In vitro digestibility of specific dsRNA by enzymes of digestive tract of shrimp Litopenaeus vannamei Digestibilidad in vitro del dsRNA específico por enzimas del tracto digestivo del camarón Litopenaeus vannamei

Objective. The digestibility of specific dsRNA by action of the enzymes of digestive tract of the whiteleg shrimp Litopenaeus vannamei was determined in vitro. Materials and methods. Digestive enzyme activity (amylase, lipase, protease, DNase and RNase) was measured in the stomach, digestive gland, and anterior, middle, and posterior intestine of juvenile shrimp, and the digestibility of DNA, RNA and the dsRNA-ORF89, specific to WSSV, was determined by in vitro assays, as well as electrophoretic and densitometric analyses. Results. The highest enzymatic activity was found in the digestive gland: amylase (81.41%), lipase (92.60%), protease (78.20%), DNase (90.85%), and RNase (93.14%). The highest digestive capacity against DNA, RNA, and dsRNA was found in the digestive gland (5.11 ng of DNA per minute, 8.55 ng of RNA per minute, and 1.48 ng dsRNA per minute). Conclusions. The highest digestibility of dsRNA-ORF89, specific to WSSV, was found in the digestive gland, whereas the lowest digestibility was observed in the posterior intestine. This is the first report regarding the digestibility of dsRNA-ORF89 by whiteleg shrimp digestive tract enzymes, with potential therapeutic importance in shrimp culture to prevent WSSV disease through balanced feed.


INTRODUCTION
One of the most powerful tools used in aquaculture against viral pathogens such as white spot syndrome virus (WSSV) is the mechanism of RNA interference (iRNA) induced by double-stranded RNA (dsRNA) (1).For example, complete inhibition of WSSV infection was achieved in the kuruma shrimp Marsupenaeus japonicus through an injection of specific dsRNA against the viral protein vp28 (2).In the fleshy prawn Fenneropenaeus chinensis, mortality was greatly reduced by applying an injection of specific dsRNA against vp281, vp28, and kinase (PK) proteins (3).Similar results have been reported for whiteleg shrimp Litopenaeus vannamei (4,5).
Recently, the long-term antiviral effects of dsRNA-vp28, dsRNA-vp26, dsRNA-wsv191, and dsRNA-ORF89 (this last one catalogued in latency functional genes) have been studied in whiteleg shrimp L. vannamei infected with WSSV.The results indicated different antiviral responses against WSSV.The whiteleg shrimp L. vannamei treated with dsRNA-ORF89 and dsRNA-vp28 achieved a 90% survival while the shrimp treated with dsRNA-vp26 and dsRNA-wsv191 achieved a survival of 79 and 17% relative to the positive control WSSV (0%) (6), which supports the theory that the antiviral effect depends on the gene to which the block is directed (7), making dsRNA-ORF89 an excellent candidate to incorporate in shrimp feed as an antiviral additive.
Currently, dsRNA can be produced by chemical synthesis, enzymatic synthesis, and by plasmid DNA vectors (8).However, the high cost involved in producing the dsRNA, the complexity of supplying the dsRNA to organisms plus maintaining the necessary concentration for their action on target cells has limited the use of dsRNA for research purposes (9).The most common methods of administering dsRNA are by injection, immersion, or oral route (per os) (10).Each of these methods has several advantages and disadvantages; For example, direct injection has been shown to have an important antiviral effect (11).However, this method of application is costly, labor intensive and considered impractical because of the large number of organisms that need to be protected in culture ponds (12).The Immersion method has the advantage of protecting a greater number of organisms; however, its use is limited to larvae or small organisms (13).The application of oral dsRNA, produced in biological systems such as E.coli HT115 (14,15) and the microalgae Chlamydomonas reinhardtii (16) make its mass application more practical by having the facility (13).La aplicación de dsRNA vía oral, producido en sistemas biológicos como es el caso de la bacteria E.coli HT115 (14,15), y la microalga Chlamydomonas reinhardtii (16), hace más práctica su aplicación masiva al tener la facilidad de incorporarlo a un alimento balanceado.Sin embargo, a la fecha, se desconoce lo que puede suceder en el tracto digestivo del camarón al incorporar dsRNA en un alimento, para funcionar como inductor del mecanismo de RNAi.
Se registró el peso de la glándula digestiva de cada uno de los camarones para estimar el índice Hepatosomático (IHS, (peso de la glándula digestiva/peso total del camarón) X100) (18).Los tejidos de cada segmento fueron pesados y homogenizados (Bio -Gen PRO200, PRO Scientific, Oxford, CT) con agua destilada fría (0 o C), a una proporción v/w de 3 mL de agua destilada g -1 de peso fresco de tejido.Los extractos crudos se clarificaron por centrifugación a 15.294 g durante 10 min a 4°C (5810R, of incorporating it into A balanced food.It is still unknown what takes place in the shrimp's digestive tract when dsRNA is added to feed to induce the iRNA mechanism. The objective of this study was to determine what digestive enzyme activity takes place in the digestive tract of the whiteleg shrimp and test digestibility of DNA, RNA, and dsRNA-ORF89, specific against WSSV, to demonstrate resistance to digestion.To ensure that the organisms used for this study were in normal physiological conditions, the determination of the digestive enzymatic activity of proteases, amylases and lipases was included to demonstrate their presence as a physiological indicator and their higher concentration in the digestive gland, as it has been reported by other authors (Alexandre et al (17).To our knowledge, this is the first study of the digestibility of dsRNA-ORF89 in whiteleg shrimp, focusing on its potential including dsRNA-ORF89 in feed to prevent WSSV disease.

MATERIAL AND METHODS
Enzymatic extracts from the digestive tract.The experiment consisted of 26 healthy juvenile whiteleg shrimp at intermolt average weight of 9.7 g (±1.7) and average size of 11.9 cm (±0.93) obtained from the culture ponds of the Centro de Investigaciones Biológicas del Noroeste (CIBNOR) (24°06' N and 110°26' W).The shrimp were fed a commercial Vimifos Bumper Crop ® diet (35% protein), a total of 3% biomass.The shrimp fasted for 24 hours before sampling.They were apparently healthy with hard and turgid bodies.Their appendices were intact and without stains or injury to the exoskeleton.Dissections were made in their digestive tract, separating stomach, digestive gland, and anterior, middle, and posterior intestines.
The weight of the digestive gland of each of the shrimp was recorded to estimate the Hepatosomatic index (IHS, weight of the digestive gland/total weight of the shrimp X 100) (18).The tissues of each segment were weighed and separately homogenized (Bio-Gen PRO200, PRO Scientific, Oxford, CT) with cold distilled water (0°C) in a v/w proportion of 3 mL distilled water g -1 fresh tissue.Raw extracts were separated by centrifugation at 15.294 g for 10 min at 4°C (5810R, Eppendorf, Hamburg, Germany).The lipid fraction was removed and the supernatant was recovered and stored at -20°C, which was considered as crude extract for protein measurement, enzymatic activity, and in vitro digestibility.

Quantification of protein.
Protein concentrations in the enzyme raw extracts were quantified by the Bradford method.In glass tubes (100 mm × 15 mm), 8 µL enzyme reagent, 792 µL distilled water, and 200 µL Bradford reagent were mixed and gently stirred by vortex.Absorbance was measured at 595 nm.Bovine serum albumin (05470, Sigma-Aldrich, St. Louis, MO) was used as the protein standard.
Determining enzymatic activity.Total activity of each enzyme was calculated as average units for each specimen for each section of the digestive tract.Enzymatic capability was expressed as percent in each section based on 100% for the entire digestive tract.The specific activity of each enzyme (proteases, amylases, lipases, RNAses and DNAses) was expressed as units.mg - protein.All measurements were made in quadruplicate; blank control samples were also measured, but enzyme reagent was added after the reaction was stopped.
Protease activity was measured by the method described by Vega-Villasante et al. (19), using azocasein (1% in Tris-HCl 50 mM at pH 7.5) as substrate.Protease activity was expressed as units of protease.mg - protein.One unit of protease was defined as the quantity of enzymes required for an increase of 0.01 absorbance units at 440 nm.min -1 .
Amylase activity was measured by the method described by Vega-Villasante et al. (19), using starch (1%, in Tris-HCl 50 mM at pH 7.5) as substrate.Amylase activitity was expressed as units of amylase.mg - protein.One amylase unit was defined as the quantity of enzymes required for an increase of 0.01 absorbance units at 550 nm.min -1 .
Lipase activity was measured by the method described by Versaw et al (20), using β-naphthyl caprylate as the substrate.Lipase activity was expressed as units of lipase.mg - protein.
One lipase unit was defined as the quantity of enzymes required for an increase of 0.01 absorbance units at 540 nm.min -1 .

WSSV, por medio de iniciadores específicos ( f o r w a r d G A A G A A G C G C A C G A AT G A C G ;
reverse GCATAATGCAGTAGCGTCAACGGC; TM 60°C), se amplificó un fragmento de 421 pb correspondientes a un gen de latencia viral.El dsRNA-ORF89 fue sintetizado utilizando un kit de transcripción in vitro (Block-iT RNAi TOPO Transcripción Kit; Invitrogen, K3500-01 and K3650-01 Invitrogen, Carlsbad, CA) siguiendo las instrucciones del fabricante: los productos de PCR del gen de latencia ORF89 se amplificaron y ligaron con el Block-iT TOPO-T7 al promotor T7.Dos reacciones de PCR secundaria se realizaron por separado combinando los iniciadores forward y reverse de T7 y ORF89 así como también forward de ORF89 y reverse de T7.Cada una de estas amplificaciones produce una cadena by vortex and incubated at room temperature (25°C) for 2 hours and stopped by adding 300 µL 1.17 M perchloric acid.It was set in an ice bath and later centrifuged at 20.817 × g at room temperature for 10 min.The supernatant, an unprecipitated hydrolyzed fraction, was quantified in a nanophotometer (P-300, Implen, Munich, Germany).The blanks were treated in the same manner, adding enzymatic extract after the perchloric acid.Nuclease activity was expressed as the number of nuclease.mg - units.One nuclease unit was defined as the amount of enzymes required to hydrolyze one nanogram of substrate.min - .
Obtaining DNA and RNA substrates for enzyme activity and in vitro digestibility.For genomic DNA extraction, the method described by Hoffman-Winston ( 22) was used and the YeaStar RNA Kit (R1002, Zymo Research, Irvine, CA) for total RNA extraction.Both nucleic acids were isolated from the yeast Yarrowia lipolytica.
In vitro digestibility of DNA, RNA, and dsRNA-ORF89 by digestive enzymes in different sections of the shrimp digestive tract.We used 2000 ng of the corresponding nucleic acid (DNA, RNA, or dsRNA-ORF89) for each 100 µL of reaction volume: 14 µL 10× buffer (500 mM of NaCl, 100 mM Tris-HCl, 100 mM MgCl 2, 10 mM DTT at pH 7.9), 28.9 µL distilled water, 50 µL enzyme reagent from the corresponding section of the shrimp digestive tract.The reaction was started by adding 7.1 µL nucleic acid substrate.The mixture was incubated at 30°C for 5 hours, taking 10 µL samples at intervals of 0, 15, 30, 60, 90, 120, 180, 240, and 300 min.Two blanks were used.The first one was similar to the procedure described, but the enzyme reagent was inactivated by heat (water bath at 95°C for 5 min).
The second blank used distilled water instead of the enzyme reagent.The samples taken at each sample time were immediately analyzed by electrophoresis in agarose gels (1% at pH 8.0) at 90 V for 30 min with molecular weight markers of 1 kb Plus marker ladder (#15615-016, Invitrogen, Carlsbad, CA).The samples were stained with nucleic acid gel stain in water (41003, Biotium, Hayward, CA) and visualized in a photodocumenter (Gel Doc EZ, Bio-Rad Laboratories, Hercules, CA).
The analyses of DNA and dsRNA-ORF89 digestion were determined with digitalized images (Image Pro Plus 7.0) Media Cybernetics, Bethesda, MD) of the total area (in pixels) of the image (23).
Nucleic acid input and auto-hydrolysis determination of enzyme reagent in the shrimp digestive tract.The reagent mixture contained 14 µL buffer 10× (500 mM NaCl, 100 mM Tris-HCl, 100 mM MgCl 2 , 10 mM DTT at pH 7.9) plus 50 µL of the corresponding enzyme reagent from sections of the shrimp digestive tract, in a final reaction volume of 100 µL, adjusted with distilled water.The reaction was started by adding the enzyme reagent and incubating at 30° C for 5 h, taking 10 µL samples at 0, 15, 30, 60, 90, 120, 180, 240, and 300 min.Each sample was immediately analyzed by electrophoresis in agarose gels (1% at pH 8.0 at 90 V, for 30 min).

Statistical analysis.
The results of enzymatic activity and digestibility of DNA and dsRNA-ORF89 of each tissue were analyzed to one-way ANOVA.Once significant differences (p<0.05) were verified, the average was determined by the Tukey multiple comparison test at 95% confidence.Statistical analyzes were performed with Statistica 7.0 (StatSoft, Tulsa, OK).

H e p a t o s o m a t i c i n d e x .
T h e a v e r a g e hepatosomatic index of the shrimp for obtaining the enzyme reagent was 0.012 (p<0.05).This index describes the condition of the organism that reflects its nutritional status also reflecting the processes of storage and transfer of proteins and lipids (24).
In contrast, the digestive activity against DNA, RNA and dsORF-89 was considerably lower in the posterior gut.The electrophoretic analysis indicated the digestion of 421bp dsRNA when it was exposed to the enzymatic reagent of the digestive tract.In the stomach a light digestion of the substrate (421bp dsRNA) was found at 300 min, indicating a relatively low RNAase III activity.In contrast, in the digestive gland a strong digestion of the substrate was observed at 30 min and its complete digestion at 120 min, indicating a relatively high hydrolytic activity on the dsRNA-ORF89.In the upper intestine, a slight hydrolysis of the substrate was observed at 240 min indicating relatively low hydrolytic activity on the dsRNA-ORF89.In the middle intestine, a slight digestion of the substrate was observed at 300 min, indicating a relatively low hydrolytic activity on the dsRNA-ORF89.Finally, in the posterior intestine, poor hydrolysis of the substrate at 300 min indicated the lowest digestive capacity on the dsRNA-ORF89 (Figure 2).In summary, in lanes 2 and 3 corresponding to the inactivated enzyme and distilled water controls respectively, in all cases the dsRNA-ORF89 substrate (421 bp) maintained intact from 0 to 300 min of treatment.In contrast, when the active enzyme reagent (lane 1) was exposed, a decrease in band intensity of the dsRNA-ORF89 was observed; Particularly with the treatment of the digestive gland.
It was also demonstrated that these nucleic acids present in the enzymatic reagents were also hydrolysed by shrimp nucleases (data not shown).

DISCUSSION
Distribution of enzyme activity in the sections of the digestive tract of whiteleg shrimp (L.vannamei) was determined as a percentage of enzyme activity of the entire digestive tract.All digestive enzymes (protease, amylase, lipase, and nucleases) are found in greater quantity in the digestive gland.These results coincide  with those reported by Alexandre et al (17), where they found a high protease activity in the digestive gland (2,300 mU), followed by the stomach (200 mU), the anterior gut (150 mU), the middle intestine MU) and the posterior gut (10 mU) (approximate values).Hernández and Murueta (25) showed that the most important proteolytic enzymes responsible for > 60% of protein digestion, which occurs in the digestive gland, are trypsin and chymotrypsin.Becerra et al (26) showed high activity of amylase and trypsin in the digestive gland.Our results on the presence and distribution of enzymatic activity in the digestive tract of shrimp showed that the shrimp used were in normal physiological conditions (27).On the other hand, since the potential form to supply the dsRNA-ORF89 to the shrimp is its inclusion in the pelleted food, the enzymatic degradation of the starches, lipids, and proteins (which constitute the food), will favor the release of the dsRNA in the shrimp digestive tract.The hepatosomatic index of the shrimp (4.12) used also indicates a normal developmental status of the shrimp, which is within the ranges reported for other marine and freshwater crustaceans, such as Litopenaeus vannamei (3.0 to 4.0) (27), Penaeus chinensis (3.4 to 6.7) (28) and Macrobrachium olfersii (1.4 to 9.9) (18).
Nucleases also participate in digestion, thus we observed that total and partial degradation of nucleic acids (DNA, RNA and dsRNA) exposed to the enzyme reagent had higher enzyme activity on the RNA (8.55 ng of ARN.minute -1 ) and to a lesser extent on the dsRNA-ORF89 (1.48 ng of dsRNA.minute - ).Other studies have reported that expression of non-specific nucleases occurs in the digestive gland of Penaeus japonicus and tiger shrimp Penaeus monodon (29), where zymographic analysis shows that they degrade as much DNA as RNA, according with our results.The results of the digestibility of dsRNA-ORF89 in the digestive tract of the shrimp, reduce the possibilities of its inclusion of naked form (without protection).This information is important to design an adequate vehicle to incorporate specific dsRNA, so that it retains its antiviral property when passing through the digestive tract.Potential vehicles for the delivery of dsRNA, to reduce their hydrolysis in the digestive tract of shrimp, include nanocarriers based on cationic liposomes (nano and micro encapsulation systems), which because of their amphipathic nature are more permeable in the Phospholipid membrane of the cells, favoring the entrance of the specific dsRNA (30,9).Another option is the preparation of microencapsulates based on cationic polysaccharides such as chitosan (15,12).presencia y distribución de la actividad enzimática en el tracto digestivo del camarón demostraron que los camarones utilizados se encontraban en condiciones fisiológicas normales (27) (29), donde el análisis zimográfico muestra que las nucleasas inespecíficas de la glándula digestiva degradan tanto ADN como ARN, en concordancia con nuestros resultados.Los resultados de la digestibilidad de dsRNA-ORF89 en el tracto digestivo del camarón, reducen las posibilidades de su inclusión de forma desnuda (sin protección).Esta información es importante para diseñar un vehículo adecuado para incorporar dsRNA específico, de modo que conserve su propiedad antiviral cuando pase a través del tracto digestivo.Los potenciales vehículos para el suministro del dsRNA, para reducir su hidrolisis en el tracto digestivo del camarón, incluye a los nanocarriers basados en liposomas (sistemas de nano-y micro-encapsulación) catiónicos, que debido a su naturaleza anfipática son más permeables en la membrana fosfolipídica de las células, favoreciendo el ingreso del dsRNA especifico (9,30).Otra opción es la preparación de microencapsulados a base de polisacáridos catiónicos como el quitosano (15,12).
The stability of dsRNA will depend on the duration of exposure to digestive enzymes.Our experimental controls (dsRNA with inactivated enzyme and dsRNA exposed to distilled water) indicated a strong stability of the (ORF89) specific dsRNA after five hours at 30° C (Figure 2 lanes 2 and 3), suggesting that only the RNAsa III activity, mainly in the digestive gland, can degrade molecules of this nature (dsRNA) (31).
It will depend on food digestion time with the dsRNA in the digestive tract of the shrimp which, depends on the species, size, and other factors, such as temperature ( 21) for example, in whiteleg shrimp, digestion, and elimination lasts ~3 hours in juveniles (and even less in smaller shrimp) (32).The amount of dsRNA-ORF89 in feed and the quantity of ingested feed will be important factors for success in a WSSV treatment (6).The amount of specific dsRNA recommended for the antiviral effect to take effect from the feed should be 5-10 times greater than that injected (11,15,33).Feed containing dsRNA-ORF89 must be capable of bringing an optimal concentration to the target organs and cells; i.e., the sites of primary WSSV replication, such as the epithelial cells in the anterior intestine in the digestive tract, and in the gills (34).To include dsRNA-ORF89 in the food, it is necessary to solve some technological problems, for example, mass production of dsRNA, its recovery, purification, encapsulation (to avoid its degradation), and inclusion in the food or to use the same biological production system (Microbial cells) as inclusion vehicle (8,9).
Hydrolysis of dsRNA-ORF89 by digestive enzymes can contribute to understanding the difference between the effectiveness of injected and orally supplied dsRNA.For example, dsRNA directed to the endogenous Gen β-actin in the giant tiger prawn P. monodon caused death (100%) when applied by injection (25 µL shrimp saline solution containing the various dsRNA purified from bacteria).In contrast, no deaths were reported when dsRNA was orally administered in food with bacteria expressing specific dsRNA (6.0 × 10 10 bacteria/g, extruded feed with β-actin-dsRNA) (35).Similar results were observed when silencing the viral gene of the associated virus of the gills (GAV) of the giant tiger prawn with bacteria expressing specific dsRNA (4.9 × 10 10 bacteria/g extruded feed of GAV-dsRNA) where survival was 97% by injection and 15-35% by oral administration (36).A similar diminished effect, by applying dsRNA-vp28 (Structural gene of the WSSV virus cell envelope) in the diet was found by Sarathi (7,8) when 25 µg VP28-dsRNA was administered intramuscularly or orally in food supplemented with chitosan and bacteria expressing VP28-dsRNA (1×10 9 bacteria/mL); Esto dependerá del tiempo de digestión de los alimentos con el dsRNA en el tracto digestivo del camarón, el cual varía de acuerdo a la especie de camarón, su tamaño y de otros factores como la temperatura; por ejemplo, en el camarón patiblanco L. vannamei la digestión total es de aproximadamente 3 horas (incluso menor en camarones pequeños) (32).La cantidad de dsRNA-ORF89 incorporado en el alimento y la cantidad ingerida puede ser un factor importante en el éxito del tratamiento para WSSV (6).La cantidad de dsRNA específico recomendada para que el efecto antiviral tenga efecto desde el alimento deberá ser 5-10 veces mayor que la inyectada (11,15,33).
Por lo anterior, nuestros resultados indican que será necesario colocar al dsRNA-ORF89 en un vehículo protector para disminuir su degradación y mantener su actividad protectora contra el WSSV.survival was 65% against challenge with WSSV using injection and 37% survival with oral treatment.Treerattrakool et al (36) silenced the gonad inhibitor hormone (GIH) in the giant tiger prawn when dsRNA-GIH was injected intramuscularly (0.3 μg dsRNA-GIH/g shrimp), but only 50% in shrimp fed a diet containing Artemia salina enriched with the bacteria E. coli dsRNA-GIH (120 OD 600 ).Although the highest antiviral effect observed occurs in organisms to which the specific dsRNA was administered by injection, its usefulness in the field is null because of the high percentage of organisms that are required to be protected, so oral administration remains the best option for dsRNA even though the antiviral effect is lower.

Agradecimientos
Due to the above, our results indicate that it will be necessary to place the dsRNA-ORF89 in a protective vehicle to decrease its degradation and maintain its protective activity against WSSV.

Figure 1 .
Figure 1.Digestibility of nucleic acids in the digestive tract sections of whiteleg shrimp L. vannamei.(A) DNA Digestion rate, (B) RNA Digestion rate, (C) dsRNA.Same letters are not statistically different (p>0.05).

.
The percentage distribution of the amylase, protease, lipase, DNase, and RNase activity in each of the tissues of the digestive tract of L. vannamei shrimp are summarized in table 1 where it is generally observed that 80% or more of the enzymatic activity is in the digestive gland.Values of 5.4 to 19.9% were found in the stomach and a significantly lower activity in the intestines.The specific activities of amylase, protease, lipase, DNase and RNase present in the digestive gland were the highest (in units.mg-1protein):amylase2183, protease 44, lipase 147 DNase 4.7, and RNase 4.7; whereas, amylase 1196, protease 28, lipase 26, DNase 1.0, and RNAse 0.69 were found in the stomach (Table2).

Table 1 .
Percentage of enzyme activity in each segment of the digestive tract of whiteleg shrimp L. vannamei.
S=stomach, DG=digestive gland, AI=anterior intestine, MI=middle intestine, (PI) posterior intestine.Same letters are not statistically different between segments of the digestive tract (p>0.05).

Table 2 .
Specific activity of amylase, protease, lipase, DNAse, and RNAse in sections of the digestive tract in whiteleg shrimp L. vannamei.