A postmeiotically bifurcated roadmap of honeybee spermatogenesis marked by phylogenetically restricted genes.

Haploid males of hymenopteran species produce gametes through an abortive meiosis I followed by meiosis II that can either be symmetric or asymmetric in different species. Thus, one spermatocyte could give rise to two spermatids with either equal or unequal amounts of cytoplasm. It is currently unknown what molecular features accompany these postmeiotic sperm cells especially in species with asymmetric meiosis II such as bees. Here we present testis single-cell RNA sequencing datasets from the honeybee (Apis mellifera) drones of 3 and 14 days after emergence (3d and 14d). We show that, while 3d testes exhibit active, ongoing spermatogenesis, 14d testes only have late-stage spermatids. We identify a postmeiotic bifurcation in the transcriptional roadmap during spermatogenesis, with cells progressing toward the annotated spermatids (SPT) and small spermatids (sSPT), respectively. Despite an overall similarity in their transcriptomic profiles, sSPTs express the fewest genes and the least RNA content among all the sperm cell types. Intriguingly, sSPTs exhibit a relatively high expression level for Hymenoptera-restricted genes and a high mutation load, suggesting that the special meiosis II during spermatogenesis in the honeybee is accompanied by phylogenetically young gene activities. Author summary: Previous histological studies suggest that spermatogenesis is concluded before adult emergence in the honeybee. We complement this histological atlas on testis development and spermatogenesis with a comprehensive dataset from the honeybee adult testes, including single-cell RNA, long-read RNA, bulk mRNA and DNA-seq data. Through the use of gene orthology between the honeybee (Apis mellifera) and Drosophila melanogaster, we show that in newly emerged drones, spermatogenesis is an ongoing process and meiosis II results in two separable clusters of spermatid cells. One of them is identified to contain small spermatids, which are consistent with previous anatomical detection of polar body-like cells in close association with regular spermatids. The characteristic features of small spermatids include an overall low transcription level, high expression of evolutionarily young genes, an enrichment of gene activities regulated by ETS-domain transcription factors, and a high level of mutation load. This work establishes the foundation for future investigations into molecular mechanisms of sperm quality assurance in the honeybee. [ABSTRACT FROM AUTHOR]