Lack of successful sexual reproduction suggests the irreversible parthenogenesis in a stick insect

Abstract

While parthenogenesis offers short-term reproductive benefits and demographic advantages, a lack of mating can result in problems such as the accumulation of deleterious mutations in the long term. Recently, cryptic sexual activity has been detected in some ancient parthenogenetic lineages (Boyer et al., 2021; Freitas et al., 2023; Vakhrusheva et al., 2020), most likely mediated by rarely occurring males. The rare males can be produced by parthenogenetic reproduction, presumably due to developmental errors, including incomplete chromosomal segregation (van der Kooi & Schwander, 2014). Surprisingly, such rare males often retain sexual functions, implying that genes expressed only in males remain functional for a long time, even in parthenogenetic lineages (van der Kooi & Schwander, 2014). The presence of rare functional males or functional male-specific genes raises questions about the asexuality of long-standing parthenogenetic lineages (Boyer et al., 2021).

Phasmida, the group of stick and leaf insects, is well-studied in terms of the evolution of parthenogenetic lineages (e.g., Bedford, 1978; Brock, 2000; Morgan-Richards et al., 2010; Pantel, 1917). Many species are recognized as obligately parthenogenetic, yet rarely occurring males have often been described (see Appendix S1: Table S1). However, the functionality of these males is rarely directly examined, due to the difficulty in sample collection. Exceptionally, rare males have been studied in ancient asexual species of Timema stick insects; these males copulate with the females of sister sexual species and successfully produce offspring (Schwander et al., 2013). Furthermore, a recent study detected evidence of rare sex in several species of asexual Timema, which should be mediated by rare males (Freitas et al., 2023). These findings suggest that rare males may be responsible for gene flow in the parthenogenetic stick insects.

Ramulus mikado is the most common stick insect in Japan and is known as a parthenogenetic species (Ichikawa, 2016; Nagashima, 2001; Yano et al., 2021). Males of R. mikado are often reported in academic journals or newspapers due to their extreme rarity, with only about 10 reported cases (Appendix S1: Table S1). Here, we were fortunate to obtain one live male, six specimens of males, and eggs after mating between rare males and females (Appendix S1: Table S2) and examined the sexual functions of rare males in these stick insects. The external morphology of these rare males was consistent with the previous reports (Appendix S1: Figure S1; Nagashima, 2001; Yano et al., 2021). No males exhibited abnormal external morphology, such as sex mosaicism (details in Appendix S1: Section S1).

First, we observed the mating behavior of a male (Male#2; Figure 1A). The male showed active mating behaviors, that is, approaching, antennal contact, mounting the female, and insertion of the penis into the female genital opening (Figure 1B; Video S1). Furthermore, we confirmed the presence of a spermatophore, which is a mass containing the spermatozoa and being formed by males in the female bursa copulatrix during mating (Appendix S1: Figure S2). Also, the help from research collaborators allowed us to confirm the successful copulation of the other three males with females (Male#1, 3, and 4, Figure 1C,D). These observations indicate that rare males retain not only their morphology but also their mating behavior.

However, our genetic analysis did not detect any male genetic contribution in the next generation. Eggs oviposited by females were collected before and after mating, and developing embryos were subjected to genetic analysis using simple sequence repeat markers (Appendix S1: Table S3; Suetsugu et al., 2023), yet no male contribution, such as male-specific alleles, was detected in embryos from eggs oviposited by females mated with males (Appendix S1: Tables S4–S10). The genotype of embryos produced by mated females matched those of virgin females, indicating that all the embryos had a parthenogenetic origin. These observed mating behaviors and parthenogenetic origins likely suggest sexual defects in males, females, or both sexes in R. mikado (regarding the origin of rare males, see Appendix S1: Section S1, Table S11, and Figure S3).

Consistent with the fruitless mating suggested above, anatomical and histological observations confirmed reproductive abnormalities in males and signs of degeneration in female reproductive organs for mating. We dissected two males: One was obtained alive (Male#2) and the other was a freshly frozen sample (Male#7). Male#2 was dissected just after the mating experiment and revealed a complete reproductive system, including a pair of testes, accessory glands, and seminal vesicles, as is typical in stick insects (Figure 2A; Appendix S1: Figure S4). However, our detailed histological observation of gametogenesis in this male revealed no mature sperm or spermatids in the testis (Figure 2B,C; Appendix S1: Section S1). Moreover, some of the nuclei were deformed/condensed and strongly stained with hematoxylin (Figure 2B), typical characteristics of apoptotic cells (Nozaki & Matsuura, 2021). We found the formation of spermatophores by the male (Appendix S1: Figure S2A), yet we could not find any sperm-like structures in the spermatophore (Appendix S1: Figure S2B,C), suggesting that no mature sperms were transferred during the ejaculation. Male#7 was also dissected and revealed to have male-type accessory glands, seminal vesicles, and a penis. Nevertheless, this male did not possess testes but ovary-like organs (Figure 2A). Based on our observations, the ovary-like organs were confirmed to be composed of several ovarioles containing oocyte-like cells (Figure 2D).

We also dissected three females that mated with Male#2 after a collection of eggs and two virgin females. Then, we confirmed the female characteristics typical in stick insects (Appendix S1: Figures S5A,B and S6). R. mikado eggs had a single micropyle, a typical characteristic of stick insects (Appendix S1: Section S1, Figures S5C and S7; Bedford, 1978). However, our histological observations showed that females had sexual organs with signs of degeneration (Appendix S1: Figure S8). The females possessed spermathecae, that is, sperm storage organs, and bursa copulatrix, that is, copulatory pouch, where the penis reaches, yet the organs exhibited a thin cuticle wall and no secretion (Appendix S1: Section S1 and Figure S8).

This study emphasizes the asexuality in the Japanese common stick insect R. mikado. Both sexes of R. mikado had seemingly complete genital organs, including a sperm reservoir in females and a penis in males, and showed active mating behaviors (Figure 1; Appendix S1: Figures S1, S2, S4–S6; Video S1). Nevertheless, the next generation produced after mating also resulted from parthenogenesis (Appendix S1: Tables S4–S10). Furthermore, the rare males lacked normal sperm or testes (Male#2 and Male#7; Figure 2), and the females showed signs of degeneration in the reproductive organs essential for mating (Appendix S1: Figure S8). All these results strongly suggest that R. mikado can no longer return to sexual reproduction. We must note that R. mikado lacks any sister sexual species in Japan, unlike Timema stick insects in North America (Schwander et al., 2013). This situation indicates a lack of safeguard mechanisms to maintain normal genes for sexual reproduction through gene flow from closely related species. Therefore, the asexuality of R. mikado likely indicates that the geological and phylogenetic isolation of parthenogenetic lineages may lead to the complete loss of sex. In this sense, the present study provides a unique insight into the evolution and loss of sex and sexual traits.

Overall, this study provides evidence for the irreversible evolution of parthenogenesis. Despite the presence of males, which exhibit both distinct external morphology and active mating behavior, these may be mere vestiges. Interestingly, R. mikado parthenogenetic lineage has been longstanding (estimated between 0.34 and 0.51 Myr) without recent sexual reproduction and cryptic gene flow (Suetsugu et al., 2023). How then do they overcome the expected long-term cost of asexuality? The mechanism of parthenogenesis in R. mikado, which requires a cytological approach to determine, can avoid the rapid loss of heterozygosity in individuals (see Appendix S1: Section S1 and Tables S4–S10). This might explain the long persistence of parthenogenesis (Schwander & Crespi, 2009). Additionally, the historical long-dispersal event (Suetsugu et al., 2023) might hint at the long history of their asexuality. Long-distance dispersal may be effective in avoiding global extinction because it allows R. mikado individuals to expand their habitat without being restricted to mountains, rivers, or the sea. Furthermore, periodic population surges in R. mikado (Yano et al., 2021) suggest that the usual disadvantages of asexual reproduction, such as higher extinction rates, might be mitigated by large effective population sizes (Ross et al., 2013). These insights underscore the complexity of parthenogenesis persistence and highlight crucial considerations for future research in this area.

Tomonari Nozaki involved in conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, project administration, resources, validation, visualization, writing—original draft, and writing—review and editing. Yasuhiko Chikami involved in conceptualization, investigation, methodology, visualization, writing—original draft, and writing—review and editing; Koki Yano involved in funding acquisition, investigation, methodology, resources, visualization, writing—original draft, and writing—review and editing; Ryuta Sato involved in data curation, formal analysis, investigation, visualization, and writing—review and editing; Kenji Suetsugu involved in conceptualization, funding acquisition, investigation, resources, validation, writing—original draft, writing—review and editing; Shingo Kaneko involved in conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, project administration, validation, visualization, writing—original draft, and writing—review and editing. All authors gave final approval for publication and agreed to be held accountable for the work performed therein.

This work was financially supported by the Japan Society for the Promotion of Science to Kenji Suetsugu (Challenging Exploratory Research number 18K19215), Tomonari Nozaki (Research Fellowship for Young Scientists number 19J01756) and Koki Yano (number 21J01422). This work was also supported by Competitive Research Funds for Fukushima University Faculty.

The authors declare no conflicts of interest.