Clownfish, particularly Amphiprion ocellaris, have been a staple in the aquarium trade for decades, with commercial producers supplying clownfish globally. However, producers face challenges that decrease profitability, such as unpredictable hatch rates potentially due to biofouling of spawning substrates or bacterial growth in larval tanks. To address these issues, it is common practice to disinfect eggs prior to hatching. Despite its widespread use, it is currently unclear how disinfection treatments directly impact the microbiome of clownfish eggs.
This study investigates the microbial and fungal communities associated with A. ocellaris eggs collected just prior to hatching, using metagenomic techniques to assess community changes in response to three commonly used disinfection treatments. Thirty eggs were harvested and frozen pre-disinfection at -80°C for baseline analysis. Eggs were divided into four groups: one control and three treated with either 1000 ppm hydrogen peroxide, 250 ppm povidone iodine, or 50 ppm formalin. Disinfectant concentrations were determined from a preliminary range-finding study that optimized survival rates and embryo hatch percentages. Following disinfection, each group was exposed to 12 hours of light in hatching tanks to inhibit hatching, after which 30 eggs from each group were frozen at -80°C for metagenomic analysis.
Microbial and fungal communities were characterized following established methods (Adamovsky et al., 2020). Samples were homogenized, and DNA was extracted using the ZymoBIOMICS DNA Miniprep kit. The 16S rRNA gene (V3-V4 region) and the fungal internal transcribed spacer (ITS) region were amplified using universal primer pairs (341F/806R and ITS1F/2), isolated with magnetic AMPure XP beads, and indexed with the Nextera XT DNA Library Preparation Kit and AccuPrime Taq DNA Polymerase. Libraries were pooled at equal concentrations, quantified using the NEBNext NGS Library Quant Kit, and sequenced on an Illumina MiSeq platform at the Interdisciplinary Center for Biotechnology Research, University of Florida.