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.