The effect of Vibrio parahaemolyticus IPNGS16 on the bacterial profile of the gut of Penaeus vannamei was assessed by 16S metagenomic analysis. The V3 hypervariable region of the bacterial 16S rDNA was amplified by PCR. Sequencing reads were generated using the 2×150 (300 cycles) for the base-read length chemistry of the Illumina MiniSeq platform. The web-based Shaman and MicrobiomeAnalyst platforms were used to analyze the sequences. The phyla Proteobacteria, Bacteroidetes, and the genera Vibrio, Ruegeria, Nautella, and Pseudoalteromonas were found among the most abundant taxonomic ranks in control, diseased, and healthy shrimp. Alpha and beta indices showed significant differences between shrimp survival in the control condition and dying shrimp (lower diversity). Metabolism (carbohydrate and amino acid metabolism-related genes and, to a lesser extent, energy, lipid, and cofactors and vitamin metabolism-related genes) of dying and surviving shrimp was affected by Vibrio infection. The top metabolic functions (cell cycle, glycine, serine, threonine, cysteine, methionine, purine, pyrimidine, pyruvate, and quorum sensing) in dying and surviving shrimp were affected by Vibrio, especially quorum sensing. The interaction network analysis showed fewer interactions in dying shrimp than control and surviving shrimp. Proteobacteria, Bacteroidetes, Vibrio, and Ruegeria predominated in all samples, and Vibrio changed bacterial diversity and metabolism in the intestine of P. vannamei. Ruegeria and Pseudoalteromonas showed negative interactions with Vibrio, suggesting their use as probiotics. This study sheds light on the Vibrio infection in the gut microbiota of shrimp.

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The effects of fulvic acid (FA) on survival and immune-related gene expression were investigated in Litopenaeus vannamei challenged with Vibrio parahaemolyticus by immersion. Shrimp were fed with different dietary FA concentrations (1, 2, 4 and 6 g/kg feed) for 20 days (first bioassay) or 8 days (second bioassay, 2 g/kg feed of FA added every 2 days) and then challenged with V. parahaemolyticus. In a third bioassay, the expression of three immune-related genes (translationally controlled tumour protein [TCTP], superoxide dismutase [SOD] and heat-shock protein 70[HSP70]) in haemocytes or hepatopancreas of experimental shrimp was measured by real-time quantitative PCR at 0, 6, 12, 24, 48, 72 and 96 hr after FA (2 g/kg feed) administration. Fulvic acid increased survival at a concentration of 2 g/kg feed supplied every two days. Interestingly, TCTP gene expression was upregulated, whereas gene expression of SOD and HSP70 was downregulated. In conclusion, dietary fulvic acid improves survival in white shrimp challenged with V. parahaemolyticus and modulates the immune response. Therefore, FA merits further evaluation as prophylactic treatment in commercial shrimp farms. K E Y W O R D SAHPND, fulvic acid, gene expression, Litopenaeus vannamei, Vibrio parahaemolyticus

The white spot syndrome virus (WSSV) remains the most devastating viral pathogen of shrimp culture worldwide. Gene silencing by RNA interference (RNAi) using double stranded RNA (dsRNA) has been considered a powerful tool for conferring protection against WSSV when viral genes are silenced, as documented in several shrimp species. However, this effect is not long lasting. Our results provide the first evidence that long-term silencing of the LvRab7 endogen produced antiviral effect against WSSV, which endured at least 21 d after dsRNA treatment (dat). Until now, the most efficient way to implement RNAi with dsRNA into the shrimp is by injection. Consequently, its application to broodstock in hatcheries is possible, minimizing the risk of vertical transmission of the virus. We show that the expression of Rab7 in hemocytes is lowest at 2 dat and finally recovers to basal status. In contrast, in gills and pleopods, gene expression silencing continued for at least 21 d. We challenged Litopenaeus vannamei broodstock with WSSV at 7, 14, or 21 dat reaching mortality rates of 0, 40, and 27%, respectively. In conclusion, the LvRab7 gene silencing is progressive and effective against WSSV. However, further studies are necessary to elucidate the functions of Rab7 in shrimp cells before applying this methodology at a commercial level.

Peraza-Gómez V. Effect of microbial immunostimulants on WSSV infection percentage and the expression of immune-related genes in white shrimp (Litopenaeus vannamei). Rev Colomb Cienc Pecu 2019; 32(3):221-231. AbstractBackground: The white spot syndrome virus (WSSV) causes high mortalities in aquaculture. The use of immunostimulants increases animal resistance. Objective: To evaluate the WSSV infection percentage and the immunostimulant effect of lactic acid bacteria and yeast (MI= microbial immunostimulants) on WSSV infected Litopenaeus vannamei. Methods: A bioassay was performed for 33 d, with treatments in triplicate. The MI was added to the feed at 8.5 mg/kg feed and offered to shrimp (9.9 ± 3.1 g) daily, every 2 days, or every 3 days. Shrimp were infected with WSSV at 9 and 19 days. The expression of four immune system-related genes was studied using qRT-PCR. Results: No significant differences were observed in growth and survival among treatments. At the end of the bioassay, WSSV infection percentage (low viral load) decreased 8.3 and 25% in treatments III and IV as compared to the control group. Treatments with MI showed significant differences in the relative expression of LvToll, transglutaminase, and prophenoloxidase genes when MI was offered daily as compared to the control group. The MI did not regulate the expression of the superoxide dismutase gene. The WSSV infection percentage decreased when feed with MI was offered every 3 d. Conclusion: The MI decrease WSSV infection percentage in L. vannamei infected with low viral load when it is offered every three days. The MI up-regulates LvToll, Tgase, and proPO genes when it is offered daily. Further research is needed regarding prophylactic treatment with microbial immunostimulants against WSSV in commercial shrimp farms. ResumenAntecedentes: El virus del síndrome de la mancha blanca (WSSV) ocasiona alta mortalidad en acuacultura. El empleo de inmunoestimulantes incrementa la resistencia de los animales. Objetivo: Evaluar el porcentaje de infección de WSSV y el efecto inmunoestimulante de bacterias ácido lácticas y levadura (MI= inmunoestimulantes microbianos) en Litopenaeus vannamei infectado con WSSV. Métodos: Se realizó un bioensayo durante 33 días con tratamientos por triplicado. Se agregó MI al alimento (8,5 mg/kg de alimento), suministrandolo al camarón (9,9 ± 3,1 g) diariamente, cada 2, o cada 3 días. Los camarones se infectaron con WSSV a los 9 y 19 días. Se estudió la expresión de cuatro genes relacionados con el sistema inmune utilizando qRT-PCR. Resultados: No hubo diferencias significativas en el crecimiento y la sobrevivencia entre los tratamientos. Al final del bioensayo, el porcentaje de infección de WSSV (baja carga viral) disminuyó un 8,3 y un 25% en los tratamientos III y IV en comparación con el grupo control. Los tratamientos con MI mostraron diferencias significativas en la expresión relativa de los genes LvToll, transglutaminasa, y profenoloxidasa cuando se suministró MI diariamente en comparación con el grupo control. Los MI no regul...