Development of microbial enrichments for simultaneous removal of sulfur and nitrogenous metabolites in saline water aquaculture.

AIM: The aim of the study was to develop microbial enrichments from the nitrifying microbial consortia and the environment for simultaneous removal of ammonia, nitrate, and sulfide in aquaculture systems at varied salinities. METHODS AND RESULTS: Sulfur and nitrogen metabolites are the major factors affecting the farmed aquatic animal species and deteriorate the receiving environments causing ecological damage. The present study reports the development of microbial enrichments from the nitrifying microbial consortia and the environment. The enrichments used thiosulfate or thiocyanate as an energy source and simultaneously removed sulfur, ammonia, and nitrite in spiked medium (125 mg/l ammonia; 145 mg/l nitrite). Further, the microbes in the enrichments could grow up to 30 g/l salinity. Metagenomic studies revealed limited microbial diversity suggesting the enrichment of highly specialized taxa, and co-occurrence network analysis showed the formation of three micro-niches with multiple interactions at different taxonomic levels. CONCLUSIONS: The ability of the enrichments to grow in both organic and inorganic medium and simultaneous removal of sulfide, ammonia, and nitrite under varied salinities suggests their potential application in sulfur, nitrogen, and organic matter-rich aquaculture pond environments and other industrial effluents.

16 S rRNA gene diversity and gut microbial composition of the Indian white shrimp (Penaeus indicus).

The endemic Indian white shrimp (Penaeus indicus) is an economically important crustacean species, distributed in the Indo-West Pacific region. Knowledge of its gut microbial composition helps in dietary interventions to ensure improved health and production. Here we analyzed V3-V4 hypervariable regions of the 16 S rRNA gene to examine intestinal microbiota in wild and domesticated farmed P. indicus. The study revealed that Proteobacteria, Fusobacteria, Tenericutes, and Bacteroidetes, were the dominant phyla in both the groups although there were differences in relative abundance. The dominant genera in case of the wild group were Photobacterium (29.5 %) followed by Propionigenium (13.9 %), Hypnocyclicus (13.7 %) and Vibrio (11.1 %); while Vibrio (46.5 %), Catenococcus (14 %), Propionigenium (10.3 %) and Photobacterium (8.7 %) were dominant in the farmed group. The results of the study suggest the role of environment on the relative abundance of gut bacteria. This is the first report characterizing gut microbial diversity in P. indicus, which can be used to understand the role of gut microbiota in health, nutrition, reproduction, and growth.

Microbial community profiling of ammonia and nitrite oxidizing bacterial enrichments from brackishwater ecosystems for mitigating nitrogen species.

Nitrogen species such as ammonia and nitrite are considered as major stressors in modern aquaculture practices. We developed enrichments of ammonia oxidising bacteria (AOB) and nitrite oxidising bacteria (NOB) for effective mitigation of nitrogenous wastes in the shrimp culture operations. The objective of this study was to understand the microbial community composition of AOB and NOB enrichments using the V3-V4 region of the 16S rDNA gene by Illumina MiSeq sequencing. The analysis revealed 2948 and 1069 OTUs at 97% similarity index and Shannon alpha diversity index of 7.64 and 4.85 for AOB and NOB enrichments, respectively. Comparative analysis showed that a total of 887 OTUs were common among AOB and NOB enrichments. The AOB and NOB enrichment were dominated by Eubacteria at 96% and 99.7% respectively. Proteobacterial phylum constituted 31.46% (AOB) and 39.75% (NOB) and dominated by α-Proteobacteria (20%) in AOB and γ-Proteobacteria (16%) in NOB. Among the species in AOB enrichment (2,948) two sequences were assigned to ammonia oxidising bacterial group belonging to Nitrosomonas, and Nitrosococcus genera and two belonged to archaeon group comprising Nitrosopumilus and Candidatus Nitrososphaeraea genera. The NOB enrichment was predominated by Nitrospiraceae and Thermodesulfovibrionaceae. Further, the data revealed the presence of heterotrophic bacteria contributing to the process of nitrification and form microcosm with the AOB and NOB. PICRUSt analysis predicted the presence of 24 different nitrogen cycling genes involved in nitrification, denitrification, ammonia and nitrogen transporter family, nitrate reduction and ammonia assimilation. The study confirms the presence of many lesser known nitrifying bacteria along with well characterised nitrifiers.

Vibrio harveyi biofilm as immunostimulant candidate for high-health pacific white shrimp, Penaeus vannamei farming.

The study was to develop Vibrio harveyi biofilm-based novel microbial product and its oral delivery for high health Penaeus vannamei farming. Yield of bacterial biofilm was optimized on chitin substrate (size: <360, 360-850 and 850-1250 µm; concentration: 0.3, 0.6 and 0.9%) in tryptone soy broth (0.15%). The biofilm was characterized by crystal violet assay, SEM and LSCM imaging; protein profiling by SDS-PAGE and LC-ESI-MS/MS. The immune stimulatory effect of the biofilm in yard experiments was evaluated by relative quantification of immune genes using real-time PCR effect on overall improvement on health status under field trials. The highest biofilm yield (6.13 ±â€¯0.2 × 107 cfu/ml) was obtained at 0.6% of <360 µm chitin substrate. The biofilm formation was stabilized by 96 h of incubation at 30 °C. Protein profiling confirmed expression of six additional proteins (SDS-PAGE) and 11 proteins were differentially expressed (LC-ESI-MS/MS) in biofilm cells over free cells of V. harveyi. Oral administration of the biofilm for 48 h confirmed to enhance expression of antimicrobial peptides, penaeidin, crustin and lysozyme in P. vannamei. Further Oral administration of biofilm for two weeks to P. vannamei (1.8 ±â€¯0.13 g) improved the growth (2.66 ±â€¯0.06 g) and survival (84.44 ±â€¯1.82%) compared to control (2.15 ±â€¯0.03 g; 70.94 ±â€¯0.66%) Nursery trials showed a significant reduction in occurrence of anatomical deformities like antenna cut (12.67 ±â€¯0.66%), rostrum cut (4.66 ±â€¯0.87%), and tail rot (3.33 ±â€¯0.88%), compared to animals fed with normal diet which was 24.33 ±â€¯2.72; 14 ±â€¯1.52 and 10.66 ±â€¯1.45% respectively. In vitro and in vivo studies suggest inactivated biofilm cells of V. harveyi on chitin substrate express additional antigenic proteins and when administered orally through feed at regular intervals stimulates immune response and improve growth, survival and health status of shrimp.

Draft Genome Sequence of Vibrio parahaemolyticus Strain VP14, Isolated from a Penaeus vannamei Culture Farm.

Here, we report the draft genome sequence of an isolate of Vibrio parahaemolyticus, VP14, recovered from the gut of Penaeus vannamei shrimp farmed in southern India. The genome of VP14 comprised 5,224,046 bp with a GC content of 45.3% and contained 5,326 genes, including 4,972 coding sequences.

Efficacy of algal Ecklonia cava extract against viral hemorrhagic septicemia virus (VHSV).

The inhibition efficacy of an extract from Ecklonia cava (E. cava) was studied to determine whether the extract and compounds exhibited inhibitory activity against VHSV in the fathead minnow (FHM) cell line and following oral administration to the olive flounder. Based on its low toxicity and effective concentration, the E. cava extract (Ext) and compounds (eckol and phlorofucofuroeckol A) were selected for further analysis. In the plaque reduction assay, simultaneous co-exposure of VHSV to Ext, eckol and phlorofucofuroeckol A showed a higher level of inhibition than the pre- and post-exposure groups. The antiviral activity in the FHM cell line was time-dependent and increased with the exposure time with the virus and Ext or the compounds. In the in vivo experiments, different Ext concentrations were orally administered to the olive flounder. In trial I, the relative percent survival (RPS) following oral administration of 500 and 50 µg/g/day of Ext was 31.25% and 12.50%, respectively. In trial II, the RPS for 1000, 500 and 50 µg/g/day of Ext was 31.57%, 0% and 0%, respectively. In trial III, the RPS after 1 and 2 weeks (1000 µg/g/day) of exposure to Ext was 26.31% and 31.57%, respectively. Oral administration of Ext (1000 µg/g/day) significantly induced inflammatory cytokine responses (IL-1ß, IL-6 and IFN-γ) at 1 and 2 days post-oral administration (dpa). Additionally, IFN-α/ß (7-12 dpa), ISG15 (2, 7 and 10 dpa) and Mx (7-12 dpa) were significantly activated in the olive flounder. In conclusion, we demonstrated an inhibitory ability of the E. cava extract and compounds against VHSV in the FHM cell line. Moreover, oral administration of the E. cava extract to the olive flounder enhanced antiviral immune responses and the efficacy of protection against VHSV, resulting in an anti-viral status in the olive flounder.

Poly (I:C) and imiquimod induced immune responses and their effects on the survival of olive flounder (Paralichthys olivaceus) from viral haemorrhagic septicaemia.

The stimulation of immune genes by polyinosinic:polycytidylic acid (poly (I:C)) and imiquimod in olive flounder (Paralichthys olivaceus) and their role in control of viral haemorrhagic septicaemia virus (VHSV) infection were examined. Poly (I:C) (100 µg/fish) treated olive flounder had very low mortality (5%) post VHSV infection, while the imiquimod treated group had 65% and 85% mortality at a dose of 100 µg/fish and 50 µg/fish, respectively. Though the imiquimod treated group had high mortality, it was lower than the untreated group, which had 90% mortality. In vivo experiments were conducted to determine effect of the two ligands on immune modulation in the head kidney of olive flounder. Poly (I:C) activated the immune genes (TLR-3, TLR-7, MDA-5, LGP-2, IRF-3, IRF-7, IL-1ß type I IFN and Mx) very early, within 1 d post stimulation, faster and stronger than imiquimod. Though Mx levels were enhanced by imiquimod, the host was still susceptible to VHSV. The poly (I:C) treated group had a high immune response at the time of infection and 1 dpi, though it decreased at later stages. The imiquimod treated group and the unstimulated group had a higher immune response to VHSV compared to the poly (I:C) treated group. The nucleoprotein copies of VHSV were very low in the poly (I:C) treated group but interestingly, were high in both untreated and imiquimod treated fish. Thus, host survival from a viral infection does not only depend on the quantity of immune response but also the time of response. Although imiquimod enhanced immune gene expression in olive flounder, a delayed response could be the reason for high mortality to VHS compared with poly (I:C), which induced the immune system effectively and efficiently to protect the host.

Impaired TLR2 and TLR7 response in olive flounder infected with viral haemorrhagic septicaemia virus at host susceptible 15 °C but high at non-susceptible 20 °C.

The olive flounder (Paralichthys olivaceus) is susceptible to viral haemorrhagic septicaemia virus (VHSV) at 15 °C but no mortality is observed at 20 °C even though the virus can grow profusely in vitro. Thus, we designed an experiment to better understand the immune response of olive flounder to VHSV when the host reared at 15 °C or 20 °C and infected with the virus. Olive flounder (18-22 g) reared at 15 ± 0.5 °C or 20 ± 0.5 °C were intra-peritoneally injected with VHSV (10(7.8) TCID50/fish) and sampled (n = 5) for head kidney at 3, 6, 12 hpi, 1, 2, 4 and 7 dpi; similarly, mock injected control groups (n = 5). Real-time PCR-based absolute quantification method was followed to quantify copies of VHSV gRNA and mRNA, while the immune gene expression of the olive flounder was quantified relative to internal control, ß-actin. Viral infection resulted in a cumulative mortality of 24% in olive flounder reared at 15 °C, but no mortality was recorded in the 20 °C group or control groups. TLR2 and TLR7 expression at 15 °C was enhanced during early-infection phase (3-6 hpi) and recovery phase (4-7 dpi) when viral transcription was low, but expression was significantly reduced (12 hpi-1 dpi) at peak-infection period. However, the 20 °C group showed low viral transcription and expressed high level of TLR7 and a moderately higher unchanged level of TLR2. In both the groups, TLR3 expression was unaffected. Nevertheless, expression of MDA5 and LGP2 increased significantly irrespective of rearing temperature at the time of peak infection, hence at 15 °C VHSV down-regulated expression of TLR2 and TLR7 but not MDA5 or LGP2. Comparatively, at 15 °C IRF3 expressed high but IRF7 remained very low. Interleukins (IL-1ß, IL-6 and IL-8) were significantly elevated in both the groups, but quicker and for a shorter period at 20 °C. In the 15 °C group, an extended period of expression of ILs could create an unsafe prolonged inflammatory condition. The olive flounders expressed high ISGs at 15 °C but were lagging by 12 h than 20 °C group. Based on these findings, we concluded that viral-mediated disruption of TLR2 and TLR7 expression in the 15 °C group could have delayed the host interferon response and provided a window for high viral growth. However, an effective host immune response at 20 °C contained VHSV from reaching the critical limit.

Temperature-dependent viral replication and antiviral apoptotic response in viral haemorrhagic septicaemia virus (VHSV)-infected olive flounder (Paralichthys olivaceus).

The olive flounder (Paralichthys olivaceus) shows a high rate of mortality to viral haemorrhagic septicaemia virus (VHSV) in the winter and spring but has zero mortality over 20 °C. In this experiment, we studied the effect of rearing temperature on viral replication, viral transcription and antiviral apoptotic immune response in VHSV-infected olive flounder by real-time polymerase chain reaction. Olive flounder were given intra-peritoneal injections of VHSV (10(7.8) TCID(50)/ml) and were reared at 15 °C or 20 °C. Five fish were randomly sampled for head kidney at 3, 6 and 12 h post-infection (hpi) and 1, 2, 4 and 7 days post-infection (dpi). Total RNA extracted from the tissue was reverse transcribed and used as template for real-time PCR. In the 15 °C group, the number of viral gRNA copies peaked after 2 dpi and remained high through 7 dpi, while in the 20 °C group, the copy number was at the highest at 1 dpi but drastically declined at later stages. Viral mRNA levels in the 15 °C group gradually increased starting at 3 hpi to reach their maximum value at 12 hpi and remained high until 2 dpi, whereas the other group showed much lower copy numbers that were undetectably low at 4 and 7 dpi. Type II IFN expression increased as the viral copies increased and the 20 °C group showed quicker and stronger expression than the 15 °C group. The MHC class I and CD8 expression was high in both the groups at early stage of infection (3-6 hpi) but at later stages (2-7 dpi) in 15 °C group expression reduced below control levels, while they expressed higher to control in 20 °C group. The expression of granzyme in 15 °C fish showed a single peak at 2 dpi, but was consistently expressing in 20 °C fish. Individuals expressed very high levels of perforin expressed very high levels of caspase 3. In 15 °C fish, TNFα, FasL and p53 expressed significantly higher than 20 °C only at initial stages of infection (3-6 hpi). Caspase 3 expression found to be low in 15 °C fish whereas it was significantly elevated in 20 °C group. Interestingly individual fish with high caspase 3 expression contained very low viral RNA. Thus, from our experiment, we can conclude that an effective apoptotic immune response in VHSV-infected olive flounder plays a crucial role in the survival of the host at higher temperatures.

Toll-like receptors and interferon associated immune factors in viral haemorrhagic septicaemia virus-infected olive flounder (Paralichthys olivaceus).

Pattern recognition receptor (PRR) toll-like receptors (TLRs), antiviral agent interferon (IFN) and the effector IFN stimulated genes (ISGs) play pivotal role in antiviral innate immunity of a host. The present in-vivo experiment was conducted to investigate the role of these innate immune factors in early phase as well as during recovery of viral haemorrhagic septicaemia virus (VHSV) infection by quantitative real-time reverse transcriptase polymerase chain reaction. A less lethal VHSV infection was generated in olive flounder (Paralichthys olivaceus) and was sampled at 3, 6, and 12h post infection (hpi), and 1, 2, 4, and 7 days post infection (dpi). At 3 hpi, the VHSV N gene was detected in three out of five fish and all five fish showed a relative fold increase of TLR 2, TLR 7, interleukin 8 (IL 8), IFN regulatory factor 3 (IRF 3), IRF 7, and ISG 15. Viral copies rapidly increased at 12 hpi then remained high until 2 dpi. When viral copy numbers were high, a higher expression of immune genes IL 1ß, IRF 3, IRF 7, Type I IFN, ISG 15 and Mx was observed. Viral copies were drastically reduced in 4 and 7 dpi fish, and also the immune response was considerably reduced but remained elevated, except for ISG 15 which found equal to control in 7 dpi fish. A high degree of correlation was observed between immune genes and viral copy number in each of the sampled fish at 12 hpi. A fish with ascites sampled at 7 dpi displayed high viral copy but under-expressed immune genes except for Mx. When viral copies were high at 1 and 2 dpi, both TLR 2 and TLR 7 were down-regulated, perhaps indicating immune suppression by the virus. The quick and prolonged elevated expression of the immune genes indicates their crucial role in survival of host against VHSV.