3.3.1. Sperm Heads: Nuclei and Acrosomes
Multiple transverse and longitudinal sections of spermatozoa and spermatids were examined in this study. The acrosome-nucleus interfaces for the four beetle species and for the strepsipteran are marked with arrows in
Figure 3. The acrosomes of the species of Meloids that are illustrated here,
Z. flava (subfamily Nemognathinae) and
H. scutellatus (subfamily Meloinae), have a simple organization (
Figure 3A,B) and lack the conspicuous subacrosomal material observed in some other beetles (
Figure 3C). A longitudinal section of a spermatid acrosome of
M. variabilis (subfamily Meloinae) also illustrates this simple organization of the acrosome in the Meloinae (inset,
Figure 3A). The organization of the rhipiphorid acrosome with its conspicuous subacrosomal material (
Figure 3C), however, is clearly an exception to the simple layered arrangement observed for meloid and strepsipteran acrosomes (
Figure 3A,B,D); by contrast, each meloid and strepsipteran acrosome has a uniformly dense interior circumscribed by a lighter electron-dense periphery and an outermost electron-dense membrane.
Figure 3.
The acrosome forms a straight interface (arrows) with the adjacent nucleus in Meloids and Strepsipteran (A, B, D). The rhipiphorid acrosome, by contrast, with its subacrosomal material (sa) forms a curved interface with the nucleus. (C) Its interface with the nucleus is marked with three arrows. The acrosome is at right in all figures. (A) Hycleus scutellatus. Inset = Mylabris variabilis. (B) Zonitis flava. (C) Macrosiagon triscuspidata. (D) Eoxenos laboulbenei. The insets in C and D show shapes of nuclei in transverse sections for M. triscuspidata and E. laboulbenei, respectively. Scale bars = 1.0 μm in A; scale bar = 0.5 μm in B; scale bars = 0.25 μm in C, D.
Figure 3.
The acrosome forms a straight interface (arrows) with the adjacent nucleus in Meloids and Strepsipteran (A, B, D). The rhipiphorid acrosome, by contrast, with its subacrosomal material (sa) forms a curved interface with the nucleus. (C) Its interface with the nucleus is marked with three arrows. The acrosome is at right in all figures. (A) Hycleus scutellatus. Inset = Mylabris variabilis. (B) Zonitis flava. (C) Macrosiagon triscuspidata. (D) Eoxenos laboulbenei. The insets in C and D show shapes of nuclei in transverse sections for M. triscuspidata and E. laboulbenei, respectively. Scale bars = 1.0 μm in A; scale bar = 0.5 μm in B; scale bars = 0.25 μm in C, D.
The nuclei of all meloid and rhipiphorid sperm are circular in transverse section (inset,
Figure 3C); the elongate nuclei of
E. laboulbenei, like the nuclei of other strepsipteran sperm [
1,
13,
14,
15], are kidney-shaped in cross section (
Figure 1D; inset,
Figure 3D).
In the ground plan of sperm architecture, the acrosomes of Coleoptera and Strepsiptera have been described as multi-layered and mono-layered respectively [
15]. The acrosome of the rhipiphorid
Pelecotoma fennica (subfamily Pelecotominae) [
14]) was described as being three-layered while the acrosome of the meloid
Lytta vesicatoria (subfamily Meloinae) was described as being two-layered [
15]. With the one exception of
Xenos vesparum examined by Dallai
et al. [
1], all other Strepsiptera examined including other
Xenos species have been reported to have acrosomes [
13,
14,
16].
3.3.2. Sperm Necks
The structure of this region shows marked differences between subfamilies in the family Meloidae:
H. scutellatus represents the subfamily Meloinae and
Z. flava represents the subfamily Nemognathinae. In the neck region of the sperm, the posterior end of the nucleus forms a straight interface with the flagellum as in
H. scutellatus (arrows,
Figure 4A); or in the case of
Z. flava,
M. tricuspidata, and
E. laboulbenei, the posterior pole of the nucleus is indented at its interface with the anterior end of the mitochondrial derivatives (double arrows,
Figure 4B–D). In these three latter images, the interface of the axoneme and the nucleus lies at a more posterior location: about 0.25 μm from the anterior end of the mitochondrial derivatives.
Figure 4.
Junction of posterior end of nucleus with axoneme and mitochondrial derivatives. Each single arrow points to posterior process of nucleus that interfaces with the axoneme. Double arrows indicate the interface of sperm nucleus and mitochondrial derivative. Arrowheads point to uncondensed chromatin. (A) Hycleus scutellatus. (B) Zonitis flava. (C) Macrosiagon triscuspidata. (D) Eoxenos laboulbenei. Scale bars = 1.0 μm in A–C; scale bar = 0.5 μm in D.
Figure 4.
Junction of posterior end of nucleus with axoneme and mitochondrial derivatives. Each single arrow points to posterior process of nucleus that interfaces with the axoneme. Double arrows indicate the interface of sperm nucleus and mitochondrial derivative. Arrowheads point to uncondensed chromatin. (A) Hycleus scutellatus. (B) Zonitis flava. (C) Macrosiagon triscuspidata. (D) Eoxenos laboulbenei. Scale bars = 1.0 μm in A–C; scale bar = 0.5 μm in D.
In the case of the sperm of
Z. flava,
M. tricuspidata, and
E. laboulbenei, a process extends posteriorly from the posterior-most end of each nucleus and interfaces with the edge of the axoneme adjacent to the mitochondrial derivatives. These nuclear processes are marked with single arrows in
Figure 4B–D. In
E. laboulbenei, this interface of the nucleus with the mitochondrial derivative of the flagellum (double arrows,
Figure 4B) is consistently occupied by a cluster of distinctive vesicles, each measuring approximately 18 nm.
In both the strepsipteran and the two beetle families, the head region of the sperm is a very elongate nucleus containing regions of uncondensed chromatin, the latter indicated with arrowheads in
Figure 4A–D.
3.3.3. Sperm Flagella: Axonemes, Mitochondrial Derivatives and Accessory Bodies
Many distinguishing characters of insect sperm lie within the flagellum: (1) the axoneme or the microtubule-based cytoskeleton of the flagellum; (2) one or two mitochondrial derivatives; and (3) one, or more commonly two, accessory bodies that represent extensions of the centriole adjunct that forms a collar at the base of the flagellum.
Figure 5A–C show two meloid axonemes (A,B) and a rhipiphorid axoneme (C). Features that are not pronounced for the axoneme of
E. laboulbenei (
Figure 5D) but are conspicuous in all the beetle axonemes (
Figure 5A–C) are the nine radial links between the two central tubules and the doublet tubules. Intertubular material [
1,
11] is observed in axonemes of the two beetle families examined as well as in axonemes of
E. laboulbenei.Mitochondrial derivatives of the four beetles examined here all contain crystalline inclusions. Each arrangement of inclusions within the mitochondrial derivatives, however, is distinctive (
Figure 5A–C, m). Mitochondrial derivatives of
E. laboulbenei, by contrast, lack conspicuous crystalline inclusions but have distinctive U-shaped configurations (
Figure 5D, m).
While sperm of Rhipiphoridae have accessory bodies (
Figure 5C, asterisk) that resemble those of sperm from a member of the meloid subfamily Nemognathinae (
Figure 5B, asterisk), spermatozoa of meloid species in the subfamily Meloinae (
Figure 5A, asterisk) more closely resemble sperm of
E. laboulbenei (
Figure 5D, asterisk) in lacking conspicuous accessory bodies.
In previous publications, a marked ultrastructural difference between Strepsiptera and other neuropteroid orders had been noted in the flagellar axoneme. The accessory tubules or peripheral singlets of the flagella in the Strepsipteran examined had 16 protofilaments (as do other neuropteroid orders) and had been described as having an incomplete “wall” resulting in a reniform or kidney shape that had not been observed in any other insect order [
11,
15]. This was considered the most compelling synapomorphy uniting the Strepsiptera. However, this structural feature of the singlets has been considered a function of the stage of sperm development and does not seem to persist in more mature accessory tubules [
17]. The circular forms of singlet or accessory tubules of the
Eoxenos axoneme of mature sperm do not match the reniform shapes of these tubules reported for the immature sperm of
Stylops sp. [
11]—a species that was incorrectly identified and is actually
Xenos sp.
The lack of conspicuous accessory bodies has been a feature noted for the sperm of all strepsipteran species (families Xenidae, Elenchidae, Halictophagidae) examined in previous reports [
11,
13,
14,
16,
18]. The flagella of the sperm of
E. laboulbenei in the family Mengenillidae likewise do not have discrete accessory bodies (
Figure 5D, asterisk). Interestingly, absence of accessory bodies is a sperm character shared with members of the entire order Diptera [
15].
Figure 5.
High magnification images of beetle and strepsipteran axonemes, mitochondrial derivatives and accessory bodies. These transverse sections of flagella are aligned so that mitochondrial derivatives (m) lie at the bottom in each image; axonemes lie at the top. Positions of distinct accessory bodies in B, C or indistinct accessory bodies in A, D are marked with small asterisks. (A) H.scutellatus (Meloidae, Meloinae). (B) Z. flava (Meloidae, Nemognathinae). (C) M. triscuspidata (Rhipiphoridae, Rhipiphorinae). (D) Eoxenos laboulbenei. Each scale bar = 0.25 μm.
Figure 5.
High magnification images of beetle and strepsipteran axonemes, mitochondrial derivatives and accessory bodies. These transverse sections of flagella are aligned so that mitochondrial derivatives (m) lie at the bottom in each image; axonemes lie at the top. Positions of distinct accessory bodies in B, C or indistinct accessory bodies in A, D are marked with small asterisks. (A) H.scutellatus (Meloidae, Meloinae). (B) Z. flava (Meloidae, Nemognathinae). (C) M. triscuspidata (Rhipiphoridae, Rhipiphorinae). (D) Eoxenos laboulbenei. Each scale bar = 0.25 μm.
Despite an earlier claim [
11] that the sperm axoneme of a Strepsipteran (
Xenos sp.) lacked both accessory bodies and “intertubular material”, a more recent publication [
1] claimed that
Xenos axonemes have both intertubular material and accessory bodies. This claim about the presence of distinct accessory bodies, however, is debatable based on careful examination of figures in this publication [
1].
As observed for representative members of other strepsipteran families, the mitochondrial derivatives of
E. laboulbenei do not have the conspicuous, well-defined crystalline inclusions [
13,
14,
16,
18]. Also like all other strepsipterans whose sperm features have been described, the mitochondrial derivatives of
Eoxenos have distinctive U-shaped configurations (
Figure 5D, m).
