Mating behavior of the jumping bristletail Petrobiellus akkesiensis (Archaeognatha: Machilidae: Petrobiellinae): Direct spermatophore transfer via genital coupling

Shodo Mtow; Ryuichiro Machida

doi:10.3897/asp.83.e159694

Research Article

Mating behavior of the jumping bristletail Petrobiellus akkesiensis (Archaeognatha: Machilidae: Petrobiellinae): Direct spermatophore transfer via genital coupling

Shodo Mtow^‡§, Ryuichiro Machida^|

‡ Fukushima University, Fukushima, Japan

§ Meijo University, Nagoya, Japan

| University of Tsukuba, Nagano, Japan

Corresponding author: Shodo Mtow ( mtow@meijo-u.ac.jp )

Corresponding author: Ryuichiro Machida ( machida@sugadaira.tsukuba.ac.jp )

Academic editor: Monika Eberhard

This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Citation:

Abstract

We examined the morphology of the male and female genitalic regions of Petrobiellus akkesiensis and describe its mating behavior as analyzed using video recordings. Petrobiellus belongs to the monogeneric subfamily Petrobiellinae (Machilidae), an enigmatic group known only from the Far East. Its male genitalic region shows remarkable specialization, leading to the inference that Petrobiellus performs direct spermatophore transfer through genital coupling. Video analysis revealed that, as expected, the male holds the female’s ovipositor using both his penis and styli on the 9^th abdominal segment: the penis grasps the basal region of the ovipositor with its hook-like tip and the styli hold the midsection of the ovipositor from above and below; the spermatophore is then discharged onto the dorsal side of the basal region of the ovipositor from the apex of the penis, and the sperm is taken into the ovipositor, thereby completing the direct sperm transfer from male to female. This is the first documented case of direct sperm (spermatophore) transfer via genital coupling in apterygote hexapods. Based on current and previous findings, we compared and characterized the mating behaviors of Archaeognatha and discuss their implications in terms of evolution of mating strategies in Hexapoda. The mating behavior of Petrobiellus represents one of the most derived modes within Machilidae and Archaeognatha, for which we propose the name “direct transfer of spermatophore by genital coupling”.

Keywords

Apterygota, Ectognatha, Entognatha, evolution, jumping bristletail, mating behavior, phylogeny, spermatophore

1. Introduction

Insects have undergone a spectacular adaptive radiation that has occurred primarily on land (Shaw 2014). In aquatic arthropods sperm fluid is not threatened by desiccation during the transfer from males to females; however, it became a major issue when insects transitioned to terrestrial environments, necessitating adaptations to prevent sperm from drying out (Heming 2003). Winged insects or Pterygota, currently representing the majority of insect species, have acquired direct sperm transfer by evolving copulatory organs that effectively prevent sperm desiccation. This has enabled Pterygota to fill a wide range of ecological niches, resulting in explosive diversification (Dallai et al. 2013; Beutel et al. 2014, 2017). Further discussion is needed to determine whether “copulation” was acquired in Pterygota as its groundplan or several times independently among winged insects (Dallai et al. 2013; Beutel et al. 2017). Although copulation is also observed in some crustaceans and myriapods (Schaller 1979; Proctor 1998; Heming 2003), copulation in insects undoubtedly evolved independently of that in crustaceans or myriapods (Dallai et al. 2013; Beutel et al. 2014).

As far as known, the “apterygote” principal lineages of Hexapoda all predominantly perform indirect sperm transfer. In this process, the male places a spherical spermatophore, i.e., a sperm spherule or a small package filled with sperm, either on the substrate, then often on a stalk or a thread secreted by the male, or somewhere on the female’s body, and the female subsequently takes it up (Schaller 1971, 1979; Heming 2003; Dallai et al. 2013; Beutel et al. 2014). Undoubtedly, indirect spermatophore transfer represents the groundplan of hexapod mating (Heming 2003; Dallai et al. 2013). Although direct transfer of free sperm occurs in some higher groups of Holometabola (Hünefeld and Beutel 2005), direct sperm transfer primarily occurs via spermatophores across Hexapoda (Schaller 1979; Heming 2003; Hünefeld and Beutel 2005; Dallai et al. 2013). As an exception, secondary indirect transfer of spermatophores occurs in rare cases within Pterygota, as reviewed by Mann (1984). Recently, a mode of indirect spermatophore transfer was reported in Zoraptera, wherein the male deposits a spermatophore on the posterior extremity of the female’s body (Dallai et al. 2013).

Apterygote hexapods, which have not acquired direct spermatophore transfer, perform indirect sperm transfer and are thus unable to leave habitats with high atmospheric humidity, such as soil (Heming 2003; Beutel et al. 2017). Various modes of mating have evolved in apterygote hexapods to ensure prompt and successful spermatophore transfer (Schaller 1971, 1979; Mann 1984; Proctor 1998). In the entognathous Collembola and Diplura, the male deposits a spherical spermatophore onto a stalk secreted by himself, and the female picks it up (as reviewed in Schaller 1971, 1979; Proctor 1998; Dallai et al. 2013; for Collembola: e.g., Betsch 1980; for Diplura: Bareth 1965). Dallai et al. (2013) suggested that this mode of indirect spermatophore transfer represents the groundplan of Hexapoda. Some symphypleonan collembolans exhibit more elaborate methods of indirect spermatophore transfer (Blancquaert and Mertens 1977; Betsch 1980). The mating behavior of the entognathous Protura has not been well studied, but they may perform some form of direct spermatophore transfer (Ewing 1940).

Indirect spermatophore transfer becomes increasingly elaborate in ectognathous apterygotes, i.e., Archaeognatha and Zygentoma, which has expanded the habitat range of these insects into more terrestrial environments (Sturm 1955, 1956, 1978, 1986, 1987, 1992, 1996, 1997; Sturm and Adis 1984; Sturm and Machida 2001; Klass and Matushkina 2018). Archaeognatha, the subject of the present study, is the most basal lineage of Ectognatha. It includes approximately 550 extant species found worldwide, except in Antarctica (Sturm and Machida 2001; Mendes 2018). Archaeognatha consists of two families: the possibly paraphyletic Machilidae with subfamilies Machilinae, Petrobiinae, and Petrobiellinae (Kaplin 1985), and the most likely monophyletic Meinertellidae, which possesses several unique, derived traits and originated in Gondwana (Sturm and Bach de Roca 1993; Sturm and Machida 2001; Klass and Matushkina 2018; Zhang et al. 2018). Furthermore, Sturm and Bach de Roca (1993) identified several ancestral archaeognathan lineages referred to as “paleoforms”, which may have arisen prior to the divergence of Machilidae and Meinertellidae. These include Charimachilis Wygodzinsky, 1939, Ditrigoniophthalmus Kaplin, 1979, and Mesomachilis Silvestri, 1911. Subsequently, Bach de Roca et al. (2013) established Turquimachilis Bach de Roca et al., 2013, as a new paleoform closely related to Charimachilis. However, the status of paleoforms remains controversial. Koch (2003) stated that the primitiveness of the paleoforms cannot be clearly established. Matushkina and Klass (2020) conducted a critical review of the paleoforms, reevaluating the morphological matrix proposed by Zhang et al. (2018), and argued that although Ditrigoniophthalmus is possibly still a paleoform, as suggested by Zhang et al. (2018), the remaining three groups of paleoforms can be more accurately placed within one of the three machilid subfamilies.

Three different modes are differentiated in the mating behavior of Archaeognatha (Sturm and Machida 2001). First, the most widespread mode, “indirect transfer of spermatophore(s) deposited on carrier thread” (mode 1), is known from Machilinae and from Pedetontus Silvestri, 1911 of Petrobiinae (all in Machilidae). After foreplay, the male stretches a taut thread, which is spun by tubular setae (referred to as grooved setae by Sturm 1991) on the parameres, between the parameres and the substrate, places one or more spherical spermatophores on the thread, and the female picks it/them up, completing spermatophore transfer. The second mode, “direct transfer of spermatophore by deposition on ovipositor” (mode 2), is known from Petrobius Leach, 1809 of Petrobiinae (Machilidae). After a brief period of foreplay, the female arches her abdomen to signal readiness to mate, and the male also arches his abdomen, depositing a relatively large spermatophore onto the female’s ovipositor, which she then takes into her body, completing spermatophore transfer. This may be repeated several times. The third mode, “indirect transfer of spermatophore deposited on stalk” (mode 3), is known only in Meinertellidae. After foreplay, the male places a spherical spermatophore with a stalk onto the substrate, and the female picks up the spermatophore from the tip of the stalk using her ovipositor, thereby completing spermatophore transfer.

Because mode 1 is the most widely known mating behavior in Archaeognatha, and thread-spinning is also involved in the mating of Zygentoma (see Sturm and Machida 2001), mode 1 is considered representative of the groundplan in Archaeognatha (see Matushkina and Klass 2020). Petrobius, which exhibits mode 2 mating behavior, lacks tubular setae on the parameres. Meinertellidae, which exhibits mode 3 behavior, lacks parameres altogether. Thus, thread-spinning does not occur in modes 2 and 3, and both are considered to have evolved from mode 1 (Sturm 1986; Sturm and Machida 2001; Klass and Matushkina 2018; Matushkina and Klass 2020).

However, it has been suggested that some archaeognathans exhibit mating behaviors that cannot be categorized within any of the modes 1, 2, 3 (Sturm and Machida 2001; Klass and Matushkina 2018; Matushkina and Klass 2020).

The first such case concerns the “paleoform” Mesomachilis. Sturm and Machida (2001) found a female of Mesomachilis californica Sturm, 1991 in which distal three-quarters part of the ovipositor was covered with a mass of spermatophore-like material, and thread-like structures were observed between the gonapophyses on both sides as well as in the spermatheca. Based on this, they proposed a possible mating behavior for this species as follows: the male first spins threads using the numerous tubular setae on the parameres (Sturm 1991) to form a net and then places a large spermatophore on it; the female then takes up the spermatophore through her ovipositor.

The second case concerns the “paleoform” Turquimachilis. Tubular setae on the parameres appear to be absent in Turquimachilis males (as inferred by Matushkina and Klass 2020), but the unsegmented parameres possess structures such as knobs, hooks, and denticulations that appear suited for grasping. Based on this, Matushkina and Klass (2020) proposed that Turquimachilis directly transfers the spermatophore by grasping the female’s genitalia with the parameres. Regarding the “paleoform” Charimachilis, Matushkina and Klass (2020) suggested that because the parameres lack tubular setae, it may perform a form of direct spermatophore transfer involving genital contact between the male and female, although no anatomical structures specifically supporting this mode have yet been identified.

One more mode of mating behavior is assumed for Petrobiellinae. This subfamily, represented by the sole genus Petrobiellus Silvestri, 1943, is considered a derived lineage within Machilidae, alongside Petrobiinae (Sturm and Bach de Roca 1993; Sturm and Machida 2001). Conversely, Kaplin (1985) regarded it as one of the ancestral lineages within Machilidae (for further phylogenetic and systematic issues related to this subfamily, see 4.1.). Several Petrobiellus species have been described from the coastal regions of Far East Asia (for details, see 4.1.). Petrobiellus males exhibit remarkable structural specialization in their genitalic regions (Uchida 1954; Sturm and Machida 2001; Klass and Matushkina 2018; Mtow and Machida 2024): the distal part of the penis curves dorsally like a hook; the styli of the 9^th abdominal segment are robust, with densely-spined, concave dorsomesal surfaces; the coxites of the 9^th abdominal segment are strongly concave mesally, and the penis is almost fully exposed between the 9^th coxites on both sides. These unique characteristics of the male genitalic region in Petrobiellus suggest that this genus performs a form of direct spermatophore transfer involving genital coupling (Sturm and Bach de Roca 1993; Sturm and Machida 2001; Klass and Matushkina 2018; Matushkina and Klass 2020). Klass and Matushkina (2018) carefully examined the male genitalic structures of Petrobiellus takunagae Silvestri, 1943 from both morphological and functional morphological perspectives and proposed two well-conceived hypotheses regarding spermatophore transfer in Petrobiellus (see 4.3.2.).

The evolution of mating behavior is of great interest in the context of hexapod terrestrialization, which played a major role in their successful colonization of land. Adaptation to terrestrial habitats accelerated with the emergence of Ectognatha, and its most basal lineage, Archaeognatha, holds particular significance in this regard. In the present study, we collected several males and females of Petrobiellus akkesiensis (Uchida, 1949) from the southern coastal region of Hokkaido, Japan, to observe and analyze their mating behavior under laboratory conditions. The results revealed that this species indeed performs direct spermatophore transfer via genital coupling, as predicted for Petrobiellus. We describe the genitalic structures of P. akkesiensis, analyze its mating behavior using video recordings, and discuss several issues related to the mating behavior and evolution of Archaeognatha.

2. Materials and methods

Twenty-five males and 40 females were collected under stones on October 19, 2021, from a mixed-sex population of Petrobiellus akkesiensis (Uchida, 1949) along the rocky coast near Cape Washibetsu, Noboribetsu, Hokkaido, Japan (42°21′12.279″N 141°03′14.705″E), and brought back to the laboratory alive. The specimens were kept separately at room temperature (14–25°C) in plastic cases (120 mm × 85 mm × 60 mm). The bottoms of the cases were lined with moistened wiping paper, on which bark covered with green algae and fallen leaves were placed as feed.

Mating experiments were conducted at room temperature on 19 occasions between December 29, 2021, and April 4, 2022. In the experiments involving top-view observations, a male–female pair was placed in a glass Petri dish (60 mm in diameter, 20 mm in height) or a plastic case (70 mm × 50 mm × 30 mm) lined with filter paper on the bottom. Matings were recorded using a Ricoh GXR digital camera (Tokyo, Japan) or a Pentax K-70 digital camera (Tokyo, Japan), both mounted on a Nikon SMZ800 stereomicroscope (Tokyo, Japan). For bottom-view observations, a male–female pair was placed in a glass Petri dish (60 mm in diameter, 20 mm in height) without a paper lining. The dish was positioned on a photographic stage constructed from a polyvinyl chloride pipe, cardboard, and a 1-mm-thick acrylic plate. Matings were recorded through the bottom of the dish using a Pentax K-70 digital camera mounted on a Nikon SMZ800 stereomicroscope with its lens barrel inverted (Fig. S1A, B).

To observe the genitalic regions of males and females, the posterior abdomen was removed, fixed in 70% ethyl alcohol, dehydrated through a graded ethyl alcohol series, air-dried after immersion in 1,1,1,3,3,3-hexamethyldisilazane, coated with gold using a Vacuum Device MSP-1S magnetron sputter (Ibaraki, Japan) or a JEOL JFC-1100 ion sputter (Tokyo, Japan), and examined under a JEOL JSM IT-100 scanning electron microscope (SEM) (Tokyo, Japan) or a Hitachi TM4000PlusII SEM (Tokyo, Japan) at an acceleration voltage of 15 kV. Among the specimens preserved in 70% ethyl alcohol, one female was found with a spermatophore attached to her ovipositor. The ovipositor of this specimen was observed using a Nikon SMZ800 stereomicroscope as well as a Hitachi TM-1000 SEM (Tokyo, Japan) at an acceleration voltage of 15 kV (non-coated, with automatic vacuum control).

In the present study, we designate abdominal structures by appending the abdominal-segment number to the structure name; for example, “stylus V” denotes the stylus of the 5^th abdominal segment.

3. Results

3.1. Genitalic regions of Petrobiellus akkesiensis

The female genitalic region of Petrobiellus akkesiensis is quite typical for Machilidae. The coxites IX and styli IX (excluding the apical spine) are nearly equal in length (Fig. 1A). The ovipositor, composed of paired gonapophyses VIII (anterior gonapophyses) and gonapophyses IX (posterior gonapophyses), is long, slender, and subparallel, extending beyond the apex of the styli IX by about twice their length (Fig. 1A, D). The terminal setae at the tips of the gonapophyses VIII and IX are long and simple (Fig. 1C, F). The chaetotaxy of the gonapophyseal articles is uniform (Fig. 1B, C, E, F).

Figure 1.

SEM micrographs of genitalic segments in Petrobiellus akkesiensis female, anterior to the top. A Venter of the abdominal segments VIII and IX, ventral view. B Enlargement of the basal part of the ovipositor, ventral view. C Enlargement of the distal part of the ovipositor, ventral view. D Venter of the abdominal segments VIII and IX, dorsal view. E Enlargement of the basal part of the ovipositor, dorsal view. F Enlargement of the distal part of the ovipositor (gonapophyses of right side only), dorsal view. — Abbreviations: Cx8, 9 – coxites VIII and IX; Gp8, 9 – gonapophyses VIII and IX; Ov – ovipositor; St8, 9 – styli VIII and IX. — Scale bars: A, D – 1 mm; B, C, E – 200 µm; F – 100 µm.

The male genitalic region of P. akkesiensis is highly specialized. Paramere VIII is absent. The coxites IX and styli IX are remarkably robust, with the styli IX (excluding the apical spine) measuring about half the length of the coxites IX (Fig. 2A). The coxites IX are mesally concave, and both the penis and parameres IX are exposed between the two coxites (Fig. 2A). A row of strong setae occurs near the posterior margin of the coxites IX (Fig. 2B, C). The styli IX are dorsomesally concave, and the concave surface is densely furnished with approximately 20 dark brown, strong spines (Fig. 2B–D; see fig. 2I in Mtow and Machida 2024). The penis is long, extending beyond the distal end of the coxites IX by 40% of the coxite length (Fig. 2A–C), composed of two articles. The proximal article, accounting for approximately 80% of the penis length, is straight, whereas the distal article is dorsally curved, terminating in a wide genital opening (phallotreme) (Fig. 2E, F). Short setae are densely distributed on the dorsal surface of the posterior part of the proximal article and the entire distal article (Fig. 2F, G). The parameres IX, located ventral to the penis, are slender and composed of I + 5–6 articles: the proximalmost article, representing 60–70% of the total paramere length (Fig. 2A, E). Tubular setae (grooved setae, as described by Sturm 1991) are observed on neither the penis nor the parameres IX (Fig. 2E–H).

In both male and female Petrobiellus, a pair of ventral sacs is present on the abdominal segments I, VI, and VII, and two pairs are present on each of the abdominal segments II to V.

Figure 2.

SEM micrographs of genitalic segments in Petrobiellus akkesiensis male, anterior to the top. A Venter of the abdominal segments VIII and IX, ventral view. B Venter of the abdominal segment IX, dorsal view. C Enlargement of the stylus IX, dorsal view. D Enlargement of a strong spine on the dorsomesally-concaved side of the stylus IX. E Penis and parameres IX, lateral view, dorsal to the right. F Enlargement of the distal article of the penis, lateral view. G Enlargement of a short seta on the dorsal side of the distal article of the penis. H Setation on the distal part of the paramere IX, lateral view. — Abbreviations: Cx8, 9 – coxites VIII and IX; Pa9 – paramere IX; Pe – penis; St8, 9 – styli VIII and IX. — Symbols: asterisk – dorsomesally-concaved side of stylus IX; star – genital opening. — Scale bars: A – 1 mm; B – 500 µm; C, E – 200 µm; F – 50 µm; D, H – 10 µm; G – 2 µm.

3.2. Mating behavior of Petrobiellus akkesiensis

We conducted 19 mating experiments with Petrobiellus akkesiensis. In 9 of these no sexual interactions occurred, whereas in the remaining 10 experiments the pairs displayed mating behavior. Of the 10 experiments involving mating interactions, 6 resulted in successful matings. In 1 of the 4 unsuccessful cases, the male failed to grasp the ovipositor; in the other 3 cases, the females unilaterally terminated genital contact. The 6 successfully completed matings are summarized in Table 1.

Table 1.

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Six experiments in which the mating was successfully complete, I – VI. The duration of mating is the approximate time from the holding of the female by the male to their separation.

Case	Direction of observation	Positioning of male against partner	Duration of mating	Figures and movies for reference
I	dorsal	right	13 min	Fig. 3A–C; File S1
II	dorsal	right	8 min	not included in the present study
III	ventral	right	25 min	not included in the present study
IV	ventral	left	30 min	not included in the present study
V	dorsal	right	25 min	Fig. 3D; File S2
VI	ventral	left	14 min	Fig. 4A–I; File S3

The mating experiment begins when a male and female P. akkesiensis are placed into the arena (glass Petri dish or plastic case). As soon as the male recognizes the female, he rushes toward the female and positions himself either to her left or right side, placing his maxillary palps on her thorax (cf. Fig. 3A). In 8 of the 10 experiments, the male stood right of the female; in the remaining two, left of her. After securing his position, the male brings his forelegs and head (particularly the clypeal region) close to the female’s body, continuing to use his maxillary palps for contact. He then begins vibrating the female, simultaneously twisting his body and pressing his postabdomen against hers. This vibration continues throughout the pairing, with occasional pauses, and may persist even after successful spermatophore transfer or after genital contact between the pair has ended.

Figure 3.

Mating behavior of Petrobiellus akkesiensis captured from movies, viewed from the top. Counters indicate the time from the commencement of the experiment, i.e., when a male and a female were placed in the experimental arena. A–C Sequential frames of mating in Case I from Table 1 (captured from File S1). A At 8 sec after the commencement of the experiment (ACE), the male positions himself to the right of the female, placing his maxillary palps on her thorax. B At 2 min 9 sec ACE, the male grasps the basal part of the ovipositor with his penis. C At 12 min 45 sec ACE, even after the penis releases the ovipositor, the male continues to hold and vibrate the female for a short while. D A single frame from Case V in Table 1 (captured from File S2), at 46 sec ACE. Soon after ovipositor-holding is established, the male’s ventral sacs begin to swell; a pair of ventral sacs in each of the abdominal segments VII and VI can be observed. — Abbreviations: MxP – maxillary palp; VS6, 7 – ventral sacs VI and VII. — Scale bars: 5 mm.

Between 10 sec and 2 min after the pair assumes the abovementioned formation (n = 9), the male protrudes his penis at a right angle from between the coxites IX on both sides to locate the female’s ovipositor. He then grasps the basal part of the ovipositor with the hook-like tip of the penis (Fig. 3B; File S1 [Movie at 02:09], File S2 [Movie at 00:27], File S3 [Movie at 01:10]). The genital opening at the distal end of the penis is hidden behind the ovipositor. The male’s styli IX also assist in holding the ovipositor. While probing for the ovipositor with the penis, the male simultaneously prepares the styli IX for holding: the stylus on the side closest to the female is bent forward, whereas the opposite stylus extends backward (e.g., Fig. 4A; File S3 [Movie at 00:55]). Once the penis grips the ovipositor, the stylus on the side nearest to the female (i.e., the right stylus IX when the male is on the left side of the female, and vice versa), which is bent forward, presses down on the middle portion of the ovipositor from above with the dorsal surface of its proximal region. Meanwhile, the opposite stylus (i.e., the left stylus IX when the male is on the left side of the female, and vice versa), which is stretched backward, pushes up on the ovipositor’s middle region from below using its dorsomesal concave area, lined with numerous strong spines. Thus, the ovipositor is secured at three points: at its base by the penis and at its middle by the styli IX on both sides. This holding occurs quickly: approximately 2 min after the male first captured the female, as shown in Fig. 3A–C (Case I in Table 1), or in < 30 sec, as shown in Fig. 3D (Case V in Table 1). The parameres do not appear to participate in mating; they are positioned ventral to the penis, although they are occasionally observed to separate from it (File S3 [Movie at 01:10–01:16]).

Figure 4.

Sequential frames of mating in Petrobiellus akkesiensis, Case VI in Table 1 (captured from File S3), viewed from the bottom. Counters indicate the time from the commencement of the experiment. Arrows indicate the outline of the spermatophores, to show their approximate dimension. A At 45 sec after the commencement of the experiment (ACE), while exploring the ovipositor with his penis, the male has already prepared the styli IX to hold the ovipositor, i.e., the stylus on the side closer to the partner (left in this frame) is bent forward, whereas the other stylus (right in this frame) is stretched backward. B At 1 min 15 sec ACE, the male protrudes his penis at a right angle and grasps the basal part of the ovipositor with the distally-curved penis. The male’s ventral sacs in the abdominal segments VII and VI begin to swell. C At 1 min 50 sec ACE, the ventral sacs in the abdominal segments V to III also swell, and the spermatophore becomes visible. D At 6 min 59 sec ACE, the spermatophore has reached its maximum size and is clearly visible at the basal part of the ovipositor. E At 8 min 3 sec ACE, the male’s ventral sacs begin to deflate. F At 8 min 42 sec ACE, all ventral sacs have deflated. G At 9 min 1 sec ACE, the spermatophore begins to diminish. H At 9 min 28 sec ACE, the male’s styli IX release the ovipositor, but the penis still maintains its hold. I At 13 min 35 sec ACE, the spermatophore has diminished to its minimum size. — Abbreviations: An – antenna; Ce – cercus; CF – caudal filament; Cx6–9 – coxites VI to IX; Ov – ovipositor; Pa9 – paramere IX; Pe – penis; Sp – spermatophore; St6–9 – styli VI to IX; T7–10 – abdominal terga VII to X; VS3–7 – ventral sacs III to VII. — Scale bars: A – 2 mm; B, C, E, H, I – 1 mm; D, F, G – 500 µm.

As soon as the ovipositor holding is established, the male’s ventral sacs begin to swell, progressing from the posterior to the anterior abdominal segments: first, a pair of ventral sacs VII and VI expands (Figs 3D, 4B; File S2 [Movie at 00:46], File S3 [Movie at 01:16]), followed by two pairs on each of the abdominal segments V and IV, and finally one of the two pairs on the abdominal segment III enlarges (Fig. 4C; File S3 [Movie at 01:44]). Simultaneously, an opaque spermatophore is discharged onto the dorsal side of the ovipositor from the genital opening at the tip of the penis (Fig. 4C; File S3 [Movie at 01:50]; cf. Fig. 4D), rapidly expanding to its maximum size, approximately 0.7 mm in diameter (approximate volume: 0.18 mm³), within a few minutes (Fig. 4D). Once the spermatophore reaches its full size, the swollen ventral sacs begin to shrink in reverse order, from the anterior to the posterior abdominal segments (Fig. 4E), and eventually retract beneath the coxites (Fig. 4F; File S3 [Movie at 08:24–08:42]). Briefly thereafter, presumably due to the uptake of sperm fluid (through the gap between gonapophyses) by the ovipositor, the spermatophore gradually diminishes in size (Fig. 4G). It is not completely absorbed, and its final size may remain as large as that shown in Fig. 4I. This marks the completion of spermatophore transfer.

After spermatophore transfer is complete, the male’s styli IX soon release the ovipositor (Fig. 4H), but it takes longer for the penis to disengage (File S1 [Movie at 09:58], File S2 [Movie at 25:36], File S3 [Movie at 13:52]). Even after the penis has released the ovipositor, the male continues to hold and vibrate the female for a short while before they eventually separate (Fig. 3C; File S1 [Movie at 12:45], File S2 [Movie at 25:48]). The entire mating behavior of P. akkesiensis, from the moment the male holds the female to their final separation, lasts approximately 10–30 min (n = 6, Table 1).

Among the specimens preserved in alcohol, we found a female with a spermatophore attached to the dorsal side of the basal region of her ovipositor. The spermatophore had a conical shape tapering posteriorly, with a base approximately 0.2 mm in diameter and a height approximately 0.4 mm (approximate volume: 0.005 mm³; Fig. 5A–C). The size of the spermatophore was nearly the same as those reduced to their minimum volume (cf. Fig. 4I). The posteriorly pointed tip of the spermatophore was inserted between the left and right gonapophyses of the ovipositor (Fig. 5C, D). Close examination of the spermatophore surface revealed numerous winding, thread-like structures, likely sperm, embedded in but partially exposed on the surface (Fig. 5E).

Figure 5.

Genitalic segments of an alcohol-preserved female specimen of Petrobiellus akkesiensis, with a spermatophore attached to her ovipositor. A Venter of the abdominal segments VIII and IX, dorsal view, anterior at the top. B–E SEM micrographs of the same specimen shown in A, dorsal view, non-coated. B The spermatophore is attached to the basal part of the ovipositor. C Enlargement of the spermatophore. D Enlargement of the area boxed in C. E Enlargement of the boxed area in D, showing numerous sperms. — Abbreviations: Cx8, 9 – coxites VIII and IX; Gp8, 9 – gonapophyses VIII and IX; Ov – ovipositor; S – sperm; Sp – spermatophore. — Scale bars: A, B – 1 mm; C – 200 µm; D – 50 µm; E – 10 µm.

4. Discussion

4.1. Systematics of Petrobiellinae and Petrobiellus

Kaplin (1985), who proposed a three-subfamily system of Machilidae, placed Ditrigoniophthalmus, which is often considered to be a paleoform of Machilidae, under Petrobiellinae, and some researchers have followed this (Zhang et al. 2018; Montagna 2020). However, this genus cannot be included in Petrobiellinae because of significant differences in diagnostic features, such as the number of abdominal styli (cf. Kaplin 1979, 2000). Other authors therefore regarded Petrobiellinae as a monogeneric subfamily consisting solely of the genus Petrobiellus (cf. Sturm and Bach de Roca 1993; Sturm and Machida 2001). In fact, after excluding Ditrigoniophthalmus from Petrobiellinae, Kaplin (2000) placed it in a newly established subfamily, Ditrigoniophthalminae, created exclusively for this genus. Petrobiellinae has generally been considered a derived lineage within Machilidae, along with Petrobiinae (Sturm and Bach de Roca 1993; Sturm and Machida 2001). However, Kaplin (1985) assigned Petrobiellinae an ancestral position within the family, and recent total evidence analyses indicate a close relationship of Petrobiellinae and Ditrigoniophthalmus (Zhang et al. 2018; Montagna 2020; cf. Matushkina and Klass 2020). Furthermore, recent molecular phylogenetic studies and total evidence analyses have indicated a close relationship between Petrobiellinae and Meinertellidae (Ma et al. 2015; Zhang et al. 2018; Montagna 2020; Guan et al. 2021), although morphological evidence to support this affinity is lacking (Sturm and Bach de Roca 1993; Sturm and Machida 2001; Klass and Matushkina 2018). Two species of Petrobiellus described by Ma et al. (2015), which are critical to the proposed relationship between Petrobiellinae and Meinertellidae, merit careful reconsideration. Ma et al. (2015) identified jumping bristletails collected from inland Yunnan, southern China (near Laos), as Petrobiellus, and named them Petrobiellus bannaensis and Petrobiellus puerensis as new species. However, as Kaplin (2020) noted, these descriptions violate Article 13.1.1 of the International Commission on Zoological Nomenclature (1999), as they are based only on molecular data without morphological descriptions. As such, these two scientific names are invalid. Moreover, the purported distribution of these two “Petrobiellus” species is problematic: they were described from inland China, whereas Petrobiellus is known only from coastal regions of the Far East (see below). Given that the molecular phylogenetics aligns these two species to the family Meinertellidae, the validity of their assignment to Petrobiellus is questionable. Notably, the subtropical and tropical rainforests near the collection sites in China are likely (if not exclusively) inhabited by meinertellid genera such as Machilellus Silvestri, 1911, Machilontus Silvestri, 1912, and Megalopsobius Silvestri, 1912. Additionally, several key diagnostic features of Petrobiellus, such as the absence of scales on the head, cephalic appendages including antennae, and legs (see below), are also shared by Meinertellidae. Therefore, the assignment of these two inland Chinese species to Petrobiellus is doubtful, as is the inferred relationship between Petrobiellinae and Meinertellidae, which was based on molecular phylogenetic analysis treating these species as representatives of Petrobiellus.

Petrobiellinae is a monogeneric subfamily represented by Petrobiellus. The genus was established by Silvestri (1943) based on several diagnostic features, the major ones being 1) the absence of scales on all parts of the antennae, mandibles, maxillae, labium, legs, and styli; 2) dumbbell-shaped lateral ocelli; 3) coxal stylets on the mid- and hindlegs; and 4) one pair of ventral sacs on the segments I, VI, and VII and two pairs on each of the segments II to V. To date, several species have been recorded from the coastal regions of the Far East, specifically Japan and Russia: Petrobiellus takunagae from Shirahama (Wakayama, Japan), Shimoda (Shizuoka, Japan), and Sado Island (Niigata, Japan) (Silvestri 1943; Machida 2020; Mtow 2021); Petrobiellus akkesiensis from Akkeshi and Noboribetsu (Hokkaido, Japan) (Uchida 1949; Mtow and Machida 2024; herein); Petrobiellus curvistylis Uchida, 1954 from Hachijo Island (Tokyo, Japan) (Uchida 1954); Petrobiellus kusakini Kaplin, 1980 from Simushir Island (Chishima Islands, Japan) (Kaplin 1980); and Petrobiellus sachalinensis Kaplin, 2020 from Sakhalin (Russia) (Kaplin 2020). Machida (2020) reported that P. curvistylis from Hachijo Island is likely a synonym of P. takunagae, which is widely distributed along the coastal regions of the Okinawa Islands to Honshu. Uchida (1954) also suggested this possibility in the original description of P. curvistylis. If this synonymy is correct, the genus Petrobiellus currently comprises four species.

Males and females are known in P. curvistylis, which was described based on two males, two females, and three juveniles, but only females are known for P. kusakini and P. sachalinensis. P. takunagae and P. akkesiensis were also initially described based only on female specimens, but males were eventually discovered, although the sex ratio in their populations is heavily biased toward females. As a result, the understanding of male morphology in this genus has progressively advanced (for P. takunagae: Sturm and Bach de Roca 1993; Sturm and Machida 2001; Klass and Matushkina 2018; for P. akkesiensis: Mtow and Machida 2024; herein).

4.2. Genitalic region of Petrobiellus

The general features of the genitalic region observed in Petrobiellus akkesiensis were consistent with those reported for other Petrobiellus species. The genitalic characteristics of Petrobiellus are summarized below.

Female. (cf. Silvestri 1943; Uchida 1954; Sturm and Bach de Roca 1993; Klass and Matushkina 2012; Kaplin 2020; Mtow and Machida 2024; herein). The female genitalic region is normal. The coxites IX and styli IX (excluding the terminal setae) are approximately equal in length. The ovipositor is long, slender, and subparallel. The chaetotaxy of the ovipositor is simple, with long terminal setae at the tips of both gonapophyses.

Male. (cf. Uchida 1954; Klass and Matushkina 2018; Matushkina and Klass 2020; Mtow and Machida 2024; herein). The male genitalic region is highly specialized. The coxites IX and styli IX are remarkably robust, with the styli IX measuring approximately half the length of the coxites IX. The coxites IX are mesally concave, and the penis is almost fully exposed between the coxites IX on both sides. A row of strong setae is present near the posterior end of the coxites IX. The styli IX are dorsomesally concave, and their concave surfaces are densely covered with strong spines. The penis consists of a long, straight proximal article and a short distal article, the latter curving dorsally and terminating in a wide genital opening. Short setae are densely distributed on the dorsal surface of the posterior part of the proximal article and on the distal article. The parameres VIII are absent. The parameres IX are slender, located ventrally to the proximal article of the penis, slightly exceeding its length, and composed of I + 3–6 articles: I + 3 in Petrobiellus takunagae (Sturm and Bach de Roca 1993; Klass and Matushkina 2018) and I + 5–6 in P. akkesiensis (Mtow and Machida 2024; herein). Uchida (1954) did not describe the articulation of the parameres in Petrobiellus curvistylis, likely due to limited observational detail. Both the penis and parameres IX lack tubular setae (referred to as grooved setae by Sturm 1991).

The ovipositor of Petrobiellus is slender and subparallel, and its chaetotaxy is regular, without setae/spines specialized for digging. It can be categorized as the primary type according to the classical classification (cf. Wygodzinsky 1941; Janetschek 1991). Sturm and Bach de Roca (1993), with further reference to Janetschek (1991), argued that the classification of the ovipositor into primary and secondary types does not reflect phylogeny, and they proposed a new system that distinguishes four types: 1) the primary type is found in Charimachilis, Mesomachilis, and some other genera of Machilinae; the ovipositor is short, not surpassing the tip of the styli IX, and is generally thickened distally; the tip of the gonapophyses VIII may be pointed, rounded, or have a pointed chitinous projection; 2) the secondary type is usually found in Machilinae and rarely in Petrobiinae and Meinertellidae; the ovipositor is also short, not surpassing the tip of the styli IX, and is generally thickened distally, but the gonapophyses VIII bear strong digging bristles and always lack chitinous teeth on the distal articles, and their tips are never pointed; 3) the tertiary type is common in Machilinae, Petrobiinae, and Meinertellidae; the ovipositor is long, usually surpassing the tip of the styli IX, and is not thickened distally; the chaetotaxy is regular and lacks specialized setae on the gonapophyseal articles; and 4) the quaternary type is known only from some genera of Meinertellidae; it resembles the tertiary type but shows an interrupted pattern in chaetotaxy on the distal articles of the gonapophyses, with setation lacking at intervals. Sturm and Bach de Roca (1993) stated that the primary type is clearly the ancestral form, and the quaternary type is the most derived. Although they did not discuss the phylogenetic implications of the secondary and tertiary types, these two are the most widely distributed among the four. The ovipositor of Petrobiellus is classified as the tertiary type.

As summarized by Sturm and Machida (2001), spermatophore transfer in Archaeognatha is classified into three modes (see 4.3.3.). The first mode, “indirect transfer of spermatophore(s) deposited on carrier thread” (mode 1), is known from Machilinae of Machilidae and Pedetontus in Petrobiinae of Machilidae, and considered the ancestral mode and the groundplan of spermatophore transfer in Archaeognatha. The other two modes are derived and involve no thread-spinning: one is the “direct transfer of spermatophore by deposition on ovipositor” (mode 2), known from Petrobius of Petrobiinae; the other is the “indirect transfer of spermatophore deposited on stalk” (mode 3), known exclusively in Meinertellidae. The mating behavior of Petrobiellus, described in 3.2. and discussed in 4.3.2., represents a unique mode that cannot be classified within any of the three previously known categories. In this newly observed behavior, the spermatophore is discharged directly onto the ovipositor, which is grasped by the penis and held by the styli IX, and transferred directly to the female. During mating, the male of Petrobiellus protrudes his penis at a right angle from between the coxites IX and grasps the basal region of the ovipositor with the distally-curved penis. The effective grasping of the ovipositor is facilitated by the suitable length of the penis, the dorsally curved, hook-like shape of its distal article, and the dense setation on the dorsal surfaces of the posterior part of the proximal article and the distal article. The styli IX on both sides also assist in holding the ovipositor in coordination with the penis. The concaved dorsomesal surfaces of the styli, furnished with many strong spines, are well-suited for this role. The coxites IX are mesally concave, creating a wide space between them, which allows the penis greater freedom of movement. Additionally, the row of strong setae near the distal ends of the coxites IX may function to prevent the ovipositor from slipping forward. Thus, the unique and specialized features of the male genitalic region in Petrobiellus are clearly associated with this specialized mode of mating and can be regarded as remarkable autapomorphies of the genus.

Klass and Matushkina (2018) argued that mating involving the spinning of threads, which is most commonly observed in Archaeognatha, the sistergroup of Dicondylia, and exclusively in all Zygentoma, the sistergroup of Pterygota, represents the groundplan of Ectognatha. They focused on the distribution pattern of tubular setae, which are specialized for spinning threads, in Archaeognatha and Zygentoma. These setae are present only on the parameres in archaeognathans that perform mode 1 mating (see Klass and Matushkina 2018); on both the parameres and the penis in Lepisma saccharinum Linnaeus, 1758 [see Sturm 1956; Matushkina and Klass 2020; Sturm (1997) mentioned their presence only on the parameres] and only on the penis in Thermobia domestica (Packard, 1873) and Tricholepidion gertschi Wygodzinsky, 1961 (see Sturm 1987, 1997; Matushkina and Klass 2020). Based on this, Klass and Matushkina (2018) inferred that the groundplan of Ectognatha may have included tubular setae on both the parameres and the penis. In this context, Mesomachilis, sometimes referred to as a paleoform of Archaeognatha, is particularly noteworthy. Mesomachilis is composed of two subgenera: Mesomachilis Sturm, 1991 and Raromachilis Sturm, 1991. In the former, tubular setae are present on both the parameres and the penis, whereas in the latter, they are found only on the parameres (Sturm 1991).

Petrobiellus possesses parameres only on abdominal segment IX, while lacking those of the abdominal segment VIII. The parameres of Petrobiellus are thin and have not been observed to actively participate in mating; they hug to the ventral side of the penis, although they are occasionally found to diverge from it. However, Sturm and Machida (2001) and Klass and Matushkina (2018) inferred that the parameres of P. takunagae, which bear numerous setae on their surfaces, may function as sensors for detecting the positional relationship between the penis and the ovipositor. Sturm and Bach de Roca (1993), Sturm and Machida (2001), and Matushkina and Klass (2020) proposed that 1) the presence of both parameres VIII and IX, 2) articulation of the distal article, and 3) possession of tubular setae represent the groundplan features in Archaeognatha; conversely, 1) the reduction of the parameres VIII (and even the parameres IX), 2) obliteration of articulation in the distal article, and 3) loss of tubular setae are considered derived features. Thus, the lacking of the parameres VIII and the absence of tubular setae in the parameres IX of Petrobiellus can be understood as derived conditions.

4.3. Sperm transfer in Archaeognatha

4.3.1. Spermatophores of Archaeognatha: droplet or typical?

Sperm transfer of the kind observed in Collembola and Diplura is considered the groundplan for Hexapoda, wherein a simple spherical spermatophore is indirectly delivered from the male to the female (Dallai et al. 2013; Beutel et al. 2014). In Archaeognatha and Zygentoma, spermatophore transfer is still performed indirectly but in a more rapid and elaborate manner; and in Pterygota, the use of spermatophores remains widespread in Pterygota as well (Schaller 1979; Heming 2003; Dallai et al. 2013).

Two types of spermatophores are distinguished (Mann 1984): the largely naked sperm-drop type, referred to as the “droplet spermatophore”, and the properly encapsulated type, referred to as the “typical spermatophore”, which may have specialized coverings or internal structures. The former is characteristic of Entognatha, whereas the latter occurs in Pterygota (Schaller 1979; Mann 1984; Heming 2003; Dallai et al. 2002, 2013). The spermatophore of Zygentoma is spherical or oval and can be categorized as a typical spermatophore, featuring a well-structured outer covering and differentiated internal organization, as also observed in Pterygota (Sturm 1956; Schaller 1979; Mann 1984; Thys 1989; Heming 2003; Dallai et al. 2013). The “typical spermatophore” may then represent a groundplan apomorphy of Dicondylia. The spermatophore of Archaeognatha, which is spherical and appears to be a naked spermatophore (see Sturm and Machida 2001; Klass and Matushkina 2018, and references therein), has traditionally been referred to as a droplet spermatophore (see Sturm and Machida 2001). However, Goldbach (2000), based on histological examination of the spermatophores of Machilis germanica Janetschek, 1953 (Machilidae) and Machilinus rupestris Lucas, 1846 (Meinertellidae), claimed that archaeognathan spermatophores should be considered typical spermatophores due to the presence of a “sheath”. However, Sturm and Machida (2001) dismissed this interpretation, at least for M. germanica, citing reasons such as the possibility that the concentration of the sperm suspension is not uniform and may be higher at the surface or that the liquid including the sperm may have coagulated during fixation, creating a sheath-like layer artifact. In the present study, we found an alcohol-preserved female specimen of a female Petrobiellus akkesiensis with a spermatophore attached to her ovipositor (Fig. 5). The spermatophore appears to have a cortical layer, so that it appears to be a typical spermatophore. However, several observations lead us to hesitate in categorizing it as such: 1) the P. akkesiensis spermatophore observed under SEM is conical (Fig. 5C), and the tip of this conical spermatophore fits precisely between the left and right gonapophyses at the base of the ovipositor; however, it is difficult to imagine a well-defined, rigid structure like a typical spermatophore fitting into such a narrow space (Fig. 5C); 2) notably, numerous sperm are embedded in and partially exposed on the surface of the spermatophore (Fig. 5D, E), supporting the idea that the spermatophore is a coagulated droplet rather than a structured capsule; it is plausible that the sperm suspension coagulated upon alcohol fixation, with sperm becoming encapsulated and partially exposed on the surface; and 3) the spermatophore of P. akkesiensis observed in the video is nearly spherical (Fig. 4D), a form common in Archaeognatha (see Sturm and Machida 2001). The unusual conical shape seen under SEM is understandable if we assume that the specimen was fixed at a stage when the sperm fluid was flowing between the gonapophyses (Fig. 4I); notably, the volume of this fixed spermatophore is approximately 0.005 mm³, which is remarkably smaller than the maximum recorded volume of the P. akkesiensis spermatophore, at approximately 0.18 mm³ (see 3.2.).

Klass and Matushkina (2018), in their review of mating behavior in Archaeognatha, stated that most spermatophores can be classified as droplet spermatophores, which they refer to as “fluid sperm”, but that the spermatophore of Mesomachilis, which is assumed to exhibit a novel form of mating behavior in Archaeognatha, and the spermatophore of Meinertellidae, in which the “indirect transfer of spermatophore deposited on stalk” mode (mode 3) is performed, are possibly typical spermatophores. The spermatophore of Neomachilellus scandens Wygodzinsky, 1978 (Meinertellidae), which has been observed to be attached to the ovipositor, is not spherical but pointed-oval in shape (Sturm and Adis 1984; Sturm and Machida 2001), and it is likely to be a typical spermatophore. However, determining whether a spermatophore is a droplet or typical type requires various lines of evidence, including histological, histochemical, and ultrastructural analyses as well as detailed study of the morphology of the male reproductive organs. Therefore, it is premature to draw definitive conclusions at this stage, as such analyses have not yet been conducted. The sperm spherule in Archaeognatha has been variously referred to as “fluid sperm”, “sperm droplet”, “droplet spermatophore”, “sperm packet”, and “typical spermatophore”, but in this section and throughout the present study, we consistently refer to it simply as the “spermatophore”, without paraphrasing.

4.3.2. Mating behavior of Petrobiellus

Klass and Matushkina (2018) and Matushkina and Klass (2020) suggested that Petrobiellus does not engage in mode 1 mating behavior “indirect transfer of spermatophore(s) deposited on carrier thread”, which involves thread-spinning and is considered the basic form of mating in Archaeognatha, because Petrobiellus males lack tubular setae on both the penis and the parameres. Furthermore, the specialized male genitalic region and the distally-hooked penis, which likely serves a grasping function, strongly indicate that Petrobiellus exhibits a form of direct spermatophore transfer that differs from any previously known modes of mating behavior in Archaeognatha (Sturm and Bach de Roca 1993; Sturm and Machida 2001; Klass and Matushkina 2018; Matushkina and Sturm 2020). In the present study, we examined in detail, for the first time, the mating behavior of this genus using Petrobiellus akkesiensis. As previously hypothesized, this species performs a direct transfer of the spermatophore involving genital coupling, a form of mating entirely distinct from those previously described in Archaeognatha. We designate this newly identified behavior as “direct transfer of spermatophore by genital coupling” (mode 4). We summarize this mating behavior in Petrobiellus.

1. The male rushes toward the female, places his maxillary palps on her thorax from the side, secures his partner by bringing his forelegs and head close to her body, and immediately begins vibrating the female. Without delay, the male twists his body and presses his postabdomen against the female’s postabdomen.

2. Soon, the ovipositor is held at three points by the male’s penis and styli IX: the male protrudes his penis at a right angle to his abdomen and grasps the basal part of the female’s ovipositor with his distally-curved penis; the male’s left and right styli IX secure the middle part of the ovipositor from above and below.

3. Once the holding of the ovipositor is established, the male’s ventral sacs begin to swell. Simultaneously, a spermatophore is ejaculated onto the dorsal side of the basal region of the ovipositor from the genital opening at the tip of the penis. The spermatophore gradually increases in size.

4. Once the spermatophore reaches its maximum size, the male’s ventral sacs begin to deflate, simultaneously the spermatophore begins to decrease in size, thereby completing the spermatophore transfer.

5. After spermatophore transfer is completed, the male releases his hold on the ovipositor, first with the styli IX, followed by the penis.

The expansion and contraction of the male’s ventral sacs during mating could be precisely observed. The increase in body fluid pressure, indicated by the swelling of the ventral sacs, appears to be directly related to the deposition of the spermatophore. In fact, the timing of ventral sac enlargement closely corresponded with the increase in spermatophore volume (Fig. 4B–D).

Klass and Matushkina (2018) carefully examined the male genitalic region of Petrobiellus takunagae from both morphological and functional morphological perspectives and proposed two hypotheses regarding the mode of spermatophore transfer in Petrobiellinae: 1) the “up-and-down hypothesis”, wherein the ovipositor is held by the penis and a pair of styli IX of the male, with the penis applying force from the ventral side of the ovipositor in the dorsal direction and the styli applying force from the dorsal side in the ventral direction; and 2) the “all-up hypothesis”, wherein the ovipositor is held through the combined action of the penis, styli IX, and the paranotal lobes of the abdominal segment X, with the penis and styli applying upward force from below and the paranotal lobes providing resistance from above. Our present study confirmed that the ovipositor is indeed held by the penis and styli IX during mating in Petrobiellus, as proposed by Klass and Matushkina (2018), but in a manner different from either of their hypotheses. Specifically, the ovipositor is securely held at three points: its basal part is grasped by the penis and its middle part is supported from above and below by the styli IX (Fig. 4B–G). Fig. 6 presents a drawing of the condition in which spermatophore delivery is nearly complete, nearly corresponding to the state depicted in Fig. 4D.

Figure 6.

Drawing showing sperm transfer in Petrobiellus akkesiensis, reconstructed based on Fig. 4D. The ovipositor is held at the three points (arrows) by the male, with the penis and the styli IX of both sides. See the text for details. Numerals indicate the abdominal segment number to which each structure belongs. — Abbreviations: Ce – cercus; CF – caudal filament; S6–8 – sternites VI to VIII; T7–10 – terga VII to X.

The basic form of mating behavior in Archaeognatha is mode 1 “indirect transfer of spermatophore(s) deposited on carrier thread”, and mating behavior involving thread-spinning is considered the groundplan of Ectognatha (Klass and Matushkina 2018). In groups that perform this mode of mating, the parameres (and rarely also the penis) bear tubular setae used for spinning. Petrobiellus (Petrobiellinae) possesses parameres only in the abdominal segment IX, which are long but not well developed, and both the parameres IX and the penis lack tubular setae. The mode 4 mating behavior “direct transfer of spermatophore by genital coupling” observed in Petrobiellus is clearly a derived condition. Similarly, Petrobius (Petrobiinae) has parameres only on abdominal segment IX, which are short, weakly developed, and devoid of tubular setae. The mode 2 mating behavior “direct transfer of spermatophore by deposition on ovipositor” performed by Petrobius (Sturm 1978; Sturm and Machida 2001) is likewise a derived form. In both mode 2 (Petrobius) and mode 4 (Petrobiellus), the spermatophore is directly transferred and placed onto the ovipositor. The key difference lies in whether the ovipositor is physically held during transfer (mode 4) or not (mode 2).

Although a shared origin is possible, the direct transfer of spermatophores in mode 2 (Petrobius) and mode 4 (Petrobiellus) is more likely to have evolved independently, because other members of Petrobiinae perform the mating behavior of mode 1, which is regarded as the groundplan in Archaeognatha. We leave this phylogenetic question open for future research and instead highlight the similarities between Petrobius and Petrobiellus (see also 4.3.3.). First, both genera are halophilic. It is plausible that direct spermatophore transfer evolved in such habitats, where exposure of spermatophores to the atmosphere must be minimized and mating must be completed quickly. Second, foreplay is extremely reduced in both. In Petrobiellus, upon encountering a female, the male immediately holds and begins vibrating her without delay. In rapid cases, the ovipositor is held and spermatophore transfer begins within 1 min of initial contact (File S2). Although vibration may be considered part of foreplay, it also occurs intermittently during and after spermatophore transfer, suggesting that it is not limited to foreplay. Similarly, in Petrobius, foreplay is minimal, and spermatophore transfer is repeated up to 10 times in rapid succession, with intervals of ≤1 min (Sturm 1978; Sturm and Machida 2001). Because mode 1, which is groundplan mating behavior in Archaeognatha, mode 3 of Meinertellidae (see Sturm and Machida 2001), and the mating behavior of Zygentoma (Sturm 1956, 1987, 1996, 1997) all involve elaborate and time-consuming foreplay, it is evident that such foreplay represents the ancestral condition. Therefore, the extreme reduction of foreplay observed in modes 2 and 4 is a derived trait. Third, in both Petrobius and Petrobiellus, the spermatophore is placed at the basal part of the ovipositor. Fourth, in both genera, the volume of a single spermatophore (Petrobiellus) is large, and the total volume of spermatophores delivered to the female in one round of mating (Petrobius) is substantial. Table 2 presents the spermatophore sizes of various archaeognathan species, as cited from previous studies, along with the calculated volumes (total volumes, in cases where multiple spermatophores are released in one mating round), including new data for P. akkesiensis (herein) and Pedetontus unimaculatus Machida, 1980 (whose mating behavior has not previously been illustrated with clear photographs; here, it is included in Fig. S2). In the case of Petrobius, although precise measurements of spermatophore size and volume are not provided, it is noted that relatively large spermatophores are repeatedly passed to the female multiple times during a single mating event (Sturm 1978; Sturm and Machida 2001), indicating that a considerable total volume is transferred. The volume of a single spermatophore in Petrobiellus (0.18 mm³) is also notably larger than those of other archaeognathan species.

Table 2.

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Spermatophores of Archaeognatha: the diameter, number of spermatophore(s), and (total) volume of spermatophore(s) deposited in one round of mating, calculated from the size and number of spermatophore(s).

Family	Subfamily	Species	Diameter (mm)	Number	Total volume (mm³)	Reference
Machilidae	Machilinae	Lepismachilis y-signata	not shown	1	―	Sturm (1955)
Machilidae	Machilinae	Machilis germanica	0.2–0.4	1–5	0.04–0.07	Sturm (1955)
Machilidae	Machilinae	Promesomachilis hispanica	0.08–0.13	14–19	0.004–0.02	Sturm (1992)
Machilidae	Petrobiinae	Pedetontus unimaculatus	0.5	1	0.07	herein
Machilidae	Petrobiinae	Petrobius maritimus	relatively large	several, < 10	voluminous	Sturm (1978), Sturm and Machida (2001)
Machilidae	Petrobiellinae	Petrobiellus akkesiensis	0.7	1	0.18	herein
Meinertellidae	―	Machilinus rupestris	0.5	1	0.07	Goldbach (2000)
Meinertellidae	―	Machiloides tenuicornis	0.3	1	0.014	Sturm (1987)
Meinertellidae	―	Neomachilellus scandens	0.5	1	0.07	Sturm and Adis (1984)

4.3.3. Mating behavior of Archaeognatha

Three modes of mating behavior, i.e., spermatophore transfer, have been identified in Archaeognatha (Sturm and Machida 2001). The first mode is “indirect transfer of spermatophore(s) deposited on carrier thread” (mode 1), which is known from Machilinae of Machilidae and from Pedetontus in Petrobiinae of Machilidae. Examples include Machilis germanica (Sturm 1955), Lepismachilis y-signata Kratochvil, 1945 (Sturm 1955), Promesomachilis hispanica Silvestri, 1912 (Sturm 1992), Dilta insulicola Wygodzinsky, 1941 (Sturm 1986), Trigoniophthalmus alternatus (Silvestri, 1904) (Sturm and Machida 2001), Pedetontus californicus (Silvestri, 1911) (Sturm and Machida 2001), Pedetontus schicki Sturm, 2001 (Sturm and Machida 2001), and Pedetontus unimaculatus (Sturm and Bach de Roca 1993; Fig. S2). Following an extended foreplay involving a patterned dance by the male and female, the male spins a taut thread composed of many fine filaments between the parameres, which bear tubular setae specialized for spinning, and the substrate. He then places one or more spermatophores on the thread and escorts the female so that she can receive the spermatophore with her ovipositor. The female then picks up the spermatophore(s) and transfers them into her body, completing the mating process. The second mode is “direct transfer of spermatophore by deposition on ovipositor” (mode 2), known from Petrobius of Petrobiinae, whose parameres (and penis) lack tubular setae for spinning. Examples include Petrobius maritimus (Leach, 1809) (Sturm 1978) and Petrobius brevistylis (Carpenter, 1913) (Hagens 1989). In this mode, foreplay is minimal. When the female abruptly lifts her abdomen, the male arches his body and places a relatively large spermatophore on the proximal half of the female’s ovipositor, which she then takes into her body. This process is repeated several times in quick succession, after which mating is complete. The third mode is “indirect transfer of spermatophore deposited on stalk” (mode 3), known only from Meinertellidae. Examples include Machiloides tenuicornis Stach, 1930 (Sturm 1986, 1987), Neomachilellus scandens (Sturm and Adis 1984), Neomachilellus adisi Wygodzinsky, 1978 (Sturm and Machida 2001), and Machilinus rupestris (Goldbach 2000). In this mode, the male initiates mating with prolonged, patterned foreplay, followed by placing a spermatophore on a stalk composed of glandular secretions originating from the vasa deferentia (Bitsch 1968). He escorts the female until her ovipositor aligns with the spermatophore, which she picks up from the tip of the stalk, completing the mating. In Meinertellidae, males lack parameres, and their penis does not bear tubular setae; therefore, threads are never spun.

The mating behavior of Zygentoma has been studied in Lepidotrichidae and Lepismatidae, including Tricholepidion gertschi (Sturm 1996, 1997), Lepisma saccharinum (Sturm 1956), and Thermobia domestica (Sweetman 1938; Sturm 1987; Inada et al. 2023). After foreplay, the male places a round or pear-shaped spermatophore on a web, thread(s), or a network of threads spun by himself, and the female picks it up with her ovipositor to complete mating (Sturm and Machida 2001). Given that thread-spinning is also involved in the mating behavior of Zygentoma, it is most reasonable to consider that mode 1 mating behavior using threads is the groundplan of Archaeognatha, whereas mode 2 in Petrobius and mode 3 in Meinertellidae are derived conditions.

It has been suggested that several groups within Archaeognatha may exhibit mating behaviors that do not fit into any of the previously known modes 1–3. The present study, focusing on one such group, Petrobiellinae, revealed that this archaeognathan performs a distinct form of mating behavior, classified here as a new mode: “direct transfer of spermatophore by genital coupling” (mode 4). Mode 4, which does not involve thread-spinning, is considered to be a derived form evolved from mode 1. Within Archaeognatha, only Petrobius (mode 2) and Petrobiellinae (mode 4) exhibit direct spermatophore transfer, and the direct spermatophore transfer behaviors of Petrobius and Petrobiellinae share several features, as discussed in section 4.3.2.

Koch (2003) and Matushkina and Klass (2020) argued that the status of the paleoforms Mesomachilis, Charimachilis, and Ditrigoniophthalmus [with Turquimachilis also included by Bach de Roca et al. (2013)] as ancestral lineages cannot be definitively established. Because Charimachilis and Ditrigoniophthalmus lack tubular setae (Matushkina and Klass 2020; in the case of Ditrigoniophthalmus, based on inference), Matushkina and Klass (2020) stated that these taxa are unlikely to perform the mode 1 mating behavior and that they may instead exhibit some form of direct spermatophore transfer, although no structures specialized for sexual coupling have been identified in them. Sturm and Machida (2001) hypothesized that Mesomachilis performs indirect spermatophore transfer using a net spun by tubular setae located on the parameres (and also on the penis, if present) (Sturm 1991). This mating behavior, as assumed for Mesomachilis, may be considered a subform of mode 1 behavior. However, considering that the presence of tubular setae on both the parameres and the penis represents the groundplan in Ectognatha as Klass and Matushkina (2018) suggested (see 4.2.), the mating behavior of Mesomachilis might reflect the ancestral state in Archaeognatha.

In the early stages of hexapod terrestrial adaptation, insemination occurred through simple indirect sperm transfer, as commonly observed in Entognatha. As evolving into the apterygote Ectognatha, their mode of indirect sperm transfer became increasingly elaborate. Eventually, direct sperm transfer associated with the acquisition of copulatory organs emerged in Pterygota. Along this evolutionary trajectory, various transitional strategies for sperm transfer likely arose. The present study reports, for the first time in apterygote hexapods, direct sperm transfer via genital coupling in a representative of Archaeognatha, specifically Petrobiellinae. This may represent one of the earliest evolutionary experiments in the innovation of sperm transfer. The mating behavior observed in Petrobius (mode 2) and the presumptive mating behavior in Turquimachilis may also be considered such transitional forms. However, within Archaeognatha, these behaviors are likely derived from mode 1, and it is unlikely, at least in a strict evolutionary sense, that they gave rise directly to copulatory mating in Pterygota, even if the early forms of copulation-based sperm transfer in Pterygota bore similarity to these archaeognathan behaviors. To more accurately identify the ancestral mating modes that led to direct sperm transfer via copulation in Pterygota, further focused investigation is needed in the basalmost clade of Dicondylia, i.e., Zygentoma, in which only indirect sperm transfer has been documented so far.

5. Acknowledgments

We are grateful to Dr. Tadaaki Tsutsumi of Fukushima University for his valuable suggestions, to Ms. Mitsuki Mtow for her kind assistance with the rearing of materials. Thanks are also due to Dr. Nikolaus Szucsich of the Naturhistorisches Museum Wien, Dr. Natalia Matushukina of the Taras Shevchenko National University of Kyiv, two anonymous reviewers, Dr. Klaus-Dieter Klass of the Senckenberg Natural History Collections Dresden, and Dr. Monika Eberhard of the Universität Hamburg for their critical review of the manuscript, and to ENAGO (www.enago.jp) for the English language review. This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI: Grant-in-Aid for JSPS Research Fellow, JP20J00039 to SM and Scientific Research (C), JP19K06821 to RM.

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Supplementary materials

Supplementary material 1

Figures S1, S2

Mtow S, Machida R (2025)

Data type: .zip

Explanation notes: Figure S1. Close-up photography device for observing the mating behavior of Petrobiellus akkesiensis from below. A Binocular stereomicroscope (Nikon SMZ800) with its lens barrel turned upside down, with lighting equipment positioned at the objective side. B Photographic stage with the microscope set. — Figure S2. Mating behavior of Pedetontus unimaculatus. A Single frame of mating in P. unimaculatus, with specimens collected in Shimoda, Shizuoka, Japan, in June 1976. B Enlargement of a spermatophore on the carrier thread spun between the parameres and substrate. – Abbreviations: Pa9 – paramere IX; Sp – spermatophore; T – carrier thread. – Scale: 1 mm.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

Download file (6.47 MB)

Supplementary material 2

Files S1–S3

Mtow S, Machida R (2025)

Data type: .zip

Explanation notes: File S1. Edited movie showing mating behavior in Petrobiellus akkesiensis, Case I in Table 1. Counters indicate the time from the commencement of the experiment, i.e., when a male and a female were placed in the experimental arena [.mov-file]. — File S2. Edited movie showing mating behavior in Petrobiellus akkesiensis, Case V in Table 1. Counters indicate the time from the commencement of the experiment, i.e., when a male and a female were placed in the experimental arena [.mov-file]. — File S3. Edited movie showing mating behavior in Petrobiellus akkesiensis, Case VI in Table 1. Counters indicate the time from the commencement of the experiment, i.e., when a male and a female were placed in the experimental arena [.mov-file].

Download file (41.82 MB)

Abstract

Keywords

﻿1. Introduction

﻿2. Materials and methods

﻿3. Results

﻿3.1. Genitalic regions of Petrobiellus akkesiensis

﻿3.2. Mating behavior of Petrobiellus akkesiensis

﻿4. Discussion

﻿4.1. Systematics of Petrobiellinae and Petrobiellus

﻿4.2. Genitalic region of Petrobiellus

﻿4.3. Sperm transfer in Archaeognatha

﻿4.3.1. Spermatophores of Archaeognatha: droplet or typical?

﻿4.3.2. Mating behavior of Petrobiellus

﻿4.3.3. Mating behavior of Archaeognatha

﻿5. Acknowledgments

﻿6. References