Probiotic potential of a novel endophytic Streptomyces griseorubens CIBA-NS1 isolated from Salicornia sp. against Vibrio campbellii infection in shrimp.

A novel endophytic Streptomyces griseorubens CIBA-NS1 was isolated from a salt marsh plant Salicornia sp. The antagonistic effect of S. griseorubens against Vibrio campbellii, was studied both in vitro and in vivo. The strain was validated for its endophytic nature and characterized through scanning electron microscopy, morphological and biochemical studies and 16SrDNA sequencing. The salinity tolerance experiment has shown that highest antibacterial activity was at 40‰ (16 ± 1.4 mm) and lowest was at 10 ‰ salinity (6.94 ± 0.51 mm). In vivo exclusion of Vibrio by S. griseorubens CIBA-NS1 was studied in Penaeus indicus post larvae and evaluated for its ability to improve growth and survival of P. indicus. After 20 days administration of S. griseorubens CIBA-NS1, shrimps were challenged with V. campbellii. The S. griseorubens CIBA-NS1 reduced Vibrio population in test group when compared to control, improved survival (60.5 ± 6.4%) and growth, as indicated by weight gain (1.8 ± 0.05g). In control group survival and growth were 48.4 ± 3.5% and 1.4 ± 0.03 g respectively. On challenge with V. campbellii, the S. griseorubens CIBA-NS1 administered group showed better survival (85.6 ± 10%) than positive control (64.3 ± 10%). The results suggested that S. griseorubens CIBA-NS1 is antagonistic to V. campbellii, reduce Vibrio population in the culture system and improve growth and survival. This is the first report on antagonistic activity of S. griseorubens isolated from salt marsh plant Salicornia sp, as a probiotic candidate to prevent V. campbellii infection in shrimps.

Vertical transmission and prevalence of white spot syndrome virus (WSSV) in the wild spawning population of the Indian white shrimp, Penaeus indicus.

White spot disease, caused by white spot syndrome virus (WSSV), has historically been the most devastating disease in shrimp aquaculture industry across the world. The mode of virus transmission is the most crucial stage in the dynamics and management of virus infection. This study explored the mechanism of vertical transmission of WSSV in Indian white shrimp, Penaeus indicus, potential native species for domestication and genetic improvement, using quantitative real time PCR (q RT PCR), light and electron microscopy, and in situ hybridization. Wild brooders of P. indicus (n = 2576) were sampled along the South east coast of India, during 2016 to 2021. Of these âˆ¼ 58 % of the brooders were positive for WSSV, and almost 50 % of infected wild brooders were at the various stages of reproductive maturation. WSSV-PCR positive brooders (n = 200) were analysed for vertical WSSV transmission. The q RT PCR studies of reproductive tissues revealed that 61 % (n = 13) of spermatophore, 54 % (n = 28) of immature ovaries and 48 % (n = 27) of ripe ovaries were infected with WSSV. The lowest level of infection was recorded in females with ripe ovaries (6.84 × 101 ± 9.79 × 100 ng genomic DNA) followed by fertilized eggs (1.59 × 102 ± 3.69 × 101 ng genomic DNA), and larvae (nauplius and zoea). The histology of gravid females with high WSSV copies showed pyknotic and karyorrhectic germinal vesicle with degenerated cortical rods. Conversely, the gravid females with low WSSV copies showed fully developed ovary without characteristic signs of WSSV infection. Transmission electron microscopic studies clearly established the presence of WSSV particles in both ovaries and spermatophores. When subjected to in situ hybridization, WSSV-specific signals were observed in connective tissues of spermatophore, although gravid ovary and fertilized eggs were failed to produce WSSV specific signals. The present study provides the first molecular and histological evidence for trans-ovarian vertical transmission of WSSV. Development of disease-free base population being the cornerstone and first step in establishing the breeding program, the present findings could be a basis for development of such programs.

A Biofloc-Based Aquaculture System Bio-augmented with Probiotic Bacteria Bacillus tequilensis AP BFT3 Improves Culture Environment, Production Performances, and Proteomic Changes in Penaeus vannamei.

Experiments were conducted to evaluate the probiotic effect of bio-augmented Bacillus tequilensis AP BFT3 on improving production, immune response, and proteomic changes of Penaeus vannamei reared in a biofloc system. Penaeus vannamei larvae (PL13) were stocked in 100-L tanks at a rate of 100 no per tank to study the effect of B. tequilensis AP BFT3 with and without biofloc (BFT-PRO and PRO). Control tanks devoid of probiotic strain were maintained in a clear water system. The growth and survival considerably increased in probiotic added biofloc reared shrimp than probiotic added clear water reared ones and control. Water quality significantly improved in probiotic added (PRO) and biofloc-probiotics (BFT-PRO) system than control. Microbiological investigations indicate increased heterotrophic bacterial load in BFT-PRO compared to the PRO and control. The quality of the isolated microbes was analyzed in terms of enzyme production, and an abundance of enzyme-producing bacterial population was observed in BFT-PRO shrimp. Immune-related genes were significantly upregulated in BFT-PRO shrimp, followed by the PRO and control. The proteomic data (2D gel electrophoresis and MALDI-TOF) of muscle tissue from the experimental animals identified 11 differentially expressed proteins. The Daxx OS and Lit v 1 tropomyosin was found upregulated in BFT-PRO shrimps. Downregulation of Na+/K+ATPase was observed in biofloc with probiotic-supplied groups. The findings revealed that the BFT system's efficacy could be improved through the addition of probiotics. The addition of B. tequilensis AP BFT3 as a probiotic in biofloc induced the expression of essential proteins, reducing contracting diseases during culture.

Impact of effluent of Pulp & Paper industry on the flora of river basin at Jaykaypur, Odisha, India and its ecological implications.

The JK Paper industry located at Rayagada discharges biologically untreated effluent more than the permissible limit prescribed by Pollution Control Board, Odisha in to the environment. The industry is seriously polluting the surrounding aquatic and terrestrial environment. No detailed intensive study was carried out by previous workers on this industry earlier. The present study aims at finding out the impact of effluent on the flora at the contaminated site. The chemically treated effluent (TE) contained significant amount of mercury and cadmium. The TE has high BOD, COD, dissolved solids and suspended solids when compared to normal river water at the site of discharge. The TE deteriorated the natural water bodies changing the physico-chemical properties of natural river water. After meeting the river water the TE was diluted after 1 km distance from the meeting point of the river. Crop plants collected from the contaminated site showed higher level of residual Hg and Cd and significant depletion in pigment was observed. Plants collected from both the sides of the treated effluent canal showed significant amount residue mercury and cadmium in the plant leaves. The plants exposed to the TE, showed variation in chlorophyll and Phaeophytin pigment content when compared to their respective control values in all terrestrial plants collected from the contaminated site. In some plant leaves little increment in the pigment level was noted but the values were not significant. The changes observed in the plant pigment might be due to heavy metal accumulation. The presence of residual Hg and Cd in crop plants and plant leaves grazed by grazing animals after absorption, accumulation and enrichment may lead to a possible biological magnification, warrants attention. Proper biological treatment, treatment of effluent by modern methods and removal of heavy metals from the effluent before discharge by the industry is suggested.

Cellular and molecular immune response and production performance of Indian white shrimp Penaeus indicus (H. Milne-Edwards, 1837), reared in a biofloc-based system with different protein levels of feed.

The present study focuses on the immunity and growth of Penaeus indicus fed with varying protein levels (25%, 30%, and 35%) in a biofloc based rearing system. A 120 days growth trial was carried out using juvenile Penaeus indicus (0.71 ± 0.01) with dietary protein level, 25% (LP), 30% (MP), and 35% (HP), and a control diet-fed with 35% acted as control group resulting in 4 treatments each with four replicates and were randomly assigned 16 tank units (7500 L each). A combination of different carbon sources (molasses, wheat flour, and rice bran in 2:1:1 ratio), yeast and a probiotic (Bacillus sp.) consortium were used for the development of biofloc. At the end of the trial, the growth parameters of shrimps viz., initial weight, feed conversion ratio (FCR), and daily growth coefficient (DGC) were computed. The results indicated that shrimp fed with medium (30%) protein (MP) diet recorded significantly (P < 0.05) improved growth performance compared to high protein fed group (35%) and low protein (25%) fed group (LP) in a biofloc system and control group (35%). The immunological parameters such as hemagglutination activity (HA) assay, serum protein, lysozyme, phenol oxidase (PO), and inhibition of superoxide dismutase (SOD) activity were observed in serum, plasma, and hemocyte lysate supernatant (HLS). The HA activity, PO activity in plasma was found to be higher in high protein fed animals, whereas medium protein resulted in enhanced PO activity in serum. Similarly, lysozyme and SOD were inhibited well in high protein fed animals compared to the low protein fed group. The vital immune genes's mRNA profiling showed a potential rise in the expressional pattern in MP and HP treatments compared to LP and control. BGBP (beta-1,3-glucan binding protein) and hemocyanin mRNA transcript levels were highly upregulated in the HP (5 fold) and moderately expressed in MP (2 fold) and LP (1-2 fold). The transcripts of peroxinectin, antimicrobial peptides like crustin showed significant upregulation in HP followed by in MP and LP and control. Likewise, other immune genes, such as SOD, prophenoloxidase (proPO), showed a similar trend in a marginal way, indicating immunomodulation in the biofloc groups. This study suggested that biofloc with high protein (35%) supplementation can substantially enhance the immune response of shrimps, although medium protein level (30%) is optimum for improving the survival, growth, and in turn economic return in Indian white shrimp.

Bioaugmentation of biofloc system with enzymatic bacterial strains for high health and production performance of Penaeus indicus.

The beneficial effects of two probiotic bacterial strains Marinilactibacillus piezotolerans and Novosphingobium sp. during the culture of Indian white shrimp, Penaeus indicus, under biofloc and clear water system were evaluated. The experimental variation were CW1 (M. piezotolerans in clear water), BFT1 (biofloc + M. piezotolerans), CW2 (Novosphingobium sp. in clear water), BFT2 (biofloc + Novosphingobium sp.) and control (without bacterial strains and biofloc). Growth and survival considerably increased in probiotic bio-augmented treatments. Probiotic incorporation significantly improved water quality, especially ammonia reduction. Microbiota analysis from gut samples taken from different treatments revealed varied microbial population structure among clear water culture, biofloc culture and control. Proteobacteria and Firmicutes were the top phyla observed in the treatments which were significantly higher in bio-augmented systems than the control. Vibrio genera were predominantly observed in control and clear water system compared to that of biofloc systems. Immune genes were significantly altered in response to probiotic gut microbial supplementation than the control. Higher gene expression profile of important immune genes was observed in the biofloc reared shrimps. Expression of digestive enzyme related genes such as trypsin, chymotrypsin, cathepsin L, cathepsin B and alpha amylase were also upregulated significantly in probiotic supplementation especially in the biofloc treatments. Proteomic analysis of hepatopancreas of shrimps from different treatments was carried out by using 2D gel electrophoresis and MALDI-TOF analysis. The proteins were mostly related to growth and stress tolerance. Eukaryotic initiation factor 4E binding protein was expressed in all the groups and it was high in biofloc treated animals followed by animals treated solely with probiotics compared to those of control groups. The results concludes that biofloc already proved as an effective culture method for healthy shrimp production and supplementation of probiotic bacterial strains registered additional benefit for growth, survival, microbial, immunological status of P, indicus culture.

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.

Effect of in-situ and ex-situ biofloc on immune response of Genetically Improved Farmed Tilapia.

The present study is aimed to investigate the effect of biofloc intake on Genetically Improved Farmed Tilapia (GIFT), developed within the system and its influence as feed supplementation on water quality, growth performance, immunological parameters, antioxidant status, immune gene expression, and its resistance to Aeromonas hydrophila infection. GIFT Tilapia juveniles of 5.1 g (±0.05) were stocked at a density of 15/m3 in lined ponds of 300 m2 in triplicates for 180 days. The experimental groups consisted of T1-biofloc developed within the culture systems (insitu), T2-biofloc supplementation in fish feed (exsitu) and C- Control without biofloc. Distillery Spent wash was used as a carbon source to maintain the C/N ratio of 10:1 for floc development in T1. Free CO2, pH, BOD, dissolved oxygen, alkalinity, Calcium and Magnesium ions, Nitrate, Nitrite and ammonia were found to be significantly different between the treatments and control throughout the experiment. The immunological (Serum protein, Respiratory burst test (RBT) and Myeloperoxidase) and antioxidant indicators (Glucose, Superoxide dismutase (SOD) and catalase) were found to be significantly higher in T1 at the end of the trial. Increased weight gain, specific growth rate, survival and decreased feed conversion ratio was found in T1 when compared with the other experimental groups. Real time quantitative PCR analysis revealed that there was no folded expression of the immunological genes such as Metallothionein gene, cathepsin L, Toll like receptor 7, Interleukin 1 ß and Tumour necrosis factor α in liver and intestine for both control and treatment. However, the upregulated expression of targeted genes except tumour necrosis factor α was found in head kidney of T1. At the end of the study, GIFT Tilapia when infected with Aeromonas hydrophila showed an improved immune response in T1 and T2 with lesser signs of infection than Control. The findings of the present study affirmed the importance of biofloc technology in triggering the immunomodulatory response of GIFT Tilapia with its upregulated immune gene expression and its role as an antimicrobial agent against Aeromonas hydrophila. This study suggests the adoption of in-situ (T1) based biofloc method to obtain better performance of GIFT Tilapia culture.

Weak correlation of bleeding scores to platelet electron microscopy: A retrospective chart review of pediatric patients with delta-storage pool disorder.

BACKGROUND: Delta granule storage pool deficiency (δ-SPD) is a rare platelet disorder in which a deficiency of platelet granules leads to poor aggregation, resulting in varying clinical bleeding phenotypes. Children with δ-SPD have variable laboratory results, making the proper diagnosis and evaluation controversial. OBJECTIVES: To describe the demographic and laboratory trends of this population and to assess the value of electron microscopy in diagnostic evaluation and its correlation to bleeding symptoms. METHODS: We performed a retrospective review of 109 pediatric patients diagnosed with δ-SPD. We collected demographic information and bleeding scores using a validated bleeding assessment tool. A descriptive and exploratory analysis was performed. RESULTS: The majority of patients were female, with an average age at diagnosis of 11.61 years. Females were diagnosed at a significantly older age presenting most often with menorrhagia, while males presented most commonly with epistaxis. The majority showed normal lumiaggregometry, the mean platelet electron microscopy (PEM) value was 2.37, and the mean bleeding score was 6. Bleeding assessment tool and PEM had a significantly weak correlation. CONCLUSIONS: Patients with more dense granules per platelet had higher bleeding scores than those with fewer dense granules per platelet. The current body of evidence does not favor the use of PEM in routine clinical practice, and results are difficult to interpret. In patients with severe mucocutaneous bleeding symptoms and normal platelet aggregation studies, consideration should be given to an alternative diagnosis and further evaluation is warranted.