Immune response of sea bass <Emphasis Type="Italic">Dicentrarchus labrax</Emphasis> to <Emphasis Type="Italic">Tenacibaculum maritimum</Emphasis> antigens

ABSTRACT:   Seawater fishes are affected by a pathology commonly called 'myxobacteriosis', caused by Tenacibaculum maritimum (formerly Flexibacter maritimus ). The disease is characterized by fin erosion and necrotic ulcers of skin and muscle, and by low but constant mortality in cultured marine fish; in Italy is one of the most important and widely spread diseases affecting sea bass Dicentrarchus labrax , gilthead seabream Sparus aurata , sharp-snouted bream Diplodus puntazzo , white bream Diplodus sargus , and six-tooted bream Dentex dentex . In order to obtain an effective vaccine against the disease, formalin killed cells (FKC), extracellular products (ECPs) and crude lipopolysaccharide (LPS) preparations were obtained from the T. maritimum strain SPVId and injected intraperitoneally twice into the sea bass. The fish immune response to the preparations was studied: agglutinating antibody titer and in vitro phagocytosis were determined after the first and second injection in order to evaluate whether the preparations are immunogenic or not and if the booster effect took place. The results show that FKC and LPS preparations increased the antibody titer after the first injection when compared to the control sea bass. Moreover, all the preparations stimulated a secondary (booster) response. In vitro phagocytosis of the total blood was significantly higher for all the preparations when compared to the controls, but the crude LPS immunized sea bass showed the highest activity.     

Effect of dietary <Emphasis Type="SmallCaps">l</Emphasis>-tryptophan on cannibalism,survival and growth in pikeperch <Emphasis Type="Italic">Sander lucioperca</Emphasis> (L.) post-larvae

The effect of supplemented commercial diets with crystalline l-tryptophan (TRP—5, 10 and 20 g TRP kg^?1) on cannibalism, survival and growth parameters of pikeperch post-larvae (Sander lucioperca) was evaluated. Fifteen-day-old pikeperch larvae (mean weight—6.8 mg) were reared during the next 28 days (20.5 °C, 16L:8D) in glass aquaria in a recirculating aquaculture system. The enzyme-linked immunosorbent assay showed that TRP-supplemented diets were effective in increasing the levels of serotonin (5-HT) in the body tissue of pikeperch. TRP supplementation resulted in a slight decrease in both types of cannibalism, although the reduction in cannibalism impact did not amount to more than a few percent. TRP treatment had no significant influence on the final survival of pikeperch post-larvae (ranged from 20.1 ± 12.4 to 29.0 ± 12.9 %). However, contrary to the earlier studies conducted on other fish species, no significant difference in the growth rates and feeding behavior of pikeperch between TRP-fed and control group were found. The final body weight and growth rate ranged from 0.211 ± 0.014 to 0.243 ± 0.016 and from 12.19 ± 0.38 to 12.76 ± 0.35 % day^?1, respectively. To our knowledge, this is the first study reporting the effects of TRP supplementation on the cannibalism–survival–growth relations in fish in the post-larval stage.     

Spatial learning-induced <Emphasis Type="Italic">egr</Emphasis>-<Emphasis Type="Italic">1</Emphasis> expression in telencephalon of gold fish <Emphasis Type="Italic">Carassius auratus</Emphasis>

The immediate-early gene (egr-1) expression was used to examine the neuron’s response in telencephalon of goldfish during spatial learning in small space. Fishes were pre-exposed in the experimental apparatus and trained to pick food from the tray in a rectangular-shaped arena. The apparatus was divided into identical compartments comprising three gates to provide different spatial tasks. After the fish learned to pass through the gate one, two more gates were introduced one by one. Fish made more number of attempts and took longer time (P < 0.05) to pass through the first gate than the gate two or three. This active learning induces the expression of egr-1 in telencephalon as established by western blot analysis. Subsequently, the fish learn quickly to cross the similar type of second and third gate and make fewer errors with a corresponding decline in the level of egr-1 expression. As the fish learned to pass through all the three gates, third gate was replaced by modified gate three. Interestingly, the level of egr-1 expression increased again, when the fish exhibit a high exploratory behavior to cross the modified gate three. The present study shows that egr-1 expression is induced in the telencephalon of goldfish while intensively acquiring geometric spatial information to pass through the gates.     

Antibacterial effect of medium-chain fatty acid: caprylic acid on gnotobiotic <Emphasis Type="Italic">Artemia franciscana</Emphasis> nauplii against shrimp pathogens <Emphasis Type="Italic">Vibrio harveyi</Emphasis> and <Emphasis Type="Italic">V. parahaemolyticus</Emphasis>

The present study was carried out to determine the antibacterial effect of caprylic acid in the culture system of Artemia franciscana nauplii inoculated with shrimp pathogens Vibrio harveyi and V. parahaemolyticus. To begin with, the antibacterial effect of different concentrations (1, 10 and 100 mM) of caprylic acid against V. harveyi and V. parahaemolyticus was assessed through bacterial growth study. This indicated that at 100 mM concentration, the growth of the pathogens was completely inhibited within 5 h, whereas, in 1.0 and 10 mM concentrations, effective inhibition was observed with the extension of time. Subsequently, the influence of pH variation on the growth inhibitory effect of 10 mM caprylic acid against V. harveyi and V. parahaemolyticus at different pH (5–7) was also made through bacterial growth study. At pH 5, the pathogen growth was very less, compared with pH 6 and 7. The Artemia nauplii (instar II) reared in 10 mM caprylic acid supplemented medium were challenged with V. harveyi and V. parahaemolyticus, and the mortality was recorded at an interval of 6 h up to 60 h. In this study, the mortality of Artemia nauplii reduced to 20.61 and 16.30% in V. parahaemolyticus and V. harveyi challenged groups, respectively. The present results provide evidence for the potential antibacterial activity of caprylic acid in aquaculture against luminescent vibrios.     

Effect of <Emphasis Type="Italic">Cratoxylum formosum</Emphasis> on innate immune response and disease resistance against <Emphasis Type="Italic">Streptococcus agalactiae</Emphasis> in tilapia <Emphasis Type="Italic">Oreochromis niloticus</Emphasis>

The effect of aqueous extract of Cratoxylum formosum on innate immune response and disease resistance in tilapia Oreochromis niloticus was investigated. The fish were fed diets containing 0% (control), 0.5% (diet 1), 1% (diet 2), and 1.5% (diet 3) of C. formosum aqueous extract for 30 days. At the end of the experimental period, parameters of innate immune response including phagocytosis of blood leukocytes, lysozyme activity in plasma, and respiratory bust activity were examined. Feeding the fish with diet 2 and diet 3 for 20 days enhanced phagocytic activity and for 30 days stimulated lysozyme and respiratory burst activities. Diet 1 increased the phagocytic activity at 30 days, but did not affect the other measured parameters. All parameters were not significantly changed (P > 0.05) in the control group throughout the experiment. Following 30 days of feeding, fish were infected with S. agalactiae. The cumulative mortalities of bacterial-infected tilapia that were fed diet 1, diet 2, and diet 3 were 56, 12, and 10%, respectively, compared with 85% in the control group. These results indicate that the aqueous extract of C. formosum may elevate the innate immune response and enhance disease resistance in tilapia.     

Use of a <Emphasis Type="Italic">Noctiluca</Emphasis>-killing bacterium <Emphasis Type="Italic">Marinobacter salsuginis</Emphasis> strain BS2 to reduce shrimp mortality caused by <Emphasis Type="Italic">Noctiluca scintillans</Emphasis>

The ability of an algicidal bacterium Marinobacter salsuginis strain BS2, isolated from shrimp pond water, to reduce shrimp mortality was investigated under laboratory conditions. When two species of shrimp (Penaeus monodon and Litopenaeus vannamei) (body length 1.5–1.8 cm) were cultured together with the dinoflagellate Noctiluca scintillans, nearly 80 % of the shrimps died within 7 days. However, when bacterial strain BS2 was also added to the culture, N. scintillans was killed within 48 h, and shrimp survival rates on the 7th day improved from 23 to 87 % for both P. monodon and L. vannamei. The bacterium BS2 alone had no effect on shrimp condition. Under conditions of increased dissolved oxygen, the effect of using BS2 was greater, and shrimp survival rates improved even more dramatically, from 26 to 98 %. These studies provide the first evidence that the use of killing bacteria, isolated from shrimp culture water, can suppress harmful algal blooms (HABs) and thus restore the efficiency of shrimp production. The control of HABs in this way in shrimp culture farms would be a major benefit for shrimp production.     

Influence of the dietary inclusion of <Emphasis Type="Italic">Gracilaria cornea</Emphasis> and <Emphasis Type="Italic">Ulva rigida</Emphasis> on the biodiversity of the intestinal microbiota of <Emphasis Type="Italic">Sparus aurata</Emphasis> juveniles

Partial substitutions of fish meal by 5, 15, or 25 % of Gracilaria cornea or Ulva rigida in experimental diets were evaluated to study their effects on biodiversity of intestinal microbiota composition in gilthead seabream (Sparus aurata) juveniles. The diets were offered to duplicate groups of 15 juvenile fish (14.0 ± 0.5 g) for 70 days, and at the end of the experiment the intestinal microbiota from four specimens of each treatment was analysed by denaturing gel gradient electrophoresis. Results showed that the substitution of fish meal by algae meal induced important modifications in the intestinal microbiota community, as a big reduction of the biodiversity when the highest percentage (25 %) of U. rigida was included. On the contrary, an increase on the number of species was detected when a 15 % of algae was included. Various Lactobacillus delbrueckii subspecies were selectively stimulated when G. cornea was included in the feed, and other bacterial species, such as those included in the Vibrio genus, were reduced.     

Cloning and characterization of <Emphasis Type="Italic">Bax1</Emphasis> and <Emphasis Type="Italic">Bax2</Emphasis> genes of <Emphasis Type="Italic">Ctenopharyngodon idellus</Emphasis> and evaluation of transcript expression in response to grass carp reovirus infection

Multidomain proapoptotic Bcl-2-associated X (Bax) protein is an essential effector responsible for mitochondrial outer membrane permeabilization, resulting in cell death via apoptosis. In this study, two Bax genes of grass carp (Ctenopharyngodon idellus), designated as CiBax1 and CiBax2, were isolated and analyzed. The obtained CiBax1 cDNA is 2058 bp long, with a 579 bp open reading frame (ORF) coding a protein of 192 amino acid residues. The full-length cDNA of CiBax2 is 1161 bp, with a 618 bp ORF coding 205 amino acids. Both CiBax1 and CiBax2 are typical members of Bcl-2 family containing conserved Bcl and C-terminal domains, and they share conserved synteny with zebrafish Bax genes despite the grass carp Bax mapping to different linkage groups. Phylogenetic analysis showed that CiBax1 was clustered with Bax from most teleost fish, and CiBax2 was close to Bax2 from teleost fish but far separated from that of Salmo salar. Quantitative real-time PCR analysis revealed broad expression of CiBax1 and CiBax2 in tissues from healthy grass carp, but the relative expression level differed. The mRNA expression of CiBax1 and CiBax2 was both upregulated significantly and peaked in all examined tissues at days 5 or 6 post-infection with grass carp reovirus. Subcellular localization indicated that CiBax1 protein was localized in both nucleus and cytosol, while CiBax2 protein only in cytosol. Moreover, CiBax2, but not CiBax1 was colocalized with mitochondrion under normal condition. Taken together, the findings would be helpful for further understanding of the function of Bax in teleost fish.     

Monitoring of <Emphasis Type="Italic">Francisella noatunensis</Emphasis> subsp. <Emphasis Type="Italic">orientalis</Emphasis> in farmed Nile tilapia (<Emphasis Type="Italic">Oreochromis niloticus</Emphasis>) in Brazil

Francisella noatunensis orientalis is a bacterium that causes emerging bacteriosis in Nile tilapia (Oreochromis niloticus) in many parts of the world, including Brazil. It is a non-motile, Gram-negative, strictly aerobic, facultative intracellular coccobacillus. This species of bacteria is responsible for low to high mortality in fish farms, causing economic losses for fish farmers. This study aimed to detect the presence of F. noatunensis orientalis using qPCR (real-time polymerase chain reaction) and to describe lesions caused by the bacterium in O. niloticus in Brazilian aquaculture. For this purpose, 360 fish from six fish farms (30 per farm) were sampled at two time points (n = 180 per sampling). Necropsies and histopathology were performed for lesion observation, in addition to qPCR and sequencing for detection and identification of Francisella species. Environmental data were collected using a multiparameter sonde YSI EXO2. All measured limnological variables were within the optimum range for cultivation of Nile tilapia. The major lesions present were melanization of the skin, splenomegaly, granulomas, and inflammatory cell responses. The prevalence of francisellosis varied from 0 to 86.66% between time periods and fish farms analyzed, and an outbreak was observed during the second sampling period. This study describes the prevalence of francisellosis in O. niloticus and reports that the lesions found are not exclusively associated with this bacterial disease.     

Effect of combination feeding of <Emphasis Type="Italic">Nannochloropsis</Emphasis> and freshwater <Emphasis Type="Italic">Chlorella</Emphasis> on the fatty acid composition of rotifer <Emphasis Type="Italic">Brachionus plicatilis</Emphasis> in a continuous culture

ABSTRACT:   A continuous culture of rotifer was conducted to investigate the effect of combination feeding of both a high density of Nannochloropsis oculata (N) and condensed freshwater Chlorella (FC) on the fatty acid composition of L-type rotifer Brachionus plicatilis in a continuous culture system. The algal feeding of the rotifers was carried out in three successive steps: N-feeding → N+FC-feeding → FC-feeding. The culture was conducted at 24°C and 25–27 psu in a 2000 mL bottle with 50% of water exchanged daily. The combination N+FC-feeding was effective in increasing rotifer density. The rotifers fed on N+FC (N+FC-R) had more non-polar lipids than polar ones, similar to those on N (N-R), opposite to the rotifers fed on FC (FC-R). N+FC-R contained higher levels of 16:2, 18:2n-6 (linoleic acid [LA]) and 20:2n-6, but lower levels of 18:1, 20:4n-6 (arachidonic acid), 20:5n-3 (eicosapentaenoic acid [EPA]) and 22:5n-3 (docosapentaenoic acid [DPA]) compared with N-R. Whereas N+FC-R contained higher levels of 16:1n-7, EPA and DPA, but lower levels of 16:2 and LA compared with FC-R. N+FC-R had more DPA in polar lipids than in non-polar ones. The Σn-6/Σn-3 ratio in N+FC-R was 0.9–1.0, significantly different from those in N-R (0.4) and FC-R (6.6–8.4). Therefore, it is inferred that the fatty acid profile of the N+FC-R cultured in a continuous culture system was affected by both N and FC. Also, the combination N+FC-feeding may be effective in manipulating the Σn-6/Σn-3 ratio in continuously cultured rotifers.     

Potential of cinnamon (<Emphasis Type="Italic">Cinnamomum verum</Emphasis>) oil to control <Emphasis Type="Italic">Streptococcus iniae</Emphasis> infection in tilapia (<Emphasis Type="Italic">Oreochromis niloticus</Emphasis>)

In this study, four essential oils—cinnamon oil, leech lime oil, lemongrass oil, and turmeric oil—were examined for their antimicrobial activities against Streptococcus iniae, a bacterium that is pathogenic in fish, in which it causes streptococcosis. Cinnamon oil was the most potent antimicrobial agent among these oils, with a minimal inhibitory concentration (MIC) of 40 μg/ml. By using gas chromatography–mass spectrometry (GC–MS), it was found that the major components of cinnamon oil were cinnamaldehyde (90.24), limonene (2.42%), cinnamyl acetate (2.03%), linalool (1.16%), and α-terpineol (0.87%). Of these compounds, only cinnamaldehyde exhibited antimicrobial activity against S. iniae, with an MIC of 20 μg/ml. In an in vivo trial, no mortality was apparent in fish fed on fish diets supplemented with 0.4% (w/w) of cinnamon oil and with 0.1% (w/w) of oxytetracycline 5 days prior to infection with S. iniae. These results indicate that cinnamon oil had a protective effect on experimental S. iniae infection in tilapia, and thus has the potential to replace the antibiotics used to control this disease.     

Characterization of <Emphasis Type="Italic">kiss2</Emphasis> and <Emphasis Type="Italic">kissr2</Emphasis> genes and the regulation of kisspeptin on the HPG axis in <Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis>

Reproduction allows organisms to produce offspring. Animals shift from immature juveniles into mature adults and become capable of sexual reproduction during puberty, which culminates in the first spermiation and sperm hydration or ovulation. Reproduction is closely related to the precise control of the hypothalamic–pituitary–gonadal (HPG) axis. Kisspeptin peptides are considered as the important regulator of HPG axis in mammalian. However, the current understanding of kisspeptin in flatfish is not comprehensive. In this study, we cloned and analyzed the kiss2 and kissr2 genes in Cynoglossus semilaevis. Interesting alternative splicing in the 5′-untranslated regions (UTR) of the Cskissr2 gene was found. The expression profiles of Cskiss2 and Cskissr2 showed relative high messenger RNA (mRNA) levels at the late gastrula stage during embryonic development, at total length = 40 mm during early gonadal differentiation, and in the brains and gonads of all investigated tissues. These results suggested that the kisspeptin system participated in embryogenesis and in the regulation of gonadal differentiation and development. Considering that the control and regulatory mechanisms of kisspeptin in the central reproductive axis are still unclear, we documented that the intramuscular injection of kisspeptin caused different sGnRH and cGnRH mRNA levels in a dose- and tissue-dependent manner. The mRNA expressions of FSH and LH were stimulated in the ovary and were inhibited in the testis under the kisspeptin treatments. These results provided foundations for understanding the roles of kisspeptin in the neuroendocrine system in fish. The manipulation of the kisspeptin system may provide new opportunities to control the gonadal development and even reproduction in fish.     

Effects of dietary microencapsulated <Emphasis Type="Italic">Cymbopogon flexuosus</Emphasis> essential oil on reproductive-related parameters in male <Emphasis Type="Italic">Rhamdia quelen</Emphasis>

In aquaculture, nutrition and supplemented diets have been shown to affect broodstock reproductive performance. In this study, we investigated the effects of dietary supplementation with Cymbopogon flexuosus essential oil (CFEO) microcapsules on reproductive-related parameters in silver catfish (Rhamdia quelen) male broodfish. Adult male broodstocks were separated into three groups according to the concentrations of supplemented CFEO (0.0?=?control; 1.0 or 3.0 mL per kg of diet). After 20 days under experimental conditions, the animals were euthanized and the gonads were harvested for gonadosomatic index, sperm analysis, oxidative stress, and histopathology; testosterone levels were measured in the plasma; gene expression of prl, smtl, pomca, and pomcb was assessed in the pituitary gland by real-time PCR. The results showed no alterations on reproductive parameters in R. quelen males treated with Cymbopogon flexuosus essential oil compared to the control-diet animals. In conclusion, CFEO microcapsules supplied for 20 days in the concentrations of 1.00 or 3.00 mL per kilogram of diet did not affect the reproduction criteria evaluated in this study in male silver catfish.     

Anti-oxidative functions of <Emphasis Type="Italic">mt2</Emphasis> and <Emphasis Type="Italic">smtB</Emphasis> mRNA expression in the gills and brain of zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) upon cadmium exposure

There were not any past studies about metallothionein isoforms (smtB and mt2) having anti-oxidative functions on zebrafish after Cd²⁺ exposure. On the other hand, the anti-oxidative enzymatic factors such as superoxide dismutase (sod), glutathione peroxidase (gpx1a), and catalase (cat) are used as references to investigate whether the smtB and mt2 have anti-oxidative responses on the gills and brain of zebrafish after 1–6 h of 0 and 1.78 μM Cd²⁺ exposure. The anti-oxidative system such as sod, cat, and gpx1a mRNA expressions demonstrated a cascade response upon Cd²⁺-induced oxidative stress in the present study. Interestingly, the smtB mRNA expression levels increased by 3.2- to 6.1-fold, and mt2 raised by 4.1- to 11.3-fold in gills at 1 and 3 h after exposure to Cd²⁺, respectively. On the other hand, the smtB mRNA levels increased by 10.6- to 58.6-fold, but mt2 mRNA levels increased by 2.3- to 11.1-fold in brain at 1 and 3 h after exposure to Cd²⁺, respectively. In addition, both tissues showed increased apoptosis levels at 3 h, and recovery after 6 h of Cd²⁺ exposure. From the results, we suggest that both mt2 and smtB play a role in anti-oxidation responses within 6 h after exposure to Cd²⁺. In conclusion, the smtB mRNA levels have a higher response than mt2 in the brain, but both mRNA expressions appear to have a similar pattern in the gill. We suggest that smtB plays an important role to defend oxidative stress in the brain of adult zebrafish upon acute Cd²⁺ exposure.     

<Emphasis Type="Italic">Streptococcus agalactiae</Emphasis> from tilapia (<Emphasis Type="Italic">Oreochromis</Emphasis> sp.) transmitted to a new host,bighead carp (<Emphasis Type="Italic">Aristichthys nobilis</Emphasis>), in China

Several outbreaks of Streptococcus agalactiae infection of bighead carp (Aristichthys nobilis) were observed in China. The molecular epidemiology and pathogenicity of S. agalactiae in bighead carp and tilapia (Oreochromis sp.) is poorly understood. In the present study, we identified S. agalactiae strains isolated from diseased bighead carp using the API 20 Strep kit and 16S rDNA sequencing and determined whether these strains came from tilapia. Of the 46 identified S. agalactiae strains, 24 strains were successfully isolated from diseased bighead carps, 20 S. agalactiae strains were isolated from tilapia, and two S. agalactiae strains were isolated from tiger frog (Hoplobatrachus chinensis). The results of molecular typing, including multilocus sequence typing, molecular serotyping, surface protein gene detection, and virulence-related gene detection showed that the 44 strains from bighead carp and tilapia were highly similar, whereas different from tiger frog GBS strains. Remarkably, the bighead carp strain Hn1404 showed high virulence in bighead carp and zebrafish. Moreover, this strain was pathogenic to Nile tilapia (Oreochromis niloticus). In addition, comparative genomic analysis showed that isolate Hn1404 had a close relationship with the bighead carp and tilapia S. agalactiae strains. All the analyses of the genetic characteristics of bighead carp and tilapia strains showed that tilapia S. agalactiae strains could be transmitted to other fish species such as bighead carp.     

Immune response of largemouth bass, <Emphasis Type="Italic">Micropterus salmoides</Emphasis>, to whole cells of different <Emphasis Type="Italic">Nocardia seriolae</Emphasis> strains

Largemouth bass Micropterus salmoides were immunized with four different N. seriolae strains—two α-glucosidase-positive (961113, KU040801) and two α-glucosidase-negative (94260, OTTS) strains—along with Freund’s incomplete adjuvant. After primary immunization (week 0), a booster was administered at weeks 4 and 8. Nonspecific immune responses to multiple immunizations with the different N. seriolae strains were determined based on serum lysozyme activity and nitroblue tetrazolium (NBT)-positive cells in peripheral blood. The serum lysozyme activity and NBT-positive cells in peripheral blood were not significantly increased even after the two booster immunizations. Specific antibody responses against N. seriolae cells were investigated by enzyme-linked immunosorbent assay. At 4 weeks after immunization, all groups immunized with N. seriolae antigens showed significant increases in their specific antibody levels. The sera from fish immunized with different N. seriolae strains exhibited reactivity with N. seriolae sonicated antigens of 28, 30, 36 and 84 kDa by western blot analysis. After two boosters, fish were challenged with live N. seriolae to assess the vaccine’s efficacy; however, multiple injections of the N. seriolae strains did not reduce mortality, irrespective of the bacterin.     

Effects of potential probiotic <Emphasis Type="Italic">Lactococcus lactis</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis> on digestive enzymatic activities of live feed <Emphasis Type="Italic">Artemia franciscana</Emphasis>

In the present study, cellulase, protease, lipase and amylase activities were performed to investigate the effects of Lactococcus lactis subsp. lactis CF4MRS bioencapsulation of Artemia franciscana. Our results show that cellulase activities (total cellulase—FPase activity, exoglucanase and endoglucanase—CMCase activity) were significantly higher (P < 0.05) in A. franciscana tissue homogenates compared to those in the control group after 8 h of L. lactic bioencapsulation. Notably, an exception case was found in β-D-glucosidase activity, whereby the cellulase activity was not significantly different (P > 0.05) compared to the control group. Administrations of L. lactis at cell concentration of 10⁸ CFU mL^?1 showed considerable improvement of other important enzymatic activities such as amylase, protease and lipase in A. franciscana. The amylase/protease ratio in probiotic-treated A. franciscana was recorded at 0.343, approximately two times higher than those without probiotic administration (0.184). In contrary, amylase/lipase ratio showed half of a reduction (0.330) in L. lactis-administrated A. franciscana compared to the control (0.614). Our study suggests that important digestive enzymes, e.g., cellulase, amylase, protease and lipase, can be enhanced through bioencapsulation of A. franciscana with L. lactis subsp. lactis, which could in turn lead to further stimulation of endogenous enzymes in the fish and shrimp larvae.