Enrichment of Artemia sp. with autochthonous probiotics at different levels in larviculture of piauçu Megaleporinus macrocephalus

The research investigated the effect of dietary supplementation with Artemia sp. enriched with the autochthonous probiotic Enterococcus faecium on growth performance, microbiota modulation, intestinal morphology, and resistance to pathogenic bacteria of Megaleporinus macrocephalus larvae. The study evaluated four treatments (C: without probiotics; T1: 1 × 10 4 ; T2: 1 × 10 6 ; and T3: 1 × 10 8 CFU·mL -1 ) in quadruplicates. The larvae (n = 160; weight = 5.3


INTRODUCTION
The piauçu Megaleporinus macrocephalus is a neotropical species cultivated in South America and the 10th most produced species in Latin America (Ramirez et al., 2017;Pereira et al., 2020).This omnivore presents rapid growth in captivity, can reach up to 500 mm of total length, and is widely accepted among consumers (Tataje and Zaniboni-Filho, 2010;Soares Junior et al., 2013).However, studies on the nutrition of the species are scarce, but fundamental for the productive intensification of the animal, especially in the early stages of life.
To increase the productivity of sustainable intensive fish farming, the use of probiotic bacteria has been adopted to promote intestinal microbiota balance, improve immunity and growth performance, as well as resistance to pathogens (Hoseinifar et al., 2018;Ringø et al., 2020).For piauçu, the only respective study performed so far focused on the allochthonous probiotic Saccharomyces cerevisiae, although it did not improve the physiology nor the production system of this species (Lima et al., 2015).
Autochthonous probiotic bacteria are more suitable as they adhere better to the intestinal epithelium, colonising it and facilitating nutrient use, in addition to having a greater potential to inhibit pathogens (Sousa et al., 2019;Yamashita et al., 2020;Yeganeh Rastekenari et al., 2021;Hossain et al., 2022).However, for these benefits to occur, probiotics need to arrive in ideal amounts in the host's intestinal tract, with food being the most viable form of inclusion.
In the case of fish larvae, the introduction of probiotics by enriched live foods is an alternative (Ghorbani Vaghei et al., 2019;Ghoname et al., 2020;Oliveira et al., 2022) as it assists in the development of the intestine, balancing the microbiota (Comabella et al., 2013;Stephens et al., 2016), immune system development, and in the synthesis and absorption of amino acids and nutrients from the diet (Chung et al., 2012;Arrieta et al., 2014).Thus, in the larviculture of aquatic organisms, studies with bioencapsulated probiotics in live diets, such as artemia and rotifers, have promising results in aquaculture (Sun et al., 2013;Bhaheerathan et al., 2020;Ghoname et al., 2020;Samat et al., 2021;Oliveira et al., 2022).
Among the probiotic bacteria with potential to feed the piauçu larvae, the genus Enterococcus is a good option because of its high capacity for adhesion, growth and colonization of the host's intestinal tract compared to heterotrophic bacteria (Dias et al., 2019;Sousa et al., 2019) and can stimulate defense cells such as T lymphocytes, antibody production (IgA), macrophages and dentritic cells in the production of compounds such as nitric oxide (Khalkhali and Mojgani, 2017).
Thus, considering the potential of the species M. macrocephalus for aquaculture, the study evaluated dietary supplementation with the autochthonous probiotic Enterococcus faecium by enriching Artemia sp.Productive performance, microbiota modulation and intestinal histomorphometry were investigated, in addition to resistance to acute challenge with Aeromonas hydrophila.

Experimental conditions
The present study was approved by the ethics committee in animal experimentation of the Universidade Federal do Pará, under the protocol CEUA no.3991300420.The autochthonous probiotic strain was isolated from M. macrocephalus (n = 15 specimens) with the weight of 0.785 ± 0.12 kg and the length of 26.59 ± 0.23 cm, obtained from extensive fish farming.The selection criteria were as follows: gram-positive, catalasenegative and affinity to aniline blue dye.After the growth of blue colonies (lactic acid bacterium), the strains were kept in culture (Man Rogosa Sharpe Broth-MRS) at 35°C for 48 h and selected by in-vitro assays in different gradients of NaCl (0, 1.5, 3 and 4.5%), pH (4, 5, 6, 8 and 9), and bile salts (2.5, 5 and 7.5% weight•volume -1 ) (Barros et al., 2022).
The probiotic bacterium was grown in Falcon tubes containing broth medium (MRS broth + NaCl 0.65%) incubated to 35°C for 24 h, centrifuged at 1,800 g for 15 min and then resuspended in sterile saline (NaCl 0.65%).To determine the experimental concentrations, the strain was maintained in tubes containing MRS broth, and serial dilution (1:10) was performed to reach the experimental concentrations (1 × 10 4 ; 1 × 10 6 and 1 × 10 8 UFC•mL -1 ), according to the methodology of Jatobá et al. (2008).For the experiment, the strains were prepared every two days.
For the experiment, 160 piauçu larvae (weight = 5.3 ± 2.3 mg; length = 3.7 ± 0.4 mm), acquired two days after hatching and acclimatized in the laboratory.The in-vivo experiment was carried out in a completely randomized design with four treatments: with Artemia without probiotics on control diet (C), and Artemia enriched the autochthonous probiotic E. faecium (T1: 1 × 10 4 ; T2: 1 × 10 6 ; and T3: 1 × 10 8 CFU•mL -1 ), in four replicates, distributed in 16 aquarium (1-L) at a density of 10 larvae•L -1 .The animals were maintained in a static system with constant aeration over a period of 20 days.The feeding rate was 150 artemia nauplii per larva•day -1 (Jomori et al., 2013), divided into four times a day (8; 11; 14 and 17 h).After the last daily feeding, food residues and excretes were removed with a siphon (± 30%) of the volume of water in each experimental unit (Abe et al., 2015;Sousa et al., 2020).

Preparation of live food and bacteriological analysis
To obtain Artemia sp.nauplii, the cysts were incubated for 24 h in 1-L aquariums with 30 g•L -1 saline water, constant aeration, and light intensity of 15 W lux (Azevedo et al., 2016).After hatching, Artemia nauplii were washed with freshwater for further enrichment with probiotics and larvae feeding.Artemia nauplii were counted for the density estimate.For counting, 1 mL of the solution containing the nauplii was sampled, they were then kept in a formalin solution (4%), and the count was performed in squared petri dishes under a stereomicroscope with a 40x magnification, in quadruplicates (Abe et al., 2015).
For enrichment, after the incubation period, the Artemia nauplii were washed with freshwater in a beaker (50 mL).The autochthonous probiotic strain E. faecium was previously cultivated in Falcon tubes (MRS Broth + 0.65% NaCl), and incubated at 35°C for 24 h (Jatobá et al., 2008).Subsequently, the strain was centrifuged at 1.800 g for 15 minutes and resuspended in sterile saline solution (SSE + 0.65% NaCl), and then reduced by 1 mL to the final concentration (1 × 10 4 ; 10 6 and 10 8 CFU•mL -1 ), representing treatments (T1, T2 and T3, respectively).Right after, the enrichment was performed for the treatments (T1: 1 × 10 4 ; T2: 1 × 10 6 ; and T3: 1 × 10 8 CFU•mL -1 ), where the concentrations were transferred to Falcon tubes with 10 mL v/v, during the period of 40 minutes.For each 1 mL, 150 Artemia nauplii were added, totaling a rate of 150 nauplii/ larva in each experimental unit for each treatment, which were administered to the larvae directly in the culture water (Vázquez-Silva et al., 2016;Sousa et al., 2020).This process was performed at all feeding times.
The microbiology of the nauplii was evaluated in CFU•g -1 after the enrichment period to determine the stable quantity of probiotics in the nauplii and confirmed as E. faecium by the MALD-TOF method.For this, the artemia were filtered through a 64-μm mesh filter, and the samples of each treatment were dried on filter paper.Subsequently, one specimen (artemia pool) was macerated in sterile 0.65% saline solution and passed through serial dilution (factor 1:10).An aliquot (100 μL) of each dilution (10 -1 , 10 -2 , 10 -3 , 10 -4 and 10 -5 CFU•mL) was seeded in petri dishes containing culture medium (MRS Agar) and incubated at 30°C for 48 h to obtain the counts of probiotic bacteria in CFU•g -1 .(adapted from Jatobá et al., 2008;Vázquez-Silva et al., 2016;Dias et al., 2022).The analyses were carried out in quadruplicate.

Productive performance
At the end of the experiment, all post-larvae were subjected to biometry to record total length, standard length, and weight.Based on these data, the parameters were determined:

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Enrichment of Artemia sp. with autochthonous probiotics at different levels in larviculture of piauçu Megaleporinus macrocephalus (100 μL) of each of the dilutions was used to inoculate petri dishes containing MRS agar for counting of probiotic bacteria, and another aliquot was seeded in tryptone soy agar (TSA) to obtain the count of total heterotrophic bacteria.The plates were then incubated at 35°C for 48 h followed by cell counting (CFU•g -1 ) in the animal's intestine (Jatobá et al., 2008;Dias et al., 2018).

Histological analysis
For the histological analysis of the intestines, eight post-larvae of each treatment were used.They were fixed in 10% formalin solution for 24 h and preserved in 70% alcohol (Azevedo et al., 2016).The intestine was removed, dehydrated in an alcohol series (70, 80, 90 and 100%), transferred to xylene and embedded in paraffin.Using a microtome, 5-μm cross sections were made and stained with hematoxylin eosin (HE).Subsequently, the histological sections were analyzed under a light microscope (Nikon, E600), and measurements of the morphometric parameters of the intestine sessions (total height, height, width, and thickness of the villi) were taken according to Silva et al. (2015).

Immersion challenge with Aeromonas hydrophila
After the supplementation period, the fish were submitted to an acute bacterial challenge by the immersion method against the pathogen A. hydrophila to assess their ability to resist infections (adapted from Nikapitiya et al., 2018).The pathogenic bacteria were cultured in BHI broth and incubated at 30°C for 24 h according to Mouriño et al. (2017) and Dias et al. (2022).The bacteria were cultured to an optical density (OD) of 600 nm, sedimented by centrifugation (3,500 rpm at 4°C for 10 min) and resuspended in sterile buffered saline (0.65%).The bacterial suspension was then dispensed in Falcon tubes with a volume of 10 mL to reach the final concentration of 10 8 CFU•mL -1 , and applied in 1-L volume post with new and innocuous water, for the challenge of infection by immersion.The control group (Ccontrol larvae) was treated with sterile saline (0.65%) (Nikapitiya et al., 2018).
The fish were kept in 1-L aquarium in a static system equipped with artificial aeration for 96 h.Every two h for four days, mortality was observed (Ina-Salwany et al., 2019).Dead fish (during the test time) and fish surviving at the end of 96 h were anesthetized by immersion in benzocaine solution 20 mg•L -1 (Pramod et al., 2010), euthanized by medullary section and submitted to microbiological analysis to determine the Koch's postulate.After the growth of bacterial colonies, they were identified by the MALDI-TOF method (Evans, 1976;Angeletti, 2017).
For this experiment, the water variables were evaluated daily, analyzing temperature, electrical conductivity, dissolved oxygen, pH, and total ammonia, with the aid of the Professional Plus YSI multiparameter.

Statistical analysis
The data was acquired, and the microbiological counts were converted into square roots, before being submitted to statistical tests.The mortality results were transformed to arcsin root (x.100 -1 ).After the data underwent normality and homoscedasticity tests (Shapiro-Wilk and Bartlett, respectively) and when heterogeneity of variance was observed, they were transformed into log 10 (x +1).Accordingly, the data were submitted to oneway analysis of variance (ANOVA), followed by Tukey's posthoc test (p < 0.05) to compare means.
The highest means (p < 0.05) of length gain and weight gain were shown for fish that had received the highest concentration of probiotic T3 (10 8 CFU•mL -1 ) in Artemia sp.(Table 1).In addition, the biomass values were the highest in T3.The probiotic promoted higher averages for treatments that had received artemia enriched with autochthonous E. faecium, with emphasis on the T3 group, with the highest average.On the contrary, the mortality rate of post-larvae was significantly increased (p < 0.0001) in the control group and in T1 (10 4 CFU•mL -1 ) at the end of the experiment (Table 1).

Microbiological analyses
The bacteria counting into Artemia nauplii showed statistic differences (p < 0.05) between groups, with the larger count in treatment T3 (8.27 ± 0.02 × 10 8 CFU•g -1 ).In contrast, higher values total heterotrophic bacterial count was observed in the control (4.34 ± 1.2 × 10 4 CFU•g -1 ) (  which differed (p < 0.0011) from treatments T2 = 1.01 ± 1.07 and T3 = 0.12 ± 0.09 log CFU•g -1 , which presented the lowest counts of colonies of heterotrophic bacteria.In the lactic acid bacteria count, there was increase in count in treatments supplemented with probiotic (T1 = 2.50 ± 0.22; T2 = 4.43 ± 0.09; and T3 = 7.11 ± 0.30 log CFU•g -1 , respectively), stood out (p < 0.0001) to the control group (C = 0.34 ± 0.25 log CFU•g -1 ) (Fig. 2).After bacterial challenge with A. hydrophila, there were differences (p < 0.05) between treatments, with the highest colony counts of probiotics found in the intestines of fish from treatment T3 (5.34 ± 0.32 × 10 7 CFU•g -1 ) and the lowest levels in the control (+).In the count of total heterotrophic bacteria in the intestines of fish after the challenge, there was also a significant difference between the groups, with lower bacterial counts in T3 compared to the other treatments.The C+ treatment showed the highest concentration of bacteria, the opposite was observed in C-(SSE 0.65%), that, in both cases, showed the lowest counts of lactic acid bacteria and total heterotrophs after acute challenge (Table 3).

Intestinal histomorphometry
Total villus height, villus height and intestinal mucosa thickness of fish fed a diet containing E. faecium at T3 (10 8 CFU•mL -1 ) were significantly higher (p < 0.01; Table 4) compared to those of the other treatments.Regarding villus width and epithelium thickness, T2 and T3 showed higher values (p < 0.01), distinguishing them from the other groups (Table 4).Thus, the intestinal villi of the larvae were altered by probiotic supplementation, with T3 prominence, so that significantly higher than other treatments (p < 0.05; Fig. 3).

Sanitary challenge
The acute infection challenge with A. hydrophila presented significant differences (p < 0.001) between groups challenged with pathogenic bacteria, with greater mortality in the control positive (93% ± 11.54%) and lower mortality for supplemented with artemia enriched with E. faecium, especially in treatment T3, 10 8 (33% ± 11.54%).Between diets supplemented with probiotics, T1 and T2, mortality rates were 66% ± 11.54% and 46 ± 11.54%, respectively, with the highest rates recorded between 48 and 72 h of the infection.For the control group negative, the survival rate was 100% (Fig. 4).
For bacterial challenge with the pathogen, Koch's postulate was confirmed by the presence of A. hydrophila in the intestines of animals challenged with the bacterium and that had died during the experimental period.After acute challenge, pathogenic strains were reisolated from infected fish and identified by the MALDI-TOF technique.
Table 3. Count of lactic acid bacterium and total heterotrophic bacterium in the intestine of fish after acute challenge with Aeromonas hydrophila.
However, to date, there are no studies on probiotic supplementation in the diet of piauçu M. macrocephalus larvae.
The findings of research indicate that the probiotic E. faecium, at 1 × 10 8 CFU•mL -1 and administered via live food in Artemia sp., improved the productive performance of the microbiota and the intestinal morphometry, in addition to increasing resistance against the pathogen A. hydrophila.After 20 days of supplementation, improvements were observed in the growth performance of piauçu that were supplemented with E. faecium at the highest concentrations, T2: 1 × 10 6 and T3: 1 × 10 8 CFU•mL -1 .
The highlight was for fish from T3, which showed greater weight gain and final length (Table 1) and higher specific weight increment (Fig. 1b).These results may be related to the greatest presence of lactic acid bacteria in the intestines of the animals.Similar results were observed for tilapia larvae supplemented with Bacillus pocheonensis at the concentration of 5 × 10 5 CFU•mL -1 in Moina micrura enrichment, conferring greater larval survival and resistance to Streptococcus agalactiae (Samat et al., 2021).The enrichment of Artemia nauplii with the probiotic Bacillus subtilis and inulin at the concentration of 7 g•L -1 for 12 days also promoted a better gain in length and total height of Pseudoplatystoma reticulatum larvae (Oliveira et al., 2022).
The administration of probiotic via live foods such as artemia and rotifers, in the feeding of fish and crustacean larvae, has gained increasing interest in aquaculture (Lobo et al., 2018;Ghoname et al., 2020;Samat et al., 2021;Oliveira et al., 2022).Previous studies showed higher growth and survival rates, as well as higher protease, amylase, and lipase activities in Macrobrachium rosenbergii post-larvae supplemented with Enterococcus durans enriched in Artemia franciscana (Bhaheerathan et al., 2020), greater resistance against the pathogen Vibrio harveyi in larvae Lates calcarifer supplemented via Artemia with Enterococcus hirae (Masduki et al., 2020) and higher n-3 HUFA levels in Solea senegalensis larvae (Lobo et al., 2018).Overall, the introduction of probiotics through enrichment of live diets showed positive results, with higher growth and survival rates (Avella et al., 2010;Bhaheerathan et al., 2020), in addition to improving the immune responses of fish (Sun et al., 2013;Ghoname et al., 2020).
The colonization of the intestinal tract of piauçu larvae was evidenced by the increase in the intestinal lactic bacteria count in fish supplemented with autochthonous bacteria, with emphasis on the T3 (7.11± 0.30 log CFU•g -1 ) and lower counts at T1 and T2 (2.50 ± 0.22 and 4.43 ± 0.09 log CFU•g -1 , respectively).This fall, in counts of lactic bacteria in T1 and T2, may be associated with insufficient amounts to maintain a stable optical density in the intestinal mucosa of the animal (Fig. 2).Opposite to the Lactobacillus sp.supplementation for Astyanax bimaculatus, where no differences were found between probiotics amounts, the effects of microbiological colonization act differently in each host (Jatobá et al., 2020).Many strains of LAB beneficially alter the intestinal tract, modulating the intestine microbiota according to the amounts of microorganisms supplemented in the diet (Dias et al., 2018).
Studies with dietary supplementation with Lactobacillus spp.via autochthonous feeding of Astyanax bimaculatus postlarvae also reported higher lactic acid bacterial counts (≥ 1 × 10 7 CFU•g -1 ) in relation to bacteria heterotrophic ones such as Vibrio spp., Pseudomonas spp.and Staphylococcus spp.(Moraes et al., 2018).The bacteria-host relation is an important factor in the viability of the selected strain, for the autochthonous bacterium have greater affinity for the adhesion of the intestinal epithelium of the host (Sousa et al., 2019;Yamashita et al., 2020;Yeganeh Rastekenari et al., 2021;Hossain et al., 2022).The report with supplementation with allochthonous and autochthonous probiotic bacteria (Lactobacillus sp. and L. lactis)  of lambari (Astyanax bimaculatus and Astyanax fasciatus) highlight the groups of indigenous bacteria in tolerant changes in the microbiota of fish, with better adhesion to the epithelium in relation to allochthonous bacteria (Jatobá and Jesus, 2022).
The intestinal probiotic colonisation with E. faecium also promoted an intestinal modulation that influenced the increase in the length and width of the intestinal villi (Table 4; Fig. 3).Similar results were found for the supplementation of Artemia sp.enriched with Bacillus subtilis at the concentration of 10 6 CFU•g -1 , in the feeding of Pseudoplatystoma reticulatum, with the highest values in total villus length of 24.4 ± 0.76 μm in the probiotic treatment compared to the control and symbiotic treatments (Oliveira et al., 2022).The application of Planococcus sp.(1 × 10 7 CFU•mL -1 ) bioencapsulated in Artemia, supplemented in the diet of Sparus aurata, significantly (p < 0.05) altered the villi length from 13 ± 30 to 35 ± 90 μm and the number of goblet cells from 20 ± 2 to 37 ± 75, but it did not affect the villus count for 20 days.Additionally, after 40 days of experiment, all parameters were changed by probiotic supplementation, to 126 ± 25 μm in villus length, 27 ± 2.5 for villus number and 96 ± 14.4 goblet cells (Ghoname et al., 2020).Thus, alteration in the villi may vary according to the period of supplementation and stage of larval development.
Changes in villus height and width are associated with the production and propagation of probiotics throughout the intestine; carbohydrates are used, generating short-chain fatty acids (SCFA) and therefore promoting the creation of peptides in the intestine and the formation of butyric acid.This balances the microbiota and maintains the integrity of the intestinal epithelial cells, facilitating nutrient absorption (Poolsawat et al., 2019;Ringø et al., 2020).Thus, probiotic supplementation increases the contact surface of the intestinal mucosa and, consequently, facilitates the use of dietary nutrients (Moraes et al., 2018;Deng et al., 2022).
The colonization, modulation and alteration of the intestinal villi reflected in improvements in the growth performance of the larvae.Similar results were reported for Epinephelus coioides larvae fed with autochthonous Bacillus clausii and Bacillus pumilus in the copepod Pseudodiaptomus annandalei, at the concentration of 1 × 10 6 CFU•mL -1 , which resulted in higher total length averages, increased total weight and the survival rate of 46.67% after 14 days of supplementation (Sun et al., 2013); however, survival was lower than that observed for piauçu larvae supplemented with E. faecium.A positive effect on growth and survival was also observed in Chirostoma jordani larvae fed with Artemia franciscana metanauplii for 40 min with Lactobacillus johnsonii (2.3 × 10 3 CFU•mL -1 ) and Bifidobacterium animalis (2.06 × 10 3 CFU•mL -1 ) (Vázquez- Silva et al., 2016).
The antagonistic activity against pathogens is one of the most important properties to be evaluated in a probiotic candidate.In the present study, the enrichment of Artemia with the autochthonous probiotic E. faecium in feed of M. macrocephalus, at concentrations T1: 10 4 , T2: 10 6 and T3: 10 8 CFU•mL -1 after 20 days of supplementation, decreased the cumulative mortality rate during 96 h of acute infection with the pathogen A. hydrophila.The highest survival rate was observed for T3 (66.67%).These results corroborate those observed for the supplementation of Nile tilapia Oreochromis sp.larvae with Bacillus pocheonensis, administered via Moina micrura at concentrations of 10 4 and 10 6 CFU•mL -1 .The authors observed survival rates of 75 and 77%, respectively, after challenge with Streptococcus agalactiae (Samat et al., 2021).A study with autochthonous E. faecium showed increase of the fish immunity and decrease for mortality from 88.29% for the control group to 73.33% in the group with probiotic in tilapia infected by S. agalactiae (Suphoronski et al., 2021).
Generally, lactic acid bacteria can activate the host's immune system in situations of stress or acute illness.Among their characteristics, there is their ability to adapt to the gastrointestinal tract, which allows them to compete for specific binding sites in the intestinal layer and to dominate the bacterial community (He et al., 2017;Li et al., 2019).In addition, they inhibit pathogens both by competitive exclusion and the secretion of inhibitory substrates, such as hydrolytic enzymes (chitinases, proteases, cellulases, and β-1,3-glutanase), with antimicrobial properties capable of degrading the cell wall components of pathogenic microorganisms (Urdaci and Pinchuk, 2004;Allameh et al., 2017).This influence of probiotic strains on adhesion by exclusion and expressive reduction of potentially pathogenic bacteria has been widely proposed as a characteristic of the probiotic effect on the host.This mechanism of exclusion of these microorganisms is linked to the production of antimicrobial in the quorum sensing (Mouriño et al., 2017;Chauhan and Singh, 2019;Dias et al., 2022).
In addition, probiotic strains of the genus Enterococcus can increase intestinal mucosal immunity through a higher concentration of pro-inflammatory factors (tumor necrosis factor-α and interleukin-β) (Standen et al., 2016).Alternatively, they can sensitize macrophage pattern recognition receptors, such as dectin-1 and β-glucan, thus activating the immune system in the face of an infection (Hoseinifar et al., 2018).Thus, the use of the probiotic E. faecium in live food in the first stage of live of M. mecrocephalus was able to potentiate the weight gain and survival of the larvae.In addition, they modulate Enrichment of Artemia sp. with autochthonous probiotics at different levels in larviculture of piauçu Megaleporinus macrocephalus the gut microbiota and villi, providing greater resistance to the pathogenic bacterium.

CONCLUSION
Thus, this is the first report on the use of autochthonous probiotic bacteria in the feeding of piauçu M. macrocepahlus larvae.Food supplementation with Artemia sp.enriched with the probiotic bacteria E. faecium 20218_1 CHB, at the concentration of 1 × 10 8 CFU•mL -1 (T3), for 20 days, promoted greater larval growth performance, modulated the gut microbiota and altered the villi intestinal tracts of supplemented fish, in addition to increasing resistance against the pathogen A. hydrophila.

Figure 3 .
Figure 3.The structure of the intestine of (a), (b), (c) and (d) Megaleporinus macrocephalus gut represents the control (C), T1, T2 and T3, respectively.Tissue sections were stained with hematoxylin and eosin with visualization in the objective (20x).