBCs for WBH samples was 28, 7 and 4 folds for guppy, molly and zebrafish, respectively. Large variability in complement activity was evident between fish species for both serum and WBHs. Evaluating the effect of freeze-thaw cycles on complement revealed significant reduction in complement activity in all tested samples. Loss of activity following three freeze-thaw cycles amounted to 48-59% when serum was tested and over 95% loss in activity for WBH. To our knowledge, this is the first study where fish WBHs were used for assaying complement activity. Our results support the suitability of this method in evaluating complement activity in small fish species or larvae, where blood cannot be obtained, as long as samples can be tested upon first thawing.

Dietary arachidonic acid affects immune function and fatty acid composition in cultured rabbitfish Siganus rivulatus.

The marbled spinefoot rabbitfish (Siganus rivulatus) is an economically valuable fish species that has potential for commercial production in aquaculture. To overcome challenges in its sustainable production, a formulated diet is required for imparting health and robustness. This study evaluates the effect of dietary supplementation with arachidonic acid (ARA; 20:4n-6) on growth, survival, immune function and fatty acid composition of red blood cells (RBCs) in rabbitfish. We conducted two feeding trials using juvenile fish (to evaluate growth and survival) and adults (to evaluate immune function and fatty acid incorporation). Fish were fed diets supplemented with three different levels of ARA (in % of total fatty acids): 0.6 (unsupplemented control), 2.6 (moderate) and 4.7 (high). The fish fed with moderate ARA levels exhibited improved (p < 0.05) growth over the control and the high ARA level groups. During an outbreak of Streptococcus iniae, fish fed with moderate ARA survived significantly (p < 0.05) better (89%) than the control and the high ARA groups (59% and 48%, respectively). Moderate ARA supplementation resulted in elevated lysozyme and complement levels in the plasma of rabbitfish. A significant increase in the total serum immunoglobulin levels was observed in both the medium and the high ARA level groups; however, a decrease in antiprotease activity was recorded in the supplemented groups as compared to the control. Fatty acid analysis in fish red blood cells revealed a significant (p < 0.05) increase in the proportion of ARA of total fatty acids in the groups fed with the medium and the high ARA level diets (9.5% and 11.2%, respectively, compared to 7.1% in the control). Concomitantly, there was a decrease in the proportion of eicosapentaenoic acid (EPA; 20:5n-3), dihomo-γ linolenic acid (DGLA; 20:3n-6) and several 18-carbon unsaturated fatty acids in these groups. In conclusion, ARA in rabbitfish feeds improved growth, survival as well as innate and acquired humoral immune functions. Thus ARA supplementation in the diet of this species could be a valuable step towards establishing the commercial culture of rabbitfish.

Impact of treated wastewater reuse and floods on water quality and fish health within a water reservoir in an arid climate.

Treated wastewater (TWW) reuse for agricultural irrigation is a well-established approach to coping with water shortages in semi-arid and arid environments. Recently, additional uses of TWW have emerged, including streamflow augmentation and aquatic ecosystem restoration. The purpose of the current study was to evaluate the water quality and fish health, in an artificial reservoir located in an arid climate (the Yeruham Reservoir, Israel), which regularly receives TWW and sporadic winter floods. The temporal distribution of water levels, nutrients and organic micropollutants (OMPs) were measured during the years 2013-2014. OMPs were also measured in sediment and fish tissues. Finally, the status of fish health was evaluated by histopathology. Water levels and quality were mainly influenced by seasonal processes such as floods and evaporation, and not by the discharge of TWW. Out of 16 tested OMPs, estrone, carbamazepine, diclofenac and bezafibrate were found in the reservoir water, but mostly at concentrations below the predicted no-effect concentration (PNEC) for fish. Concentrations of PCBs and dioxins in fish muscle and liver were much lower than the EU maximal permitted concentrations, and similar to concentrations that were found in food fish in Israel and Europe. In the histopathological analysis, there were no evident tissue abnormalities, and low to moderate infection levels of fish parasites were recorded. The results from the Yeruham Reservoir demonstrated a unique model for the mixture effect between TWW reuse and natural floods to support a unique stable and thriving ecosystem in a water reservoir located in an arid region. This type of reservoir can be widely used for recreation, education, and the social and economic development of a rural environment, such as has occurred in the Yeruham region.

The prawn Macrobrachium vollenhovenii in the Senegal River basin: towards sustainable restocking of all-male populations for biological control of schistosomiasis.

Early malacological literature suggests that the outbreak of schistosomiasis, a parasitic disease transmitted by aquatic snails, in the Senegal River basin occurred due to ecological changes resulting from the construction of the Diama dam. The common treatment, the drug praziquantel, does not protect from the high risk of re-infection due to human contact with infested water on a daily basis. The construction of the dam interfered with the life cycle of the prawn Macrobrachium vollenhovenii by blocking its access to breeding grounds in the estuary. These prawns were demonstrated to be potential biological control agents, being effective predators of Schistosoma-susceptible snails. Here, we propose a responsible restocking strategy using all-male prawn populations which could provide sustainable disease control. Male prawns reach a larger size and have a lower tendency to migrate than females. We, therefore, expect that periodic restocking of all-male juveniles will decrease the prevalence of schistosomiasis and increase villagers' welfare. In this interdisciplinary study, we examined current prawn abundance along the river basin, complemented with a retrospective questionnaire completed by local fishermen. We revealed the current absence of prawns upriver and thus demonstrated the need for restocking. Since male prawns are suggested to be preferable for bio-control, we laid the molecular foundation for production of all-male M. vollenhovenii through a complete sequencing of the insulin-like androgenic gland-encoding gene (IAG), which is responsible for sexual differentiation in crustaceans. We also conducted bioinformatics and immunohistochemistry analyses to demonstrate the similarity of this sequence to the IAG of another Macrobrachium species in which neo-females are produced and their progeny are 100% males. At least 100 million people at risk of schistosomiasis are residents of areas that experienced water management manipulations. Our suggested non-breeding sustainable model of control-if proven successful-could prevent re-infections and thus prove useful throughout the world.

Evaluation of guppy (Poecilia reticulata Peters) immunization against Tetrahymena sp. by enzyme-linked immunosorbent assay (ELISA).

Analysis of the effectiveness of guppy (Poecilia reticulata Peters) immunization based on measurements of antibody (Ab) titers suffers from a shortage of reagents that can detect guppy antibodies (Abs). To overcome this problem, we immunized mice with different preparations of guppy immunoglobulins (Igs) and used the mouse antisera to develop a quantitative enzyme-linked immunosorbent assay (ELISA). The most efficient immunogen for mouse immunization was guppy Igs adsorbed on protein A/G beads. Antisera from mice boosted with this immunoglobulin (Ig) preparation were highly specific and contained high Ab titers. They immunoreacted in a Western blot with Ig heavy and light chains from guppy serum, and Ig heavy chain from guppy whole-body homogenate. The mouse anti-guppy Ig was applied in an ELISA aimed at comparing the efficiency of different routes of guppy immunization against Tetrahymena: (i) anal intubation with sonicated Tetrahymena (40,000 Tetrahymena/fish in a total volume of 10 µL) mixed with domperidon, deoxycholic acid and free amino acids (valine, leucine, isoleucine, phenylalanine and tryptophan), or (ii) intraperitoneal (i.p.) injection of sonicated Tetrahymena in complete Freund's adjuvant (15,000 Tetrahymena/fish in total a volume of 20 µL). Negative control fish were anally intubated with the intubation mixture without Tetrahymena, or untreated. ELISA measurement of anti-Tetrahymena Ab titer revealed a significantly higher level of Abs in i.p.-immunized guppies, compared to the anally intubated and control fish. In addition, the efficiency of immunization was tested by monitoring guppy mortality following (i) i.p. challenge with Tetrahymena (900 Tetrahymena/fish) or (ii) cold stress followed by immersion in water containing 10,000 Tetrahymena/mL. Fish mortality on day 14 post-Tetrahymena infection by i.p. injection exceeded 50% in the control and anally intubated fish, compared to 31% in i.p.-immunized fish. Immunization did not protect from pathogen challenge by immersion. The results suggest a direct correlation between the anti-Tetrahymena Ab response and fish resistance to i.p.-injected Tetrahymena, but not to infection by immersion preceded by cold stress.

New pathological condition in cultured mulloway Argyrosomus japonicus: histopathological, ultrastructural and molecular studies.

Mulloway Argyrosomus japonicus is a native fish species in Western Australia, for which aquaculture production has recently been developed. A single cohort was stocked in a cage offshore at Geraldton, Western Australia, at a water depth of 6 m. Fish appeared healthy before stocking. Routine histological analysis was carried out from 10 mo post stocking and until completion of harvest (about 2.5 yr post stocking). No gross pathology was evident. Microscopically, however, granulomatous lesions were present in the kidneys of almost 100% of the fish examined. Enclosed in the granuloma was an aggregate of organisms, 4.2 to 5.4 µm in diameter. Kidney granulomas appeared as multi-focal aggregates. Granulomas at different stages of formation and finally fibrosing granulomas were observed. Granulomas also appeared infrequently in other organs: a few granulomas were found in the liver and spleen and a single granuloma in the heart of one fish. Transmission electron microscopy (TEM) revealed that the organism was composed of 2 cells, an outer cell enclosing an inner cell. The inner cell was surrounded by a double membrane and the outer cell by a single membrane. Cellular material, presumably of parasitic nature, surrounded the outer cell. The organism contained primitive mitochondria and abundant free ribosomes. Small subunit ribosomal DNA (SSU rDNA) sequence obtained by PCR revealed an 84% sequence identity with the myxosporean Latyspora scomberomori. Based on TEM and preliminary molecular results, we suggest that the organism is the extrasporogonic developmental stage of a myxozoan parasite, which failed to form spores in the mulloway host.

Optimization and validation of a colorimetric assay for Tetrahymena sp. survival.

A colorimetric assay based on the reduction of 3-(4,5-dimethyl-2-thiazol-2-yl)-2,5-diphenyl-2h-tetrazolium bromide (MTT), for the quantification of Tetrahymena sp. survival is described. An increase in the concentration of Tetrahymena sp. cells from 0 to 1 x 10(6) cells/ml produced a linear (R(2)=0.9965) increase in the optical density (OD, 570-630 nm), and dead cells (pre-exposed to 250 mg/l formalin for 4 h) did not produce a background reading. Cells exposed to sublethal concentrations to formalin (100 mg/l or less for 4 h) recovered their growth. Using the MTT assay, we determined that Tetrahymena sp. is sensitive to formalin, chloramine-T, hydrogen peroxide, copper sulfate and NaCl. The sensitivity increased with increasing chemical concentrations and exposure time. Tetrahymena sp. was resistant to bromex and malachite green. The use of this assay in drug screening for the development of treatments for tetrahymenosis and as a bioassay to evaluate the toxicity of environmental toxicants is discussed.