Fig. 1.

Extraction and single-cell sequencing of testes from four adult beetles. a and b) Microscope pictures of adult beetle testes dissected. a) Stereomicroscope image of dissected beetle testes in water. b) DAPI stained slide squash of adult beetle testes. I, Distal end of one teste organ. II, Proximal end of same teste organ; and III, Seminary tubule. c) Magnified views of cell populations for GSC (upper panel) and spermatocytes and spermatids (lower panel). d) Diagram of beetle testes with estimated expression levels of previously validated markers; β2-tubulin (TC009035), Rad50 (TC006703), and Enolase (TC011729). Estimated expression patterns were derived through examination of promoter driven GFP expression in Khan et al., (2021). Numbers represent the number of cells at each stage of mitotic/meiotic division. Spermatids are represented with 2⁹ cells even though there is no division due to each cyst encompassing 2 bundles of antiparallel sperm. The distal end of testes is at the bottom of diagram and proximal end is at the top. e) Expression of previously validated markers for spermatogenic staging across all cells. f) Expression of homologs to tissue markers used to stage spermatogenesis in D. melanogaster and A. gambiae (Table 1). Expression of genes displayed as rows of heatmap with cell expression in each column. g) UMAP projection of weighted principal components showing stage specific clusters. h–j) Expression of previously validated stage specific markers in cells displayed as UMAP projection. k) Switch-like mechanics of β₂-tubulin, Rad50, and Enolase for determining pseudotime status of each cell.