Transcriptome profiling of the spermatheca identifies genes potentially involved in the long-term sperm storage of ant queens

Abstract

Females of social Hymenoptera only mate at the beginning of their adult lives and produce offspring until their death. In most ant species, queens live for over a decade, indicating that ant queens can store large numbers of spermatozoa throughout their long lives. To reveal the prolonged sperm storage mechanisms, we identified enriched genes in the sperm-storage organ (spermatheca) relative to those in body samples in Crematogaster osakensis queens using the RNA-sequencing method. The genes encoding antioxidant enzymes, proteases, and extracellular matrix-related genes, and novel genes that have no similar sequences in the public databases were identified. We also performed differential expression analyses between the virgin and mated spermathecae or between the spermathecae at 1-week and 1-year after mating, to identify genes altered by the mating status or by the sperm storage period, respectively. Gene Ontology enrichment analyses suggested that antioxidant function is enhanced in the spermatheca at 1-week after mating compared with the virgin spermatheca and the spermatheca at 1-year after mating. In situ hybridization analyses of 128 selected contigs revealed that 12 contigs were particular to the spermatheca. These genes have never been reported in the reproductive organs of insect females, suggesting specialized roles in ant spermatheca.

Figure 1

Sample preparation schematic: Differential gene expressions patterns were compared between (a) the spermatheca and the body, (b) the spermatheca from virgin and inseminated queens, and (c) the spermatheca at 1 week and 1 year after mating.

Figure 2

Plots of differentially expressed genes between the spermatheca and the body samples at 1 week (a) and 1 year (b) after mating. Red dots indicate differentially expressed genes with false discovery rates (FDR) of <0.01and |log₂ fold change| ≥ 1.

Figure 3

Plots of differentially expressed genes between the spermathecae of inseminated vs. virgin ant queens (a) and between the spermathecae at 1 week and 1 year after mating (b). Red dots indicate differentially expressed genes (FDR < 0.01 and |log₂ fold change| ≥ 1).

Figure 4

Heat map of selected genes that were regulated by mating status or sperm storage period in the spermathecae. Reads per million (RPM) values of the spermatheca and the body samples were calibrated to Z-scores.

Figure 5

Longitudinal section of the spermatheca stained with hematoxylin and eosin (a) and expression patterns of selected highly expressed contigs in the spermatheca (b–o). Schematic indications of spermatheca morphology are shown in the upper right corner (a). Details of the spermathecal gland are shown in the bottom left corner (n). Scale bar, 100 µm; GC, genital chamber; HE, hilar columnar epithelium of the spermathecal reservoir; R, reservoir; SD, spermathecal duct; SG, spermathecal gland.

Figure 6

Expression patterns of 12 contigs that were only expressed in the spermatheca. Scale bar, 100 µm; HE, hilar columnar epithelium of the spermathecal reservoir; R, reservoir; SD, spermathecal duct; SG, spermathecal gland.