# Examining the Effects: Lack of Impact by Endolysin and Phage Treatment on Rotifer and Larvae Microbiota

## TL;DR

This study shows that phage and phage-derived enzymes used to control harmful bacteria in aquaculture do not significantly disrupt the microbiota of water, rotifers, or zebrafish larvae.

## Contribution

The study demonstrates the microbiota-safe application of phage and lysin under hatchery-relevant conditions.

## Key findings

- Phage and lysin treatments preserved microbial richness and diversity across all tested matrices.
- RNA-based analyses revealed treatment effects in rotifer and water microbiota not seen in DNA data.
- Zebrafish larvae microbiota structure remained largely unaffected by the treatments.

## Abstract

Background: Bacteriophages and phage-derived lytic enzymes are increasingly considered to be targeted antimicrobial tools in aquaculture; however, their compatibility with non-target microbial communities under hatchery-relevant conditions remains insufficiently characterized. Objectives This study evaluates the impact of a lytic phage (CH20) and a phage-derived lysin (LysVp1), applied under previously validated conditions for rapid Vibrio control, on the microbiota associated with seawater, rotifers, and zebrafish larvae challenged with Vibrio alginolyticus GV09. Methods: Treatments were independently applied to each biological matrix using short exposure times representative of hatchery practices, intentionally capturing the critical window during which microbial transfer from live feed to larvae occurs. Microbial communities were analyzed using 16S rRNA gene sequencing, with DNA- and RNA-derived datasets evaluated separately. Results: Alpha diversity indices were compared using appropriate statistical tests, while beta diversity was assessed using Aitchison distance, PERMANOVA, and dispersion analyses, and differential abundance was evaluated using ANCOM-BC2. Alpha diversity metrics showed no significant differences among treatments across all matrices, indicating the preservation of microbial richness and diversity. Beta diversity patterns differed according to the nucleic acid source, with RNA-based analyses revealing treatment-associated shifts in rotifer and water microbiota that were not consistently detected at the DNA level. In zebrafish larvae, neither phage nor lysin treatment significantly altered overall community structure, although dispersion effects reflected limitations related to sample size. Conclusions: Overall, these results indicate that phage CH20 and lysin LysVp1 exert minimal impact on alpha diversity and limited, context-dependent effects on microbial community structure, supporting their microbiota-safe potential for aquaculture applications.