Granular Hemocytes Research Articles

The N-terminal peptide generated after activation of prophenoloxidase affects crayfish hematopoiesis

The circulating hemocytes of invertebrates are important mediators of immunity, and hemocyte homeostasis is of high importance for survival and health of crustaceans. The prophenoloxidase (proPO)-activating system is one of the most essential immune reactions, which can be activated by pattern recognition proteins from microorganisms. Activation of proPO by the proPO activating enzyme generates an N-terminal peptide, with cleavage site after Arg176, as well as the active enzyme phenoloxidase, which is the key enzyme for melanization. In the present study we demonstrate a role for the N-terminal proPO-peptide in hematopoiesis. Injection of this proPO-peptide increased the number of circulating hemocytes and especially granular hemocytes. We also show that the reactive oxygen species (ROS) production in the anterior proliferative center was enhanced after proPO peptide injection, which is a prerequisite for rapid hemocyte release from the hematopoietic tissue. Moreover, this peptide had an effect on ROS production in in vitro cultured hematopoietic cells and induced spreading of these cells within 72 h. Taken together, our findings show a role of the N-terminal proPO peptide in stimulation of hematopoiesis in crayfish, Pacifastacus leniusculus.

Bio-augmentation of heterotrophic bacteria in biofloc system improves growth, survival, and immunity of Indian white shrimp Penaeus indicus

Effect of bio-augmentation of Bacillus spp in biofloc on growth, survival and immunity in Indian white shrimp Penaeus indicus was evaluated. Nine Bacillus strains were isolated and screened individually as well as in the form of a consortia. To maintain a C:N ratio of 12:1 a blend of carbohydrate sources was used. Bio-augmentation with bacterial consortium and Virgibacillus sp. produced improved growth and immunity. Shrimp survival ranged from 80 to 95% among treatments. Production was higher (35%) in the biofloc tanks with an average body weight (ABW) of 10.89 ± 1.2 g. On evaluating the immune responses, it was found that trypsin significantly (P < 0.05) enhanced Prophenoloxidase (PO) activity in Lysinibacillus, Bacillus cereus, Bacillus licheniformis and Bacillus subtilis bio-augmented groups. Laminarin induced PO activity was observed in groups supplemented with Oceanobacillus sp., Bacillus sp.and Bacillus megaterium. The lysozyme (LZ) activity was significantly (P < 0.05) higher in B. cereus and Microbial Consortia (MC), while other treatments were less effective. Total hemocyte count (THC) significantly (P < 0.05) increased in all treatment groups compared to the control. Hyaline hemocyte (HH) count was significantly (P < 0.05) higher in the control group (14.43%). Semi granular hemocytes (SGH) was higher in groups treated with Lysinibacillus, Bacillus sp., B. licheniformis and B. subtilis. The granular hemocyte (GH) count was significantly (P < 0.05) higher in Virgibacillus sp., B. cereus, B.megaterium and Oceanobacillus sp. The biofloc alone (BF), treated and augmented with B. megaterium significantly (P < 0.05) increased phagocytic activity. Highly significant phagocytic index (PI) was observed in bio-augmented groups, BF and MC. The relative expression levels of immune genes were found to be significantly up-regulated in shrimps grown in bio-augmented groups. Enhanced immunological parameters implies that bio-augmentation of biofloc with Bacillus spp. improved immunity in shrimps. Hence, bio-augmentation of probiotics in biofloc may be useful in improving culture conditions to produce P. indicus.

A Type IIa crustin from the pink shrimp Farfantepenaeus paulensis (crusFpau) is constitutively synthesized and stored by specific granule-containing hemocyte subpopulations

Crustins are cysteine-rich antimicrobial peptides (AMPs) widely distributed across crustaceans. From the four described crustin Types (I to IV), crustins from the subtype IIa are the most abundant and diverse members found in penaeid shrimp. Despite the critical role of Type IIa crustins in shrimp antimicrobial defenses, there is still limited information about their synthesis and antimicrobial properties. Here, we report the subcellular localization and the antibacterial spectrum of crusFpau, a Type IIa crustin from the pink shrimp Farfantepenaeus paulensis. The recombinantly expressed crusFpau showed antimicrobial activity against both Gram-positive and Gram-negative bacteria at low concentrations. Results from immunofluorescence using anti-rcrusFpau antiserum revealed that crusFpau is synthetized and stored by both granular and semigranular hemocytes, but not by hyaline cells. Interestingly, not all granular and semigranular hemocytes stained for crusFpau, revealing that this crustin is produced by specific granule-containing hemocyte subpopulations. Finally, we showed that the granule-stored peptides are not constitutively secreted into the plasma of healthy animals.

In vivo administration of LPS and β-glucan generates the expression of a serum lectin and its cellular receptor in Cherax quadricarinatus

In crustaceans, it has been suggested that specific protection against pathogens could be triggered by vaccines and biological response modifiers; although the specific mechanisms of this protection have not been clarified yet. In the crayfish Cherax quadricarinatus, a humoral lectin (CqL) binds its own granular hemocytes through a specific receptor (CqLR) and increases the production of reactive oxygen species (ROS). In the present study, we challenged in vivo crayfishes with immunostimulants, β-glucan (200 μg/kg) or LPS (20 μg/kg), and identified the participation of cellular and humoral mechanisms. The stimulants generated a complex modification in the total hemocytes count (THC), as well as in the proportion of hemocyte subsets. At 2 h after the challenge, the largest value in THC was observed in either challenged crayfishes. Furthermore, at the same time, hyaline hemocytes were the most abundant subset in the hemolymph; after 6 h, granular hemocytes (GH) were the most abundant hemocyte subset. It has been observed that a specific subset of GH possesses a CqLR that has been related to ROS production. After 2 and 6 h of the β-glucan challenge, a significant increase in CqLR expression was observed in the three circulating hemocyte subsets; also, an increased expression of CqL was detected in a granular hemocytes sub-population. After 2 and 6 h of stimulation, the specific activity of the serum lectin challenged with β-glucan was 250% and 160% higher than in the LPS-treated-group, respectively (P < 0.05). Hemocytes from challenged crayfishes were stimulated ex vivo with CqL, ROS production was 180% higher in hemocytes treated with β-glucan + CqL than in hemocytes treated with LPS + CqL (P < 0.05). The results evidence the effectivity of immune stimulators to activate specific crayfish defense mechanisms, the participation of CqL and its receptor (CqLR) could play an important role in the regulation of immune cellular functions, like ROS production, in Cherax quadricarinatus.

The Role of Bacillus amyloliquefaciens on Litopenaeus vannamei During the Maturation of a Biofloc System

Biofloc technology is a sustainable aquaculture production system which uses microorganisms to maintain water quality and to increase productivity. In this system, probiotics can enhance the positive effects of bioflocs on the cultured species. The objective of this research is to study the role of the probiotic bacterium Bacillus amyloliquefaciens during the formation of a biofloc system for the culture of Litopenaeus vannamei. Two doses of probiotic were assayed and applied directly to the water. The experiment was developed in nine tanks distributed as follows: Three control tanks with no probiotic, three tanks with a probiotic dose of 103 cfu/mL, and three tanks with a dose of 104 cfu/mL. Water quality, microbial activity, growth parameters and the immune system state of shrimps were monitored throughout the maturation process. The results indicate a positive effect upon the shrimp immune system throughout the study period, where specifically there was an increase in granular hemocytes in the shrimp hemolymph. During the immature biofloc phase, granular hemocytes were 5% higher in tanks supplemented with the probiotic. During the mature biofloc phase, granular hemocytes were 7% higher in those same tanks. During the maturation of the biofloc, environmental conditions are more unfavorable for shrimp growth, due to the accumulation of nitrites. So, the effect of the probiotic is especially important during this stage when the shrimp are stressed and are more vulnerable to diseases. However, the effects on microbial activity, water quality and Litopenaeus vannamei growth did not increase the benefits of the biofloc system.

Isolation and molecular characterization of hemocyte sub-populations in kuruma shrimp Marsupenaeus japonicus

Crustacean hemocytes, which have usually been classified morphologically based on methods using Giemsa or May-Giemsa stains, have recently been categorized using monoclonal antibodies or marker genes. However, these latter techniques are not yet widely used, and different classification methods are used for hemocytes among laboratories. Therefore, we aimed to develop a molecular classification method that can be widely used by researchers. The method we have developed uses lectins and magnetic-activated cell sorting (MACS) to isolate sub-populations of hemocytes. Two lectins, wheat germ agglutinin (WGA) and tomato lectin (Lycopersicon esculentum lectin; LEL), characteristically bind to hemocytes, which allows them to be classified into three sub-populations. Furthermore, different sub-populations of hemocyte can be isolated by using LEL and MACS. These sub-populations were characterized as non-granular and granular hemocytes, and the accumulation patterns of the gene transcripts were consistent with the results of a functional analysis reported previously. The lectin-based hemocyte isolation method developed in this study has good reproducibility.

Effects of chronic lead and cadmium exposure on the oriental river prawn (Macrobrachium nipponense) in laboratory conditions

During the past decade, the total population and overall reproductive performance of the oriental river prawn (Macrobrachium nipponense) in the Anzali international wetland and its basin rivers has been severely reduced. This seems to be caused by an increase in heavy metal pollution from industrial wastewaters. We investigated the effects of chronic levels of lead (Pb) and cadmium (Cd) on reproductive success, hemato-immunology, crustacean hyperglycemic hormone (cHH), Pb/Cd bioaccumulation and histopathology of hepatopancreas of oriental river prawn. The present study was separately carried out using four chronic treatments for Pb (0 (control), 0.024, 0.072 and 0.12 μM/l) and Cd (0 (control), 0.026, 0.053 and 0.08 μM/l) in experimental tanks during 60 days. The broodstock were fed by commercial shrimp diet, ad libitum, twice a day. The results revealed that reproductive activities ceased when Pb levels were 0.072 and 0.12 μM/l and also when Cd levels reached 0.08 μM/l. At 0.024 μM/l Pb, 0.026 and 0.053 μM/l Cd, the prawns showed initial reproductive activity but after spawning, the egg-clutches were found detached from the pleopods of the brood females. Hemato-immunology results showed that by increasing the Pb and Cd concentrations, large granular hemocyte (LGH) and cHH values also increased. However, small granular hemocyte (SGH) count was inversely affected. A significant increase in mortality (P < 0.05) was observed as both Pb and Cd concentrations increased. Histological observations are indicative of increased necrosis, lumen size, melanization, vacuolation and abnormal lumen shape, as the heavy metals concentrations increased. In conclusion, obtained results can help to elucidate effects of Pb and Cd on freshwater decapod crustaceans reproductive performance and related physiological parameters.

Growth, antioxidant system, and immunological status of shrimp in bioflocs and clear water culture systems

Abstract: The objective of this work was to evaluate the effect of the traditional culture system in clear water and of the one in microbial flocs on the antioxidant and immunological status of Litopenaeus vannamei shrimp. Samples (gill, hemolymph, and hepatopancreas) were collected on days 15, 30, and 60 of the experimental period. The following immunological parameters were analyzed at each evaluation time: granular and hyaline hemocytes, total protein, and apoptosis. Assays on glutamate-cysteine ligase (GCL) activity and on the concentrations of reduced glutathione (GSH) and thiobarbituric acid reactive substances (TBARS) were also performed. The evaluated immunological parameters did not differ significantly between treatments. Shrimp reared in clear water showed higher levels of lipid peroxidation in the gills and of GCL activity in the hemolymph on days 15 and 30. Shrimp in microbial floc had a higher survival rate, and the water did not have to be renewed during the experimental period. The microbial floc system improves production levels and provides a healthier culture environment.

Effect of probiotics on immune competence of giant freshwater prawnMacrobrachium rosenbergii

This study evaluates the effect of probiotics, Zymetin, on the immune efficiency of Macrobrachium rosenbergii juvenile against pathogenic Vibriospp. and Aeromonasspp. The experiment was conducted in glass aquaria with same level of feeding under different treatments, that is, negative control (Cn), positive control with Vibrio spp. (Cv) and Aeromonasspp. (Ca), prawn juveniles fed with probiotics (Zymetin) at 5 g/kg of feed but without pathogen (T1), probiotic fed prawn challenged with Vibrio spp. (T2) and Aeromonasspp. (T3). The results demonstrated that T1 showed higher survival rate, total haemocyte count, non-granular haemocyte count, phagocytic activity, and clearance efficiency compared to other treatment groups. In contrast, decreased number of small and large granular haemocyte was observed in T1. Despite that, THC was found to be significantly different (p < 0.05) among all the controls and lowest was in Cv, followed by Ca. Besides, phagocytic activity and clearance efficiency against Vibrio spp. and Aeromonasspp. decreased significantly in Cn, Ca, and Cv fed M. rosenbergiijuvenile, while the values were found to be higher in T1 followed by T2 and T3, in that order. In addition, the challenge test showed an increasing trend of total and beneficial bacterial density as well as declining trend of some harmful bacteria in the water and gut of prawn in all the groups tested (p < 0.05).

A comparative global proteomic analysis of the hematopoietic lineages in the crustacean Pacifastacus leniusculus

In crustaceans as in other arthropods, the circulating hemocytes are vital for protecting the animal against attacking microorganisms. As many hemocytes are destroyed early during an infection, new hemocytes must fast get in place to prevent disperse of a pathogenic microbe, In order to understand the hematopoietic process in more detail we here report a complete proteomic analysis from purified cell types from the APC of the hematopoietic tissue, via the remaining parts of the HPT to the mature semigranular and granular hemocytes. Several possible cell type specific proteins are detected and new putative biomarkers within the crayfish hematopoietic lineage that can be used to increase the understanding of how the differentiation process is regulated is described.

The effect of the lectin from Cherax quadricarinatus on its granular hemocytes

In crustaceans, lectins and hemocytes of the innate immune system provide the first line of defense. Although evidence points to the potential role of lectins in regulating hemocyte activity, the processes underlying the lectin activation have not been evaluated. In the present study, the receptor for CqL, a humoral lectin from Cherax quadricarinatus specific for galactose/sialic acid, was identified in a granular subset of hemocytes. The CqL receptor (CqLR) is a 490-kDa glycoprotein, composed of four identical 120-kDa subunits. As shown by immunohistochemistry, CqL at 7.5 μg/mL as optimal dose, after 2 min, induced, specifically on granular hemocytes, increased phosphorylation of serine (152%), threonine (192%), and tyrosine (242%) as compared with non-treated hemocytes; moreover, CqL induced increased generation of reactive oxygen species (ROS). Specific kinase inhibitors showed inhibition (P < 0.001) of ROS production induced by CqL. These results strongly suggest that CqL actively participated in the generation of ROS through kinases induced by a CqLR in a subset of granular hemocytes of the crayfish C. quadricarinatus. The results provide strong evidence that CqL activates, through specific granular hemocytes, receptors that modulate cellular functions in C. quadricarinatus.

Effects of X-ray irradiation on haemocytes of Procambarus clarkii (Arthropoda: Decapoda) males

The red swamp crayfish Procambarus clarkii is an invasive alien species spreading worldwide. The sterile male release technique (SMRT) is among the methods used to contrast the growth of P. clarkii populations within invaded areas. In this study males underwent X-ray sterilisation with a dose of 40 Gy and their immunocompetence was analysed in comparison to untreated animals to ascertain whether radiation can affect welfare parameters other than reproductive organs. The present research investigated the immune function in P. clarkii males in term of (1) morphological haemocyte characterisation by transmission electron microscopy to identify the main phagocyting haemocyte after in vivo artificial non-self challenge with latex beads; (2) total and differential haemocyte counts; and (3) basal and total phenoloxidase activities as components of the humoral defence. Three types of circulating haemocytes were characterised via transmission electron microscopy: hyaline, semigranular and granular haemocytes. The ultrastructural features of haemocyte granules allowed the characterisation of a fourth type of haemocyte, the medium granule haemocyte. In vivo artificial non-self-challenge with latex beads identified the semigranular haemocytes as primarily involved in phagocyting activity. Circulating haemocytes of males irradiated with a dose of 40 Gy, after 20 days, showed a significantly lower diameter in the granules of hyaline and semigranular haemocytes, but no other evident ultrastructural alterations in comparison with un-irradiated animals were found. Irradiated males showed a significant decrease of about 80% of circulating haemocytes and an increase in frequency of semigranular and granular haemocytes. No significant differences in basal and total phenoloxidase activity were recorded and this could, in part, explain the good survival level of irradiated males despite the drastic decline of the haemocyte number. This study represents the basis to appraise whether SMRT affects important functions, such as those of the immune system, in addition to altering the gonad tissue.

In Situ Subcellular Imaging of Copper and Zinc in Contaminated Oysters Revealed by Nanoscale Secondary Ion Mass Spectrometry.

Determining the in situ localization of trace elements at high lateral resolution levels in the biological system is very challenging, but critical for our understanding of metal sequestration and detoxification. Here, the cellular and subcellular distributions of Cu and Zn in contaminated oysters of Crassostrea hongkongensis were for the first time mapped using nanoscale secondary ion mass spectrometry (nanoSIMS). Three types of metal-containing cells were revealed in the gill and mantle of oysters, including Cu-specific hemocytes, Cu and Zn-containing granular hemocytes, and Cu and Zn-containing calcium cells. Obvious intercellular distribution of Cu was found in the gill tissue, indicating the potential role of hemolymph in the transportation of Cu in oysters. The distribution of Cu showed a strong colocalization with sulfur and nitrogen in Cu-specific hemocyte and intercellular hemolymph. In the Cu and Zn-containing granular hemocytes and calcium cells, the co-occurrence of Cu and Zn with phosphorus and calcium was also found. Different relationships of distributions between Cu/Zn and macronutrient elements (nitrogen, sulfur and phosphorus) implied the differential metal complexation in oysters. Interestingly, quantitative analysis of the ratios of 32S-/12C14N- and 31P-/12C14N- of metal-deposited sites suggested the dynamic process of transfer of Cu and Zn from the metabolized protein pool to a more thermodynamically stable and detoxified form.

Hemocytes and hemocytic responses in the mole crab Emerita emeritus (Linnaeus 1767).

The mole crab, Emerita emeritus, collected from the sandy shores of a Chennai beach, was investigated for cellular immune responses based on the morphology and defensive reactions of the circulating haemocytes. Three haemocyte morphotypes were identified using light and electron microscopy, and separated in a discontinuous percoll gradient. A phagocytosis study using human B erythrocyte as a target cell under phase-contrast optics showed that granular and semi-granular haemocytes were phagocytic, and this response was enhanced by using serum (opsonin)-coated human B erythrocyte in unfractionated and fractionated haemocytes. Observation of TEM image of phagocytosis revealed that the initial recognition and binding of the target cell was restricted to granular and semigranular haemocytes, which were lacking with hyaline cells. However, the encapsulation of DEAE Sepharose CL 6B beads, either untreated or coated with serum (opsonin), was restricted to hyaline cells. This suggests the occurrence of two cell lines in haemocytes, based on the differences observed in the response of haemocytes to bind target cells for phagocytosis or encapsulation. The present study also differentiated the activation of PO in the plasma, serum, and haemocyte lysate supernatant (HLS).

Pathogenicity profile of Vibrio parahaemolyticus in farmed Pacific white shrimp, Penaeus vannamei

A pathobiological study was conducted using Vibrio parahaemolyticus (VP) strain isolated from vibriosis affected shrimp (Penaeus vannamei) farms in Kancheepuram and Thiruvallur districts of Tamil Nadu during August 2014 to February 2015. The isolate was identified based on the morphological, physiological, biochemical and molecular characters. LD50 value with intramuscular injection was determined as 2.6 × 104 cfu/shrimp and sequential pathology was studied giving 6.1 × 103 cfu/shrimp (LD25). Total plate count (TPC) and total Vibrio count (TVC) in water, pond sediment, haemolymph, muscle, HP and gut were found significantly (P < 0.01) higher in natural cases than the experimental set up. Clinical signs and lesions observed in the natural and experimental cases were anorexia, lethargy, cuticle softening, loose shells, abdominal muscle cramp, red discoloration, opaque and whitish abdominal and tail musculature, necrosis of exoskeleton or splinter burns, reddish pleural borders of antennae, uropods and telson, swollen tail fan, ulcers, moribund shrimp sinking to bottom, and mortalities with shrunken discoloured HP with empty gut. Total haemocyte count (THC), small nongranular haemocyte (SNGH), large nongranular haemocyte (LNGH), small granular haemocyte (SGH) and large granular haemocyte (LGH) counts lowered significantly (P < 0.01) at 3, 6, 12, 24, 48, 96 and 192 h post injection (p.i). No LGH were found after 96 h of challenge. The post injection qPCR analyses of haemocytes showed up-regulations of penaeidin-3a, lysozyme, prophenoloxidase I, prophenoloxidase II and serine protein at 3 and 6 h of infection. There was total down-regulation of crustin from 3 to 192 h p.i. There was a remarkable elevation in the level of proPO I with concomitant depletion of proPO II. The pattern of up- and down-regulations in proPO I and SP were similar. The post infection qPCR analyses showed that these immune related genes could be used as markers for assessing the immune status of P. vannamei. Major histopathological manifestations observed were haemocyte infiltration/nodule in the epidermis, skeletal and cardiac muscles, atrophy of the excretory organ, and disrupted HP tubules with diffuse interstitial edema and haemocytic infiltration. Further HP showed that there was thickening of intertubular space, karyomegaly with prominent nucleoli, rounding and sloughing of HP tubular epithelium, many mitotic figures with bacterial colonies and apoptotic bodies, separation of shrunken tubule epithelium from myoepithelial fibers, regeneration of tubules, cystic, dilated and vacuolated appearance of HP tubules, hypoplastic changes in the tubules with no B, R and F cells, granuloma formation, concretions in tubules, calcification, necrosis, and washed out appearance with complete loss of architecture. The progression of the degenerative changes in the HP tubular epithelial cells was from proximal to distal end. In haematopoietic organ, increased mitotic activities with focal to extensive depletion and degeneration were observed. Degeneration of the stromal matrix with spheroid formation in lymphoid organ was observed among the Vp infected natural and experimental animals. Degeneration of glandular structures in the prehensile appendages with bacterial colonies, melanization and loss of epithelial layer in oesophagus, swelling and loss of architecture with mucinous secretion in the stomach, degeneration of peritrophic membrane in the lumen of intestine were observed in field cases but not in the experimental studies. Further, this study established the pathobiology of the Vp isolate to P. vannamei.

Activation of phenoloxidase activity by humoral lectin in hemocytes of freshwater crab Paratelphusa jacquemontii

The lectin, Pjlec isolated from the hemolymph of the freshwater crab Paratelphusa jacquemontii hemagglutinated (HA) with mice, rabbit and rat erythrocytes. However, the lectin failed to agglutinate neraminidase treated asialylated erythrocytes showing its sialic acid specificity. The poyacyrlamide gel electrophoresis of lectin yielded 310kDa proteins, on sodium sulphate dodecyl (SDS) gel appeared as a tetramer with subunits of 76kDa. The observation of in vitro phagocytosis in granular hemocytes of lectin opsonized rabbit erythrocyte by Transmission electron microscopy (TEM) showed the release of lytic vesicles by exocytosis prior to engulfment. The Pjlec lectin also showed an ability to oxidize L-3, 4 dihydroxyphenylalanine (L-DOPA) and in hemocyte lysate preparation (HLS) was enhanced on reduction with SDS and on proteolytic cleavage with trypsin. The lectin appeared to have a regulatory role in activation of enzyme activity associated with phagocytosis and melanin formation.

Assessment of the cytotoxic and genotoxic effects of TiO2 nanoparticles in A549 cells

The mole crab, Emerita emeritus, collected from the sandy shores of a Chennai beach, was investigated for cellular immune responses based on the morphology and defensive reactions of the circulating haemocytes. Three haemocyte morphotypes were identified using light and electron microscopy, and separated in a discontinuous percoll gradient. A phagocytosis study using human B erythrocyte as a target cell under phase-contrast optics showed that granular and semi-granular haemocytes were phagocytic, and this response was enhanced by using serum (opsonin)-coated human B erythrocyte in unfractionated and fractionated haemocytes. Observation of TEM image of phagocytosis revealed that the initial recognition and binding of the target cell was restricted to granular and semigranular haemocytes, which were lacking with hyaline cells. However, the encapsulation of DEAE Sepharose CL 6B beads, either untreated or coated with serum (opsonin), was restricted to hyaline cells. This suggests the occurrence of two cell lines in haemocytes, based on the differences observed in the response of haemocytes to bind target cells for phagocytosis or encapsulation. The present study also differentiated the activation of PO in the plasma, serum, and haemocyte lysate supernatant (HLS).

Dietary Supplementation with Butyrate and Polyhydroxybutyrate on the Performance of Pacific White Shrimp in Biofloc Systems

AbstractThis study aimed to evaluate the effects of dietary supplementation with sodium butyrate or polyhydroxybutyrate (PHB) on growth performance, as well as changes in intestinal microbiota and hemato‐immunological parameters, of Litopenaeus vannamei reared under a superintensive biofloc system. Twelve 800‐L tanks were each stocked with 250 shrimp/m3 (3.96 ± 0.04 g mean initial weight) and reared over a 6‐wk period. The basal diet and two test diets supplemented with 2% of each feed additive. At the conclusion of the growth trial shrimp fed with the butyrate‐supplemented diet, as compared with the control shrimp, showed higher survival and productivity and lower total bacterial and Thiosulfate‐citrate‐bile salts‐sucrose Agar (TCBS) counts in the intestine. However, no differences were observed in other performance parameters analyzed. Shrimp fed with both supplementation regimens also showed an increase in total and granular hemocytes, as well as an increase in serum agglutination titer. Shrimp offered diets supplemented with sodium butyrate had higher counts of hyaline cells. Thus, for L. vannamei reared in a superintensive biofloc system, it can be concluded that dietary supplementation of sodium butyrate, more so than PHB, acted as an immune system modulator by reducing the concentration of pathogenic bacteria in shrimp gut, thereby increasing survival and productivity.

Development of Indigenous Bio-sensing Methodology for Rapid and Low Cost Endotoxin Detection System

Endotoxin is a signature molecule of gram-negative bacteria and is clinically significant as the agent of gramnegative sepsis, a disease condition with high mortality. This study describes an enzyme-substrate reaction using the distilled water lysate of the granular hemocytes (amebocytes) of the mangrove horseshoe crab (Tachypleus gigas), a native of the Bay of Bengal, and a chromogenic peptide which results in the production of yellow spectroscopically monitored product in the presence of endotoxin. The assay is complete within 30 min and shows a lower detection limit of 0.2 EU/mL. The novelty of our approach is the use of mangrove horseshoe crabs are the source of amebocyte lysate instead of the American horseshoe crab (Limuluspoly phemus).The anticipated methodology offers advantages for the South Asian market that include a low cost due to use of indigenous reagents. This method shows exquisite sensitivity and provides a rapid assessment of LPS concentrations. We assume that the commercial development of our assay will reduce the cost of a single chromogenic endotoxin detection test as compared to the cost of present imported kits presently available in Asian and Indian market.

Histological changes and antioxidant enzyme activity in signal crayfish (Pacifastacus leniusculus) associated with sub-acute peracetic acid exposure.

Peracetic acid (PAA) is a powerful disinfectant recently adopted as a therapeutic agent in aquaculture. A concentration of 10mgL(-1) PAA effectively suppresses zoospores of Aphanomyces astaci, the agent of crayfish plague. To aid in establishing safe therapeutic guideline, the effects of PAA on treated crayfish were investigated through assessment of histological changes and oxidative damage. Adult female signal crayfish Pacifastacus leniusculus (n=135) were exposed to 2mgL(-1) and 10mgL(-1) of PAA for 7 days followed by a 7 day recovery period in clean water. Superoxide dismutase activity was significantly lower in gill and hepatopancreas after three days exposure to 10mgL(1) PAA than in the group treated with 2mgL(-1) PAA and a control in only clean water. Catalase activity in gill and hepatopancreas remained unaffected by both exposures. Glutathione reductase was significantly decreased in gill of 10mgL(-1) PAA treated crayfish and increased in group exposed to 2mgL(-1) compared to control after 7 days exposure. Antioxidant enzyme activity in exposed groups returned to control values after recovery period. Gill, hepatopancreas, and antennal gland showed slight damage in crayfish treated with 2mgL(-1) of PAA compared to the control group. The extent and frequency of histological alterations were more pronounced in animals exposed to 10mgL(-1). The gill was the most affected organ, infiltrated by granular hemocytes and displaying malformations of lamella tips and disorganization of epithelial cells. After a 7 day recovery period, the infiltrating cells in affected tissues of the exposed crayfish began to return to normal levels. Results suggested that the given concentrations could be applied to signal crayfish against crayfish plague agent in aquaculture; however, further studies are required for safe use.