Use of brome mosaic virus-like particles in feed, to deliver dsRNA targeting the white spot syndrome virus vp28 gene, reduces Penaeus vannamei mortality.

Numerous strategies have been investigated to combat viral infections in shrimp, specifically targeting the white spot syndrome virus (WSSV) that has caused outbreaks worldwide since the 1990s. One effective treatment involves intramuscular application of dsRNA-mediated interference against the viral capsid protein VP28. However, this approach presents challenges in terms of individual shrimp management, limiting its application on a large scale. To address this, our study aimed to evaluate the efficacy of oral delivery of protected dsRNA using chitosan nanoparticles or virus-like particles (VLPs) synthesized in brome mosaic virus (BMV). These delivery systems were administered before, during, and after WSSV infection to assess their therapeutic potential. Our findings indicate that BMV-derived VLPs demonstrated superior efficiency as nanocontainers for dsRNA delivery. Notably, the treatment involving vp28 dsRNA mixed in the feed and administered simultaneously to shrimp already infected with WSSV exhibited the highest survival rate (48%), while the infected group had a survival rate of zero, suggesting the potential efficacy of this prophylactic approach in commercial shrimp farms.

Agavin induces beneficial microbes in the shrimp microbiota under farming conditions.

Prebiotics and probiotics have shown a number of beneficial impacts preventing diseases in cultured shrimps. Complex soluble carbohydrates are considered ideal for fostering microbiota biodiversity by fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAPS). Here we evaluated the growth performance and microbiota composition of the white shrimp Litopenaeus vannamei after dietary intervention using agavin as a FODMAP prebiotic under farming conditions. Adult L. vannamei were raised at a shrimp farm and the effect of agavin supplemented at 2% (AG2) or 10% (AG10) levels were compared to an agavin-free basal diet (BD). After 28 days-trial, the feed conversion ratio, total feed ingested, and protein efficiency ratio was significantly improved on animals fed with AG2. At the same time, no effect on growth performance was observed in AG10. Surprisingly, after sequencing the V3-V4 regions of the 16S rRNA gene a higher microbial richness and diversity in the hepatopancreas and intestine was found only in those animals receiving the AG10 diet, while those receiving the AG2 diet had a decreased richness and diversity, both diets compared to the BD. The beta diversity analysis showed a clear significant microbiota clustering by agavin diets only in the hepatopancreas, suggesting that agavin supplementation had a more substantial deterministic effect on the microbiota of hepatopancreas than on the intestine. We analyzed the literature to search beneficial microbes for shrimp's health and found sequences for 42 species in our 16S data, being significantly increased Lactobacillus pentosus, Pseudomonas putida and Pseudomonas synxantha in the hepatopancreas of the AG10 and Rodopseudomonas palustris and Streptococcus thermophiles th1435 in the hepatopancreas of the AG2, both compared to BD. Interestingly, when we analyzed the abundance of 42 beneficial microbes as a single microbial community "meta-community," found an increase in their abundance as agavin concentration increases in the hepatopancreas. In addition, we also sequenced the DNA of agavin and found 9 of the 42 beneficial microbes. From those, Lactobacillus lactis and Lactobacillus delbrueckii were found in shrimps fed with agavin (both AG2 and AG10), and Lysinibacillus fusiformis in AG10 and they were absent the BD diet, suggesting these three species could be introduced with the agavin to the diet. Our work provides evidence that agavin supplementation is associated with an increase of beneficial microbes for the shrimp microbiota at farming conditions. Our study provides the first evidence that a shrimp prebiotic may selectively modify the microbiota in an organ-dependent effect.

Antiviral therapy in shrimp through plant virus VLP containing VP28 dsRNA against WSSV.

The white spot syndrome virus (WSSV), currently affecting cultured shrimp, causes substantial economic losses to the worldwide shrimp industry. An antiviral therapy using double-stranded RNA interference (dsRNAi) by intramuscular injection (IM) has proven the most effective shrimp protection against WSSV. However, IM treatment is still not viable for shrimp farms. The challenge is to develop an efficient oral delivery system that manages to avoid the degradation of antiviral RNA molecules. The present work demonstrates that VLPs (virus-like particles) allow efficient delivery of dsRNAi as antiviral therapy in shrimp. In particular, VLPs derived from a virus that infects plants, such as cowpea chlorotic mottle virus (CCMV), in which the capsid protein (CP) encapsidates the dsRNA of 563 bp, are shown to silence the WSSV glycoprotein VP28 (dsRNAvp28). In experimental challenges in vivo, the VLPs- dsRNAvp28 protect shrimp against WSSV up to 40% by oral administration and 100% by IM. The novel research demonstrates that plant VLPs, which avoid zoonosis, can be applied to pathogen control in shrimp and also other organisms, widening the application window in nanomedicine.

Microbiome of Pacific Whiteleg shrimp reveals differential bacterial community composition between Wild, Aquacultured and AHPND/EMS outbreak conditions.

Crustaceans form the second largest subphylum on Earth, which includes Litopeneaus vannamei (Pacific whiteleg shrimp), one of the most cultured shrimp worldwide. Despite efforts to study the shrimp microbiota, little is known about it from shrimp obtained from the open sea and the role that aquaculture plays in microbiota remodeling. Here, the microbiota from the hepatopancreas and intestine of wild type (wt) and aquacultured whiteleg shrimp and pond sediment from hatcheries were characterized using sequencing of seven hypervariable regions of the 16S rRNA gene. Cultured shrimp with AHPND/EMS disease symptoms were also included. We found that (i) microbiota and their predicted metagenomic functions were different between wt and cultured shrimp; (ii) independent of the shrimp source, the microbiota of the hepatopancreas and intestine was different; (iii) the microbial diversity between the sediment and intestines of cultured shrimp was similar; and (iv) associated to an early development of AHPND/EMS disease, we found changes in the microbiome and the appearance of disease-specific bacteria. Notably, under cultured conditions, we identified bacterial taxa enriched in healthy shrimp, such as Faecalibacterium prausnitzii and Pantoea agglomerans, and communities enriched in diseased shrimp, such as Aeromonas taiwanensis, Simiduia agarivorans and Photobacterium angustum.

Metabolism of Seriola lalandi during Starvation as Revealed by Fatty Acid Analysis and Compound-Specific Analysis of Stable Isotopes within Amino Acids.

Fish starvation is defined as food deprivation for a long period of time, such that physiological processes become confined to basal metabolism. Starvation provides insights in physiological processes without interference from unknown factors in digestion and nutrient absorption occurring in fed state. Juveniles of amberjack Seriola lalandi were isotopically equilibrated to a formulated diet for 60 days. One treatment consisted of fish that continued to be fed and fish in the other treatment were not fed for 35 days. The isotopic signatures prior to the beginning of and after the starvation period, for fish in the starvation and control treatments, were analysed for lipid content, fatty acid composition and isotopic analysis of bulk (EA-IRMS) and of amino acids (compound specific isotope analysis, CSIA). There were three replicates for the starvation group. Fatty acid content in muscle and liver tissue before and after starvation was determined to calculate percent change. Results showed that crude lipid was the most used source of energy in most cases; the PUFAs and LC-PUFAs were highly conserved. According to the protein signature in bulk (δ15N) and per amino acid (δ13C and δ15N), in muscle tissue, protein synthesis did not appear to occur substantially during starvation, whereas in liver, increases in δ13C and δ15N indicate that protein turnover occurred, probably for metabolic routing to energy-yielding processes. As a result, isotopic values of δ15N in muscle tissue do not change, whereas CSIA net change occurred in the liver tissue. During the study period of 35 days, muscle protein was largely conserved, being neither replenished from amino acid pools in the plasma and liver nor catabolized.

Protein retention assessment of four levels of poultry by-product substitution of fishmeal in rainbow trout (Oncorhynchus mykiss) diets using stable isotopes of nitrogen (δ15N) as natural tracers.

This is second part from an experiment where the nitrogen retention of poultry by-product meal (PBM) compared to fishmeal (FM) was evaluated using traditional indices. Here a quantitative method using stable isotope ratios of nitrogen (δ(15)N values) as natural tracers of nitrogen incorporation into fish biomass is assessed. Juvenile rainbow trout (Oncorhynchus mykiss) were fed for 80 days on isotopically distinct diets in which 0, 33, 66 and 100% of FM as main protein source was replaced by PBM. The diets were isonitrogenous, isolipidic and similar in gross energy content. Fish in all treatments reached isotopic equilibrium by the end of the experiment. Two-source isotope mixing models that incorporated the isotopic composition of FM and PBM as well as that of formulated feeds, empirically derived trophic discrimination factors and the isotopic composition of fish that had reached isotopic equilibrium to the diets were used to obtain a quantitative estimate of the retention of each source of nitrogen. Fish fed the diets with 33 and 66% replacement of FM by PBM retained poultry by-product meal roughly in proportion to its level of inclusion in the diets, whereas no differences were detected in the protein efficiency ratio. Coupled with the similar biomass gain of fishes fed the different diets, our results support the inclusion of PBM as replacement for fishmeal in aquaculture feeds. A re-feeding experiment in which all fish were fed a diet of 100% FM for 28 days indicated isotopic turnover occurred very fast, providing further support for the potential of isotopic ratios as tracers of the retention of specific protein sources into fish tissues. Stable isotope analysis is a useful tool for studies that seek to obtain quantitative estimates of the retention of different protein sources.

Intestinal absorption of amino acids in the Pacific bluefin tuna (Thunnus orientalis): in vitro lysine-arginine interaction using the everted intestine system.

The interaction between lysine (Lys) and arginine (Arg) in the proximal intestinal region of Pacific bluefin tuna (Thunnus orientalis) was evaluated using the everted intestine method. This in vitro intestinal system has been shown to be an effective tool for studying the nutrient absorption without the need to handle the tuna fish in marine cages as needed for digestibility and amino acid (AA) absorption. We used a factorial design with two sets of variables: low and high Lys concentration (10 and 75 mM) and four different Arg concentrations (3, 10, 20, and 30 mM). Both amino acids were dissolved in marine Ringer solution with a basal amino acidic composition consisting of a tryptone solution (9 mg mL(-1)). No interaction was observed between the absorption of Lys and Arg during the first 10 min of the experiment when low concentration of Lys and Arg was used in the hydrolyzate solution. However, there seemed to be a positive effect on Lys absorption when both amino acids were at high concentrations (30 and 75 mM, respectively). This type of studies will led us to test different formulations and/or additives to better understand the efficiency of AA supplementation as an alternative to in situ studies that are difficult to follow to design with the Pacific Bluefin Tuna.

Laccase-mediated transformations of endocrine disrupting chemicals abolish binding affinities to estrogen receptors and their estrogenic activity in zebrafish.

Endocrine disrupting chemicals (EDCs) are known to mainly affect aquatic organisms, producing negative effects in aquaculture. Transformation of the estrogenic compounds 17ß-estradiol (E2), bisphenol-A (BPA), nonylphenol (NP), and triclosan (TCS) by laccase of Coriolopsis gallica was studied. Laccase is able to efficiently transform them into polymers. The estrogenic activity of the EDCs and their laccase transformation products was evaluated in vitro as their affinity for the human estrogen receptor alpha (hERα) and for the ligand binding domain of zebrafish (Danio rerio) estrogen receptor alpha (zfERαLBD). E2, BPA, NP, and TCS showed higher affinity for the zfERαLBD than for hERα. After laccase treatment, no affinity was found, except a marginal affinity of E2 products for the zfERαLBD. Endocrine disruption studies in vivo on zebrafish were performed using the induction of vitellogenin 1 as a biomarker (VTG1 mRNA levels). The use of enzymatic bioreactors, containing immobilized laccase, efficiently eliminates the endocrine activity of BPA and TCS, and significantly reduces the effects of E2. The potential use of enzymatic reactors to eliminate the endocrine activity of EDCs in supply water for aquaculture is discussed.

Efecto de la adición de tres pigmentos carotenoides comerciales (astaxantina, cantaxantina y β-caroteno) en la dieta sobre la coloración de la concha o nácar de juveniles de abulón rojo Haliotis rufescens / Effect of commercial carotene pigments (astaxanthin, cantaxanthin and β-carotene) in juvenile abalone Haliotis rufescens diets on the color of the shell or nacre

With the aim to observe any color effect on the shell or nacre of juvenile red abalone (Haliotis rufescens), three diets were formulated adding carotene pigments (astaxanthin, cantaxanthin and β-carotene) and one control diet without pigments. Juvenile abalone (n = 504) with a shell length and weight of 5.46 ± 0.87 mm and 0.03 ± 0.16 g, respectively, were utilized. The abalones were randomly distributed in twelve buckets (20 L) connected to a recirculation system under controlled temperature and constant water flow. Each treatment was done in triplicate. After 90 days of experimentation, the organisms fed on diets with inclusion of pigments showed a length growth rate of 53.06 ± 6.91 μm/day and weight of 1.34 ± 0.24 mg, whereas the juveniles fed with the control diet showed a growth rate of 74.93 ± 14.63 μm/day and weighed 2.13 ± 0.40 mg. The formation of shell and color recorded resulted in a minor color change compared to the control diet. However, in spite of these changes the supplementation of pigment at this point is not recommended. Nevertheless, more efforts should be made to research the shell color manipulation.

Con el propósito de observar cambios en la coloración de la concha y nácar de juveniles de abulón rojo (Haliotis rufescens), se formularon tres dietas a las que se les agregaron pigmentos carotenoides (astaxantina, cantaxantina y β-caroteno) y una dieta testigo sin pigmentos. Se obtuvieron juveniles de abulón (n = 504) de 5.46 ± 0.87 mm y 0.03 ± 0.16 g de longitud promedio de la concha y peso, respectivamente. Los abulones se distribuyeron aleatoriamente en 12 cubetas de 20 L, conectadas a un sistema cerrado de recirculación, con temperatura y flujo constante. Cada tratamiento se realizó por triplicado. Después de 90 días de experimentación, los organismos alimentados con dietas con inclusión de pigmentos presentaron una tasa de crecimiento en longitud de 53.06 ± 6.91 μm/dia y peso 1.34 ± 0.24 mg, mientras que los abulones de la dieta testigo crecieron a razón de 74.93 ±14.63 μm/dia y pesaron 2.13 ± 0.40 mg, sin que se observaran diferencias significativas entre los tratamientos (α = 0.05) experimentales. La formación de la concha y la coloración se registraron mediante imágenes fotográficas y con ayuda de una paleta de colores se observó un ligero cambio en la coloración de la parte exterior de la concha hacia tonalidades amarillas de los abulones alimentados con dietas que incluían pigmentos, siendo más intensa para aquellos que contenían β-caroteno. Sin embargo, a pesar de estos cambios no se recomienda la incorporación de pigmentos para abulón en ese momento, pero será necesario investigar más sobre la manipulación del color de sus conchas.

Nonylphenol algal bioaccumulation and its effect through the trophic chain.

Nonylphenol is a metabolic intermediate from the microbial transformation of detergents used worldwide. While nonylphenol shows some acute toxicity, it is also able to mimic important hormones resulting in the disruption of several processes by interfering with the signals that control the overall physiology of the organism. The effect of the pollutant nonylphenol (NP) through the trophic chain was studied. Microalgae Isochrysis galbana was able to bioconcentrate NP 6940 times, where 77% of initial NP (100microgl(-1)) is accumulated intracellularly after 1-h incubation. Crustacean Artemia fransiscana showed 25% higher growth when fed with NP-rich algae. However, Artemia metabolized almost all NP ingested and only traces of NP could be found in the organism, eliminating future NP effects. Zebrafish (Brachydanio rerio) were affected by the presence of 171microgg(-1) of NP in the diet, showing higher levels of the hormone vitellogenin and lower levels of cytochrome P450 activity. These results showed that organisms placed in the first level of trophic chain are able to significantly bioconcentrate the pollutant and endocrine disruptor NP. These grassed organisms affect the growth of crustacean. Moreover, the organisms placed on the top of some trophic chains, such as fish, could be affected by the presence of NP in their food, in both the hormone levels and metabolic enzymes. This work shows that the environmental presence of NP should be considered as a risk for the organisms living in an ecosystem.