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Effect of medicinal plants on the survival of white shrimp (Penaeus vannamei) challenged with WSSV and Vibrio parahaemolyticus.

The negative impact of viral (white spot syndrome virus, WSSV) and bacterial (acute hepatopancreatic necrosis disease, AHPND) diseases on shrimp production demands continuous innovation to increase productivity (Leu et al., 2013; Tran et al., 2013; Nunan et al., 2014). Viral diseases are not curable, and there are only antibiotics to treat bacterial diseases, which cause resistance and environmental impact (Chanu et al., 2012; Medina-Beltran et al., 2012). Wich has led to the use of natural products, such as medicinal plants, which are a good alternative in the prevention and treatment of diseases because they are deposits and sources of safe and cheap chemical products and do not generate resistance in pathogens (Chanu et al., 2012). The plants Aegle marmelos, Cynodon dactylon, Lantana camara, Momordica charantia, Phyllanthus amarus and fucoidan extracted from Sargassum wightii showed antiviral activity against WSSV and immunostimulated Penaeus monodon (Balasubramanian et al., 2007; Immanuel et al., 2012). In this sense, Allium sativum (garlic) contains dietary fiber, 32% sugars (included inulin), flavonoids, pectin, and the organic sulfur compounds alliin (allicin, ajoenes, vinyldithiins, and sulfides), scordinin A and B, and garlicnins (Swiderski et al., 2007; Nohara et al., 2013). Zingiber officinale (ginger) contains sesquiterpens and phenolic compounds (gingerol and shogaol) (Hasan et al., 2012). Echinacea purpurea (Echinacea) is one of the most important and well-known medicinal plants in the world. It is highly composed of alkamides, caffeic acid derivatives, and polysaccharides; however, compounds like flavonoids (quercetin, kaempferol, isorhamnetin and their free phenolic acids, including p-coumaric, p-hydroxybenzoic, and protocatechuic acids), amides, and alkaloids have also been isolated (Spelman & Wetschler, 2009; Manayi et al., 2015). Ocimum sanctum (purple basil) contains volatile oils (eugenol, carvacrol, ursolic acid, linalool, limatrol, caryophyllene, and sitosterol) (Mondal et al., 2009; Pattanayak et al., 2010), phenolic compounds (apigenin, rosmarinic acid, cirsilineol, cirsimaritin, isotimusin, and isotimonin) (Yanpallewar et al., 2004; Pattanayak et al., 2010), flavonoids (orientin, vicenin, luteolin, and molludistin) (Gupta et al., 2002), tannins, and a series of sesquiterpenes and monoterpenes (bornyl acetate, b-elemene, neral, A-pinene, b-pinene, camfeno, campesterol, and stigmasterol) (Jaggi et al., 2003).

The study aimed to determine the effect of medicinal plantas on the survival of Penaeus vannamei challenged with WSSV and Vibrio parahaemolyticus by ingestion and immersion.

Shrimp were obtained from a commercial farm with the hatchery, transported, and maintained at the Centro Interdisciplinario de Investigacion para el Desarrollo Integral Regional, Unidad Sinaloa. A white spot disease analysis was made in 12 shrimp, using PCR (nested PCR, Kimura, et al., 1996), to know the percentage of WSSV-infected shrimp.

Farm shrimp (5-6 g) with symptoms of white spot disease were collected to obtain an inoculum in saline solution (2.5% NaCl) and a paste of macerated abdominal muscle and branchial lamella. A white spot disease analysis was made by PCR (nested and single PCR). An inoculum of V. parahaemolyticus was prepared according to Lopez-Leon et al. (2016).

The mixture of powdered plants (PP), A. sativum (40%), Z. officinale (20%), E. purpurea (20%), and O. sanctum (20%) was added to commercial feed Camaronina[R] (Purina, 35% protein), which was reconstituted in a meat mill, dried at room temperature with a fan for 24 h, and stored at 4[degrees]C. This proportion of powdered plants was based on previous works and on the cost of the plants since Echinacea is very expensive.

Before each V. parahaemolyticus challenge (bioassays one and two), a bioassay (4 d) was conducted to determine the [LC.sub.50] (lethal concentration, 50%) using animals weighing 528 [+ or -] 31.7 mg and 452 [+ or -] 50 mg. Experimental glass aquariums (6 L) contained 4 L of filtered sea water under constant aeration. Every bioassay consisted of five treatments, each one in triplicate (30 shrimps, 10 per tank): I) control without Vibrio; II) Vibrio (1x[10.sup.3] CFU [mL.sup.-1]); III) Vibrio (1x[10.sup.4] CFU [mL.sup.-1]); IV) Vibrio (1x[10.sup.5] CFU [mL.sup.-1]); and V) Vibrio (5x[10.sup.5] CFU [mL.sup.-1]). Shrimp were fed (35% protein feed) twice daily at 09:00 and 17:00 h. Bioassays were conducted under the natural photoperiod. No cleaning of the tanks was made during the challenge period, and the temperature was maintained between 29.5 and 30.5[degrees]C to favor shrimp infection. Salinity was maintained at 30. Mortality was recorded three times daily, and final data were used to calculate the [LC.sub.50] using Probit analysis (Finney, 1952) with StatPlus[R] 2009 professional 5.8.4.

The first bioassay was conducted for 10 d with 10 shrimp (528 [+ or -] 31.7 mg) per glass aquarium with 4 L of seawater under constant aeration. The challenge was done by adding bacteria ([LC.sub.50] = 15.9x[10.sup.4] CFU [mL.sup.-1]) and infected shrimp tissue (WSSV, 500 mg, low viral load) to each glass aquarium at day seven once only. The bioassay consisted of five treatments each one in triplicate (10 shrimps per tank): I) control without pathogens; II) control with WSSV + Vibrio [LC.sub.50]; III) PP (1 g kg [feed.sup.-1]) + WSSV + Vibrio [LC.sub.50]; IV) PP (2 g kg [feed.sup.-1]) + WSSV + Vibrio [LC.sub.50]; and V) PP (4 g kg [feed.sup.-1]) + WSSV + Vibrio [LC.sub.50]. Shrimp were fed (35% protein feed) twice daily during 10 d (09:00 and 17:00 h) with commercial feed alone or commercial feed with PP. The bioassay was conducted under the natural photoperiod. The temperature was maintained between 29.5 and 30.5[degrees]C. Uneaten food and waste material were removed (except days 7-10 of the challenge to avoid eliminating the vibrio) by siphoning every three days before feeding, and 50% of the water was exchanged. During the bioassay, mortality was recorded daily. Shrimp from the stock were WSSV-free.

The second bioassay was conducted for 20 d with 10 shrimp (452 [+ or -] 50 mg) per glass aquarium with 4 L of seawater and constant aeration. The challenge was done by adding bacteria ([LC.sub.50] = 6.5x[10.sup.4] CFU [mL.sup.-1]) and infected shrimp tissue (WSSV, 200 mg, high viral load) to each glass aquarium at day nine once only. The bioassay consisted of five treatments each one in triplicate (10 shrimps per tank): I) control without pathogens; II) control with WSSV + Vibrio [LC.sub.50]; III) PP (4 g kg [feed.sup.-1]) daily + WSSV + Vibrio [LC.sub.50]; IV) PP (4 g kg [feed.sup.-1]) every 2 days + WSSV + Vibrio [LC.sub.50]; and V) PP (4 g kg [feed.sup.-1]) every 3 days + WSSV + Vibrio [LC.sub.50]. Shrimp were fed previously mentioned. The bioassay was conducted under the natural photoperiod. The temperature was maintained between 29.5 and 30.5[degrees]C. Uneaten food and waste material were removed (except days 9-12 of the challenge to avoid eliminating pathogens) by siphoning every three days before feeding, and 50% of the water was exchanged. During the bioassay, mortality was recorded daily. Shrimps from the stock were WSSV-free.

Survival data in percentage were arcsine transformed. One-way variance analysis (ANOVA) was applied to examine the differences in survival. Where significant ANOVA differences were found, a Tukey's HSD test was used to identify these differences at P < 0.05.

In shrimp fed PP at different concentrations, the highest survival was observed in treatment IV (96.67 [+ or -] 3.33%) and the lowest in treatment II (6.67 [+ or -] 3.33%). The negative control group and treatments with PP showed significant differences (P < 0.05) as compared with treatment II in which the shrimps were only challenged with pathogens (Fig. 1).

High survival (96.7-100%) was observed in shrimp fed PP at different frequencies. The negative control group and treatments with PP showed significant differences (P < 0.05) as compared with treatment II in which the shrimps were only challenged with pathogens. Live and dead shrimp positive to WSSV showed low viral load (nested PCR). WSSV prevalence was similar in all treatments challenged with both pathogens (Fig. 2).

Plants are a source of secondary metabolites (Chanu et al., 2012) of interest for aquaculture to prevent and treat diseases and it is known that combinations of medicinal plants can show a better biological effect than an individual medicinal plant since the bioactive compounds of each one can act in synergy (Martinez et al., 2013; Mas Toro et al., 2017). For the above, we worked with a combination of garlic, ginger, basil, and Echinacea to know their effect on the survival of P. vannamei challenged with pathogens such as V. parahaemolyticus and WSSV. However, it is important to mention that plants, in addition to antimicrobial and immunostimulant molecules, contain molecules such as phytic acid and tannins that can affect cultured organisms (Bairagi et al., 2002; El-Adawy, 2002; Flores-Miranda et al., 2014); thus, the amount and frequency of application in the feed must be determined.

In this work, survival of shrimp challenged with WSSV and V. parahaemolyticus and fed with 4 g kg [feed.sup.-1] of PP at different frequencies was higher (50-90%) than the survival of shrimp not fed with PP. Results were consistent with those reported by Huynh et al. (2011) who found that P. vannamei showed increased resistance to infection with V. alginolyticus and WSSV when they were immersed in seawater containing Sargassum hemiphyllum var. chinense powder and its extract. In the same way, Trejo-Flores et al. (2016) found a protective effect of Aloe vera powder (1 g kg [feed.sup.-1] every two days) in P. vannamei challenged with the same pathogens of this work.

Regarding WSSV prevalence in live and dead shrimp, it was similar in all treatments challenged with both pathogens, indicating that in shrimp challenged with the low viral load, mortality occurred mainly due to V. parahaemolyticus (Rubio-Castro et al., 2016), even though, Medina-Beltran et al. (2012) and Peraza-Gomez et al. (2014) observed that E. purpurea and Uncaria tomentosa have antiviral (WSSV) effects that contribute to obtaining better survival in cultured white shrimp infected with WSSV.

Results showed that the tested PP protects shrimp against WSSV and V. parahaemolyticus. Therefore, further research about the effect of garlic, ginger, Echinacea, and basil is necessary for commercial shrimp farms.

ACKNOWLEDGEMENTS

The authors thank the Secretaria de Investigacion y Posgrado del Instituto Politecnico Nacional (Mexico) for financial support (20160629). Jesus A. Fierro-Coronado thanks CONACYT for the doctoral fellowship (237352).

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Jesus Arturo Fierro-Coronado (1,2), Antonio Luna-Gonzalez (1), Carlos Juventino Caceres-Martinez (2) Cesar A. Ruiz-Verdugo (2), Ruth Escamilla-Montes (1), Genaro Diarte-Plata (1) Maria del Carmen Flores-Miranda (3), Pindaro Alvarez-Ruiz (1) &Viridiana Peraza-Gomez (4)

(1) Instituto Politecnico Nacional, Centro Interdisciplinario de Investigacion para el Desarrollo Integral Regional, Unidad Sinaloa, Guasave, Sinaloa, Mexico

(2) Postgrado en Ciencias Marinas y Costeras, Universidad Autonoma de Baja California Sur La Paz, B.C.S., Mexico

(3) Departamento de Estudios para el Desarrollo Sustentable de Zonas Costeras Centro Universitario de la Costa Sur, Universidad de Guadalajara, San Patricio Melaque, Jalisco, Mexico

(4) Escuela Nacional de Ingenieria Pesquera, Universidad Autonoma de Nayarit San Blas, Nayarit, Mexico

Corresponding author: Antonio Luna-Gonzalez (aluna@ipn.mx)

Corresponding editor: Mauricio Laterca

Received: 20 February 2018; Accepted: 11 February 2019

DOI: 10.3856/vol47-issue2-fulltext-20
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Title Annotation:Short Communication
Author:Fierro-Coronado, Jesus Arturo; Luna-Gonzalez, Antonio; Caceres-Martinez, Carlos Juventino; Ruiz-Verd
Publication:Latin American Journal of Aquatic Research
Date:May 1, 2019
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