Genome Characterization and Infectivity Potential of Vibriophage-ϕLV6 with Lytic Activity against Luminescent Vibrios of Penaeus vannamei Shrimp Aquaculture

Shrimp aquaculture, especially during the hatchery phase, is prone to economic losses due to infections caused by luminescent vibrios. In the wake of antimicrobial resistance (AMR) in bacteria and the food safety requirements of farmed shrimp, aqua culturists are seeking alternatives to antibiotics for shrimp health management, and bacteriophages are fast emerging as natural and bacteria-specific antimicrobial agents. This study analyzed the whole genome of vibriophage-ϕLV6 that showed lytic activity against six luminescent vibrios isolated from the larval tanks of P. vannamei shrimp hatcheries. The Vibriophage-ϕLV6 genome was 79,862 bp long with 48% G+C content and 107 ORFs that coded for 31 predicted protein functions, 75 hypothetical proteins, and a tRNA. Pertinently, the vibriophage-ϕLV6 genome harbored neither AMR determinants nor virulence genes, indicating its suitability for phage therapy. There is a paucity of whole genome-based information on vibriophages that lyse luminescent vibrios, and this study adds pertinent data to the database of V. harveyi infecting phage genomes and, to our knowledge, is the first vibriophage genome report from India. Transmission electron microscopy (TEM) of vibriophage-ϕLV6 revealed an icosahedral head (~73 nm) and a long, flexible tail (~191 nm) suggesting siphovirus morphology. The vibriophage-ϕLV6 phage at a multiplicity of infection (MOI) of 80 inhibited the growth of luminescent V. harveyi at 0.25%, 0.5%, 1%, 1.5%, 2%, 2.5%, and 3% salt gradients. In vivo experiments conducted with post-larvae of shrimp showed that vibriophage-ϕLV6 reduced luminescent vibrio counts and post-larval mortalities in the phage-treated tank compared to the bacteria-challenged tank, suggesting the potentiality of vibriophage-ϕLV6 as a promising candidate in treating luminescent vibriosis in shrimp aquaculture. The vibriophage-ϕLV6 survived for 30 days in salt (NaCl) concentrations ranging from 5 ppt to 50 ppt and was stable at 4 °C for 12 months.


Introduction
Aquaculture, the farming of aquatic animals, significantly contributes to meeting the global demand for animal protein and generates large-scale employment opportunities for millions of people worldwide [1]. Shrimp farming is a major aquaculture activity in several countries, and farmed shrimp have a significant trade value across the globe. However, bacterial infections caused by vibrio species are extremely hazardous for sustainable shrimp aquaculture. Vibrio species such as V. parahaemolyticus, V. harveyi, V. alginolyticus, V. campbelli, V. penaeicida, V. splendidus, V. fluvialis, and V. tubiashii cause infections in aquatic animals. V. harveyi is the most important bacterial pathogen of penaeid shrimp that causes mortalities, especially in larval shrimp, and causes huge losses to shrimp aquaculture [2][3][4][5], which have

Isolation of Luminescent V. harveyi Hosts
Water samples collected from P. vannamei shrimp hatcheries (n = 20) and aquaculture farms (n = 12) were screened for the occurrence of luminescent vibrios by spread plating on nutrient agar supplemented with 3% salt [41] and thiosulfate citrate bile sucrose agar (TCBS agar). The plates were incubated at 28 • C for 18 h and observed in the dark to view the luminescence. The luminescent colonies were isolated and purified by streaking on nutrient agar supplemented with 3% salt. The well-isolated colony was re-streaked and observed for luminescence. The luminescent bacteria were assigned to the Vibrio genus based on the results of growth on TCBS agar, Gram's staining, nitrate reduction, oxidase production, and the Hugh and Leifson glucose oxidation/fermentation test [42] and used as bacterial hosts. The luminescent vibrios were further tested for V. harveyi based on biochemical tests, viz., sugar fermentation (arabinose, cellobiose, dulcitol, galactose, glucose, m-inositol, maltose, mannose, raffinose, rhamnose, salicin, sorbitol, and sucrose), amino-acid decarboxylase/dihydrolase (arginine, ornithine, and lysine), salt tolerance (0%, 0.5%, 1%, 3%, 6%, 8%, and 10% NaCl), amylolytic, proteolytic, and lipolytic DNAase activities, and luminescence production [43]

Isolation of Vibriophage
Water samples from P. vannamei shrimp hatcheries (n = 20) and aquaculture farms (n = 12) of Andhra Pradesh, India, and the sewage treatment plant of Visakhapatnam, India, were collected and screened for lytic vibriophages against luminescent vibrios by employing the single-host enrichment method. Briefly, 47.5 mL of water sample was mixed with 12.5 mL of overnight culture of luminescent V. harveyi-LV6 as the host strain, which was added to 15 mL of 5×nutrient broth with 3% salt (peptone 5 g L −1 , beef extract 3 g L −1 , NaCl 30 g L −1 ) and incubated for 6 h at 28 ± 2 • C. Post-enrichment, the phage-enriched culture was centrifuged (10,000 rpm for 20 min at 4 • C), filtered through a 0.22 µm sterile syringe filter to remove residual bacterial host cells, and the filtrate was tested for vibriophages by spotting 10 µL of the filtrate on NA + 3% salt plates seeded separately with overnight cultures of each of the luminescent vibriohosts. The appearance of clearance at the spotted area indicated the presence of lytic vibriophages.

Purification and Precipitation of Vibriophage
Vibriophage was purified by employing a single-agar method [44], in which 1 mL of phage (filtrate) and 1 mL of luminescent vibrio host (V. harveyi-LV6) were mixed and added to 8 mL of molten and cooled soft nutrient agar supplemented with 3% salt (peptone 5 g L −1 , sodium chloride 30 g L −1 , and agar-agar 8 L −1 ) and finally poured onto a sterile petri plate. The plates were incubated at 28 • C ± 2 • C for 8 h to obtain plaques. The phage filtrate was serially diluted in SM buffer and analyzed separately. The phage titer is expressed as pfu mL −1 and calculated using the following formula: Number of phages (pfu mL −1 ) = Total number of plaques × Dilution factor (1) The isolated plaques were picked, and the process was repeated three times to obtain purified vibriophage with consistent plaque morphology. Phage precipitation was performed by treating purified vibriophage (25 mL) with polyethylene glycol (10% w/v PEG 8000 and 1 M NaCl) at 4 • C for 1 h, followed by overnight incubation at −20 • C for 24 h and centrifugation at 10,000 rpm for 20 min at 4 • C [45,46]. The pellet was resuspended in SM buffer (100 mM NaCl, 8 mM MgSO 4 .7H 2 O, 50 mM Tris-Cl, pH 7.5), which constituted the purified and enriched vibriophage, and stored at 4 • C for downstream analysis.

TEM Morphology of Vibriophage-φLV6
For morphological analysis, 10 µL of purified and enriched vibriophage (~10 8 pfu mL −1 ) was loaded on a 200-mesh copper grid, stained with Uranyless 22409, and examined under 15,000× nm magnification at an accelerated voltage of 120 kV using a transmission electron microscope (JEOL Japan) at the National Institute of Animal Biotechnology, Hyderabad, India.

DNA Extraction and Whole Genome Sequencing of Vibriophage-φLV6
Vibriophage-φLV6 DNA was extracted and purified with Qiagen's DNeasy Blood & Tissue Kit [47]. Initially, PEG-precipitated vibriophage (500 µL) was treated with 1. 25 µL DNase and RNase (20 mg mL −1 ) and incubated at 37 • C for 1 h; then treated with 1.25 µL proteinase (20 mg mL −1 ) and 25 µL of 10% SDS and incubated at 60 • C for 1 h. The vibriophage-φLV6 DNA was extracted as per the kit manufacturer's instructions and finally suspended in TE buffer. The quality and concentration of the extracted DNA were assessed using the Qubit ® dsDNA HS Assay Kit, and the integrity of the DNA was determined by electrophoresis on 1% agarose gel. The whole genome sequencing libraries were prepared using the NEBNext ® Ultra TM II FS DNA Library Prep Kit for Illumina at ClevergeneBiocorp Private Limited, Bangalore, India. The QC-passed library was diluted to 2 nM and sequenced on the Illumina HiSeq 4000. The high-quality reads were used to assemble the genomes using the HGA genome assembler [48].
Multiple phage genomes belonging to siphoviruses were compared and visualized using BRIG (Blast Ring Image Generator) with default settings. Comparative genome analysis was performed using ViPTree [52]. The phage genome was categorized into structural modules, DNA metabolism modules, packaging modules, lysis modules, hypothetical modules, and additional functional modules [31,53,54]. Data pertaining to the major capsid protein (n = 16) terminase large subunit (n = 18) of vibriophages and other phages related to vibriophage-φLV6 were downloaded from the NCBI database and aligned using the MUS-CLE algorithm. Phylogenetic analysis was performed using MEGA 10.0.5 software [55] based on the major capsid protein and terminase large subunit using the neighbor-joining method with robust 1000 bootstrap replicates. The genome map of vibriophage-φLV6 was drawn using Proksee (https://proksee.ca/ (accessed on 2 December 2022)).

In Vitro Determination of Optimum Multiplicity of Infection (MOI) for Determining Phage Infectivity Potential
The multiplicity of infection, i.e., the ratio of the number of vibrophage-φLV6 required to lyse luminescent V. harveyi, was determined employing the 2-step microtiter plate assay [56]. Briefly, in the 2-step microtiter assay, a broad range of MOIs (ranging from MOI-0.0001 to MOI-10000) were initially tested for their ability to inhibit the growth of target bacteria. A narrow range of effective MOIs from the first step was selected to determine the optimum MOI in the second step. The optimum MOI, i.e., the lowest number of phages required to inhibit the growth of the target bacteria, was determined in the second step. The optimum MOI of vibriophage-φLV6 against the luminescent V. harveyi-LV6 host was 79 and was previously determined [56]. On similar lines, the optimum MOIs of vibriophage-φLV6 against five other luminescent vibrio hosts, viz., LV36, LV38, LV40, LV44, and LV45, isolated from shrimp hatcheries, were determined. Glass tanks filled with 27 ppt seawater (10 L) and P. vannamei post-larvae of PL-11 size (n = 250) per tank were used for the in vivo study. Tank-1 (control) contained only shrimp post-larvae; Tank-2 (bacteria control) was spiked with luminescent V. harveyi-LV6 at an 8.6 × 10 6 cfu mL −1 concentration. Tank-3 (phage control) was inoculated with vibriophage-φLV6 (8.0 × 10 6 pfu mL −1 ). Tank-4 (treatment tank) was spiked with luminescent V. harveyi-LV6 and simultaneously treated with vibriophage-φLV6 at an MOI of 80 (80 pfu phage to 1 cfu bacteria). All the tanks were kept at ambient temperature An in vivo experiment was conducted in glass tanks containing 25 ppt salinity sea water (1 L) and P. vannamei post-larvae of PL-3 size (n = 100) per tank. The bacteria control tanks were spiked with six luminescent Vibrio spp. isolates (LV36, LV38, LV40, LV44, LV45, and LV6; 10 9 cfu mL −1 ). The vibriophage-treatment tanks were spiked with six luminescent vibrio bacteria at a concentration of 10 9 cfu mL −1 (each bacteria) and simultaneously treated with vibriophage-φLV6 at pre-determined MOIs (MOI-79 for the LV6 host, MOI-41.5 for the LV40 host, MOI-33.6 for the LV36 host, MOI-29.3 for the LV38 host, MOI-1.5 for the LV45 host, and MOI-0.7 for the LV44 host). The control tanks were spiked with neither bacteria nor bacteriophage. All the tanks were kept at ambient temperature (28-30 • C) under illuminated conditions with continuous aeration. Feed was not provided to the post-larvae during the experiment period. The total vibrio counts and post-larvae survivability were checked for 48 h.

Lytic Ability of Vibriophage-φLV6 under Different Salinity Conditions
In the shrimp aquaculture system of India, the salinity of the water in the shrimp hatcheries is maintained between 25 and 35 ppt (2.5 and 3.5%) but the farming of P. vannamei shrimp in aquaculture farms is carried out at different salinities ranging from 4 ppt to 45 ppt [57]. In order to check the applicability of the vibriophage-φLV6, both in hatcheries and different farming conditions of P. vannamei, a salt gradient experiment on determining the lytic activity of vibriophage was taken up. Tubes containing nutrient broth with different salt concentrations, viz., 0.5%, 1%, 2%, 3%, 4%, and 5%, were inoculated with vibriophage-φLV6 at a 10% level and incubated at 28 ± 2 • C. The tubes were taken out every 3 days for 30 days and checked for the lytic activity of vibriophage-φLV6 by the spotting method.
Three controls, viz., bacterial control (without phage), phage control (without bacteria), and media control (without bacteria and phage), were also introduced in triplicate wells at the same salt gradients. The OD 600 readings were taken at 30 min intervals for 4 h.

Storage Stability of Vibriophage
The vibriophage-φLV6 suspended in SM buffer was stored at 4 • C for one year. Samples were drawn intermittently, and the counts (pfu mL −1 ) of vibriophage-φLV6 were determined by performing the single-agar method [44].

Statistical Analysis
The results of post-larval survivability and vibrio counts in phage-treatment tanks and bacterial-challenged tanks were analyzed statistically employing an unpaired t-test and a chi-square test to test the difference at a 5% level of significance using InVivoStat, Version 4.7 [58].

Isolation of Luminescent Vibrios
A total of 27 luminescent vibrios were isolated from P. vannamei shrimp hatchery water samples that showed characteristic luminescence activity. Luminescent vibrios were not detected in water samples from shrimp aquaculture farms. One luminescent vibrio (LV6) that was isolated from a luminescent vibriosis-infected P.s vannamei post-larvae tank was identified as V. harveyi based on the results of biochemical and morphintorial analysis. The remaining luminescent vibrios were identified to the genus level and categorized as luminescent Vibrio spp. (Figure 1a,b). The vibriophage-ɸLV6 suspended in SM buffer was stored at 4 °C for one year. Samples were drawn intermittently, and the counts (pfu mL −1 ) of vibriophage-ɸLV6 were determined by performing the single-agar method [44].

Statistical Analysis
The results of post-larval survivability and vibrio counts in phage-treatment tanks and bacterial-challenged tanks were analyzed statistically employing an unpaired t-test and a chi-square test to test the difference at a 5% level of significance using InVivoStat, Version 4.7 [58].

Isolation of Luminescent Vibrios
A total of 27 luminescent vibrios were isolated from P. vannamei shrimp hatchery water samples that showed characteristic luminescence activity. Luminescent vibrios were not detected in water samples from shrimp aquaculture farms. One luminescent vibrio (LV6) that was isolated from a luminescent vibriosis-infected P.s vannamei postlarvae tank was identified as V. harveyi based on the results of biochemical and

Isolation and Lytic Spectrum of Vibriophage
A lytic phage named vibriophage-ɸLV6 was isolated from the water of the post-larvae tank of a luminescent vibriosis-infected P. vannamei shrimp hatchery. Vibriophage-ɸLV6 showed a clear lytic zone against luminescent V. harveyi-LV6 ( Figure 2) after single-host enrichment and yielded pinpointed plaques with a diameter of <1 mm on the single-agar method. The vibriophage count after purification, PEG precipitation, and suspension in SM buffer was 3.42 × 10 10 pfu mL −1 . PEG precipitation increased the concentration of the vibriophage 100 times. None of the remaining 19 water samples collected from different shrimp hatchery sites, 20 water samples from shrimp aquaculture farms, and one water sample from a sewage treatment plant revealed the presence of vibriophage against luminescent V. harveyi. The vibriophage-ɸLV6 showed lytic activity against one luminescent V. harveyi (LV6) and five isolates of luminescent Vibrio spp. from shrimp hatcheries, viz., LV36, LV38, LV40, LV44, and LV45.

Isolation and Lytic Spectrum of Vibriophage
A lytic phage named vibriophage-φLV6 was isolated from the water of the post-larvae tank of a luminescent vibriosis-infected P. vannamei shrimp hatchery. Vibriophage-φLV6 showed a clear lytic zone against luminescent V. harveyi-LV6 ( Figure 2) after single-host enrichment and yielded pinpointed plaques with a diameter of <1 mm on the single-agar method. The vibriophage count after purification, PEG precipitation, and suspension in SM buffer was 3.42 × 10 10 pfu mL −1 . PEG precipitation increased the concentration of the vibriophage 100 times. None of the remaining 19 water samples collected from different shrimp hatchery sites, 20 water samples from shrimp aquaculture farms, and one water sample from a sewage treatment plant revealed the presence of vibriophage against luminescent V. harveyi. The vibriophage-φLV6 showed lytic activity against one luminescent V. harveyi (LV6) and five isolates of luminescent Vibrio spp. from shrimp hatcheries, viz., LV36, LV38, LV40, LV44, and LV45.

Vibriophage-ɸLV6 Genome Characterization and Phylogenetic Analysis
The genome size of the vibriophage-ɸLV6 was 79,862 bp with a G+C content of The coding sequences (CDS) accounted for 93% of the vibriophage genome (74,178 and coded for proteins with known functions (41%), and hypothetical proteins (52%

TEM Morphology of Vibriophage-φLV6
The morphology of vibriophage-φLV6 observed under transmission electron microscopy ( Figure 3) revealed that the phage had an icosahedral head (~73 nm) and a long filamentous non-contractile tail (~191 nm), respectively, suggesting that the vibriophage-φLV6 might belong to siphoviruses.

Vibriophage-ɸLV6 Genome Characterization and Phylogenetic Analysis
The genome size of the vibriophage-ɸLV6 was 79,862 bp with a G+C content of The coding sequences (CDS) accounted for 93% of the vibriophage genome (74,178 and coded for proteins with known functions (41%), and hypothetical proteins (52% total of 107 putative ORFs were predicted in the genome of vibriophage-ɸLV6 with g

Vibriophage-φLV6 Genome Characterization and Phylogenetic Analysis
The genome size of the vibriophage-φLV6 was 79,862 bp with a G+C content of 48%. The coding sequences (CDS) accounted for 93% of the vibriophage genome ( and coded for proteins with known functions (41%), and hypothetical proteins (52%). A total of 107 putative ORFs were predicted in the genome of vibriophage-φLV6 with gene lengths ranging between 129 and 4146 bases. The details of the ORFs and the predicted proteins are presented in Figure 4 and Table 1. Out of 107 ORFs, 31 of the ORFs in the vibriophage-φLV6 genome had predicted functions, and 75 ORFs had currently unknown functions, i.e., hypothetical proteins. One tRNA coding for lysine was found in the genome of vibriophage-φLV6. No antibiotic resistance genes or bacterial virulence genes were found in the genome of vibriophage-φLV6. functions, i.e., hypothetical proteins. One tRNA coding for lysine was found in the genome of vibriophage-ɸLV6. No antibiotic resistance genes or bacterial virulence genes were found in the genome of vibriophage-ɸLV6.    Phylogenetic trees were constructed targeting single gene analysis of major capsid protein ( Figure 5a) and terminase large subunit protein (Figure 5b). Results from the two phylogenetic trees indicated that vibriophage-φLV6 is closely related to vibriophage-V-YDF132, vibriophage-VH2_2019, and vibriophage-vB_VpS_PG28.

Whole Genome Comparison
The whole genome sequences of the vibriophages used in phylogenetic tree construction of terminase subunit and major capsid protein were obtained from NCBI and compared with the genome of vibriophage-ɸLV6. The results were similar to the phylogenetic analysis, i.e., vibriophage-V-YDF132 (isolated from a group fish farm in China and active against V. harveyi), vibriophage-VH2_2019 (isolated from Hatches Creek, USA, and active against V. natriegens), and vibriophage-vB_VpS_PG28 (isolated from sewage at a seafood market in China and active against V. parahaemolyticus) shared more similarity with vibriophage-ɸLV6 compared to other phages (Figure 7).

Whole Genome Comparison
The whole genome sequences of the vibriophages used in phylogenetic tree construction of terminase subunit and major capsid protein were obtained from NCBI and compared with the genome of vibriophage-φLV6. The results were similar to the phylogenetic analysis, i.e., vibriophage-V-YDF132 (isolated from a group fish farm in China and active against V. harveyi), vibriophage-VH2_2019 (isolated from Hatches Creek, USA, and active against V. natriegens), and vibriophage-vB_VpS_PG28 (isolated from sewage at a seafood market in China and active against V. parahaemolyticus) shared more similarity with vibriophage-φLV6 compared to other phages (Figure 7).
The vibriophage-ɸLV6 genome was negative for antimicrobial-resistance determinants (ARGs), integrase, and bacterial virulence genes, making it a suitable candidate for in vivo phage applications to control luminescent vibriosis in shrimp aquaculture.   Table 2). The G+C content of vibriophage-φLV6 (48%) was comparable to that of the previously reported vibriophages (43.6% to 47.6%). The ORFs with predicted function were relatively higher in vibriophage-φLV6 (138) compared to other vibriophages with similar genome sizes (121-127 ORFs). The jumbo-sized vibriophage-vB_VhaM_pir03, with a genome size larger than 200 kb, had 137 ORFs. Moreover, tRNA was reported only in vibriophage-φLV6 that encoded for lysine.
The vibriophage-φLV6 genome was negative for antimicrobial-resistance determinants (ARGs), integrase, and bacterial virulence genes, making it a suitable candidate for in vivo phage applications to control luminescent vibriosis in shrimp aquaculture.

Vibriophage-φLV6 Proteome
Vibriophage-φLV6 has six ORFs, i.e., ORF 3, ORF 6, ORF 7, ORF 8, ORF 9, and ORF 11, predicted for functions related to the structural composition of the phage. Vibriophage-φLV6 has dedicated machinery for 7 ORFs, i.e., ORF 62, ORF 68, ORF 72, ORF 86, ORF 92, ORF 94, and ORF 96, involved in phage DNA metabolism. ORF 82, which encodes for the terminase large subunit, plays a vital role in viral genome packaging. The terminase large subunit has two subunits: a smaller subunit involved in viral DNA packaging and a larger subunit involved in ATPase and endonuclease activities.

Determination of Optimum Multiplicity of Infection
The optimization of MOI is important to determine the lowest number of phages required to inhibit the growth of a specific bacteria. The luminescent V. harveyi isolates that were susceptible to vibriophage-φLV6 in the spot assay were selected for MOI determination. The optimum MOI for the isolate LV6 was previously reported by us at MOI-79 [56]. Similarly, the optimum MOI of vibriophage-φLV6 to inhibit the growth of the remaining five susceptible luminescent Vibrio spp. isolates, viz., LV36, LV38, LV40, LV44, and LV45, was determined using the two-step microtiter plate assay. In the two-step microtiter assay, a narrow range of MOIs were selected in the first step, and the optimum MOI was determined in the second step (Figure 9a,b). The narrow range of MOIs out of the nine MOIs (0.0001 to 10,000) that were selected in the first step ranged between 6.725 and 672.5 for LV36; 5.854 and 585.4 for LV38; 8.3 and 83 for LV40; 0.01 to 1.42 for LV44; and 0.03 to 3 for LV45. The optimum MOIs of vibriophage-φLV6 obtained in the second step were 41.5 for LV40, 33.6 for LV36, 29.3 for LV38, 1.5 for LV45, and 0.7 for LV44. These optimized MOIs were applied for challenge studies in glass tanks. 11, predicted for functions related to the structural composition of Vibriophage-ɸLV6 has dedicated machinery for 7 ORFs, i.e., ORF 62, ORF 68, O 86, ORF 92, ORF 94, and ORF 96, involved in phage DNA metabolism. ORF encodes for the terminase large subunit, plays a vital role in viral genome pac terminase large subunit has two subunits: a smaller subunit involved in packaging and a larger subunit involved in ATPase and endonuclease activiti

Determination of Optimum Multiplicity of Infection
The optimization of MOI is important to determine the lowest numbe required to inhibit the growth of a specific bacteria. The luminescent V. harv that were susceptible to vibriophage-ɸLV6 in the spot assay were selecte determination. The optimum MOI for the isolate LV6 was previously report MOI-79 [56]. Similarly, the optimum MOI of vibriophage-ɸLV6 to inhibit the the remaining five susceptible luminescent Vibrio spp. isolates, viz., LV36, L LV44, and LV45, was determined using the two-step microtiter plate assay. step microtiter assay, a narrow range of MOIs were selected in the first st optimum MOI was determined in the second step (Figure 9a,b). The narrow ran out of the nine MOIs (0.0001 to 10,000) that were selected in the first step rang 6.725 and 672.5 for LV36; 5.854 and 585.4 for LV38; 8.3 and 83 for LV40; 0.01 LV44; and 0.03 to 3 for LV45. The optimum MOIs of vibriophage-ɸLV6 obta second step were 41.5 for LV40, 33.6 for LV36, 29.3 for LV38, 1.5 for LV45, and 0 These optimized MOIs were applied for challenge studies in glass tanks.

Challenge Studies to Test the In Vivo Lytic Ability of Vibriophage-ɸLV6
Vibriophage-ɸLV6 was employed at an optimized MOI to control the luminescent V. harveyi and luminescent Vibrio spp. in tanks containing postvannamei shrimp that were spiked with either single or multiple isolates of l Vibrio spp.

Effectiveness of Vibriophage-ɸLV6 Treatment at an Optimized MOI of 8 Single Luminescent V. harveyi-LV6
There was a continuous increase in OD600 in the bacteria-control tank uncontrolled proliferation of bacteria, whereas the vibriophage-treated tank negligible increase in OD600 until 6 h of exposure. The luminescent bacteria cou high in the bacterial-control tank (1.02 × 10 8 cfu mL −1 ) after 4 h of exposure; h luminescent bacteria count was less than 300 cfu mL −1 in vibriophage-ɸLV6 tr ( Figure 10). The vibriophage count ranged between 3.6 × 10 9 and 3.7 × 10 9 phage-treated tanks, whereas no vibriophage was detected in bacteria-con There was a continuous increase in the OD600 values of the water in the bact tank, indicating uncontrolled proliferation of bacteria. However, the vibriop treated tank showed a negligible increase in OD600 values, lower luminesce counts, but very high counts of vibriophage and higher post-larvae survival. of the tank (10 L) indicated the effectiveness of employing the vibriopha controlling the growth of luminescent V. harveyi, and the optimum MOI det the two-step microtiter plate method was sufficient to control the growth for 6

Challenge Studies to Test the In Vivo Lytic Ability of Vibriophage-φLV6
Vibriophage-φLV6 was employed at an optimized MOI to control the growth of luminescent V. harveyi and luminescent Vibrio spp. in tanks containing post-larvae of P. vannamei shrimp that were spiked with either single or multiple isolates of luminescent Vibrio spp.
3.8.1. Effectiveness of Vibriophage-φLV6 Treatment at an Optimized MOI of 80 against a Single Luminescent V. harveyi-LV6 There was a continuous increase in OD 600 in the bacteria-control tank, indicating uncontrolled proliferation of bacteria, whereas the vibriophage-treated tanks showed a negligible increase in OD 600 until 6 h of exposure. The luminescent bacteria count was very high in the bacterial-control tank (1.02 × 10 8 cfu mL −1 ) after 4 h of exposure; however, the luminescent bacteria count was less than 300 cfu mL −1 in vibriophage-φLV6 treated tanks ( Figure 10). The vibriophage count ranged between 3.6 × 10 9 and 3.7 × 10 9 pfu mL −1 in phage-treated tanks, whereas no vibriophage was detected in bacteria-control tanks. There was a continuous increase in the OD 600 values of the water in the bacteria-control tank, indicating uncontrolled proliferation of bacteria. However, the vibriophage-φLV6 treated tank showed a negligible increase in OD 600 values, lower luminescent bacterial counts, but very high counts of vibriophage and higher post-larvae survival. The results of the tank (10 L) indicated the effectiveness of employing the vibriophage-φLV6 in controlling the growth of luminescent V. harveyi, and the optimum MOI determined by the two-step microtiter plate method was sufficient to control the growth for 6 h.

Effect of Vibriophage-ɸLV6 on Inhibiting the Growth of Multiple Luminescent Vibrio Hosts (n = 6)
The shrimp post-larvae mortality was higher in bacteria-challenged tanks (37.5% ± 3%) compared to phage-treated tanks (9.5% ± 3%) and control tanks (8% ± 1%). In other words, significantly higher survivability of the shrimp post-larvae was observed in the phage-treated tank ( Figure 11) compared to the bacteria-challenged group (unpaired ttest and chi-square test, p< 0.05). The sucrose non-fermenting vibrio loads were distinctly higher in bacteria-spiked tanks (3,57,100 cfu mL −1 ) compared to vibriophage-treated tanks (1000cfu mL −1 ). Phage activity was detected only in vibriophage-treated tanks but not in control or bacteria-spiked tanks. The results indicate that Vibriophage-ɸLV6 effectively reduced the numbers of multiple luminescent vibrios and reduced the mortality of shrimp post-larvae. Figure 11. Survival of P. vannamei post-larvae in vibriophage-ɸLV6 treated tanks. The shrimp post-larvae mortality was higher in bacteria-challenged tanks (37.5% ± 3%) compared to phage-treated tanks (9.5% ± 3%) and control tanks (8% ± 1%). In other words, significantly higher survivability of the shrimp post-larvae was observed in the phage-treated tank ( Figure 11) compared to the bacteria-challenged group (unpaired t-test and chi-square test, p < 0.05). The sucrose non-fermenting vibrio loads were distinctly higher in bacteria-spiked tanks (357,100 cfu mL −1 ) compared to vibriophage-treated tanks (1000 cfu mL −1 ). Phage activity was detected only in vibriophage-treated tanks but not in control or bacteria-spiked tanks. The results indicate that Vibriophage-φLV6 effectively reduced the numbers of multiple luminescent vibrios and reduced the mortality of shrimp post-larvae. The shrimp post-larvae mortality was higher in bacteria-challenged tanks (37.5% ± 3%) compared to phage-treated tanks (9.5% ± 3%) and control tanks (8% ± 1%). In other words, significantly higher survivability of the shrimp post-larvae was observed in the phage-treated tank ( Figure 11) compared to the bacteria-challenged group (unpaired ttest and chi-square test, p< 0.05). The sucrose non-fermenting vibrio loads were distinctly higher in bacteria-spiked tanks (3,57,100 cfu mL −1 ) compared to vibriophage-treated tanks (1000cfu mL −1 ). Phage activity was detected only in vibriophage-treated tanks but not in control or bacteria-spiked tanks. The results indicate that Vibriophage-ɸLV6 effectively reduced the numbers of multiple luminescent vibrios and reduced the mortality of shrimp post-larvae.

Survivability of Vibriophage under Different Salinity Conditions
The vibriophage-φLV6 survived under different salt conditions of 5 ppt, 10 ppt, 20 ppt, 30 ppt, 40 ppt, and 50 ppt, indicating their applicability in brackish water and marine waters. Vibriophage-φLV6 survived and exhibited its lytic activity for 30 days (maximum period tested) at both 28 • C and 35 • C.
3.10. Stability Testing of Vibriophage-φLV6 Activity against Luminescent V. harveyi Host LV6 at Different Salt Gradients V. harveyi-LV6 (bacteria controls) did not show any growth at 0%, very weak growth at 0.25% salt concentration, and relatively weak growth at 0.5% salt concentration. The growth of V. harveyi-LV6 was optimal at salt concentrations between 1% and 3%, yielding an OD 600 value of~0.3 to 0.4 ( Figure 12). This growth pattern vis-à-vis salt concentration was on expected lines, as V. harveyi is a halophilic organism and salt is integral to its growth [30]. It was pertinent to note that the lytic activity of vibriophage-φLV6 when applied at an MOI of 80 against V. harveyi-LV6 was not affected by the different salt concentrations (0% to 3%) and efficiently halted the growth of V. harveyi-LV6 as evidenced by lower (~0.1) OD 600 that were similar to the media controls and phage controls at the end of 240 min of incubation ( Figure 12). V. harveyi-LV6 (bacteria controls) did not show any growth at 0%, very weak growth at 0.25% salt concentration, and relatively weak growth at 0.5% salt concentration. The growth of V. harveyi-LV6 was optimal at salt concentrations between 1% and 3%, yielding an OD600 value of ~0.3 to 0.4 ( Figure 12). This growth pattern vis-à-vis salt concentration was on expected lines, as V. harveyi is a halophilic organism and salt is integral to its growth [30]. It was pertinent to note that the lytic activity of vibriophage-ɸLV6 when applied at an MOI of 80 against V. harveyi-LV6 was not affected by the different salt concentrations (0% to 3%) and efficiently halted the growth of V. harveyi-LV6 as evidenced by lower (~0.1) OD600 that were similar to the media controls and phage controls at the end of 240 min of incubation ( Figure 12).

Storage Stability of Vibriophage-ɸLV6
Concentrated suspension of vibriophage-ɸLV6 stored at 4 °C did not show any reduction in phage numbers for 9 months of storage as the plaque counts obtained on

Storage Stability of Vibriophage-φLV6
Concentrated suspension of vibriophage-φLV6 stored at 4 • C did not show any reduction in phage numbers for 9 months of storage as the plaque counts obtained on single agar remained almost similar (10 10 pfu mL −1 ), but a slight reduction in the phage numbers (less than one log) was observed at the end of 12 months of storage at 4 • C (Table 3).

Discussion
The use of bacteriophages as therapeutic agents in aquatic animal-health management has gained renewed interest due to the emergence of resistance in pathogenic bacteria towards antibiotics and safety issues related to antibiotic residues in food products. Globally, shrimp farming is increasingly contributing to animal protein requirements, but farm productivity is adversely affected by diseases caused by bacteria of the genus Vibrio. V. harveyi is the major causative agent of luminescent vibriosis in shrimp hatcheries and aquaculture farms. In the present study, a vibriophage, named vibriophage-φLV6, was isolated from the water of a P. vannamei shrimp hatchery. Vibriophage-φLV6 showed in vitro lytic activity against luminescent V. harveyi (LV6) that was isolated from a shrimp hatchery affected with luminescent vibriosis. Vibriophage-φLV6 produced pinpoint plaques on soft-agar plates seeded with the bacterial host. Misol et al. (2020) also reported that vibriophage-vB-VhaM-pir03 produced pin-hole plaques with a diameter of 0.27 ± 0.05 mm on V. harveyi-seeded plates. A 100-fold increase in the concentration of vibriophage-φLV6 was achieved by PEG precipitation, and a similar increase in the concentration of coliphages by PEG precipitation was reported [46]. The host spectrum of vibriophage-φLV6 (six luminescent Vibrios) was relatively lower compared to recently reported V. harveyi bacteriophages. Vibriophage-Virtus, isolated from the water of a fish brood stock section in Crete, Greece, could infect 8 of the 16 strains of V. harveyi [39], and vibriophage-B_VhaM_pir03, isolated from the water of the Port of Piraeus, Greece, showed lytic activity against 31 AMR strains of V. harveyi, V. alginolyticus, V. campbellii, and V. owensii [38]. Most of the phages that were isolated against V. harveyi are reported to be lytic, but two bacteriophages, viz., VHML [4] and VHS1 [60], were found to be temperate.
The genome analysis of vibriophage-φLV6 indicated a genome size of 79.8 kb with a G+C content of 48% that was comparable to the previously reported vibriophages (43.6% to 47.6%). The genome of vibriophage-φLV6 was highly functional. The ORFs with predicted function were relatively higher in vibriophage-φLV6 (138) compared to other vibriophages with similar genome sizes (121-127 ORFs). Even the jumbo-sized vibriophage-vB_VhaM_pir03, with a genome size larger than 200 kb, had only 137 ORFs. Vibriophage-φLV6 harbored replication, regulation, structural, and packaging modules. Though the vibriophage-φLV6 genome did not reveal a lysis module, it carried a sufficient number of genes that encode for early DNA metabolism, which play an essential role in early viral infection similar to vibriophage-V-YDF132 [39]. Vibriophage-φLV6 possesses an ORF7 that encodes for a structural protein (tail tube measure protein). Wu et al. (2020) stated that tail tubular protein, encoded by vibriophage-PcB-1G, plays a critical role in bacterial-cell lysis [32], suggesting that Vibriophage-φLV6 can mediate bacterial-cell lysis through the tail tubular system. Vibriophage-φLV6 possessed a single tRNA that encoded lysine. Many vibriophages, such as Vibrio parahaemolyticus phage-seahorse and KVP-40 carried high numbers of tRNAs, which may provide the phage with a small degree of autonomy when it comes to the translation of its own genes [71]. It is pertinent to note that more than half of the 107 ORFs of vibriophage-φLV6 code for hypothetical proteins whose function is currently unknown. Research efforts are needed to decipher the true function of these hypothetical proteins, as the viral genome machinery is relatively small and has no reason to burden itself with unwanted proteins.
Phylogenetic trees constructed with conserved proteins in the bacteriophage genomes, viz., the major capsid protein and the terminase large subunit protein [72], indicated that vibriophage-φLV6 is closely related to vibriophage-V-YDF132, vibriophage-VH2_2019, and vibriophage-vB_VpS_PG28, asserting that their origin is from a common ancestor. Multiple genome alignments showed that vibriophage-φLv6 had genome sequence similarity of 75% to vibriophage-vB_VpS_PG28, vibriophage-V-YDF132, and vibriophage-VH2_2019. The proteome of vibriophage-φLV6 had a dedicated machinery of ORFs involved in phage DNA metabolism and viral genome packaging. The proteome of vibriophage-φLV6 is similar to the proteome of vibriophage-V-YDF132 [31]. Vibriophage-φLV6 possessed an auxiliary metabolic gene that encodes for pyruvate phosphate dikinase (PPDK), an essential component of the Embden-Meyerhof-Parnas (EMP) glycolytic pathway that was also reported in the vibriophages belonging to siphovirus [69,73]. Genomes of the marine vibriophages isolated from nutrient-deficient environments were abundant in auxiliary metabolic genes compared to those isolated from nutrient-rich environments [59].
Horizontal gene transfer (HGT) occurs between phages and bacterial populations through either generalized or specialized transductions [70,74]. Prior to their therapeutic application, the profiling of vibriophages for genomic traits is an essential pre-requisite to ward off an unwanted increase in the virulence of their hosts [16,59,70]. Vibriophage-φLV6 appears to be a suitable candidate phage for in vivo phage applications to control luminescent vibriosis as it does not carry antimicrobial resistance determinants or bacterial virulence genes. Moreover, it does not have an integrase gene associated with phage lysogeny. The vibriophage-φLV6 produces clear plaques against the host bacteria and reduced the counts of host bacteria in microtiter plate assays, and the reduction in growth was proportional to the number of phages used (i.e., lower bacterial growth at higher MOIs). These results indicate the vibriophage-LV6 was lytic and not lysogenic. Siphophage-VHS was reported to carry a shrimp haemocyte agglutination gene [55]. Siphophage-VHS1 and vibriophage-φLV6 were tested on V. harveyi in Pacific white shrimp (P. vannamei) tanks, while vibriophage-vB_VhaM_pir03 was tested in brine shrimp (Artemia) culture. On the other hand, vibriophage-VB_VhaS_PcB-1G and vibriophage-virtus were tested on V. harveyi in finfish tanks.
Several studies have demonstrated the effectiveness of vibriophages in treating vibriosis in a variety of animal models [17,29,38,39,[75][76][77][78][79]. Karunasagar et al. (2007) reported that vibriophages resulted in higher survival rates (80%) of black tiger shrimp (P. monodon) in hatcheries compared to survival achieved by conventional antibiotic treatment (40%). vB_VhaM_pir03 when applied to Artemia naupli, increased the survival rates of larvae in the phage-treated group to 15-20% in 48 h than the V. harveyi bacterial control group [38]. Droubogiannis, and Katharios, reported 35% survival of gilthead seabream larvae in a single dose of phage application, while Vinod et al. (2006) reported higher survival of P. monodon in double dose (80%) phage treated groups compared to the V. harveyi chal-lenged control group. Misol et al. (2020) observed that during vibriophage treatment, the bacterial population infected at MOI-10 showed the lowest growth. Droubogiannis and Raveearios (2022) reported that the growth of bacteria was inhibited within 2 h of post-infection with vibriophage at an MOI of 100 but took longer time at lower MOIs of 0.1, 1, and 10. Here we report that the application of vibriophage-φLV6 to post-larvae of P. vannamei challenged with V. harveyi at an optimized MOI of 80 resulted in a steep decrease in the luminescent V. harveyi counts in phage-treated tanks compared to counts in bacterial-challenged tanks. Shrimp post-larvae survivability was higher in phage-treated tanks compared to bacteria-spiked tanks. As vibriophage-φLV6 phage suspension resulted in higher post-larval survival, decreased luminescent vibrio loads, and decreased sucrose non-fermenting vibrio counts, it can be considered therapeutic to control luminescent vibriosis in hatcheries and aquaculture systems. However, employing a cocktail of phages can overcome possible phage resistance and simultaneously inhibit several strains of luminescent vibrios. The survivability of vibriophage-φLV6 under different salt conditions (0.5% to 5%) indicates its applicability in shrimp hatcheries and growth ponds.

Conclusions
The present study demonstrates the isolation and genomic characterization of vibriophage-φLV6 and assesses its in vitro and in vivo lytic ability against luminescent Vibrio harveyi. There is a paucity of complete genome data on vibriophages against V. harveyi available in the NCBI database, and in this context, the genomic information of vibriophage-φLV6 adds new information from India. The vibriophage-φLV6 genome codes for many hypothetical proteins, and research efforts are needed to elucidate their function for a complete understanding of the vibriophage. In vitro and in vivo inhibition trials with vibriophage-φLV6, indicated a decrease in luminescent vibrio loads and higher shrimp post-larval survival in phage-treated tanks compared to bacteria-control tanks, suggesting that vibriophage-φLV6 can be a potential alternative to antibiotics in reducing luminescent vibriosis in shrimp aquaculture. Prior to phage therapy becoming a common practice for aquatic animal health management in aquaculture, issues such as mass production of bacteriophages, the designing of phage cocktails for warding off phage resistance, the creation of phage repositories, etc., must be addressed.