Franciscana Nauplii Research Articles

Salinity tolerance, growth and survival of three Artemia franciscana (Kellogg, 1906) populations under laboratory conditions

AbstractIn the 1980s, Artemia franciscana from San Francisco Bay (SFB) was introduced into Kenyan saltworks, where it has colonized and established stable populations. However, little is known about its biology, particularly with respect to its parental SFB population. This study compared the salinity tolerances of Kenyan (KEN) population, their SFB progenitors and those of Great Salt Lake (GSL) populations. Growth and survival of these A. franciscana populations were evaluated under varying salinity levels in a laboratory set up. A. franciscana nauplii were cultured at a rate of 1 nauplii/mL in 36 Erlenmeyer flasks and fed microalgae (Chaetoceros sp.) at 1.5 × 106 cells/animal/day for 8 days. Survival was evaluated daily and survivors were fixed in individual vials with Lugol solution. The total length of each fixed A. franciscana nauplii specimen was measured under a compound microscope. All populations were susceptible to salinities greater than 100 g/L. Compared with the parental SFB population, the KEN population exhibited significantly reduced survival and growth at 140 g/L, suggesting a narrower salinity tolerance range. These findings underscore the need for further studies focusing on other physiological parameters, abiotic factors and genetic characterization to confirm whether the KEN population is experiencing ecological adaptation. This will contribute to the optimization of Artemia practices in various salinity environments as a result of climate change.

ArHsp90 is important in stress tolerance and embryo development of the brine shrimp, Artemia franciscana.

Females of the extremophile crustacean, Artemia franciscana, either release motile nauplii via the ovoviviparous pathway or encysted embryos (cysts) via the oviparous pathway. Cysts contain an abundant amount of the ATP-independent small heat shock protein that contributes to stress tolerance and embryo development, however, little is known of the role of ATP-dependent molecular chaperone, heat shock protein 90 (Hsp90) in the two processes. In this study, a hsp90 was cloned from A.franciscana. Characteristic domains of ArHsp90 were simulated from the deduced amino acid sequence, and 3D structures of ArHsp90 and Hsp90s of organisms from different groups were aligned. RNA interference was then employed to characterize ArHsp90 in A.franciscana nauplii and cysts. The partial knockdown of ArHsp90 slowed the development of nauplius-destined, but not cyst-destined embryos. ArHsp90 knockdown also reduced the survival and stress tolerance of nauplii newly released from A.franciscana females. Although the reduction of ArHsp90 had no effect on the development of diapause-destined embryos, the resulting cysts displayed reduced tolerance to desiccation and low temperature, two stresses normally encountered by A.franciscana in its natural environment. The results reveal that Hsp90 contributes to the development, growth, and stress tolerance of A.franciscana, an organism of practical importance as a feed source in aquaculture.

Toxicity Evaluation of Iodine Disinfectant Inclusion on Mortality and Growth of Artemia franciscana Nauplii

Artemia franciscana nauplii is a source of nutrients and pathogen vectors due to the non-selective nature of the filter feeder. Prevention of pathogens is done by adding iodine disinfectant to suppress the emergence of bacteria. One way to find out if a substance is toxic is by testing the toxicity and lethal concentration (LC50) on A. franciscana nauplii. The aim of this study was to evaluate the disinfectant effect of iodine and determine the lethal concentration of iodine in 50% of A. franciscana nauplii (LC50). Brine Shrimp Lethality Test (BSLT) method using Artemia as test animal. The results of the toxicity test with several doses of iodine showed that the toxic threshold value for 96 hours was 20.31 ppm. The results were supported by the presence of stunted growth at a dose of 100 ppm by measuring the length and weight. The conclusion of this study is that the administration of iodine at several concentrations is toxic to A. franciscana nauplii if it exceeds 20.31 ppm and affects the growth of A. franciscana nauplii.

Replacement of Artemia franciscana Nauplii by Moina minuta Neonates as Live Food on the Larviculture of Angelfish (Pterophyllum scalare -Schultze, 1823) and Severum (Heros severus - Heckel, 1840)

The present study aimed to assess the potential substitution of Artemia franciscana nauplii with Moina minuta neonates as the initial feed for angelfish (Pterophyllum scalare) and severum (Heros severus), evaluating the fish growth performance, live food cost, and intestinal histomorphometry. To achieve this goal, two separate experiments were carried out for each species during 20 days, using 200 postlarvae. Both experimental designs consisted of five treatments, namely, 100% A. franciscana nauplii (A100-M0); 75% A. franciscana nauplii and 25% M. minuta neonates (A75-M25); 50% A. franciscana nauplii and 50% M. minuta neonates (A50-M50); 25% A. franciscana nauplii and 75% M. minuta neonates (A25-M75); and 100% M. minuta neonates (A0-M100), with four replications (10 postlarvae per repetition). Angelfish postlarvae fed with A100-M0, A75-M25, A50-M50, and A25-M75 had better values of final length (13.31 mm, 14.15 mm, 13.32 mm, and 13.32 mm), gain in length (7.91 mm, 8.75 mm, 7.92 mm, and 7.92 mm), and length-specific growth rate (4.51% day−1, 4.82% day−1, 4.51% day−1, and 4.51% day−1). In the case of severum postlarvae, the highest length growth performance occurred in those fed with A100-M0 (15.86 mm, 8.90 mm, and 4.12% day−1), A75-M25 (15.45 mm, 8.49 mm, and 3.99% day−1), and A50-M50 (14.78 mm, 7.82 mm, and 3.76% day−1). Both species (angelfish and severum postlarvae) fed with A100-M0 and A75-M25 presented higher values of final weight (28.88 mg and 34.49 mg; 55.84 mg and 53.95 mg), gain in weight (27.56 mg and 33.08 mg; 51.03 mg and 49.14 mg), and weight-specific growth rate (15.42% day−1 and 16.30% day−1; 12.26% day−1 and 12.09% day−1). Live food cost for angelfish and severum postlarvae was higher in A100-M0 (US$ 0.92 and US$ 1.08) and lower for the ones in A0-M100 (US$ 0.36 and US$ 0.35). Severum postlarvae fed with A100-M0 (57.20 µm), A75-M25 (55.66 µm), and A50-M50 (54.27 µm) presented the highest intestinal villi heights. Thus, partial replacement of A. franciscana nauplii by M. minuta neonates in the proportion of A75-M25 or 100A-0M is viable and effective during the first feed of these fish species.

Short-term cold stress and heat shock proteins in the crustacean Artemia franciscana.

In their role as molecular chaperones, heat shock proteins (Hsps) mediate protein folding thereby mitigating cellular damage caused by physiological and environmental stress. Nauplii of the crustacean Artemia franciscana respond to heat shock by producing Hsps; however, the effects of cold shock on Hsp levels in A. franciscana have not been investigated previously. The effect of cold shock at 1°C followed by recovery at 27°C on the amounts of ArHsp90, Hsp70, ArHsp40, and ArHsp40-2 mRNA and their respective proteins in A. franciscana nauplii was examined by quantitative PCR (qPCR) and immunoprobing of western blots. The same Hsp mRNAs and proteins were also quantified during incubation of nauplii at their optimal growth temperature of 27°C. qPCR analyses indicated that the abundance of ArHsp90, Hsp70, and ArHsp40 mRNA remained relatively constant during both cold shock and recovery and was not significantly different compared with levels at optimal temperature. Western blotting revealed that ArHsp90, ArHsp40, and ArHsp40-2 were generally below baseline, but at detectable levels during the 6h of cold shock, and persisted in early recovery stages before declining. Hsp70 was the only protein that remained constant in quantity throughout cold shock and recovery. By contrast, all Hsps declined rapidly during 6h when nauplii were incubated continuously at 27°C optimal temperature. Generally, the amounts of ArHsp90, ArHsp40, and ArHsp40-2 were higher during cold shock/recovery than those during continuous incubation at 27°C. Our data support the conclusion that low temperature preserves Hsp levels, making them available to assist in protein repair and recovery after cold shock.

Cyst viability and stress tolerance upon heat shock protein 70 knockdown in the brine shrimp Artemia franciscana.

Females of the brine shrimp Artemia franciscana produce either free-swimming nauplii via ovoviviparous pathway of reproduction or encysted embryos, known as cysts, via oviparous pathway, in which biological processes are arrested. While previous study has shown a crucial role of ATP-dependent molecular chaperone, heat shock protein 70 (Hsp70) in protecting A. franciscana nauplii against various abiotic and abiotic stressors, the function of this protein in diapausing embryos and cyst development, however, remains unknown. RNA interference (RNAi) was applied in this study to examine the role of Hsp70 in cyst development and stress tolerance, with the latter performed by desiccation and freezing, a common method used for diapause termination in Artemia cysts. Hsp70 knockdown was apparent in cysts released from females that were injected with Hsp70 dsRNA. The loss of Hsp70 affected neither the development nor morphology of the cysts. The time between fertilization and cyst release from Artemia females injected with Hsp70 dsRNA was delayed slightly, but the differences were not significant when compared to the controls. However, the hatching percentage of cysts which lacks Hsp70 were reduced following desiccation and freezing. Taken together, these results indicated that Hsp70 possibly plays a role in the stress tolerance but not in the development of diapause-destined embryos of Artemia. This research makes fundamental contributions to our understanding of the role molecular chaperone Hsp70 plays in Artemia, an excellent model organism for diapause studies of the crustaceans.

Beta-glucan's varying structure characteristics modulate survival and immune-related genes expression from Vibrio harveyi-infected Artemia franciscana in gnotobiotic conditions

β-Glucans have long been used as an immunostimulant in aquaculture. However, the relationship of its structure to its immunomodulatory properties are poorly understood. In this study, the particle size and chemical structure of β-glucans extracted from wild-type strain of baker's yeast (Saccharomyces cerevisiae) and its null-mutant yeasts Gas1 were characterised. Using Sigma β-glucan as a reference, the immunomodulatory properties of these polysaccharides in the germ-free Artemia franciscana model system in the presence of Vibrio harveyi bacterial challenge were investigated. The survival of the A. franciscana nauplii, upon challenge with V. harveyi, was significantly higher in all three glucan-treated groups compared to the control. The glucan Gas1 with a lower degree of branching and shorter side chain length had the most prominent V. harveyi-protective effects. The particle size did not affect the nauplii survival when challenged with V. harveyi. Results also showed that the salutary effect of the tested glucans was associated with the upregulation of innate immune genes such as lipopolysaccharide and β-1,3-glucan-binding protein (lgbp), high mobility group box protein (hmgb), and prophenoloxidase (proPO). Interestingly, the up-regulation of superoxidase dismutase (sod) and glutathione-s-transferase (gst) was only observed in Gas1 treated group, indicating that Gas1 could function to induce higher reactive oxygen species and stronger immunomodulatory function in A. franciscana, and therefore higher survival rate. The expression of heat shock protein 70 (hsp70), peroxinectin (pxn), and down syndrome cell adhesion molecule (dscam) remain unaltered in response to glucan treatment. Taken together, this study provides insights into the structure-function relationship of β-glucan and the results confirmed that β-glucan can be an effective immunostimulant in aquaculture, especially the Gas1 glucan.

Effect of Streptomyces sp. RL8 on the survival of Artemia franciscana nauplii and resistance to Vibrio parahaemolyticus

Artemia franciscana nauplii were enriched with Streptomyces sp. RL8 in order to assess its effect on the survival rate of Artemia and its protective effects against Vibrio parahaemolyticus infection. Streptomyces sp. RL8 showed antagonistic activity against the opportunistic pathogenic Vibrio species of interest for aquaculture and decreased the growth of V. parahaemolyticus Collection of Aquatic Important Microorganisms (CAIM) 170 during in vitro co-culture experiments. Two experimental groups of nauplii, each replicated three times, were enriched with strain Streptomyces sp. RL8 at 106 and 108 colony-forming units (CFU)/ml; an unfed group served as the control. The experimental groups were challenged 24 h post-feeding with the strain V. parahaemolyticus CAIM 170 at 105 and 107 CFU/ml, and the survival of A. franciscana recorded at 24 and 48 h post-inoculation. Nauplii survival was significantly higher (88.75% and 86.25%) in the groups treated with Streptomyces sp. RL8 at 106 and 108 CFU/ml than in the control group (70.71%). These results confirm the probiotic properties of Streptomyces sp. RL8 and suggest that A. franciscana enriched with this strain can be fed to fish and shrimp larvae.

The 9H-Fluoren Vinyl Ether Derivative SAM461 Inhibits Bacterial Luciferase Activity and Protects Artemia franciscana From Luminescent Vibriosis.

Vibrio campbellii is a major pathogen in aquaculture. It is a causative agent of the so-called “luminescent vibriosis,” a life-threatening condition caused by bioluminescent Vibrio spp. that often involves mass mortality of farmed shrimps. The emergence of multidrug resistant Vibrio strains raises a concern and poses a challenge for the treatment of this infection in the coming years. Inhibition of bacterial cell-to-cell communication or quorum sensing (QS) has been proposed as an alternative to antibiotic therapies. Aiming to identify novel QS disruptors, the 9H-fluroen-9yl vinyl ether derivative SAM461 was found to thwart V. campbellii bioluminescence, a QS-regulated phenotype. Phenotypic and gene expression analyses revealed, however, that the mode of action of SAM461 was unrelated to QS inhibition. Further evaluation with purified Vibrio fischeri and NanoLuc luciferases revealed enzymatic inhibition at micromolar concentrations. In silico analysis by molecular docking suggested binding of SAM461 in the active site cavities of both luciferase enzymes. Subsequent in vivo testing of SAM461 with gnotobiotic Artemia franciscana nauplii demonstrated naupliar protection against V. campbellii infection at low micromolar concentrations. Taken together, these findings suggest that suppression of luciferase activity could constitute a novel paradigm in the development of alternative anti-infective chemotherapies against luminescent vibriosis, and pave the ground for the chemical synthesis and biological characterization of derivatives with promising antimicrobial prospects.

Effect of Food Enrichment on the Survival of Hippocampus Ingens Girard, 1858 Alevine under Semicontroled Conditions

Due to the decline in seahorse populations generated by exploitation and degradation of their habitat, an attempt has been made to obtain the closed cycle of cultivation of different species of the genus, includingH. ingens, the unique specie located in the Eastern Pacific. In the majority of the studies carried out, the mainlimitation has been the provision of a diet that supplies an adequate growth and survival of the organisms. The objective of this research was to determine the effect of nutritional enrichment of Brachionus plicatilis and Artemiafranciscana, used as food in the survival of H. ingens. Fifteen specimens of wild adult seahorses were capturedin Bahía de La Paz, and generated four couples and 17 successful batches. To assess the enrichment from thethird to the seventh day, each batch was divided into six groups and placed in 60-liter aquaria, feeding on rotifersmaintained with six different treatments. Three Artemia franciscana nauplii enrichment treatments were thentested to evaluate the effect on the survival of the juveniles in the period from day eight to day 60. From day 61and on seahorses were fed with adult Artemia franciscana also enriched with the three different treatments. Theseresults show that the enrichment of the food offered in the first months of life of seahorses has a significant effecton their survival.

Bioencapsulation and Colonization Characteristics of Lactococcus lactis subsp. lactis CF4MRS in Artemia franciscana: a Biological Approach for the Control of Edwardsiellosis in Larviculture.

Predominance of beneficial bacteria helps to establish a healthy microbiota in fish gastrointestinal system and thus to reduce emerging pathogen. In this study, the colonization efficacy of Lactococcus lactis subsp. lactis CF4MRS in Artemia franciscana and its potential as a probiotic in suppressing Edwardsiella sp. infection were investigated in vivo. The colonization extent of the bioencapsulated L. lactis was established through visualization of gfp gene-transformed L. lactis in A. franciscana. Here, we demonstrate that when A. franciscana is administrated with L. lactis at 108CFUmL-1 for 8h, the highest relative percentage of survival (RPS = 50.0) is observed after inoculation with Edwardsiella sp. The total counts of L. lactis entrapped in Artemia were the highest (ranged from 3.2 to 5.1 × 108CFUmL-1), when 108-109CFUmL-1 of L. lactis was used as starting inoculum, with the bioencapsulation performed within 8-24h. Fluorescent microscopy showed gfp-transformed L. lactis colonized the external trunk surfaces, mid-gut and locomotion antennules of the A. franciscana nauplii. These illustrations elucidate the efficiency of colonization of L. lactis in the gastrointestinal tract and on the body surfaces of Artemia. In conclusion, L. lactis subsp. lactis CF4MRS shows a good efficacy of colonization in Artemia and has the potential for biocontrol/probiotic activity against Edwardsiella sp. infection.

A workflow management system for early feeding of the European hake

Diversification of marine species has emerged as a priority in the aquaculture agenda of many countries due to its large industrial potential and as an alternative to overharvested fisheries. Aquaculture diversification entails new challenges during early life stages of candidate species such as survival bottlenecks or body malformations, many of them due to uncoupling between classic diets and early nutritional requirements. Monospecific diets are common in fish aquaculture, e.g. beginning with a rotifer-based diet, followed by a mixed diet of rotifer and artemia nauplii and ending with artemia nauplii and metanauplii until weaning. Despite some success was reported using such protocol in early hake feeding the massive mortality observed as approaching 25dph makes optimization of early feeding and larval management a current challenge for the domestication of this species. The main goal of this study was to design and test a workflow management system for early feeding of the European hake as a candidate species. The null hypothesis tested was that optimization of rearing settings had no effect on early growth and survival up to 30dph as compared to classic culture protocols using commercial prey. Absence of prey in 6dph hake larvae stomachs indicates that their external feeding at 14°C begins just after that age. Early feeding preference depends on prey size (<500μm before 9dph) as well as on pigmentation and behavior e.g. those with poor escape reactivity such as A. franciscana Nauplii. Significant feeding specialization on wild zooplankton such as P. intermedius and T. longicornis occurred after 9dph (Chesson selectivity index=0.11). Feeding activity was maximal in darkness (D) and medium light intensity (600lx, MLI) as compared to the lethal light intensity of 1700lx (HLI). Rotifer-based diets entailed low larvae growth and hake culture unviability after 15dph but inclusion of wild zooplankton in early diets doubled growth of 30dph larvae regarding artemia-based diets. The adaptive prey-size diet designed (MiACop) by combining stages of copepods (nauplii, copepodite and adult), rotifer and commercial nauplii of artemia was five-fold superior to the artemia/zooplankton diet all along the first 30dph larvae culture. The massive cannibalism observed from 25dph on was related to the absence of an adequate prey size such as that of mysids and euphausiids in combination with semi-dry feed to trigger weaning. Current workflow design for early feeding of the European hake can be helpful to assuring a larger proportion of juveniles entering the weaning phase. Statement of relevanceFirst feeding of the European hake.

Influence of Mortierella alpina enrichedArtemia franciscana on distal intestinal histology of sea bass larvae (Dicentrarchus labrax)

The present study evaluated the effect of Mortierella alpina and M. alpina plus oil emulsion enriched Artemia franciscana on distal intestine histology of seabass larvae (Dicentrarchus labrax). D. labrax larvae (3.20 mg) were fed on A. franciscana nauplii enriched with 1ml/L M. alpina (MA), 1m/L M. alpina combined with 1ml/L of the following oils: black seed oil, grape seed oil, linseed oil or safflower oil for 28days. D. labrax larvae fed on unenriched A. franciscana served as a control. The results indicated that D. labrax larvae fed A. franciscana enriched with M. alpina or M. alpina in combination with black seed oil, linseed oil or safflower oil showed normal intestinal histology. D. labrax larvae fed A. franciscana enriched with M. alpina – grape seed oil showed minor fat infiltration in enterocytes. The present results suggest that a dietary combination between M. alpina and vegetable oils such as black seed, linseed or safflower may not alter intestinal histomorphology of D. labrax larvae.

Knockdown of heat shock protein 70 (Hsp70) by RNAi reduces the tolerance of Artemia franciscana nauplii to heat and bacterial infection

RNA interference (RNAi) was used to assess the role of the molecular chaperone Hsp70 in protecting nauplii of the crustacean, Artemia franciscana, against abiotic and biotic stressors. As revealed respectively by RT-PCR and immunoprobing of Western blots, the injection of A. franciscana females with Hsp70 dsRNA eliminated Hsp70 mRNA and protein in nauplii released from these animals. The loss of Hsp70 had no apparent effect on either embryo development or the viability of A. franciscana nauplii. The survival of nauplii lacking Hsp70, as compared to those containing the Hsp, was reduced 41% by heat stress and 34% upon Vibrio campbellii infection. A role for Hsp70 in protecting A. franciscana nauplii against heat stress and bacterial infection was demonstrated for the first time by using RNAi to knock down the protein. The results suggest that Hsp70 assisted in maintaining protein homeostasis within stressed cells while potentially stimulating the innate immune system of A. franciscana. Information of the type generated in this work has potential for application in the culture of A. franciscana and other commercially important aquatic invertebrates.

An examination of the medicinal potential ofPlanchonella queenslandica: toxicity, antibacterial, and antiviral activities

Planchonella queenslandica (family Sapotaceae) is an Australian native plant with a history of indigenous Australian medicinal usage. Its antimicrobial activity was investigated by disc diffusion assays against a bacterial panel. Antiviral activity was determined using an MS2 bacteriophage plaque reduction model system. Toxicity was determined using the Artemia franciscana nauplii bioassay. Methanol, water and ethyl acetate leaf extracts displayed antibacterial activity. The methanol and ethyl acetate extracts had the broadest specificity, inhibiting the growth of 9 of the 14 bacteria tested (64 %) respectively. The water extract inhibited the growth of 6 of the 14 bacteria tested (43 %). All extracts were more effective against Gram-positive than Gram-negative bacteria. The methanol and ethyl acetate extracts each inhibited the growth of 100 % of the Gram-positive species tested and 5 of the 9 (56 %) Gram-negative bacteria. The water extract inhibited 3 of the 4 (75 %) Gram-positive bacteria and 3 of the 9 (33 %) of the Gram-negative bacteria tested. The methanol, water and ethyl acetate extracts also displayed antiviral activity in the MS2 plaque reduction assay, inhibiting 90.8 ± 2.4 %, 50.7 ± 5.8 and 71.7 ± 4.4 % of MS2 plaque formation respectively. All extracts were also shown to be non-toxic or of low toxicity in the Artemia fransiscana bioassay. The low toxicity of the P. queenslandica extracts and their inhibitory bioactivity in the MS2 bacteriophage reduction bioassay and against a panel of bacteria validate Australian Aboriginal usage as an antiseptic agent and confirms their medicinal potential.

An Evaluation of the Antibacterial, Anti-Giardial, Anticancer and Toxicity Properties of Selected Nut Extracts

Background: Foods with high oil and fatty acid contents have been linked with a variety of medicinal properties including bacterial growth inhibition, anti-Giardial activity and the inhibition of cancer cell proliferation. Almond, cashew, hazelnut and walnut contain very high fatty acid contents. Despite this, these nuts have not been adequately screened for medicinal properties. Materials and Methods: Almond, cashew, hazelnut and walnut powders were extracted and tested for antimicrobial activity using modified disc diffusion and MIC methods. Inhibitory activity against the gastrointestinal protozoal parasite Giardia duodenalis and against CaCo2 cancer cells were evaluated using colorimetric cell proliferation assays. Toxicity was evaluated using an Artemia franciscana nauplii bioassay. Results: The methanolic almond and walnut solvent extractions displayed broad spectrumgrowth inhibitory activity, inhibiting the growth of 100 % and 7 of the 11 (64 %) bacterial strains tested respectively. Themethanolic walnut extract was a particularly potent growth inhibitor, with MIC values of ~ 1000 µg/mL, 700 µg/mL and 800 µg/mL against A. baylyi, P. mirabilis and P. vulgaris respectively. The methanolic almond extract also had moderate to inhibitory activity against E. coli (MIC ~ 2000 µg/mL), P. mirabilis and P. vulgaris (both ~ 2500 µg/mL). The methanolic cashew and hazelnut extracts were moderate inhibitors of E. coli growth (~ 2500 and 1250 µg/mL respectively). The methanolic almond extract was also a potent inhibitor of G. Duodenalis proliferation (IC50 878 µg/mL). All extracts were ineffective at blocking the growth of the colorectal cancer cell line CaCo2. Instead, most of the extracts substantially stimulated proliferation. All of the nut extracts were non-toxic in the Artemia nauplii bioassay. Conclusion: The bacterial growth inhibitory activities of the methanolic almond and walnut extracts, the anti-Giardial activity of the methanolic almond extract and their lack of toxicity indicates the potential of these extracts in the discovery and development of new natural antibiotic agents.

Antiviral Effect of Fucoidan Extracted from the Brown Seaweed, Sargassum wightii, on Shrimp Penaeus monodon Postlarvae against White Spot Syndrome Virus

This study was carried out to determine the effect of seaweed polysaccharide, fucoidan from brown seaweed, Sargassum wightii, on Penaeus monodon postlarvae (PL) against white spot syndrome virus (WSSV). The fucoidan was extracted from S. wightii, and the yield was observed as 2.832 ± 0.204%. Artemia franciscana nauplii were enriched with extracted fucoidan at four different concentrations (100, 200, 300, and 400 mg/L) and fed to shrimp P. monodon PL for 20 d. After feeding experiment, the P. monodon PL were challenged with WSSV, and the mortality percentage was recorded daily up to 21 d. During the challenge test, the control PL showed 100% cumulative mortality within 9 d, but the fucoidan‐enriched Artemia nauplii fed groups of PL exhibited 60–94% cumulative mortality within 21 d. The reduction in mortality percentage of experimental groups of PL over control PL was ranged between 33.71 and 61.65%. The polymerase chain reaction analysis confirmed the concentration‐dependent variation in WSSV infection.

Treatment of vibriosis in European sea bass larvae, Dicentrarchus labrax L., with oxolinic acid administered by bath or through medicated nauplii of Artemia franciscana (Kellogg): efficacy and residual kinetics

European sea bass larvae were challenged by bath with Listonella anguillarum strain 332A, 2.5×10(7) CFUmL(-1) for 1h. Fish either received no treatment or oral treatment with Artemia franciscana (Kellog) nauplii enriched with oxolinic acid, or bath treatments with oxolinic acid. Medication commenced 1day following challenge and was performed on days 1, 3 and 5 post-challenge at a dosage of 20mgL(-1) for 2h for bath treatments, while two doses each of 750 nauplii per fish were administered daily for five consecutive days in oral treatments. Cumulative mortality reached 96% for the unmedicated challenged group, 32% in the group receiving bath treatments and 17% in the group receiving medicated nauplii. Pharmacokinetic parameters of oxolinic acid were calculated in sea bass larvae, for both treatments. Steady-state concentrations of oxolinic acid of 48.0 and 75.2μgg(-1) were achieved for bath treatment and oral treatment, respectively, while the elimination half-life was calculated to be 25.1h for bath treatment and 21.7h for oral treatment.

Bioprospection of Microalgae and Cyanobacteria as Biocontrol Agents Against Vibrio campbellii and Their Use in White Shrimp Litopenaeus vannamei Culture

Vibrio spp. are the most common and harmful shrimp pathogenic bacteria; however, microalgae and cyanobacteria have the ability to produce antimicrobial substances against these species. In this study, the organic and aqueous extracts of 28 species of marine microalgae and cyanobacteria were screened against Vibrio campbellii M1. Two of these phytoplankton species with antibacterial activity in aqueous extracts (Dunaliella tertiolecta and Skeletonema costatum) and nontoxic to brine shrimp Artemia franciscana nauplii were used to evaluate their anti‐Vibrio effect when used as green‐water cultures in Vibrio‐challenged white shrimp Litopenaeus vannamei cultures. No differences in mortality of juvenile L. vannamei were observed between treatments tested, suggesting that the pathogenicity of V. campbellii could be related to the growth stage of shrimp. The proximal composition of D. tertiolecta and S. costatum was in the recommended range for penaeid shrimp nutrition, allowing shrimp supplemented with these microalgae to have significantly greater total length and weight than control shrimp. Shrimp supplemented with S. costatum presented the highest values of organic mass (11.48 mg/organism) and growth rate (0.31 mg/d) in comparison to D. tertiolecta. These results indicate that microalgae are not only capable of producing antibacterial compounds against Vibrio but can also help shrimp nutrition.

Light and transmission electron microscopy of Vibrio campbellii infection in gnotobiotic Artemia franciscana and protection offered by a yeast mutant with elevated cell wall glucan

Luminescent vibrios are amongst the most important pathogens in aquaculture, affecting almost all types of cultured organisms. Vibrio campbellii is one of these most important pathogens. In this study, the effects of feeding mnn9 yeast cell wall mutant and wild type yeast strain were investigated in the digestive tract of brine shrimp nauplii, Artemia franciscana, after experimental infection with V. campbellii (LMG 21363). Gnotobiotic A. franciscana nauplii were fed daily with dead Aeromonas hydrophila LVS3, and with either wild type strain of baker's yeast, Saccharomyces cerevisiae, or mutant strain mnn9, of which the cell wall contains elevated chitin and glucan and lower mannose levels. After three days of feeding, some nauplii were challenged with V. campbellii. Mean survival (%), individual length (mm) and total length (mm) at one day and two days after challenge were significantly higher in the group fed mnn9 than in the group fed wild type yeast (81±1.50 and 63±0.49, 1.56±0.07 and 1.13±0.02, 38.21±3.11 and 21.26±0.81 respectively for one day and 50±2.37 and 20±1.41, 2.33±0.01 and 1.24±0.04, 34.97±5.56 and 7.45±1.63 for two days after challenge). Histological examination revealed that the luminal diameter and enterocyte height of both mid- and hindgut were larger in the mnn9-fed group. Colonization of the gut lumen by V. campbellii could be observed by transmission electron microscopy for the group of nauplii fed with wild type yeast. Furthermore, it was observed that V. campbellii caused damage to the gut epithelium including shortening and disappearance of the microvilli, destruction of the apical cell membrane and cell lysis in the nauplii fed wild type yeast. The gut epithelium remained intact in challenged nauplii fed mnn9 yeast. The morphological findings of the present study further substantiate previous studies reporting a protective effect of this yeast cell wall mutant.