Microbial Associations
The four metagenomic bins identified using Autometa match to
the bacterial genera Spiroplasma, Acinetobacter, Enterobacter,and Rhodococcus. A BUSCO analysis to confirm completeness
indicated that the Acinetobacter, Enterobacter, andRhodococcus genomes were not high quality (3.2%, 0%, and 0%
complete, respectively) to continue downstream analysis. The draftSpiroplasma sp. NR genome contained one 1,414,835bp genomic
contig and a 28,794bp plasmid, for a total length of 1,443,729bp, an
average GC content of 26.08%, 2,085 open reading frames, and 29 tRNA
encoding genes. The BUSCO analysis of 151 markers conserved among
Mollicutes indicated that the S. sp. NR contained 148 complete
and single-copy BUSCOs (98%), one fragmented BUSCO (0.7%), and two
missing BUSCOs (1.3%). A CheckM analysis with 228 markers in
the Mollicutes lineage found similar quality, with 97.12% of the genome
being complete.
To determine the relationship of S. sp. NR to otherSpiroplasma species, we generated a ML tree for the16S rRNA gene
from 38 Spiroplasma isolates, spanning the known major clades ofSpiroplasma (Figure 3A ). Spiroplasma sp. NR and
three isolates from Harpalus pensylvanicus (Coleoptera:
Carabidae) form a monophyletic group within the lineage ofSpiroplasma associated with insects. Annotation with KEGG
indicates that S. sp. NR can import and phosphorylate sugars via
a phosphoenolpyruvate phosphotransferase system and can use G6P and
fructose-6P through a complete glycolytic pathway and near-complete
pentose phosphate pathway (Figure 3B; Figure S8 ). Similar to
other Spiroplasma species, S. sp. NR is predicted to
produce lactate, acetate, and propanoate. Spiroplasma sp. NR is
capable of oxidative phosphorylation as it encodes an F-type ATPase, and
it contains most of the genes (SecD/F, SecY, SecG, YidC, SecA, FtsY,
ffh) that encode the preprotein translocation Sec-SRP system, although
its genome is missing the gene encoding the essential stabilizing SecE
component. Notably, S. sp. NR does not have the amino acid
biosynthesis pathways or genes for the citric acid cycle, which has been
observed in other Spiroplasma species. Annotations using
antiSMASH and CRISPrfinder indicated the absence of
biosynthetic gene clusters and CRISPR regions. No RIPs were detected inS. sp. NR, but there is evidence of loci similar to the
male-killing gene, spaid . A total of 23 ORFs exhibited
significant alignment (E-value <1E-05) to spaid , but
only one ORF remained as a significant alignment when the
TrEMBL database was used. This ORF alignment has only 10%
query coverage and 49.57% identity to spaid . Using a HMM based
approach, four ORFs significantly aligned to the ankyrin-repeat HMM
marker and one ORF significantly aligned to the Ovarian Tumour-like
deubiquitinase domain (OTU) HMM marker, with no overlap between the
ORFs. Both markers are characteristic of spaid. Finally, analysis
of RNAseq data from N. riversi revealed the presence of expressedSpiroplasma reads, supporting transcriptional activity ofSpiroplasma in the egg, larval, and adult life stages, for both
the adult female and male (Table S12 ).