4.2 RNA virus study results and discussion
Amplicon sequencing of the dinoRNAV mcp gene produced a total of 7.4 million raw reads across 19 samples representing three potential reservoirs of dinoRNAV diversity across the reef. The 7.4 million raw reads were processed and reduced to 2.8 million merged reads at the expected amplicon length of 420 bases. Merged mcp amplicons dereplicated into 1.1 million unique sequences from which 481 ASVs and 191 aminotypes were identified. The ASV-level results indicated a potential trend of higher dinoRNAV richness in corallivore feces relative to coral colonies (Kruskal-Wallis H test: p-value = 0.14, Figure 5-I). Aminotype results, however, revealed that dinoRNAV richness is significantly higher in corallivore feces, relative toPocillopora coral colonies (Figure 5-II; Kruskal-Wallis H test: p-value = 0.005; Wilcoxon signed-rank test: Pocillopora vs. corallivore, p = 0.01, Pocillopora vs. Acropora , p = 0.04). We interpret that a biological difference in richness likely does exist between dinoRNAV communities in corallivore feces versus those in at least some species of coral holobionts, and this difference may be more readily detected with aminotype-based analyses (as ASV-based analyses may contain more “noise” due to errors arising during RNA virus replication). This use case illustrates the potential benefits of running nucleotide and protein-based amplicon analyses in tandem when testing hypotheses regarding virus community diversity and dynamics. Furthermore, both ASV and aminotypes differed significantly in composition according to dinoRNAV reservoir (anosim with Bray Curtis distances, R=0.99, p<0.01; Figure 5-III, IV), although some overlap (14%, 26 of 190 aminotypes) among dinoRNAV communities was observed (Supplemental Figure S6). Overall, this vAMPirus-based analysis of RNA virus amplicon sequencing data further corroborates that dinoRNAV communities differ across reef reservoirs (Grupstra et al. 2022, Montalvo-Proano et al. 2017, Howe-Kerr et al. 2022, Figure 5) and generates a new hypothesis to be tested in future studies: corallivorous fishes are environmental hotspots of dinoRNAV diversity on reefscapes.