Termite queens close the sperm gates of eggs to switch from sexual to asexual reproduction Toshihisa Yashiro and Kenji Matsuura1 Laboratory of Insect Ecology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan Edited by Bert Hölldobler, Arizona State University, Tempe, AZ, and approved October 16, 2014 (received for review July 2, 2014) Males and females are in conflict over genetic transmission in the ants (11–13) and termites (14–16) selectively use both sexual re- evolution of parthenogenesis, because it enhances female re- production and thelytoky when mating with males. In these social productive output but deprives the males’ genetic contribution. insects, queens produce new queens asexually by thelytokous par- For males, any trait that coerces females into sexual reproduction thenogenesis, but they produce workers through sexual re- should increase their fitness. However, in the termite Reticulitermes production. This conditional switching of reproductive tactics allow speratus, queens produce their replacements (neotenic queens) par- queens to increase the transmission of their genes to the next thenogenetically while using normal sexual reproduction to pro- generation and maintain genetic diversity in the worker force, but it duce other colony members. Here, we show that termite queens inevitably provokes queen–male sexual conflict by reducing the produce parthenogenetic offspring in the presence of kings by clos- males’ genetic contribution to future generations (17). This system ing the micropyles (sperm gates; i.e., openings for sperm entry) of provides an ideal opportunity to study how and why females switch their eggs. Our field survey showed that termite eggs show large from sexual to asexual reproduction, even in the presence of males. variation in numbers of micropyles, with some having none. Micro- The breeding system of the subterranean termite Reticulitermes satellite analysis showed that embryos of micropyleless eggs de- velop parthenogenetically, whereas those of eggs with micropyles speratus is characterized by asexual queen succession (AQS) (Fig. 1 are fertilized and develop sexually. Surveys of eggs among queens and SI Text). Termite colonies are typically founded by a monoga- of different age groups showed that queens begin to lay micro- mous pair of primary reproductives (adult winged forms): a king pyleless eggs when they are older and thus, need to produce their and queen. In Reticulitermes termites, primary queens live more EVOLUTION replacements parthenogenetically. In addition, we found clear sea- than 11 y (18). As the primary queen senesces and thus, her egg sonality in new neotenic queen differentiation and micropyleless production becomes insufficient to maintain the colony, sec- egg production. This micropyle-dependent parthenogenesis is the ondary queens (i.e., neotenic queens) differentiate within the first identification, to our knowledge, of the mechanism through colony and supplement egg production, eventually replacing the which females control egg fertilization over time in diploid animals, primary queen (Fig. 1 and Fig. S1) (18, 19). In AQS species, implying a novel route of the evolution of parthenogenesis in favor queens produce their neotenic replacement queens asexually but of female interests without interference from males. use normal sexual reproduction to produce other colony mem- bers (14–16). Because sex determination in Reticulitermes termites is thelytoky | micropyle | asexual queen succession | egg fertilization | male heterogamy (20), queens cannot produce secondary kings by Isoptera parthenogenesis (17). This AQS system enables founding queens to increase their reproductive output while retaining the same trans- he near ubiquity of sexual reproduction is one of the most mission rate of their genes to future generations. Therefore, the Tenduring puzzles in evolutionary biology, because all else founder queen can be considered genetically immortal until the being equal, asexual populations have a twofold fitness advan- colony dies, because neotenic queens are also replaced by sub- tage over their sexual counterparts and should rapidly out- sequent cohorts of parthenogenetically produced neotenic queens. number sexual populations (1, 2). A proposed major advantage of sexual reproduction is that it promotes genetic variability Significance across generations, facilitating adaptation to local ecological conditions (3–5). However, mathematical models have revealed that evolution need not favor sexual reproduction, even when it To clarify the evolution of parthenogenesis, given the potential sexual conflict over genetic transmission, identifying the does increase variability that is beneficial, and thus, the preva- mechanism regulating egg fertilization in females is essential. lence of sexual reproduction remains an enigma (6, 7). Some In the termite Reticulitermes speratus, queens produce their biologists have approached this question by considering how loss replacements (neotenic queens) parthenogenetically but use of sex can be achieved in a population of sexual organisms (8– sexual reproduction to produce other colony members. We 10). The evolution of parthenogenesis may be no exception in discovered that queens of the termite close micropyles (open- that it creates sexual conflict, given that parthenogenetic re- ings for sperm entry) of their eggs to produce parthenogenetic production enhances female reproductive output but deprives offspring in the presence of kings. Furthermore, we found that the males’ genetic contribution to future generations. Therefore, queens control the proportion of micropyleless eggs by regu- any trait in males that coerces parthenogenetic females into sexual lating of the number of micropyles over time. This study reproduction should increase their fitness. A recent theoretical describes a novel route of the evolution of parthenogenesis in study showed that the evolution of male coercion substantially favor of females’ interests without interference from males. favors the maintenance of sexual reproduction, although a fe- male barrier against the coercion can evolve (10). Author contributions: K.M. designed research; T.Y. and K.M. performed research; K.M. If male counteradaptations interfere with parthenogenesis, contributed new reagents/analytic tools; T.Y. and K.M. analyzed data; and T.Y. and K.M. females would unlikely be able to switch from sexual to asexual wrote the paper. reproduction unless isolated from males. Identifying the mecha- The authors declare no conflict of interest. nism regulating egg fertilization is critical to better understand the This article is a PNAS Direct Submission. evolutionary routes of parthenogenesis given the potential sexual 1To whom correspondence should be addressed. Email: [email protected]. conflict involved. Unlike facultative parthenogenesis, where This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. females use thelytoky only in the absence of males, queens of some 1073/pnas.1412481111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1412481111 PNAS Early Edition | 1of6 Downloaded by guest on September 27, 2021 Incipient colony Mature colony Queen replacement PK PQ PK SQ PQ SQ SQ PQ SQ PK SQ PK PQ PK SQ AB C D AB C CD D fertilized eggs terminal fusion fertilized eggs terminal fusion ACBCADBD CC ACBCADBD CCDD AC BCADBD DD AC BCADBD CCDD Sexually produced offspring Parthenogens Sexually produced offspring Parthenogens Differentiation into Differentiation into Differentiation into Differentiation into alates and workers secondary queens alates and workers secondary queens Fig. 1. Colony development and asexual queen succession in the termite R. speratus. As the primary queen senesces, secondary queens produced asexually by the primary queen differentiate within the colony and supplement egg production, eventually replacing the primary queen (details described in SI Text). Squares indicate males, and circles represent females. PK, primary king; PQ, primary queen; SQ, secondary queen. (Scale bars: 3 mm.) How can queens of termites (i.e., diploid social insects) control of 3.23 μm(±0.15 SEM, n = 10) in diameter (Fig. 2B), and the egg fertilization? In haplodiploid social Hymenoptera, unfertil- average number of micropyles for all samples (n = 6,000) was ized eggs become males, and fertilized eggs produce females 9.48 (±0.04 SEM, range = 0–33) (Fig. 2 C and D). Significant (21), giving queens a potentially powerful mechanism for con- differences were observed in the number of micropyles among trolling fertilization (22, 23). However, in diploid insects, sperm colonies (F59, 5,940 = 27.35, P < 0.0001; one-way ANOVA), and release is generally activated through a neural loop whenever an 7 of 60 colonies (11.7%) had micropyleless eggs (Table S1). egg passes the genital chamber, with no control over fertilization Unmated R. speratus queens lay eggs, and the unfertilized eggs (24). In this study, we focused on micropyles of eggs to identify develop parthenogenetically, with a comparably high hatching a possible mechanism for controlling fertilization in diploid insects. rate as fertilized eggs (29). All micropyleless eggs showed normal A micropyle is a channel that extends from the external gateway embryonic development as observed in previous studies on par- through the chorion and ends in the vitelline membrane, serving thenogenesis in R. speratus. To test whether the embryos of as the route of sperm entry into a mature oocyte (25–27).