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.

Thelytokous parthenogenesis in which diploid females are produced from unfertilized eggs, was recently reported for some ant species. Here, we document thelytokous reproduction by queens in the polygynous species Pyramica membranifera. Queens that emerged in the laboratory were kept with or without workers under laboratory conditions. Independent colony founding was successful for a few queens if prey was provided. All artificial colonies, which started with a newly emerged queen and workers produced new workers and some of the colonies also produced female sexuals. Some of the female sexuals shed their wings in the laboratory and started formation of new polygynous colonies. Workers had no ovaries and thus, were obligatorily sterile.

We here show an example of morphological novelties, which have evolved from insect wings into the specific structures controlling social behaviour in an ant species. Most ant colonies consist of winged queen(s) and wingless workers. In the queenless ponerine ant Diacamma sp. from Japan, however, all female workers have a pair of small thoracic appendages, called "gemmae", which are homologous to the forewings and acts as an organ regulating altruism expression. Most workers, whose gemmae are clipped off by other colony members, become nonreproductive helpers, while only a single individual with complete gemmae becomes functionally reproductive. We examined histologically the development of gemmae, and compared it with that of functional wings in males. Female larvae had well-developed wing discs for both fore- and hindwings. At pupation, however, the wing discs started to evaginate and later degenerate. The hindwing discs completely degenerated, while the degeneration of forewing discs was incomplete, leading to the formation of gemmae. The degeneration process involved apoptotic cell death as confirmed by TUNEL assay. In addition, glandular cells differentiated from the epithelial cells of the forewing buds after completion of pupation. The mechanism of developmental transition from wing to gemma can be regarded as an evolutionary gain of new function, which can be seen in insect appendages and vertebrate limbs.

Gotoh, A., Billen, J., Tsuji, K., Sasaki, T. and Ito, F. 2011. Histological study of the spermatheca in three thelytokous parthenogenetic ant species, Pristomyrmex punctatus, Pyramica membranifera and Monomorium triviale (Hymenoptera: Formicidae). —Acta Zoologica (Stockholm) 00:1–8. The evolution of obligate parthenogenesis may induce the degeneration of female mating ability and subsequently affect the morphology of the female reproductive organs related to mating and/or sperm storage. Here, we investigated the size and structure of the sperm storage organ, the spermatheca, in three thelytokous parthenogenetic myrmicine ant species, Pristomyrmex punctatus, Pyramica membranifera and Monomorium triviale, and compared it with that of their related sexually reproducing species. So far, mated individuals have never been found in these three species, which appears to be in line with their parthenogenetic status. Although the spermatheca appears to be useless in these species, we could not find any evidence on the degeneration in size and morphology of their spermathecae. The spermathecal reservoir still has the columnar hilar epithelium, which is one of the major features for a functional spermatheca in ants.

Reproductive division of labor is one of the crucial features in social insects, however, the developmental mechanisms leading to modifications in the reproductive apparatus of workers are still not very clear. Ants show a remarkable diversity in the morphological specialization of the worker's reproductive apparatus, that allows to distinguish four types, type 1: workers that have ovaries and a functional spermatheca, and that reproduce like queens, type 2: workers have ovaries and a vestigial spermatheca, type 3: workers have ovaries but no spermatheca, and type 4: workers lost both ovaries and spermatheca. We investigated morphogenesis of the worker spermatheca in 28 ant species by histological examination. In workers of type 1, the morphogenesis of the spermatheca is very similar to that in ant queens. In type 2, the spermathecal disc also differentiates, however, the development is interrupted and remains vestigial. In types 3 and 4, the absence of the spermatheca in the adult phase is caused by a degeneration after initial formation of the spermathecal disc or by a complete lack of the spermathecal discs. The timing of these interruption and degeneration events varies among species. The species exhibiting an earlier interrupting point of spermatheca formation in workers have a larger queen-worker dimorphism, that seems to be independent from ant phylogeny.