Establishment of a rapid, cost-effective, and accurate method for assessing insect sperm viability.

High-quality sperm cells are crucial to reproductive success for both males and post-mating females in animals. Sperm viability, defined as the proportion of viable sperm cells, is used as a sperm quality index and this method has provided new insights into research on reproductive strategies. Sperm viability has been assessed by fluorescent staining of sperm cells. However, current staining protocols could potentially underestimate viability due to cell damage caused by cell treatments such as high dye concentration and long time for post-mounting. In this study, we established a method that enables rapid sperm viability assessment, has low sperm cell toxicity, and provides precise results regardless of operator expertise, and cost-effective using sperm cells from an ant, Crematogaster osakensis (Hymenoptera). First, to shorten the time for observation of a sufficient number of sperm cells, the volume per field of view was increased by height elevation between the glass slide and the coverslip, thereby we increased the number of sperm cells in a field of view. Second, to reduce sperm cell toxicity, we optimized the minimum dye concentration and incubation time using acridine orange (AO) and Hoechst in addition to SYBR 14 and propidium iodide (PI), which has been used in most previous studies. We determined the optimal protocol to be 1 µg/mL AO and 150 µM PI without incubation. Besides, we automated counting sperm cells with ImageJ software and combined with manual correction for more accurate results. We employed the improved method for sperm samples from mealworm beetles (Tenebrio molitor) and silkmoths (Bombyx mori). This method, established through our study, will advance research on reproductive strategies, including sperm competition and sperm quality maintenance in females.

Near-anoxia induces immobilization and sustains viability of sperm stored in ant queens.

After copulation, insect females store sperm in a spermatheca for some duration until fertilization. At the beginning of their adult lives, ant queens can preserve numerous viable sperm cells from copulation for over ten years. However, the key factors influencing long-term sperm storage have not been identified. Here we show that the spermathecal environment is nearly anoxic, which induces sperm immobilization. Furthermore, mitochondrial respiratory inhibitors suppress sperm motility, suggesting that sperm immobilization may be caused by a shortage of ATP generated from only glycolysis under near-anoxic conditions. Sperm immobilization is not induced by acidification via glycolytic metabolism because the spermathecal fluid is not acidic. Finally, we show that artificial anoxic conditions rather than aerobic conditions sustain viable sperm cells. Therefore, near-anoxia is a key factor influencing long-term sperm storage in ant queens. The viability of sperm cells under artificial anoxia, however, is lower than that of those dissected immediately from queens. Moreover, the immotile sperm cells under more than 4 h of anoxia do not begin swimming after aerobic exposure, unlike those under anoxic conditions for less than 2 h. This finding indicates that factors other than anoxia are also necessary for long-term sperm preservation.

Morphology of immatures of the thelytokous ant, Monomorium triviale Wheeler (Formicidae: Myrmicinae: Solenopsidini) with descriptions of the extraordinary last-instar queen larvae.

The ant genus Monomorium is one of the most species-rich but taxonomically problematic groups in the hyperdiverse subfamily Myrmicinae. An East Asian species, M. triviale Wheeler, produces both reproductive queens and sterile workers via obligate thelytokous parthenogenesis. Here, we describe the immature forms of M. triviale based on light and scanning electron microscopy observations, with a note on the striking caste dimorphism in the last larval instar. The last-instar queen larvae were easily recognized by their large size, aphaenogastroid body shape, and rows of doorknob-like tubercles on the lateral and dorsal body surface. This type of queen-specific structure has not been found in ants in general, let alone congeneric species found in Japan. In stark contrast to the queen larvae, worker larvae showed a pheidoloid body shape and a body surface similar to other ants. The worker larvae were estimated to have three instars, consistent with previously described congeners. The pupae of both castes had no cocoon, a characteristic commonly described in other Myrmicinae species. In total, the developmental period from egg to adult worker averaged 59 days under 25C. We discuss possible functions of the tubercles of queen larvae based on previous studies.

Journey of sperms from production by males to storage by queens in Crematogaster osakensis (Hymenoptera: Formicidae).

Ants show a unique reproductive system among insects. Males finish sperm production, and their testes degenerate at a young stage. After copulation, spermatozoa are transferred into the queens, who store the received sperm cells throughout their long lifespan without additional mating. In the present study, we investigated the reproductive biology of Crematogaster osakensis from male sexual maturation to sperm transfer, and sperm storage in queens. The sperm production was completed by eclosion and all produced spermatozoa had migrated to the seminal vesicle and the testes shrank until 10 days after eclosion. Sperm were not connected with bundles in mature males. The sperm cells were immobilized in the seminal vesicle. The sperm cells with the spermatophore were ejaculated into the bursa copulatrix and remained immotile during transfer from the bursa copulatrix to the spermathecal reservoir via the spermathecal duct. These findings provide significant insights into the importance of sperm immobilization, which prevents sperm damage and/or production of reactive oxygen species rather than swimming faster competed with rival male spermatozoa to reach into the sperm storage site, even in the polyandrous species, C. osakensis. Immobilization was also observed in the spermathecal reservoir 5 years after mating. This observation suggests that sperm immobilization is one of the important factors for successful long-term sperm storage and maintaining low levels of sperm metabolism.

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

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.

Degeneration patterns of the worker spermatheca during morphogenesis in ants (Hymenoptera: Formicidae).

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.

Thelytokous parthenogenesis by queens in the dacetine ant Pyramica membranifera (Hymenoptera: Formicidae).

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.

Evolution of specialized spermatheca morphology in ant queens: insight from comparative developmental biology between ants and polistine wasps.

In many ant species, the queens can keep spermatozoa alive in their spermatheca for several years, which goes along with unique morphological characteristics of the queen's spermatheca. The relative spermatheca size in ant queens is prominently larger than that in social wasps. Furthermore, the epithelium lining the spermatheca reservoir of ants consists of columnar cells in the hilar region and squamous cells in the distal region, whereas it is formed by columnar cells only in social wasps. To study the evolution of the unique spermatheca morphology in ant queens, we compared the various processes during spermatheca development between two ponerine ant species of the genus Pachycondyla (=Brachyponera) and three polistine wasp species of the genus Polistes. From histological observations, we can define four developmental events in the ant queens: (1) invagination of the spermatheca primordium, (2) the reservoir wall thickness becomes unequal, (3) the reservoir diameter doubles as the lining epithelial cells become flattened except for the hilar region, and (4) the increase in thickness of the reservoir epithelium is limited to the hilar region which doubles in thickness. In polistine wasps, the second and the third developmental events are absent and the entire epithelium of the spermatheca wall becomes thick in the final step. We therefore conclude that for ant queens the second and third steps are crucial for the enlargement of the spermatheca size, and that the second to the fourth steps are crucial for the specialization of the reservoir wall structure.

Comparison of spermatheca morphology between reproductive and non-reproductive females in social wasps.

Social wasps show an obvious evolution of the differentiation in behavior and external size between reproductive and non-reproductive females, with no clear differences in the Stenogastrinae, via overlap in the Polistinae, to clear differences in the Vespinae. In this study, we examined the morphological appearance of the spermatheca in representative species of these three subfamilies. The general anatomical organization of the spermatheca comprises a reservoir, a duct and two spermathecal glands, and is in line with its common structure in other social Hymenoptera. All examined wasp species have a spermathecal reservoir with uniform wall thickness, which is similar to the situation in the bees, but differentiates them from the ants. Within the wasps, the shape of the reservoir, the shape of the spermathecal glands and their attachment site to the spermatheca differs among the Stenogastrinae, Polistinae and Vespinae. The reservoir wall is thick in the Polistinae and Vespinae, while in the Stenogastrinae, it varies from thin in Parischnogaster to thick in Eustenogaster, with an intermediate situation in Liostenogaster. In all examined species, we found no differences in the spermathecal development between reproductive and non-reproductive wasps.