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 ).