Black Tiger Shrimp Penaeus Monodon Research Articles

The immune response of the C-Jun in the black tiger shrimp (Penaeus monodon) after bacterial infection

The transcription factor C-Jun widely exists in vertebrates and invertebrates and plays an important role in various kinds of stimulus response. In this study, PmC-jun gene was first cloned from Penaeus monodon. The full-length cDNA of PmC-jun was 1857 bp in length and included an 879 bp open reading frame (ORF), which encoded 293 amino acids. qRT-PCR analysis results showed that PmC-jun mRNAs were ubiquitously expressed in all the examined tissues. The highest expression level was observed in gill, followed by hepatopancreas. The expression patterns of PmC-jun after Vibrio harveyi and Streptococcus agalactiae injections were studied by qRT-PCR experiment. PmC-jun increased obviously in the gill and hepatopancreas. The expression pattern of PmC-jun in the hepatopancreas was further studied using in situ hybridization (ISH) method. The mRNA expression level of PmC-jun significantly increased in the hepatopancreas after bacterial infection. The expression sites of PmC-jun were almost unchanged. PmC-jun played a regulatory role in pathogen invasion.

Laboratory cohabitation challenge model for shrimp hepatopancreatic microsporidiosis (HPM) caused by Enterocytozoon hepatopenaei (EHP).

Background Enterocytozoon hepatopenaei (EHP) causes hepatopancreatic microsporidiosis (HPM) in shrimp. It is probably endemic in Australasia and was first characterized and named from the giant or black tiger shrimp Penaeus monodon from Thailand in 2009. Later, it was also found to infect exotic Penaeus vannamei imported for cultivation in Asia. HPM is not normally associated with shrimp mortality, but information from shrimp farmers indicates that it is associated with significant growth retardation that is not clearly noticeable until 2–3 months of cultivation. In order to study modes of HPM transmission and to test possible control measures, a laboratory challenge model was needed that would mimic the mode of infection in shrimp ponds.ResultsWe describe successful transmission in a cohabitation model with natural E. hepatopenaei (EHP)-infected shrimp in closed, perforated plastic containers placed in aquaria together with free-swimming, uninfected shrimp. After a period of 14 days all the free-swimming shrimp tested positive by PCR (approximately 60% with heavy infections evident by 1-step PCR positive test results) and gave positive histological and in situ hybridization results for E. hepatopenaei (EHP) in the hepatopancreas.ConclusionsA laboratory cohabitation model for studying E. hepatopenaei (EHP) has been developed and used to confirm that E. hepatopenaei (EHP) can be directly transmitted horizontally among shrimp via water. The model will facilitate studies on methods to prevent the E. hepatopenaei (EHP) transmission.

A novel C-type lectin in the black tiger shrimp Penaeus monodon functions as a pattern recognition receptor by binding and causing bacterial agglutination

C-type lectins are pattern recognition proteins that play important roles in innate immunity in invertebrates by mediating the recognition of pathogens. In this study, a novel C-type lectin gene, PmCLec, was cloned and characterized from the black tiger shrimp Penaeus monodon. The open reading frame of PmCLec is 657 bp in length. It encodes a predicted protein of 218 amino acids with a calculated molecular mass and an isoelectric point of 24086 Da and 4.67, respectively. Sequence analysis of PmCLec showed similarity to members of the C-type lectin gene superfamily. The deduced protein contains a single carbohydrate recognition domain (CRD) and four conserved cysteine residues (Cys58, Cys126, Cys141, Cys149) that are involved in the formation of disulfide bridges. PmCLec transcripts are expressed in various tiger shrimp tissues, with the highest expression in the lymphoid organ. RNAi-mediated silencing of PmCLec resulted in higher cumulative mortality of knockdown shrimp after Vibrio harveyi infection compared to the control groups. Recombinant PmCLec was successfully expressed in the E. coli system. In the presence of Ca2+, purified rPmCLec protein binds and agglutinates Gram-positive bacteria (Staphylococcus aureus, S. hemolyticus), but only slightly binds and agglutinates E. coli and could not bind to the Gram-negative bacteria Bacillus megaterium and Vibrio harveyi. These results suggest that PmCLec functions as a pattern recognition receptor that is implicated in shrimp innate immunity.

Methodological approach for the collection and simultaneous estimation of greenhouse gases emission from aquaculture ponds.

Global warming/climate change is the greatest environmental threat of our time. Rapidly developing aquaculture sector is an anthropogenic activity, the contribution of which to global warming is little understood, and estimation of greenhouse gases (GHGs) emission from the aquaculture ponds is a key practice in predicting the impact of aquaculture on global warming. A comprehensive methodology was developed for sampling and simultaneous analysis of GHGs, carbon dioxide (CO2), methane (CH4), and nitrous oxide (N2O) from the aquaculture ponds. The GHG fluxes were collected using cylindrical acrylic chamber, air pump, and tedlar bags. A cylindrical acrylic floating chamber was fabricated to collect the GHGs emanating from the surface of aquaculture ponds. The sampling methodology was standardized and in-house method validation was established by achieving linearity, accuracy, precision, and specificity. GHGs flux was found to be stable at 10±2°C of storage for 3days. The developed methodology was used to quantify GHGs in the Pacific white shrimp Penaeus vannamei and black tiger shrimp Penaeus monodon culture ponds for a period of 4months. The rate of emission of carbon dioxide was found to be much greater when compared to other two GHGs. Average GHGs emission in gha-1day-1 during the culture was comparatively high in P.vannamei culture ponds.

Characterization of molecular properties and regulatory pathways of CrustinPm1 and CrustinPm7 from the black tiger shrimp Penaeus monodon

CrustinPm1 and crustinPm7 are the two most abundant isoforms of crustins identified from the hemocytes of the black tiger shrimp, Penaeus monodon. CrustinPm1 inhibits only Gram-positive bacteria, while crustinPm7 acts against both Gram-positive and Gram-negative bacteria. This work aims to characterize the molecular properties of recombinant crustinPm1 and crustinPm7, and the regulatory pathways of these two crustins. Circular dichroism spectroscopy revealed that crustinPm1 contained 40.81% alpha-helix and 22.34% beta-sheet, whereas crustinPm7 is made up of 32.86% alpha-helix and 27.53% beta-sheet. CrustinPm1 and crustinPm7 bound to phosphatidic acid (PA) with positive cooperativity of Hill slope (H) > 2, indicating that at least two molecules of crustins bind with one PA molecule. It is worth noting that both crustins bound to PA with significantly higher affinity than to lipoteichoic acid (LTA) and lipopolysaccharide (LPS). We speculate that crustin might also achieve antimicrobial activity by targeting PA, a signaling lipid. Regulatory pathways of crustinPm1 and crustinPm7 were investigated by knockdown of PmRelish and PmMyD88. This study demonstrated that crustinPm1 is mediated through the Toll signaling pathway, while crustinPm7 is regulated via both Toll and Imd pathways.

Parentage determination in black tiger shrimp (Penaeus monodon) based on microsatellite DNA markers

Parentage determination was a kind of important technology of selective breeding for aquaculture species. In this study, fourteen microsatellite markers were isolated and chosen to estimate the parentage of 100 progeny in ten black tiger shrimp (Penaeus monodon) full-sib families. Simulation analysis indicated that the power of five loci to exclude false parents was 98.9 % and that of six loci over 99.9 % based on allele frequency data. Moreover, the cumulative assignment success rate was 99 % when no parent information was available using six most informative microsatellite markers. Furthermore, the marker panel tested in mixed families, 89.0 % of progeny was correctly assigned to their parental pairs. Based on the microsatellite analysis, it could be quickly and effectively identified different genealogy and sources of P. monodon; moreover, it provided for selective breeding and multiplication release in this species.

A snake-like serine proteinase (PmSnake) activates prophenoloxidase-activating system in black tiger shrimp Penaeus monodon

Clip domain serine proteinases (ClipSPs) play critical roles in the activation of proteolytic cascade in invertebrate immune systems including the prophenoloxidase (proPO) activating system. In this study, we characterized a snake-like serine protease, namely PmSnake, from the shrimp Penaeus monodon which has previously been identified based on the subtractive cDNA library of proPO double-stranded RNA (dsRNA)-treated hemocytes. An open reading frame of PmSnake contains 1068 bp encoding a predicted protein of 355 amino acid residues with a putative signal peptide of 22 amino acids and two conserved domains (N-terminal clip domain and C-terminal trypsin-like serine proteinase domain). Sequence analysis revealed that PmSnake was closest to the AeSnake from ant Acromyrmex echinatior (53% similarity), but was quite relatively distant from other shrimp PmclipSPs. PmSnake transcript was mainly expressed in shrimp hemocytes and up-regulated after systemic Vibrio harveyi infection indicating that it is an immune-responsive gene. Suppression of PmSnake expression by dsRNA interference reduced both transcript and protein levels leading to a reduction of the hemolymph phenoloxidase (PO) activity (36%), compared to the control, suggesting that the PmSnake functions as a clip-SP in shrimp proPO system. Western blot analysis using anti-PmSnake showed that the PmSnake was detected in hemocytes but not in cell-free plasma. In vitro PO activity and serine proteinase activity assays showed that adding rPmSnake into the shrimp hemolymph could increase PO activity as well as serine proteinase activity suggesting that the rPmSnake activates the proPO system via serine proteinase cascade.

Effect of desiccation on oxidative stress and antioxidant response of the black tiger shrimp Penaeus monodon

In the present study, the oxidative stress and antioxidant response in hepatopancreas of the black tiger shrimp Penaeus monodon under desiccation stress were studied, such as activities of antioxidant enzymes (SOD, CAT, GPx and POD), oxidative damage to lipid and protein which indexed by contents of LPO, MDA, protein carbonyl (PC) and ROS production, and the expression of HSP70 and ferritin gene. The duration of desiccation significantly influenced the shrimp survival, and the mortality rates were 10% and 55.0% after desiccation 0.5 h and 3 h, respectively. Compared with the control group, after exposed to desiccation stress, the content of LPO, MDA, PC and ROS production in hepatopancreas increased significantly. SOD, CAT and POD activity in hepatopancreas increased significantly at 0.5 h, but decreased markedly at 1 h. GPx activity in hepatopancreas increased significantly at 0.5 h and 1 h, then decreased significantly at 3 h. The transcript levels of HSP70 and ferritin gene in hepatopancreas increased significantly at 1 h. HE staining showed that desiccation induced damage symptoms in hepatopancreas of P. monodon. These results revealed that desiccation could induce oxidative stress and antioxidant response via confusion of antioxidant enzymes activity and gene transcript level in hepatopancreas of P. monodon, and the time of shrimp under desiccation should lower than 0.5 h.

De novo assembly and transcriptome characterization of major growth-related genes in various tissues of Penaeus monodon

Black tiger shrimp (Penaeus monodon) is an economically important species for aquaculture in Vietnam; however, very little information is available regarding its transcriptome. In this study, we sequenced four transcriptome libraries from mRNA of heart, muscle, hepatopancreas, and eyestalk tissues of P. monodon individuals using the Illumina MiSeq® platform, yielding 78,575,346 raw paired-end reads. After quality control, a total of 62,327,726 paired-end reads were de novo assembled into 69,089 unigenes with an average size of 447.89bp, N50 of 481bp, and transcriptome size of 30.94Mb. Comparisons of the assembled unigenes against five public protein databases identified 165 unigenes related to growth and development, and expression analysis revealed that 14,229 unigenes were differentially expressed among tissues. In addition, we detected 12,768 simple sequence repeats (SSRs) in 11,173 unigenes. This work provides candidate genes for trait mapping, marker-assisted breeding, genetic studies and functional genome annotation of P. monodon. Statement of relevanceThis paper is relevant for the Shrimp Industry.

Experimental culture of black tiger shrimp Penaeus monodon Fabricius, 1798 in open sea floating cage

An experiment was designed and conducted to assess the feasibility for culture of Penaeus monodon in 6 m dia HDPE circular floating cage, installed at 10-12 m depth off Visakhapatnam in the Bay of Bengal. The cage was fixed with three cylindrical nets; inner net of 2 mm mesh (6 m dia×5.5 m height) to rear post-larvae from stock to 80th day; middle net of 10 mm mesh (6 m×5.5 m) to rear juveniles from day 81 onwards and an outer net of 40 mm mesh (8 m dia×4.5 m height) to prevent entry of predators. Post-larvae (PL23) of P. monodon (mean total length 16.1±3.8 mm), were stocked in the cage at a density of 1179 PL m-3). Bottom water parameters viz., salinity, temperature, dissolved oxygen, pH and ammonia (NH3-N) recorded in the cage site during the culture period were 34-35 ppt, 26-32°C, 3.9-4.6 ml l-1, 8.1-8.3 and 0.05-0.07 mg l-1, respectively. Shrimp feed varying in size suitable to different growth stages, was used during the trial. Feed was given twice during the first 20 days and thrice during 21-99 days. Feeding rate per day ranged from 133.3% of the biomass of shrimp at stocking time to 3.1% of the biomass of shrimp at preharvest. P. monodon registered a growth rate of 0.87 mm (TL)/0.018 g (wt) per day during first 30 days; 0.89 mm/0.069 g per day during 31-60 days and 0.37 mm/0.051 g per day during 61-90 days. On day 100, about 207 kg of shrimp (production rate, 1.62 kg m-3) was harvested and the mean size of harvested shrimp was 81.54±15.20 mm TL/4.50±2.80 g wt. Survival recorded was 31.4% and the feed conversion ratio (FCR) was 3.97. Present study demonstrated the possibility for culture of P. monodon in open sea floating cage. The possible causes for low survival and high FCR are discussed.

Experimental culture of black tiger shrimp Penaeus monodon Fabricius, 1798 in open sea floating cage

Experimental culture of black tiger shrimp Penaeus monodon Fabricius, 1798 in open sea floating cage

Response of a Mu-class glutathione S-transferase from black tiger shrimp Penaeus monodon to aflatoxin B1 exposure.

Glutathione S-transferases (GSTs) are a family of multifunctional phase II enzymes that are involved in the detoxification of exogenous and endogenous compounds. In this study, a full-length cDNA of Mu-class GST (PmMuGST) was isolated from the hepatopancreas of Penaeus monodon using rapid amplification of cDNA ends method. The full length cDNA of PmMuGST is 867 bp, contains an open read frame of 660 bp, and encodes a polypeptide of 219 amino acids with a molecular mass of 25.61 kDa and pI of 6.15. Sequence analysis indicated that the predicted protein sequence of PmMuGST was very similar to (86 %) that of Litopenaeus vannamei. A conserved domain of GST_N_Mu_like (PSSM: cd03075) and GST_C_family_superfamily_like (PSSM: cl02776) was indentified in PmMuGST. Real time quantitative RT-PCR analysis indicated that PmMuGST was present in all of the tested tissues. PmMuGST transcripts both in the hepatopancreas and in the muscle were significantly induced after 14 days of treatment with a low dosage of AFB1 (50 μg/kg) exposure and were significantly inhibited after 42 and 56 days of a high dosage of AFB1 (1000, 2500 μg/kg AFB1) exposure. Taken together, the Mu-class GST from P. monodon was inducible and was involved in the response to AFB1 exposure.

Differentially expressed transcripts in stomach of Penaeus monodon in response to AHPND infection

Acute Hepatopancreatic Necrosis Disease (AHPND) is an emerging disease in aquacultured shrimp caused by a pathogenic strain of Vibrio parahaemolyticus. As with several pathogenic bacteria, colonization of the stomach appeared to be the initial step of the infection for AHPND-causing Vibrio. To understand the immune responses in the stomach of black tiger shrimp (Penaeus monodon), differentially expressed transcripts (DETs) in the stomach during V. parahaemolyticus strain 3HP (VP3HP) infection was examined using Ion Torrent sequencing. From the total 42,998 contigs obtained, 1585 contigs representing 1513 unigenes were significantly differentially expressed with 1122 and 391 unigenes up- and down-regulated, respectively. Among the DETs, there were 141 immune-related unigenes in 10 functional categories: antimicrobial peptide, signal transduction pathway, proPO system, oxidative stress, proteinases/proteinase inhibitors, apoptotic tumor-related protein, pathogen recognition immune regulator, blood clotting system, adhesive protein and heat shock protein. Expression profiles of 20 of 22 genes inferred from RNA sequencing were confirmed with the results from qRT-PCR. Additionally, a novel isoform of anti-lipopolysaccharide factor, PmALF7 whose transcript was induced in the stomach after challenge with VP3HP was discovered. This study provided a fundamental information on the molecular response in the shrimp stomach during the AHPND infection that would be beneficial for future research.

Molecular cloning and expression of chitin deacetylase 1 gene from the gills of Penaeus monodon (black tiger shrimp).

Chitin deacetylases have been identified and studied in several fungi and insects but not in crustaceans. These glycoproteins function in catalyzing the conversion of chitin to chitosan by the hydrolysis of N-acetamido bonds of chitin. Here, for the first time, the full length cDNA of chitin deacetylase (CDA) gene from crustaceans was fully cloned using a partial fragment obtained from a transcriptome database of the gills of black tiger shrimp Penaeus monodon that survived White Spot Syndrome Virus (WSSV) infection employing Rapid Amplification of cDNA Ends (RACE) PCR. The shrimp CDA, named PmCDA1, was further characterized by in silico analysis, and its constitutive expression determined in apparently healthy shrimp through reverse transcription PCR (RT-PCR). Results revealed that the P.monodon chitin deacetylase (PmCDA1) is 2176 bp-long gene with an open reading frame (ORF) of 1596bp encoding for 532 amino acids. Phylogenetic analysis revealed that PmCDA1 belongs to Group I CDAs together with CDA1 and CDA2 proteins found in insects. Moreover, PmCDA1 is composed of a conserved chitin-binding peritrophin-A domain (CBD), a low-density lipoprotein receptor class A domain (LDL-A) and a catalytic domain that is part of CE4 superfamily, all found in group I CDAs, which are known to serve critical immune function against WSSV. Finally, high expression of PmCDA1 gene in the gills of apparently healthy P.monodon was observed suggesting important basal function of the gene in this tissue. Taken together, this is a first report of the full chitin deacetylase 1 (CDA1) gene in crustaceans particularly in shrimp that exhibits putative immune function against WSSV and is distinctly highly expressed in the gills of shrimp.

ENVIRONMENTAL ASSESSMENT OF POLYCULTURE FARMING PRACTICE BASED ON MACROBENTHIC ASSEMBLAGES: A STUDY CASE AT COASTAL AREA OF KALIWUNGU, KENDAL (CENTRAL JAVA, INDONESIA)

The purpose of this study was to determine environmental quality parameters using number of species, diversity and similarity of macrobenthic communities. This study was conducted at two locations, the Location I was a polyculture farming area, farming milkfish (Chanos Chanos) and black tiger shrimp (Penaeus monodon) and seaweed Gracilaria sp. in the coastal area of Mororejo, Kendal District, Central Java. Location II was the coastal area of PT. Plywood Indonesia, which is located adjacent to industrial activities as well as directly affected the tide. Systematic random sampling was employed, measuring physical-chemical parameters of water and sediment. Samples of macrobenthos were taken from the sediment. Data was analysed using diversity and evenness indices approach. Samples of macrobenthos were taken from the sediment using Eckman grab, then was analysed using diversity and evenness indices approach. Results showed that the Location I was dominated by Cerithidea cingulata and Terebralia sulcata (Potamididae), Minima batillaria (Batillaridae). The Location II was dominated by Cirratulus sp., Cirriformia sp. and Aphelochaeta (Cirratulidae) and Prionospio sp. (Spionidae), which are considered as indicators of disturbed area. This results implied that the use of area for both polyculture and industrial activities may lead to environmental disturbance, thus environmental coastal management need to be applied in regular basis, both temporally and spatially.

Molecular cloning and expression analysis of MAT1 gene in black tiger shrimp (Penaeus monodon).

MAT1 (ménage à trois 1), an assembly factor and targeting subunit of the CDK-dependent kinase (CAK), can regulate the cell cycle, transcription, and DNA repair. This study was intended to investigate the role of MAT1 in the reproductive maturation of black tiger shrimp (Penaeus monodon). In this study, the P. monodon MAT1 (PmMAT1) gene was identified and characterized. The full-length cDNA of PmMAT1 was 1490 bp in length with an open-reading frame of 993 bp corresponding to 330 amino acids. The temporal expression of PmMAT1 in various tissues was measured by quantitative real-time PCR with the highest expression observed in ovaries. In the ovaries, the PmMAT1 gene was continuously but differentially expressed during the maturation stages. Comparative analyses of MAT1, CDK7, and cyclin H in the CAK complex of P. monodon indicated that the expression of CDK7 and cyclin H coincided with that of MAT1 during the ovary maturation stages. Serotonin (5-HT) injection promoted the expression level of PmMAT1 in the ovaries of shrimp at 6-48 h post-injection. These results indicate that PmMat1 plays a prominent role in the process of ovarian maturation.

Improvement in Nutritional Quality of Shrimp Meal with Autoclave and Chemical Treatments: an in vitro Study.

The present study was conducted to improve the nutritional quality of shrimp meal (SM) comprising of heads with hulls of black tiger shrimp (Penaeus monodon) waste by autoclaving and chemical treatments. The sun-dried SM was divided into 5 treatment groups, such as 1) control (untreated), 2) autoclaved (autoclaved at 121°C for 10 min), 3) NaOH (treated with 3% NaOH), 4) HCl (treated with 3% HCl) and 5) formic acid (treated with 3% formic acid) groups. After treatment, they were ground to pass through 1.0 mm mesh screen and then used for analyses of chemical composition and in vitro dry matter (DM) and CP digestibilities. Data were subjected to one-way ANOVA and differences among treatment means (P<0.05) were distinguished with Tukey's test. There were no significant difference in chemical composition and in vitro DM and CP digestibilities between control and autoclaved groups, except ether extract level (P<0.05), suggesting that autoclaving affected the nutritional quality of SM little. NaOH group exhibited significantly decreased CP level and in vitro DM digestibility, increased crude ash (CA) level and unchanged in vitro CP digestibility, comparing with control group. These results suggest that NaOH treatment affected the nutritional quality of SM adversely. HCl and formic acids groups showed significantly increased CP level and in vitro digestibilities of DM and CP, and decreased CA level, showing that acid treatment can improve nutritional quality of SM: formic acid treatment may be more effective because of the greater values in CP level and digestibilities and decreased crude fibre level which was not observed in HCl group (P<0.05). The results obtained here suggest acid, especially formic acid, treatment is promising to improve the nutritional quality of SM but autoclaving and NaOH treatments.

Molecular cloning and functional characterization of cyclin E and CDK2 from Penaeus monodon.

Reduced reproductive performance of the black tiger shrimp (Penaeus monodon) has caused economic losses and hampered the fishing industry. Detailed investigation of the molecular mechanism by which the cell cycle is regulated in this organism is needed to understand the development and maturation of ovaries and oocytes, with a view to improving reproductive capacity. Cell cycle progression is mainly determined by cyclin-dependent kinase (CDK) and cyclin complexes, the cyclin E/CDK2 complex playing a key role in G1/S transition. However, knowledge of the interplay between cyclin E and CDK2 in invertebrates remains limited. In this study, full-length P. monodon cyclin E (Pmcyclin E) and CDK2 (PmCDK2) sequences were cloned. The open reading frame of Pmcyclin E was 1263 bp in length and encoded a 47.9-kDa protein, while that of PmCDK2 was 921 bp, encoding a protein of 34.9 kDa. Recombinant cyclin E and CDK2 proteins were expressed in Escherichia coli and purified by Ni-chelating affinity chromatography. In addition, a pull-down assay was performed to identify any interaction between Pmcyclin E and PmCDK2. This research provides a basis for the study of the functional mechanisms of the cyclin E/CDK2 complex in shrimp, further enriching our knowledge of invertebrate cell cycle regulation.

Reproductive biology, maturation size and sex ratio of black tiger shrimp (Penaeus monodon Fabricius, 1798) from fishing grounds of Digha coast, West Bengal, India.

The present paper studies the reproductive biology, maturation size and sex ratio of Penaeus monodon collected from Digha fishing grounds, India during 2011-2013. A total of 633 individual of P. monodon were examined and among them 242 were males and 391 were females. The overall yearly sex ratio was observed to be 1:1.6 (males: females). Based on the results, the spawning season of P. monodon was mainly in January-February and was extended up to June. The first maturity was observed at 163.5 mm length. The estimated number of ova in the mature ovary ranged from 120155 to 961240 in P. monodon .

Effects of biofloc under different carbon sources and protein levels on water quality, growth performance and immune responses in black tiger shrimpPenaeus monodon(Fabricius, 1978)

A 75-day 2 × 3 factorial experiment was conducted to evaluate the effect of two levels of dietary protein (32 and 40%) and two different carbon sources (rice flour–R and molasses-M), and without carbohydrate (control–C) in black tiger shrimp Penaeus monodon juveniles (3.37 ± 0.04 g) at 100 nos. m−3 in 100 L fibre reinforced plastic tanks. Biofloc volume and total suspended solid were higher in molasses added groups (32 + M and 40 + M) followed by rice flour (32 + R and 40 + R) and controls (32C and 40C). Molasses and rice flour addition significantly reduced (P < 0.01) the total ammonia–N compared to controls. The highest Vibrio, Bacillus and Lactobacillus counts were recorded in 40 + M, 32 + M and 32 + R respectively. Among the treatments, significantly higher (P < 0.01) final body weight was recorded in 40 + R (8.5 ± 0.3 g), 40 + M (7.8 ± 0.3 g) and 32 + R (7.5 ± 0.4 g) compared to control groups, 32C (6.1 ± 0.3 g), 40C (6.4 ± 0.3 g) and molasses added group, 32 + M (5.7 ± 0.4 g). Rice flour supplementation significantly increased (P < 0.01) the total haemocyte count (×106 cells mL−1) in 32 + R (45.7 ± 3.7) and 40 + R (44.3 ± 3.1) compared to controls, 32C (27.3 ± 3.4) and 40C (25.8 ± 0.9). Similarly, higher superoxide dismutase, catalase, serum protein and glucose were recorded in the rice flour added groups, 40 + R followed by 32 + R. Among the treatments, the highest level of prophenoloxidase (OD 490 nm, 0.3 ± 0.0) and survival after challenge with Vibrio harveyi (55.6%) was observed in 32 + R. The study elucidates that rice flour addition produces optimum level of biofloc with better growth and immune responses compared to molasses and control. Furthermore, rice flour addition at 32% protein level could replace 40% protein feed.