Effect of microencapsulated phenolic compound extracts of Maclura tinctoria (L.) Steud on growth performance and humoral immunity markers of white leg shrimp ( Litopenaeus vannamei , Boone, 1931) juveniles

Aim of study: : The effect of microencapsulated phenolic compound extracts of Maclura tinctoria (MTBE) on growth performance and humoral immunity markers of the white leg shrimp Litopenaeus vannamei juveniles (0.5 ± 0.2 g initial weight) was studied. Area of study: M. tinctoria was collected from Hampolol, Campeche, and Arroyo del Agua, Culiacan, Sinaloa, Mexico. Material and methods: Three MTBE inclusions (0.5, 1, and 2.5 g MTBE/kg, Purina® ) were compared with a control commercial feed (Purina® ) during 30 days. Nine phenolic acids, nine flavonols, four dihydro-flavonoids, four flavones, and seven unidentified phenolic compounds were determined fin the MTBE using a Perkin Elmer ® HPLC chromatograph and diode array-detection. Main results: The mean concentrations of total phenolic compounds, total flavonoid compounds, and condensed tannins were 198.05 ± 5.59 mg gallic acid equivalent (GAE) g-1 dw, 78.57 ± 1.80 quercetin equivalent g -1 , and 28.32 ± 0.33 mg epicatechin equivalent g -1 , respectively. The ferric reducing antioxidant power and the total antioxidant capacity, respectively, averaged 28.32 mg GAE mL -1 and 10.9 mg ascorbic acid equivalent mL -1 . Survival, weight gain, and specific growth rate of L. vannamei were similar among the experi mental diets. The dietary inclusion of MTBE at 0.5 g/kg of food showed significant higher ( p < 0.05) plasma hemocyte lysate protein (1.35 ± 0.055 µg mL -1 ), prophenoloxidase (0.47 ± 0.15, Abs. 492 nm), and superoxide anion (O 2 .-) activity (0.21 ± 0.07, Abs. 630 nm). Research highlights: The supplementation of MTBE at 0.5 g/kg of food could be considered as a potential alternative additive for L. vannamei diet in the juvenile production, since it improved the response of the humoral immunity markers at post larval life stages, when cultivated shrimp are more susceptible to be infected by pathogens.


Introduction
Shrimp aquaculture is one of the main economic activities in tropical and subtropical areas, which supply this product at a world scale reaching more than four million tons. The success of white shrimp (Litopenaeus vannamei Boone 1931) production depends on reducing the impact of diseases and epizootics (Zhang et al., 2016;Dewangan et al., 2017), mainly during its larval and juvenile stages. Among the most important pathogens that cause diseases in larval and juvenile stages of P. vannamei are White Spot Syndrome Virus (WSSV) and Taura Syndrome Virus (TSV), and Vibrio species (Cheng et al., 2011;Rajkumar et al., 2017). Intensification of culture applications has led to diseases causing up to 80% economic losses in hatcheries (mainly within 60-80 days post-stocking) (Kibenge, 2019)); e.g. total losses caused just by WSSV have amounted to 3 billion US dollars per year (Yang et al., 2016). For decades, the use of antibiotics has been a common practice for the prevention of shrimp diseases (Bermúdez-Almada & Espinosa-Plascencia, 2012); however, it is well documented that the application of these chemicals is a management practice that involves dangers and risks to the environment and human health (Thornber et al., 2020). Therefore, the search for alternative compounds that are environmentally-friendly, harmless to human health, and do not compromise shrimp production are a priority (Romero et al., 2012).
The use of alternative substances and microorganisms with antimicrobial and immunostimulant activities to reduce diseases impact (Peraza-Gómez et al., 2014;Trejo-Flores et al., 2016) and diminish the application of antibiotics (Cabello, 2006) in the shrimp industry has been increasing. Allelopathic powders and plant extracts are immunomodulator and immunostimulant additives commonly used in safeguarding animal health (Tiwari et al., 2018). Among these products, the phenolic compounds-based extracts have been described as potential immunostimulants, antipathogenic, and antistress agents in aquaculture (Chakraborty & Hancz, 2011).
The Maclura tinctoria tree is a deciduous, semideciduous, and even perennial arboreal native species of dry and wet tropical regions from Mexico to Argentina (Berg, 2001). Although antibacterial, antioxidant, and immunostimulant benefits of M. tinctoria extracts have been proved in laboratory assays (Lamounier et al., 2012;Matson-Robles et al., 2015), their chemical properties are commonly reduced by temperature, light, and environmental conditions (Santos-Buelga et al., 2019). The microencapsulation technique represents a viable alternative (Ozkan et al., 2019) to preserve the allopathic properties of M. tinctoria' extracts. There are few studies on the use of some phenolic compounds in shrimp culture (Sudheer et al., 2011;Chandran et al., 2016;Tomazelli-Júnior et al., 2018), but so far, supplementation of microencap-sulated phenolic extracts of M. tinctoria in diets for L. vannamei juvenile has not been described yet. In the present work, we studied the effect of microencapsulated phenolic compound extracts of M. tinctoria on growth performance (final weight gain, specific growth rate, and survival) and humoral immunity markers (protein concentration in plasma and hemocytes, phenoloxidase activity, and superoxide anion concentration) of white shrimp L. vannamei juveniles.

Extraction, identification, quantification, and microencapsulation of M. tinctoria phenolic compounds
To obtain the MTBE microcapsules, the bark powder was mixed with ethanol, stirred, and kept in the dark for 24 h. Thereafter, a solution was prepared with 183 g of bark powder and 1 L of absolute ethanol (50%) FAGA-LAB ® . The solution was stirred for 24 h, filtered through an analytical sieve with a mesh (90 μm) (WS Tyler ® , USA) to remove solids, and subsequently kept in amber jars (Almaraz-Abarca et al., 2013).
The microencapsulation technique was developed in the Aquaculture Laboratory of the CIIDIR-IPN Sinaloa Research Center, and is being processed for a patent (MX/a/2016/007504) at the Instituto Mexicano de la Propiedad Intelectual, IMPI (González-Ocampo et al., 2016).

Phenolic content
The phenolic composition of MTBE was determined through high performance liquid chromatography, HPLC (Perkin-Elmer ® , Series 200 HPLC system, Shelton, CT, 3 Antioxidant additive shrimp feeds USA) using a Perkin Elmer ® Brownlee Analytical C18 column (4.6 × 250 mm, 5 µm) and diode array-detection (DAD) system (Perkin Elmer Series 200, USA), with a modified gradient method (Campos & Markham, 2007). Maximal absorbance was achieved employing a gradient method with mobile phase, which consisted of a mixture of water, 0.05% orthophosphoric acid (2.5 pH, Baker . Spectral information was obtained from the phenolic compounds' spectral data described by Mabry et al. (1970) and Campos & Markham (2007).

Antioxidant capacity
The total antioxidant capacity (TAC) was calculated by the ferric reducing antioxidant power (FRAP) method (Oyaizu, 1986) and the molybdate VI reduction to molybdate V assay, modified by Prieto et al. (1999). The FRAP assay is based on the reduction power of the plant extracts. The ferric ion (Fe 3+ ) is reduced to ferrous ion (Fe 2+ ) forming the blue complex (Fe2+ TPTZ-1), which increases the absorption at 593 nm; samples and standard (ascorbic acid) were read after 30 min. Results are expressed as the extract concentration required to reach an absorbance value of 0.5 EC 50 (effective concentration at 50%) in equivalent milligrams of ascorbic acid per milliliter of extract (mg EAA mL -1 ) and equivalent milligrams of ascorbic acid per gram of microcapsules (mg EAA g -1 ). The calibration curve of ascorbic acid (Eq. 1) was prepared with four concentrations of ascorbic acid (10-40 µL combined with a volume of methanol to reach 1 mL as final volume). The absorbance values of the samples were substituted in the equation and the results were adjusted to the dilution factor used and expressed in equivalent milligrams of gallic acid per gram of microcapsules (mg GAE g -1 ). TAC was determined by the method of reducing molybdate VI to molybdate V, following the procedure described Prieto et al. (1999). Each sample of 100 μL was mixed with 1000 μL of molybdate solution and incubated in a thermoblock (Thermolyne ® Dry Bath DB28125) for 90 min at 95 °C. Once all samples had been cooled to room temperature (in the dark), the absorbance at 695 nm was recorded. A calibration curve (A695 = 0.0729-0.0747 [gallic acid], correlation coefficient r = 0.9947) (Eq. 2) was prepared with different concentrations of gallic acid (0.5, 1.0, 2.5, 5.0, 7.5. and 10 mg mL -1 ).
The absorbance values of the sample were substituted in the equation, and the results were adjusted to the used dilution factor and expressed in mg GAE mL -1 .

Bioassay
Juveniles of L. vannamei (0.5 ± 0.2 g) were obtained from the "Cuate Mechado" shrimp farm (Guasave, Sinaloa, Mexico). Experimental units consisted of 120-L plastic tanks with 80 L of seawater (34-35 mg L -1 ) supplied with constant aeration. Stocking density was adjusted at 10 shrimp per tank. MTBE treatments were tested as a feeding additive with three replicates and coded as follows: CTRL, control group fed with commercial feed (Purina ® , 35% protein); T1, 0.5 g MTBE/kg of feed; T2, 1 g MTBE/ kg of feed; and T3, 2.5 g MTBE/kg of feed. Shrimps were fed twice a day (9:00 and 17:00 h) at a feeding ration of 7% of total biomass per tank, for 30 days. The tanks were cleaned (residues of food and wastes) by siphoning the bottom every three days. Clean seawater was added to replace water loss by evaporation and siphoning up to 120 L from each thank every five days.

Growth and survival of L. vannamei
The final weight gain, specific growth rate, and survival of Litopenaeus vannamei juveniles were evaluated at the end of the study. Final weight gain (%) was obtained following the procedure of Amaya et al. (2007). The specific growth rate (SGR) (% d-1) was calculated in percentage using the Ziaei-Nejad et al.
where t is the bioassay period (d), lnW 0 is the natural logarithm of the initial shrimp´s weight (g), and lnW t is the natural logarithm of the final weight (g). Survival (%) was calculated according to Ziaei-Nejad et al. (2006).

Hemolymph analysis
Hemocytes from plasma and lysate supernatant (HLS) were obtained following the method of Vargas-Albores et al. (1993). Protein concentration in HLS was determined using the Bradford (1976) method using bovine serum albumin (BSA, Sigma ® ) as standard. The Bradford method involves the binding of Coomassie Brilliant Blue G-250 to protein and the absorbance is determined at 595 nm.

Phenoloxidase, prophenoloxidase, and superoxide anion
Humoral response of L. vannamei juvenile was determined with the activity of phenoloxidase (PO) and prophenoloxidase (proPO) enzymes and the superoxide anion concentration. PO for small samples was measured spectrophotometrically (490 nm min -1 g protein -1 ) according to Hernández-López et al. (1996), by recording the formation of dopachrome produced from L-dihydroxyphenylalanine (L-DOPA) and the amount of inactive proPO in samples containing also PO activity was calculated as total available proPO minus PO activity before trypsin treatment. The superoxide anion (O 2 -) was quantified (630 nm) using the methodology of Song & Hsieh (1994) based on SIC-EDTA buffer to wash hemocyte pellets; the cytoplasmic formazan was read at 630 nm in a Thermo Spectronic Genesys 2 spectrophotometer.

Physicochemical seawater parameters
Salinity, pH, dissolved oxygen, and temperature were determined in each tank before cleaning, siphoning, and water replacement. Nitrites, nitrates, and ammonium (Strickland & Parsons, 1972) were measured at the beginning and end of the bioassay and throughout the study remained within the optimum levels for shrimp culture (Brock & Main, 1994).

Statistical analysis
ANOVA was applied to determine the differences of protein in plasma and HLS, PO and proPO activities, SGR, and superoxide anion concentration (mean ± SD) among treatments. Results of SGR were log 10 transformed to normalize its distribution. If significant differences were found (p < 0.05), a post-hoc Tukey honestly significant difference (HSD) with multiple comparisons of Scheffé, Bonferroni and Holm was used to identify the differences.

Antioxidant capacity
The antioxidant capacity of the microcapsules, containing the phenolic compound extracts of M. tinctoria, averaged 28.32± 0.33 mg GAE mL -1 and 10.9 ± 0.44 mg ascorbic acid equivalent mL -1 for TAC and FRAP, respectively (Table 1).

Plasma, HLS and total protein
The T1 group displayed a significantly higher (p < 0.05) HLS protein content (1.35 ± 0.55 µg mL -1 ) than T2 and T3 groups (Fig. 3a), however, none of the groups showed statistically significant differences when compared to the control group. Total and plasma protein showed no significant differences (p > 0.05) (Figs. 3b and 3c).

Concentrations of superoxide anion and PO (proPO) activity
Superoxide anion concentration showed no significant difference between T1 and T2 groups. T1 group had a higher superoxide anion concentration (0.21, Abs. 630 nm) than T3 groups, but none of the groups was different from the control group (Fig. 4) (p ˂ 0.05).
The activity of PO (activated proPO) in HLS (Fig. 5A) and total PO (Fig. 5B) was significantly higher (p < 0.05) in T1 as compared to treatments T2 and T3.

Discussion
Numerous studies have established the economic impact due to diseases and mortality caused by diverse pathogens during larval and postlarval stages in L. vannamei aquaculture (Doyle, 2016;de Torres Bandeira et al., 2019), and have focused on the use of diverse plant extracts for shrimp immunostimulation (Chakraborty & Hancz, 2011).
In this study, the phenolic compounds detected in the fruits (glycosides, flavanones, flavonoids, and xanthones) are similar to previous reports using wood, bark and  leaves from Maclura genus (Lamounier et al., 2012;Matson-Robles, et al., 2015), which independently of the plant tissue used, the phenolic compounds showed antioxidant, antibacterial, and immunostimulant properties. The MTBE supplied to shrimp feeds have not affected the L. vannamei juvenile growth and survival, as resulted in the present study. Similar observations were reported by Subramanian et al. (2013). These authors reported a higher SGR in white shrimp fed with a dietary inclusion of the Korean black raspberry, Bokbunja Rubus coreanus extract during four weeks. As M. tinctoria, the Korean black raspberry contains total phenolic compounds (TPC) and total flavonoids (Yang & Choi, 2017), which are directly related to the antioxidant activity and improve the growth in earlier stages of aquaculture species (Hussain et al., 2020).
Survival showed no significant differences (p > 0.05) between treatments and the control group. This survival pattern of L. vannamei had been reported earlier when adding other phenolic extracts from species such as Rhodiola rosea (Wang et al., 2017b), Yucca schidigera, and Quillaja saponaria (Hernández-Acosta et al., 2016) to the shrimp's diet. The humoral system could be able to respond to the treatments since shrimps infected with pathogens and treated with phenolic extracts have shown higher survival rates (Júnior, et al., 2018).
The plasma protein includes protein from enzymes, hormones and metabolites. Increment of proteins in the plasma has been described to be caused by a diverse factors such as the stress or the presence of pathogens in the plasma (Gholamhosseini et al., 2020). In the present study, no significant higher plasma protein was determined among CTRL and treatments. This might be because the MTBE supplied in the feed was not detected as a threat by the immune system of the L. vannamei juve-niles. Nevertheless, the protein content in the HLS was significantly higher at 0.5 mg kg -1 but not in the plasma and total protein content. High protein in HLS has been reported to be a response of the humoral system that increases the production of enzymatic proteins (Valentim-Neto et al., 2015;Wang et al., 2017a); commonly, crustaceans with a good immune system present a high protein content in hemocytes (Lamela et al., 2008). In the present study, supplementation with MTBE in the treated L. vannamei juveniles could have activated their humoral response. Live pathogen challenge bioassay, together with immune gene expression need to be considered to demonstrated the effectiveness of microencapsulated MTBE in shrimp juvenile culture.
Although the superoxide anion (SO) showed no significant differences (p>0.05) among treatments, it decreased at higher MTBE concentrations, being significantly lower (p<0.05) in the T3 than T1 and T2. It is suggested that the highest MTBE supplementation (2.5 mg of MTBE per kilogram of feed) could reduce the SO anion production. The antioxidant system of L. vannamei has been previously stimulated with alcoholic plant extracts (Esquer-Miranda et al., 2016;Júnior et al., 2018), and, in some cases, polyphenols have ameliorated the phagocytosis of the retinal pigment of epithelial cells (Liu et al., 2018). Supplementation with MTBE could  Bars are means ± SD. Different letters indicate significant differences (p < 0.05). stimulate the antioxidant system of L. vannamei juvenile as seen in T1, and increase its PO activity, which agrees with the reports by Vanichkul et al. (2010), Hsieh et al. (2013), and Wang et al. (2017b). Extracts from the species Uncaria tormentosa showed lower Trolox equivalent (3.610 ± 0.054 mmol g -1 ) (Júnior et al., 2018) than any of the extracts tested in the present study. Their lower TAC but higher TPC (554.60 ± 0.015 mg GAE g -1 ) was reported accompanied by a significant higher survival rate of L. vannamei at 2 and 4% of U. tormentosa to eliminate the clinical signs of WSSV. Similar high survival rates were reported by Sudheer et al. (2011), who added Rhizophora mucronata, and Sonneratia spp. to the feed of Penaeous monodon challenged with WSSV, and Ceriops tagal extracts to the shrimp diet. The microencapsulated phenolic compound extracts doses used in the present study could be high in relation to the shrimp's weight. It is necessary to calculate the MTBE dose based on the concentration of phenolic extracts to properly determine this critical effect and correlate it with shrimp survival and growth rate.
In L. vannamei, the proPO system plays a key role in the humoral response against pathogen infections and the production of proPO is increased some days after infection (Wu et al., 2017). The significantly higher pro-PO activity (p < 0.05) found in HLS could be attributed to the stimulation by the microencapsulated phenolic compounds supplied; although survival showed no significant differences (p > 0.05), the shrimp juvenile in T2 and T3 showed 100% survival. Phenolic extracts increase the gene expression related to the immune system and the production of antioxidant enzymes (Subramanian et al., 2013). The 33 encapsulated phenolic compounds determined in the present study may have stimulated the humoral system and antioxidant capacity, increasing survival and growth in shrimps. These phenolic compounds have been proven to possess the ability to trap superoxide anions and hydroxyl radicals Hamed et al., 2017).
In conclusion, MTBE supplementation did not show negative effects on shrimp survival and growth. There were no significant differences in any of the tested humoral immunity markers of the white leg shrimp (L. vannamei) (HLS protein, superoxide anion, proPO, and phenoloxidase). In this sense, further research is needed considering lower MTBE concentrations, feeding periods, and the effects on other humoral agents, like total hemocytes count, immune gene expressions, and/or antioxidant enzymatic processes in L. vannamei, including catalase and superoxide dismutase. To the best of our knowledge, this is the first report on the effect of microencapsulated phenolic compound extracts of M. tinctoria on phenoloxidase activity, protein content, survival, growth specific rate, and anion superoxide concentration in L. vannamei juveniles.