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RETRACTED: Sperm ultrastructure in two species of Panorpa and one Bittacus (Mecoptera)
Micron 41 (2010) 622–632
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Sperm ultrastructure in two species of Panorpa and one Bittacus (Mecoptera) Sha Xie, Baozhen Hua ∗ Key Laboratory of Plant Protection Resources and Pest Management, Ministry of Education, Northwest A&F University, Yangling, Shaanxi 712100, China
a r t i c l e
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Article history: Received 23 January 2010 Received in revised form 22 March 2010 Accepted 22 March 2010 Keywords: Insecta Mecoptera Spermatozoa Structure Panorpidae Bittacidae
a b s t r a c t The sperm ultrastructure of the scorpionflies Panorpa liui and P. longihypovalva in Panorpidae and the hangingfly Bittacus planus in Bittacidae were investigated using transmission electron microscopy. The common features of the spermatozoa shared by all the mecopterans examined include a bilayered acrosome with a central perforatorium, an elongated homogeneously condensed nucleus, and a long flagellum with a 9 + 2 axoneme pattern and two mitochondrial derivatives. The two species of Panorpa possess a fossa at the posterior end of the nucleus, and differ from B. planus by lacking both the globular units running laterally from the head to the flagellum and the Golgi complex-derived membrane present in the flagellum. P. liui has pear-shaped mitochondrial derivatives and two small accessory bodies, while P. longihypovalva has elliptical mitochondrial derivatives and only one accessory body. The marked differences of the sperm structure among the Panorpa examined further confirm the paraphyly of this genus. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Mecoptera is an intriguing minor order of insects, with approximately 600 extant described species placed in nine families and 35 genera (Cai et al., 2008; Penny and Byers, 1979; Tan and Hua, 2009a,b). It was regarded as one of the basal lineage in Holometabola based on their larvae having a pair of compound eyes on the head and the fossil record (Byers and Thornhill, 1983). Mecoptera was once argued to be paraphyletic with Siphonaptera based on molecular data (Whiting, 2002). After detailed comparative analysis of the sperm structure, however, Dallai et al. (2003) concluded that Mecoptera is still monophyletic as traditionally defined and treated Boreus hyemalis as a member of Mecoptera rather than of Siphonaptera. As the two most species-rich families in the Mecoptera, Panorpidae and Bittacidae comprise 90% of mecopteran species (Byers and Thornhill, 1983; Kaltenbach, 1978). The Panorpidae, which are distributed in the northern hemisphere, are commonly called scorpionflies because their ninth abdominal segment (genital bulb) in males is enlarged and curves anterodorsally, superficially resembling the stinger of a scorpion. The cosmopolitan Bittacidae are commonly known as hangingflies due to their adults being unable to stand on a surface but suspending from the edges of leaves or from twigs by their forelegs. Panorpa Linneaus was considered by Willmann (1989) to be paraphyletic with Neopanorpa Weele based
on morphological characters. This argument recently received support from molecular data (Misof et al., 2000). Similarly, Bittacus Latreille, the most speciose genus in Bittacidae, was also regarded to be paraphyletic (Lambkin, 1988; Whiting, 2002). Spermatozoa are characterized by patterns of rapid and divergent morphological evolution to the extent that variation in sperm morphology provides a useful tool for the reconstruction of phylogenies in various insect groups, including the Hexapoda, Paraneoptera, Polyneoptera, Neuropteroid orders, Diptera, etc. (Baccetti, 1987, 1998; Jamieson et al., 1995). In Mecoptera, however, the sperm structure is still poorly documented to date. In Panorpidae, spermatozoal ultrastructure has only been surveyed in the European Panorpa annexa and P. germanica (Baccetti, 1972; Baccetti et al., 1969; Dallai et al., 2003), and the North American P. nuptialis (Gassner et al., 1972). In Bittacidae the sperm ultrastructure has only been reported in Bittacus apicalis (Breland et al., 1966), which was transferred into the genus Hylobittacus by Byers (1979). Thus with respect to the genus Bittacus, the sperm structure has not been really studied hitherto. Our objective of this study is to compare the sperm ultrastructure of two species in Panorpa and one species in Bittacus, hoping to provide spermatological data for the future generic revision of these two paraphyletic genera.
2. Materials and methods ∗ Corresponding author at: Entomological Museum, Northwest A&F University, Yangling, Shaanxi 712100, China. Tel.: +86 29 87091342; fax: +86 29 87091342. E-mail addresses: [email protected] (S. Xie), [email protected] (B. Hua). 0968-4328/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2010.03.007
2.1. Specimen collection Male adults of Panorpa liui Hua, 1997 were obtained from the Dongling Park (41◦ 50 N, 123◦ 35 E, 90 m), Shenyang City, Liaoning
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Fig. 1. Panorpa liui spermatozoa. Light micrographs (A) and transmission electron micrographs (B–I) showing late spermatids through testes. (A) Spermatids dissociated from a spermatogonial cyst; (B) gourd-shaped nuclei (N) with two deep lateral grooves (arrows); (C) cross-section through the acrosome, showing a bilayered acrosome (a) and a central perforatorium (p); (D–F) cross-sections of nucleus (N), showing the fossa (arrows) with the tip of the larger mitochondrial derivative (md1) inserted; (G–I) cross-sections in the centriole adjunct region. In (G), observe the larger and the smaller mitochondrial derivatives (md1 and md2, respectively); in (H), two mitochondrial derivatives (md1, md2) and the centriole (c) are surrounded by the centriole adjunct (ca); in (I), the two mitochondrial derivatives (md1, md2) enlarge in volume, with the centriole (c) surrounded by centriole adjunct (ca). Scale bars: A = 16 m; B = 1 m; C–I = 0.25 m.
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Province, northeastern China in early August 2009. Male adults of P. longihypovalva Hua and Cai, 2009 were collected in the Huoditang Forest Farm (33◦ 25 N, 108◦ 30 E, 1550 m), Ningshan County, Shaanxi Province in early June 2009. Males of Bittacus planus Cheng, 1949 were captured in the Zhuque Forest Park (33◦ 47 N, 108◦ 35 E, 1620 m), Huxian County, Shaanxi Province in mid July 2009. 2.2. Sample preparation After the live male adults were anesthetized in ethyl ether, their testes were dissected out rapidly in the Ringer’s solution (NaCl:CaCl:KCl:dH2 O = 10 g:0.34 g:0.3 g:1100 ml) and then fixed in a mixture of 2% paraformaldehyde and 2.5% glutaraldehyde in the phosphate buffered saline (PBS, 0.1 M, pH 7.2) for 12 h, to which 1.8% of sucrose was added. The fixed specimens were kept separately with small ice block in a vacuum bottle before being stored at 4 ◦ C. 2.3. Transmission electron microscopy (TEM) The fixed specimens were rinsed with PBS, and post-fixation was performed in 1% osmium tetroxide for 2 h at 4 ◦ C. After other four times 15-min rinse in the same buffer, the samples were dehydrated through a graded ethanol series, and infiltrated with araldite (Histochoice) and embedded in Epon 812. Ultrathin sections were obtained with a diamond knife on an ultramicrotome (Reichert Ultracut E), routinely stained with uranyl acetate and lead citrate, and observed with a JEOL JEM-1230 transmission electron microscope at 80 kV. 3. Results 3.1. Panorpa liui Late spermatids in the deformation zone have a shuttle-shaped head (Fig. 1A). The mature spermatozoon is elongated and filiform, composed of a short acrosome, an elongated nucleus, and a long tail, which consists of a centriole adjunct and a flagellum. The acrosome is very short, about 2.5 m in length (Fig. 2A) and 0.5 m in diameter, conical with two components: a central perforatorium and an outer acrosomal vesicle (Fig. 1C). In longitudinal section, the acrosome has a diameter equal to that of the nucleus at their junction (Fig. 2B and C). The nucleus contains homogeneously condensed chromatin, generally gourd-like in cross-section through the anterior region because of two deep, symmetrical, lateral grooves of the nuclear envelope which extend along the entire length of the nucleus and end in the centriole adjunct region. At this level, the nucleus is about 1.0 m in diameter (Fig. 1B). Posteriorly, there is a small deep cavity in the inner of the nucleus (the nuclear fossa), into which the tip of the larger mitochondrial derivative is adapted (Fig. 1D). Then, the cavity expands with the larger mitochondrial derivative oblique from the centre to one side of the nucleus (Fig. 1E and F). In slightly more posterior region, the smaller mitochondrial derivative appears and extends parallel along the larger one (Fig. 1G). At the basal end of the neck region, a centriole adjunct surrounds the centriole and the two mitochondrial derivatives (Fig. 1H), and connects the sperm head with the tail. Posteriorly the two mitochondrial derivatives are of unequal size (Fig. 1I). The flagellum consists of an axoneme, two accessory bodies, and two mitochondrial derivatives. The axoneme and the two mitochondrial derivatives are of helicoidal array (Fig. 2D and E). The two centriole adjuncts are of crescent form and are located on one side of the two mitochondrial derivatives (Fig. 3A). The axoneme
Fig. 2. Panorpa liui spermatozoa in longitudinal sections through different regions. (A–C) Head of sperm showing the bilayered acrosome, constituted by a central perforatorium (p) and a conical acrosomal vesicle (a) and the long nucleus (N). (D–F) The helicoidal array of mitochondrial derivatives (md1, md2), axoneme (ax) and accessory bodies (arrows). Scale bars: A, C = 1 m; B, D–F = 0. 5 m.
begins to exhibit its outline at this level although its nine microtubular doublets and two central tubules are not clear yet (Fig. 3A). At the anterior region of the sperm flagellum, the smaller mitochondrial derivative becomes larger, almost reaching the same size as the larger one, both being trapezoidal in cross-section (Fig. 3B). Beside the axoneme, near the cell membrane are two small cylindrical accessory bodies, facing the two mitochondrial derivatives (Fig. 3B). In more posterior region, the centriole adjunct disappears. The two mitochondrial derivatives contain dense materials inside, are twice as large as those at the anterior tip, pear-shaped in crosssection, and occupy more than three quarters in volume of the entire cell (Fig. 3C). The accessory bodies are quite small, and one of them becomes smaller and smaller until disappears along with the progressive vanishing of one mitochondrial derivative (Fig. 3D). The accessory body left alone is located on the same side with the single large mitochondrial derivative (Fig. 3D). The
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Fig. 3. Panorpa liui spermatozoa. Cross-sections through the centriole adjunct and the flagellar regions. (A) At the level of neck region, the crescentiform centriole adjunct (ca) partly surrounds the mitochondrial derivatives (md) and the axoneme (ax). (B) The basal region of the centriole adjunct, showing the condensed sector-shaped centriole adjunct (ca), the trapeziform mitochondrial derivatives (md), the two accessory bodies (ab), and the axoneme (ax). (C) Anterior part of the flagellum, showing the axoneme (ax), two accessory bodies (ab), and two pear-shaped mitochondrial derivatives (md). (D) Different levels of the sperm flagellum: accessory bodies (ab) and one mitochondrial derivative (md) progressively diminished (arrows) until only one ab and one md are left (black arrowhead), eventually only md and the axoneme (ax) remain (white arrowhead). (E) Through the flagellar axoneme at the same level with (D), showing two mitochondrial derivatives (md), two accessory bodies (ab) and the axoneme (ax). (F) Magnification of the axoneme in E, showing complete A-subtubule (arrow) and incomplete B-subtubule (arrowhead). Scale bars: A = 0.25 m; B–D = 0.5 m; E = 0.15 m; F = 0.05 m.
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Fig. 4. Panorpa longihypovalva spermatozoa. Cross-section through a sperm cyst (sc). (A) Acrosome (a) with a central perforatorium (p) accompanying the nucleus (N). (B) The dumbbell-shaped nuclei (N) with two wide lateral grooves and the two lateral lamellar bodies (la) in sperm cyst (sc). (C) A sperm cyst (sc) with 126 spermatozoa. (D) Different levels of the spermatozoa. Anteriorly the nucleus has a cloverleaf structure (arrowheads) (upper left); posteriorly the centriole (c) and mitochondrial derivatives (md) are surrounded by the centriole adjunct (ca), and two lateral lamellar bodies (la) accompanying the centriole adjunct (middle left); slightly posteriorly the kidney-shaped centriole adjunct progressively diminished until one terminates (arrow). Scale bars: A = 0.2 m; B = 2 m; C = 2.5 m; D = 1 m.
axoneme and the two mitochondrial derivatives run longitudinally in a helicoidal fashion, which is more regular than before (Fig. 2E). The structure of the axoneme is clearly visible with the simple 9 + 2 microtubule pattern, lacking accessory tubules (Fig. 3E). The
two central microtubules are hollow, their walls each being composed of 13 protofilaments (Fig. 3F). Doublet microtubules exhibit thick radial spokes directed towards the central complex. The complete A-subtubule is provided with 13 protofilaments and carries two dynein arms towards its neighboring doublet, whereas the
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Fig. 5. Panorpa longihypovalva spermatozoa. Cross-sections through the neck and the flagellum regions. (A) At the neck region, the centriole (c) with nine microtubular doublets and two mitochondrial derivatives (md) are surrounded by the centriole adjunct (ca) (upper right); at the flagellum region, a flagellum consists of an axoneme (ax), two md, and two kidney-shaped ca located on the outside of the two md (bottom left). Around the lateral lamellae (la) and the ca, there appear some dispersed microtubules (arrow). (B) Fossa of the nucleus (white arrow) and centriole adjunct (ca) progressively diminished (arrowhead). (C) The axoneme of 9 + 2 pattern. (D) A single accessory body (ab) between the axoneme (ax) and the two mitochondrial derivatives (md). (E) The progressively diminished mitochondrial derivatives (md) and the accessory body (ab). (F) Only one mitochondrial derivative (md) and the axoneme can be seen in the posterior end of the flagellum. Scale bars: A, B, F = 0.5 m; C = 0.05 m; D, E = 0.25 m.
incomplete B-subtubule shows 10 protofilaments in its tubular wall (Fig. 3F). Close to the posterior end, the flagellum is characterized by the absence of all the accessory bodies. The single mitochondrial derivative also diminished, with a size similar to the axoneme (Figs. 2F and 3D). 3.2. Panorpa longihypovalva A sperm cyst consists of 126 spermatozoa in a compact, equidirectional distribution, with different structures being observed at different levels (Fig. 4C). The bilayered acrosome and the homogeneous nucleus adhere to each other at the anterior tip of the sperm (Fig. 4A).
Two wide symmetrical lateral grooves run along the nucleus, giving it a dumbbell-shape (Fig. 4B). Two small lateral lamellar bodies are observed to run along the nucleus (Fig. 4B and D). Posteriorly, the dumbbell-shaped nucleus invaginates at its top side away from the lateral lamellae, being cloverleaf-shaped in the cross-section (Fig. 4D). At the centriole adjunct region, the nucleus has a deep fossa, into which the centriole is embedded (Fig. 5B). The centriole adjunct begins to expand slightly posteriorly, surrounding the centriole and the two mitochondrial derivatives (Fig. 4D). Around the lateral lamellae and the centriole adjunct are some dispersed microtubules, whose size is similar to the tubulets of the centriole, about 30 nm in diameter (Fig. 5A). Further posteriorly, the kidney-shaped centriole adjuncts are located on one side of the two mitochon-
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drial derivatives, and progressively diminish until one of them terminates just before the other (Fig. 4D). Eventually both of them disappear at the anterior flagellar region. The flagellum consists of an axoneme, a pair of mitochondrial derivatives and a single accessory body (Fig. 5D). The lateral lamel-
lae that run along from the nucleus to the centriole adjunct region disappeared. The axoneme is characterized by a 9 + 2 microtubular pattern, with nine outer doublets and two central microtubules (Fig. 5C). The lumen of the two central microtubules is hollow as in the A- and B-subtubules of the doublets. In the cross-section, the
Fig. 6. Bittacus planus spermatozoa. Cross (A, B, F), oblique (E), and longitudinal (C, D) sections through the acrosome and nucleus region. (A) Acrosome, showing a threelayered plasma membrane (arrows) and the central perforatorium (p). (B) The nucleus (N) has two symmetrical lateral indentations (arrows). (C) Globular units (arrows) run longitudinally in a helicoidal fashion at the anterior region of the nucleus. (D) The whole acrosome with the perforatorium (p) surrounded by a series of parallel, regularly spaced globular units (arrows). (E) The posterior end of the nucleus (N) accompanying globular units (arrow). (F) Nucleus portion of spermatozoa in a sperm cyst (sc). Scale bars: A = 0.3 m; B, D = 0.5 m; C = 0.25 m; E = 1 m; F = 2 m.
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Fig. 7. Bittacus planus spermatozoa. (A) Cross-section through anterior tip of the nucleus (N), showing the globular units (arrow). (B) Longitudinal section at nucleus region, some globular units (arrows) are located near the nucleus (N). (C) Longitudinal section at posterior end of the nucleus (N) with the centriole (c) and the anterior part of the axoneme (ax). (D, E) Cross-section through the flagellum. In (D), showing the anterior tip with the axoneme (ax), two accessory bodies (ab), and two mitochondrial derivatives (md); in (E), showing the posterior region with two accessory bodies (ab) situated between the axoneme (ax) and the mitochondrial derivatives (md). (F) Cross-section through the axoneme, showing a simple 9 + 2 pattern. Scale bars: A = 0.5 m; B–F = 0.2 m.
interior of the two mitochondrial derivatives are occupied by the central crystalline structure. At this level, the two mitochondrial derivatives are roughly cylindrical, about 0.3 m in diameter while the accessory body is smaller, about 0.15 m (Fig. 5E). Towards the posterior flagellar region, the accessory body transforms from a somewhat crescent to an elliptical shape in cross-section (Fig. 5D and E). The slightly posterior tail region is characterized by only one mitochondrial derivative, which is tightly invaded by the axoneme from one side (Fig. 5F). 3.3. Bittacus planus In a sperm cyst, 118 spermatozoa are observed to be oriented in the same direction, aligned side-by-side in register and transected through the nucleus (Fig. 6F). The acrosome of a mature spermatozoon is a bilayered structure, and shows a helicoidal array in appearance (Fig. 6D). The cylindrical perforatorium and the acrosomal vesicle are surrounded by a distinct three-layered plasma membrane (Fig. 6A), the thickness of which varies from 20 to 100 nm (Fig. 6B). Beneath the membrane at this area, the spermatozoon is seen to contain a series of parallel, regularly spaced globular units of 0.1–0.2 m in diameter (Fig. 6C and D). The hollow globular units extend from the acrosome through the entire nucleus to the flagellum region (Fig. 7A and D), in which they gradually disappear (Fig. 7E). They are located on one side of the nucleus at the nucleus region (Figs. 6E and 7A,B), and occupy almost half of the volume at the anterior end of the flagel-
lum (Fig. 7D). The entire spermatozoon shows a plasma membrane with an unusual regular glycocalyx organized as ridges or lamellae (Fig. 7D). The flagellum is composed of an axoneme, two mitochondrial derivatives, two accessory bodies, and a layer of Golgi complexderived membrane (Fig. 8A). The axoneme is derived from the postnuclear centriole (Fig. 7C). The two mitochondrial derivatives are of unequal size, with the larger one elliptical and the smaller one cylindrical in shape (Fig. 7D). They are accompanied each by a semi-lunar shaped accessory body (Fig. 7D). More posteriorly they are similar in size and shape (Fig. 7E). The axoneme has a 9 + 2 microtubular pattern. Each of the two central tubules is about 35 nm in diameter, with 13 protofilaments. Each doublet tubule consists of a complete A-subtubule with 13 protofilaments and an uncomplete B-subtubule with 10 protofilaments (Fig. 7F). A-subtubule is provided with an inner radial spoke directed towards the axoneme center and two outer arms towards its neighboring doublet (Fig. 7F). More posteriorly, a pronounced Golgi complex-derived membrane is observed between the accessory bodies and the mitochondrial derivatives (Fig. 8A). At this level, the smaller mitochondrial derivative expands its diameter, gradually approaching to that of the larger one; the two accessory bodies are almost of equal size (Fig. 8A). At slightly posterior region, the Golgi complexderived membrane is absent, the two mitochondrial derivatives are cylindrical and similar in size, and the two accessory bodies become more circular in shape (Fig. 8C).
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Fig. 8. Bittacus planus spermatozoa. Cross (A–C) and longitudinal (D–F) sections through the sperm flagellum. (A) The Golgi complex-derived membrane (arrow) located between the two mitochondrial derivatives (md) and two accessory bodies (ab); ax: axoneme. (B) The two accessory bodies (ab) are triangular, lying between the single mitochondrial derivative (md) and the axoneme (ax). (C) Flagellum consists of two mitochondrial derivatives (md), two accessory bodies (ab), and the axoneme (ax), with the Golgi complex-derived membrane disappeared. (D) A helicoidal array of the axoneme (ax) around two mitochondrial derivatives (md1, md2). (E) Some globular units (arrows) are near the axoneme (ax) and localized in the plasma membrane. (F) At the end of flagellum tail, the mitochondrial derivatives, accessory bodies, and the axoneme all disappeared. Scale bars: A, B, D = 0.5 m; C = 1 m; E, F = 0.25 m.
Close to the posterior end, only one mitochondrial derivative is left in the spermatozoon. The axoneme maintains its regular circular configuration. The two accessory bodies are concentrated, smaller in volume and triangular in shape (Fig. 8B).
The axoneme and the two mitochondrial derivatives run longitudinally also in a helicoidal fashion (Fig. 8D). The axoneme is accompanied in some place by a few globular units, which are surrounded by the plasma membrane (Fig. 8E). By the end of the tail
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region, the flagellum is devoid of the mitochondrial derivatives and the accessory bodies as well as the axoneme (Fig. 8F). 4. Discussion Sperm ultrastructure has only been surveyed for the European Panorpa germanica, P. annexa, and Boreus hyemalis (Baccetti, 1972; Baccetti et al., 1969; Dallai et al., 2003; Gassner et al., 1972), the North American P. nuptialis (Gassner et al., 1972) and Hylobittacus apicalis (Breland et al., 1966) in Mecoptera to date. In this paper, we add three Chinese species, including two in Panorpa (Panorpidae) and one in Bittacus (Bittacidae) for their spermatozoal ultrastructure. Based on the results of these limited investigations, we tentatively conclude that the Mecoptera might share the following common feature on their sperm ultrastructure: a bilayered acrosome, an elongated nucleus, a short centriole adjunct region, a very long flagellum with two developed mitochondrial derivatives, two accessory bodies, and an axoneme of a 9 + 2 microtubular pattern, lacking accessory tubules that are common for majority of the other insects. The testis follicles of sexually mature male insects are full of sperm cysts, within which spermatogenesis proceeds. A cyst is a group of germ cells surrounded by an epithelium. The proximally situated cysts contain spermatogonia, whereas the distal ones comprise spermatids and spermatozoa. The number of spermatids per cyst is generally equal to an integral power of 2 (Jamieson et al., 1999). In the mecopteran species we examined, a cyst contains 126 spermatozoa in P. longihypovalva and 118 in B. planus, indicating that the theoretical spermatid number per cyst should be 128 or 26 . This result has not been reported previously. The reason why the actual number is less than 128 per cyst may result from the degenerating spermatogonia that were phagocytosed by the somatic cells of the cyst. Breland et al. (1966) claimed that the sperm tail of the North American Hylobittacus apicalis has a 9 + 9 + 2 pattern of axoneme microtubules, but their published figures are not convincing as was pointed out by Jamieson et al. (1999). Similarly, in a few spermatid axonemes of P. germanica, Dallai et al. (2003) also observed two microtubules outside the nine axonemal doublets, speculating that the two additional microtubules as the result of an abortive formation of accessory tubules. This seems to present a reasonable explanation to the claim of 9 + 9 + 2 axoneme pattern in H. apicalis (Breland et al., 1966). In the European P. annexa and P. germanica, the outer chromatin consists of tubular filaments (Baccetti et al., 1969) or orthogonally arrayed parallel nuclear fibres (Dallai et al., 2003). Similar tubular filaments were not observed in our investigation on the Chinese P. liui and P. longihypovalva, as well as in the North American P. nuptialis (Gassner et al., 1972). A deep fossa was observed at the posterior end of the nucleus both in P. liui and P. longihypovalva. Moreover, P. liui also has a postnuclear rather than prenuclear location of the centriole and two mitochondrial derivatives, a plesiomorphy character present in P. nuptialis but not in P. annexa and P. germanica (Dallai et al., 2003; Gassner et al., 1972; Jamieson, 1987; Jamieson et al., 1999). This indicates that P. liui is close to P. nuptialis, both being at the basal position in the phylogeny of Panorpa. Significant features of the spermatozoon that P. longihypovalva differ from P. liui lie in the presence of the lateral lamellae extending from the anterior region of the nucleus to the flagellum and the possession of only one accessory body. These two features also exist in P. germanica (Dallai et al., 2003) and P. nuptialis (Gassner et al., 1972), though the accessory body was called fibrous rod by Gassner et al. (1972). All the data discussed above on the sperm ultrastructure further confirm the paraphyly of the genus Panorpa reached by external morphology (Cai et al., 2008; Willmann, 1989)
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and molecular data (Misof et al., 2000), as well as the eggs (Ma et al., 2009) and the female reproductive system (Hou and Hua, 2008). The spermatozoon of B. planus is peculiar by the presence of the globular units from the acrosome through the nucleus to the flagellum. The globular structure has not been described previously, and their function, if any, is unknown. Another peculiarity in B. planus mature spermatozoon lies in the presence of the Golgi complex-derived membrane, which was argued to be involved in the crystallization of insect mitochondria during a complicated process of rearrangement and fusion among spermatid mitochondria (André, 1962). The occurrence of Golgi complex-derived membrane has not been described previously in the spermatozoa of Mecoptera. Although some fascinating discoveries were found on the spermatozoal ultrastructure of Mecoptera in our present study and some early researches (Baccetti et al., 1969; Dallai et al., 2003; Gassner et al., 1972), only eight species in three families have been examined in this interesting order so far. More work on the spermatological research of Mecoptera is desperately needed.
Acknowledgements We thank Dr. Yankai Li for providing the spermatid light micrograph of P. liui. Our thanks are also due to Guoyun Zhang and Qingmei Han for their technical assistance in transmission electron microscopy observation. Special thanks are due to two anonymous reviewers for their helpful and valuable comments on the revision of our manuscript. This research was financially supported by the National Natural Science Foundation of China (grant nos. 30670255 and 309703863) and the Research Fund for the Doctoral Program of Higher Education (grant no. 20090204110004).
References André, J., 1962. Contribution à la connaissance du chondriome: étude de ses modifications ultrastructurales pendant la spermatogénèse. J. Ultrastruct. Res. Suppl. 3, 1–185. Baccetti, B., 1972. Insect sperm cells. Adv. Insect Physiol. 9, 315–397. Baccetti, B., 1987. Spermatozoa and phylogeny in orthopteroid insects. In: Baccetti, B. (Ed.), Evolutionary Biology of Orthopteroid Insects. Ellis Horwood Limited, Chichester, UK, pp. 12–112. Baccetti, B., 1998. Spermatozoa. In: Harrison, F.W., Locke, M. (Eds.), Microscopic Anatomy of Invertebrates. Vol. 11C. Insecta. Wiley-Liss, New York, pp. 843–894. Baccetti, B., Dallai, R., Rosati, F., 1969. The spermatozoon of Arthropoda. III. The lowest holometabolic insects. J. Micros. 8, 233–248. Breland, O.P., Gassner, G., Riess, R.W., Biesele, J.J., 1966. Certain aspects of the centriole adjunct, spermiogenesis, and the mature sperm of insects. Can. J. Genet. Cyt. 8, 759–773. Byers, G.W., 1979. Hylobittacus, a new genus of North American Bittacidae (Mecoptera). J. Kans. Entomol. Soc. 52, 402–404. Byers, G.W., Thornhill, R., 1983. Biology of the Mecoptera. Annu. Rev. Entomol. 28, 203–228. Cai, L.J., Huang, P.Y., Hua, B.Z., 2008. Sinopanorpa, a new genus of Panorpidae (Mecoptera) from the Oriental China with descriptions of two new species. Zootaxa 1941, 43–54. Cheng, F.Y., 1949. New species of Mecoptera from northwest China. Psyche 56, 139–173. Dallai, R., Lupetti, P., Afzelius, B.A., Frati, F., 2003. Sperm structure of Mecoptera and Siphonaptera (Insecta) and the phylogenetic position of Boreus hyemalis. Zoomorphology 122, 211–220. Gassner, G., Breland, O.P., Biesile, J.J., 1972. The spermatozoa of the scorpionfly Panorpa nuptialis: a transmission electron microscope study. Ann. Entomol. Soc. Am. 65, 1302–1309. Hou, X.Y., Hua, B.Z., 2008. Structures of the female reproductive systems in Panorpidae (Mecoptera) with remarks on their taxonomic significance. Acta Zootaxon. Sin. 33, 427–434. Hua, B.Z., 1997. A new species of the genus Panorpa (Mecoptera: Panorpidae) from northeast China. Entomotaxonomia 19, 213–215. Hua, B.Z., Cai, L.J., 2009. A new species of the genus Panorpa (Mecoptera: Panorpidae) from China with notes on its biology. J. Nat. Hist. 43, 545–552. Jamieson, B.G.M., 1987. The Ultrastructure and Phylogeny of Insect Spermatozoa. Cambridge University Press, Cambridge, pp. 205–208. Jamieson, B.G.M., Ausio, J., Justine, J.L., 1995. Advances in spermatozoal phylogeny and taxonomy. Mém. Mus. natn. Hist. nat. 166, 1–565.
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Jamieson, B.G.M., Dallai, R., Afzelius, B.A., 1999. Insects: Their Spermatozoa and Phylogeny. Science Publishers, Enfield, NH, pp. 313–320. Kaltenbach, A., 1978. Mecoptera (Schnabelhafte, Schnabelfliegen). Handbuch. Zool. 4, 1–111. Lambkin, K.J., 1988. An Australian species of the genus Bittacus Latreille (Mecoptera: Bittacidae). Mem. Queensland Mus. 25, 439–444. Ma, N., Cai, L.J., Hua, B.Z., 2009. Comparative morphology of the eggs in some Panorpidae (Mecoptera) and their systematic implication. Syst. Biodivers. 7, 403–417. Misof, B., Erpenbeck, D., Sauer, K.P., 2000. Mitochondrial gene fragments suggest paraphyly of the genus Panorpa (Mecoptera, Panorpidae). Mol. Phylogenet. Evol. 17, 76–84.
Penny, N.D., Byers, G.W., 1979. A check-list of the Mecoptera of the world. Acta Amazon. 9, 365–388. Tan, J.L., Hua, B.Z., 2009a. Bicaubittacus, a new genus of the Oriental Bittacidae (Mecoptera) with descriptions of two new species. Zootaxa 2221, 27–40. Tan, J.L., Hua, B.Z., 2009b. Terrobittacus, a new genus of the Chinese Bittacidae (Mecoptera) with descriptions of two new species. J. Nat. Hist. 43, 2937–2954. Whiting, M.F., 2002. Mecoptera is paraphyletic: multiple genes and phylogeny of Mecoptera and Siphonaptera. Zool. Scr. 31, 93–104. Willmann, R., 1989. Evolution und Phylogenetisches System der Mecoptera (Insecta: Holometabola). Abh. Senckenberg. Naturforsch. Ges. 544, 1–153.
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Sperm ultrastructure in two species of Panorpa and one Bittacus (Mecoptera) Sha Xie, Baozhen Hua ∗ Key Laboratory of Plant Protection Resources and Pest Management, Ministry of Education, Northwest A&F University, Yangling, Shaanxi 712100, China
a r t i c l e
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Article history: Received 23 January 2010 Received in revised form 22 March 2010 Accepted 22 March 2010 Keywords: Insecta Mecoptera Spermatozoa Structure Panorpidae Bittacidae
a b s t r a c t The sperm ultrastructure of the scorpionflies Panorpa liui and P. longihypovalva in Panorpidae and the hangingfly Bittacus planus in Bittacidae were investigated using transmission electron microscopy. The common features of the spermatozoa shared by all the mecopterans examined include a bilayered acrosome with a central perforatorium, an elongated homogeneously condensed nucleus, and a long flagellum with a 9 + 2 axoneme pattern and two mitochondrial derivatives. The two species of Panorpa possess a fossa at the posterior end of the nucleus, and differ from B. planus by lacking both the globular units running laterally from the head to the flagellum and the Golgi complex-derived membrane present in the flagellum. P. liui has pear-shaped mitochondrial derivatives and two small accessory bodies, while P. longihypovalva has elliptical mitochondrial derivatives and only one accessory body. The marked differences of the sperm structure among the Panorpa examined further confirm the paraphyly of this genus. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Mecoptera is an intriguing minor order of insects, with approximately 600 extant described species placed in nine families and 35 genera (Cai et al., 2008; Penny and Byers, 1979; Tan and Hua, 2009a,b). It was regarded as one of the basal lineage in Holometabola based on their larvae having a pair of compound eyes on the head and the fossil record (Byers and Thornhill, 1983). Mecoptera was once argued to be paraphyletic with Siphonaptera based on molecular data (Whiting, 2002). After detailed comparative analysis of the sperm structure, however, Dallai et al. (2003) concluded that Mecoptera is still monophyletic as traditionally defined and treated Boreus hyemalis as a member of Mecoptera rather than of Siphonaptera. As the two most species-rich families in the Mecoptera, Panorpidae and Bittacidae comprise 90% of mecopteran species (Byers and Thornhill, 1983; Kaltenbach, 1978). The Panorpidae, which are distributed in the northern hemisphere, are commonly called scorpionflies because their ninth abdominal segment (genital bulb) in males is enlarged and curves anterodorsally, superficially resembling the stinger of a scorpion. The cosmopolitan Bittacidae are commonly known as hangingflies due to their adults being unable to stand on a surface but suspending from the edges of leaves or from twigs by their forelegs. Panorpa Linneaus was considered by Willmann (1989) to be paraphyletic with Neopanorpa Weele based
on morphological characters. This argument recently received support from molecular data (Misof et al., 2000). Similarly, Bittacus Latreille, the most speciose genus in Bittacidae, was also regarded to be paraphyletic (Lambkin, 1988; Whiting, 2002). Spermatozoa are characterized by patterns of rapid and divergent morphological evolution to the extent that variation in sperm morphology provides a useful tool for the reconstruction of phylogenies in various insect groups, including the Hexapoda, Paraneoptera, Polyneoptera, Neuropteroid orders, Diptera, etc. (Baccetti, 1987, 1998; Jamieson et al., 1995). In Mecoptera, however, the sperm structure is still poorly documented to date. In Panorpidae, spermatozoal ultrastructure has only been surveyed in the European Panorpa annexa and P. germanica (Baccetti, 1972; Baccetti et al., 1969; Dallai et al., 2003), and the North American P. nuptialis (Gassner et al., 1972). In Bittacidae the sperm ultrastructure has only been reported in Bittacus apicalis (Breland et al., 1966), which was transferred into the genus Hylobittacus by Byers (1979). Thus with respect to the genus Bittacus, the sperm structure has not been really studied hitherto. Our objective of this study is to compare the sperm ultrastructure of two species in Panorpa and one species in Bittacus, hoping to provide spermatological data for the future generic revision of these two paraphyletic genera.
2. Materials and methods ∗ Corresponding author at: Entomological Museum, Northwest A&F University, Yangling, Shaanxi 712100, China. Tel.: +86 29 87091342; fax: +86 29 87091342. E-mail addresses: [email protected] (S. Xie), [email protected] (B. Hua). 0968-4328/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2010.03.007
2.1. Specimen collection Male adults of Panorpa liui Hua, 1997 were obtained from the Dongling Park (41◦ 50 N, 123◦ 35 E, 90 m), Shenyang City, Liaoning
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Fig. 1. Panorpa liui spermatozoa. Light micrographs (A) and transmission electron micrographs (B–I) showing late spermatids through testes. (A) Spermatids dissociated from a spermatogonial cyst; (B) gourd-shaped nuclei (N) with two deep lateral grooves (arrows); (C) cross-section through the acrosome, showing a bilayered acrosome (a) and a central perforatorium (p); (D–F) cross-sections of nucleus (N), showing the fossa (arrows) with the tip of the larger mitochondrial derivative (md1) inserted; (G–I) cross-sections in the centriole adjunct region. In (G), observe the larger and the smaller mitochondrial derivatives (md1 and md2, respectively); in (H), two mitochondrial derivatives (md1, md2) and the centriole (c) are surrounded by the centriole adjunct (ca); in (I), the two mitochondrial derivatives (md1, md2) enlarge in volume, with the centriole (c) surrounded by centriole adjunct (ca). Scale bars: A = 16 m; B = 1 m; C–I = 0.25 m.
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Province, northeastern China in early August 2009. Male adults of P. longihypovalva Hua and Cai, 2009 were collected in the Huoditang Forest Farm (33◦ 25 N, 108◦ 30 E, 1550 m), Ningshan County, Shaanxi Province in early June 2009. Males of Bittacus planus Cheng, 1949 were captured in the Zhuque Forest Park (33◦ 47 N, 108◦ 35 E, 1620 m), Huxian County, Shaanxi Province in mid July 2009. 2.2. Sample preparation After the live male adults were anesthetized in ethyl ether, their testes were dissected out rapidly in the Ringer’s solution (NaCl:CaCl:KCl:dH2 O = 10 g:0.34 g:0.3 g:1100 ml) and then fixed in a mixture of 2% paraformaldehyde and 2.5% glutaraldehyde in the phosphate buffered saline (PBS, 0.1 M, pH 7.2) for 12 h, to which 1.8% of sucrose was added. The fixed specimens were kept separately with small ice block in a vacuum bottle before being stored at 4 ◦ C. 2.3. Transmission electron microscopy (TEM) The fixed specimens were rinsed with PBS, and post-fixation was performed in 1% osmium tetroxide for 2 h at 4 ◦ C. After other four times 15-min rinse in the same buffer, the samples were dehydrated through a graded ethanol series, and infiltrated with araldite (Histochoice) and embedded in Epon 812. Ultrathin sections were obtained with a diamond knife on an ultramicrotome (Reichert Ultracut E), routinely stained with uranyl acetate and lead citrate, and observed with a JEOL JEM-1230 transmission electron microscope at 80 kV. 3. Results 3.1. Panorpa liui Late spermatids in the deformation zone have a shuttle-shaped head (Fig. 1A). The mature spermatozoon is elongated and filiform, composed of a short acrosome, an elongated nucleus, and a long tail, which consists of a centriole adjunct and a flagellum. The acrosome is very short, about 2.5 m in length (Fig. 2A) and 0.5 m in diameter, conical with two components: a central perforatorium and an outer acrosomal vesicle (Fig. 1C). In longitudinal section, the acrosome has a diameter equal to that of the nucleus at their junction (Fig. 2B and C). The nucleus contains homogeneously condensed chromatin, generally gourd-like in cross-section through the anterior region because of two deep, symmetrical, lateral grooves of the nuclear envelope which extend along the entire length of the nucleus and end in the centriole adjunct region. At this level, the nucleus is about 1.0 m in diameter (Fig. 1B). Posteriorly, there is a small deep cavity in the inner of the nucleus (the nuclear fossa), into which the tip of the larger mitochondrial derivative is adapted (Fig. 1D). Then, the cavity expands with the larger mitochondrial derivative oblique from the centre to one side of the nucleus (Fig. 1E and F). In slightly more posterior region, the smaller mitochondrial derivative appears and extends parallel along the larger one (Fig. 1G). At the basal end of the neck region, a centriole adjunct surrounds the centriole and the two mitochondrial derivatives (Fig. 1H), and connects the sperm head with the tail. Posteriorly the two mitochondrial derivatives are of unequal size (Fig. 1I). The flagellum consists of an axoneme, two accessory bodies, and two mitochondrial derivatives. The axoneme and the two mitochondrial derivatives are of helicoidal array (Fig. 2D and E). The two centriole adjuncts are of crescent form and are located on one side of the two mitochondrial derivatives (Fig. 3A). The axoneme
Fig. 2. Panorpa liui spermatozoa in longitudinal sections through different regions. (A–C) Head of sperm showing the bilayered acrosome, constituted by a central perforatorium (p) and a conical acrosomal vesicle (a) and the long nucleus (N). (D–F) The helicoidal array of mitochondrial derivatives (md1, md2), axoneme (ax) and accessory bodies (arrows). Scale bars: A, C = 1 m; B, D–F = 0. 5 m.
begins to exhibit its outline at this level although its nine microtubular doublets and two central tubules are not clear yet (Fig. 3A). At the anterior region of the sperm flagellum, the smaller mitochondrial derivative becomes larger, almost reaching the same size as the larger one, both being trapezoidal in cross-section (Fig. 3B). Beside the axoneme, near the cell membrane are two small cylindrical accessory bodies, facing the two mitochondrial derivatives (Fig. 3B). In more posterior region, the centriole adjunct disappears. The two mitochondrial derivatives contain dense materials inside, are twice as large as those at the anterior tip, pear-shaped in crosssection, and occupy more than three quarters in volume of the entire cell (Fig. 3C). The accessory bodies are quite small, and one of them becomes smaller and smaller until disappears along with the progressive vanishing of one mitochondrial derivative (Fig. 3D). The accessory body left alone is located on the same side with the single large mitochondrial derivative (Fig. 3D). The
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Fig. 3. Panorpa liui spermatozoa. Cross-sections through the centriole adjunct and the flagellar regions. (A) At the level of neck region, the crescentiform centriole adjunct (ca) partly surrounds the mitochondrial derivatives (md) and the axoneme (ax). (B) The basal region of the centriole adjunct, showing the condensed sector-shaped centriole adjunct (ca), the trapeziform mitochondrial derivatives (md), the two accessory bodies (ab), and the axoneme (ax). (C) Anterior part of the flagellum, showing the axoneme (ax), two accessory bodies (ab), and two pear-shaped mitochondrial derivatives (md). (D) Different levels of the sperm flagellum: accessory bodies (ab) and one mitochondrial derivative (md) progressively diminished (arrows) until only one ab and one md are left (black arrowhead), eventually only md and the axoneme (ax) remain (white arrowhead). (E) Through the flagellar axoneme at the same level with (D), showing two mitochondrial derivatives (md), two accessory bodies (ab) and the axoneme (ax). (F) Magnification of the axoneme in E, showing complete A-subtubule (arrow) and incomplete B-subtubule (arrowhead). Scale bars: A = 0.25 m; B–D = 0.5 m; E = 0.15 m; F = 0.05 m.
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Fig. 4. Panorpa longihypovalva spermatozoa. Cross-section through a sperm cyst (sc). (A) Acrosome (a) with a central perforatorium (p) accompanying the nucleus (N). (B) The dumbbell-shaped nuclei (N) with two wide lateral grooves and the two lateral lamellar bodies (la) in sperm cyst (sc). (C) A sperm cyst (sc) with 126 spermatozoa. (D) Different levels of the spermatozoa. Anteriorly the nucleus has a cloverleaf structure (arrowheads) (upper left); posteriorly the centriole (c) and mitochondrial derivatives (md) are surrounded by the centriole adjunct (ca), and two lateral lamellar bodies (la) accompanying the centriole adjunct (middle left); slightly posteriorly the kidney-shaped centriole adjunct progressively diminished until one terminates (arrow). Scale bars: A = 0.2 m; B = 2 m; C = 2.5 m; D = 1 m.
axoneme and the two mitochondrial derivatives run longitudinally in a helicoidal fashion, which is more regular than before (Fig. 2E). The structure of the axoneme is clearly visible with the simple 9 + 2 microtubule pattern, lacking accessory tubules (Fig. 3E). The
two central microtubules are hollow, their walls each being composed of 13 protofilaments (Fig. 3F). Doublet microtubules exhibit thick radial spokes directed towards the central complex. The complete A-subtubule is provided with 13 protofilaments and carries two dynein arms towards its neighboring doublet, whereas the
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Fig. 5. Panorpa longihypovalva spermatozoa. Cross-sections through the neck and the flagellum regions. (A) At the neck region, the centriole (c) with nine microtubular doublets and two mitochondrial derivatives (md) are surrounded by the centriole adjunct (ca) (upper right); at the flagellum region, a flagellum consists of an axoneme (ax), two md, and two kidney-shaped ca located on the outside of the two md (bottom left). Around the lateral lamellae (la) and the ca, there appear some dispersed microtubules (arrow). (B) Fossa of the nucleus (white arrow) and centriole adjunct (ca) progressively diminished (arrowhead). (C) The axoneme of 9 + 2 pattern. (D) A single accessory body (ab) between the axoneme (ax) and the two mitochondrial derivatives (md). (E) The progressively diminished mitochondrial derivatives (md) and the accessory body (ab). (F) Only one mitochondrial derivative (md) and the axoneme can be seen in the posterior end of the flagellum. Scale bars: A, B, F = 0.5 m; C = 0.05 m; D, E = 0.25 m.
incomplete B-subtubule shows 10 protofilaments in its tubular wall (Fig. 3F). Close to the posterior end, the flagellum is characterized by the absence of all the accessory bodies. The single mitochondrial derivative also diminished, with a size similar to the axoneme (Figs. 2F and 3D). 3.2. Panorpa longihypovalva A sperm cyst consists of 126 spermatozoa in a compact, equidirectional distribution, with different structures being observed at different levels (Fig. 4C). The bilayered acrosome and the homogeneous nucleus adhere to each other at the anterior tip of the sperm (Fig. 4A).
Two wide symmetrical lateral grooves run along the nucleus, giving it a dumbbell-shape (Fig. 4B). Two small lateral lamellar bodies are observed to run along the nucleus (Fig. 4B and D). Posteriorly, the dumbbell-shaped nucleus invaginates at its top side away from the lateral lamellae, being cloverleaf-shaped in the cross-section (Fig. 4D). At the centriole adjunct region, the nucleus has a deep fossa, into which the centriole is embedded (Fig. 5B). The centriole adjunct begins to expand slightly posteriorly, surrounding the centriole and the two mitochondrial derivatives (Fig. 4D). Around the lateral lamellae and the centriole adjunct are some dispersed microtubules, whose size is similar to the tubulets of the centriole, about 30 nm in diameter (Fig. 5A). Further posteriorly, the kidney-shaped centriole adjuncts are located on one side of the two mitochon-
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drial derivatives, and progressively diminish until one of them terminates just before the other (Fig. 4D). Eventually both of them disappear at the anterior flagellar region. The flagellum consists of an axoneme, a pair of mitochondrial derivatives and a single accessory body (Fig. 5D). The lateral lamel-
lae that run along from the nucleus to the centriole adjunct region disappeared. The axoneme is characterized by a 9 + 2 microtubular pattern, with nine outer doublets and two central microtubules (Fig. 5C). The lumen of the two central microtubules is hollow as in the A- and B-subtubules of the doublets. In the cross-section, the
Fig. 6. Bittacus planus spermatozoa. Cross (A, B, F), oblique (E), and longitudinal (C, D) sections through the acrosome and nucleus region. (A) Acrosome, showing a threelayered plasma membrane (arrows) and the central perforatorium (p). (B) The nucleus (N) has two symmetrical lateral indentations (arrows). (C) Globular units (arrows) run longitudinally in a helicoidal fashion at the anterior region of the nucleus. (D) The whole acrosome with the perforatorium (p) surrounded by a series of parallel, regularly spaced globular units (arrows). (E) The posterior end of the nucleus (N) accompanying globular units (arrow). (F) Nucleus portion of spermatozoa in a sperm cyst (sc). Scale bars: A = 0.3 m; B, D = 0.5 m; C = 0.25 m; E = 1 m; F = 2 m.
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Fig. 7. Bittacus planus spermatozoa. (A) Cross-section through anterior tip of the nucleus (N), showing the globular units (arrow). (B) Longitudinal section at nucleus region, some globular units (arrows) are located near the nucleus (N). (C) Longitudinal section at posterior end of the nucleus (N) with the centriole (c) and the anterior part of the axoneme (ax). (D, E) Cross-section through the flagellum. In (D), showing the anterior tip with the axoneme (ax), two accessory bodies (ab), and two mitochondrial derivatives (md); in (E), showing the posterior region with two accessory bodies (ab) situated between the axoneme (ax) and the mitochondrial derivatives (md). (F) Cross-section through the axoneme, showing a simple 9 + 2 pattern. Scale bars: A = 0.5 m; B–F = 0.2 m.
interior of the two mitochondrial derivatives are occupied by the central crystalline structure. At this level, the two mitochondrial derivatives are roughly cylindrical, about 0.3 m in diameter while the accessory body is smaller, about 0.15 m (Fig. 5E). Towards the posterior flagellar region, the accessory body transforms from a somewhat crescent to an elliptical shape in cross-section (Fig. 5D and E). The slightly posterior tail region is characterized by only one mitochondrial derivative, which is tightly invaded by the axoneme from one side (Fig. 5F). 3.3. Bittacus planus In a sperm cyst, 118 spermatozoa are observed to be oriented in the same direction, aligned side-by-side in register and transected through the nucleus (Fig. 6F). The acrosome of a mature spermatozoon is a bilayered structure, and shows a helicoidal array in appearance (Fig. 6D). The cylindrical perforatorium and the acrosomal vesicle are surrounded by a distinct three-layered plasma membrane (Fig. 6A), the thickness of which varies from 20 to 100 nm (Fig. 6B). Beneath the membrane at this area, the spermatozoon is seen to contain a series of parallel, regularly spaced globular units of 0.1–0.2 m in diameter (Fig. 6C and D). The hollow globular units extend from the acrosome through the entire nucleus to the flagellum region (Fig. 7A and D), in which they gradually disappear (Fig. 7E). They are located on one side of the nucleus at the nucleus region (Figs. 6E and 7A,B), and occupy almost half of the volume at the anterior end of the flagel-
lum (Fig. 7D). The entire spermatozoon shows a plasma membrane with an unusual regular glycocalyx organized as ridges or lamellae (Fig. 7D). The flagellum is composed of an axoneme, two mitochondrial derivatives, two accessory bodies, and a layer of Golgi complexderived membrane (Fig. 8A). The axoneme is derived from the postnuclear centriole (Fig. 7C). The two mitochondrial derivatives are of unequal size, with the larger one elliptical and the smaller one cylindrical in shape (Fig. 7D). They are accompanied each by a semi-lunar shaped accessory body (Fig. 7D). More posteriorly they are similar in size and shape (Fig. 7E). The axoneme has a 9 + 2 microtubular pattern. Each of the two central tubules is about 35 nm in diameter, with 13 protofilaments. Each doublet tubule consists of a complete A-subtubule with 13 protofilaments and an uncomplete B-subtubule with 10 protofilaments (Fig. 7F). A-subtubule is provided with an inner radial spoke directed towards the axoneme center and two outer arms towards its neighboring doublet (Fig. 7F). More posteriorly, a pronounced Golgi complex-derived membrane is observed between the accessory bodies and the mitochondrial derivatives (Fig. 8A). At this level, the smaller mitochondrial derivative expands its diameter, gradually approaching to that of the larger one; the two accessory bodies are almost of equal size (Fig. 8A). At slightly posterior region, the Golgi complexderived membrane is absent, the two mitochondrial derivatives are cylindrical and similar in size, and the two accessory bodies become more circular in shape (Fig. 8C).
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Fig. 8. Bittacus planus spermatozoa. Cross (A–C) and longitudinal (D–F) sections through the sperm flagellum. (A) The Golgi complex-derived membrane (arrow) located between the two mitochondrial derivatives (md) and two accessory bodies (ab); ax: axoneme. (B) The two accessory bodies (ab) are triangular, lying between the single mitochondrial derivative (md) and the axoneme (ax). (C) Flagellum consists of two mitochondrial derivatives (md), two accessory bodies (ab), and the axoneme (ax), with the Golgi complex-derived membrane disappeared. (D) A helicoidal array of the axoneme (ax) around two mitochondrial derivatives (md1, md2). (E) Some globular units (arrows) are near the axoneme (ax) and localized in the plasma membrane. (F) At the end of flagellum tail, the mitochondrial derivatives, accessory bodies, and the axoneme all disappeared. Scale bars: A, B, D = 0.5 m; C = 1 m; E, F = 0.25 m.
Close to the posterior end, only one mitochondrial derivative is left in the spermatozoon. The axoneme maintains its regular circular configuration. The two accessory bodies are concentrated, smaller in volume and triangular in shape (Fig. 8B).
The axoneme and the two mitochondrial derivatives run longitudinally also in a helicoidal fashion (Fig. 8D). The axoneme is accompanied in some place by a few globular units, which are surrounded by the plasma membrane (Fig. 8E). By the end of the tail
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region, the flagellum is devoid of the mitochondrial derivatives and the accessory bodies as well as the axoneme (Fig. 8F). 4. Discussion Sperm ultrastructure has only been surveyed for the European Panorpa germanica, P. annexa, and Boreus hyemalis (Baccetti, 1972; Baccetti et al., 1969; Dallai et al., 2003; Gassner et al., 1972), the North American P. nuptialis (Gassner et al., 1972) and Hylobittacus apicalis (Breland et al., 1966) in Mecoptera to date. In this paper, we add three Chinese species, including two in Panorpa (Panorpidae) and one in Bittacus (Bittacidae) for their spermatozoal ultrastructure. Based on the results of these limited investigations, we tentatively conclude that the Mecoptera might share the following common feature on their sperm ultrastructure: a bilayered acrosome, an elongated nucleus, a short centriole adjunct region, a very long flagellum with two developed mitochondrial derivatives, two accessory bodies, and an axoneme of a 9 + 2 microtubular pattern, lacking accessory tubules that are common for majority of the other insects. The testis follicles of sexually mature male insects are full of sperm cysts, within which spermatogenesis proceeds. A cyst is a group of germ cells surrounded by an epithelium. The proximally situated cysts contain spermatogonia, whereas the distal ones comprise spermatids and spermatozoa. The number of spermatids per cyst is generally equal to an integral power of 2 (Jamieson et al., 1999). In the mecopteran species we examined, a cyst contains 126 spermatozoa in P. longihypovalva and 118 in B. planus, indicating that the theoretical spermatid number per cyst should be 128 or 26 . This result has not been reported previously. The reason why the actual number is less than 128 per cyst may result from the degenerating spermatogonia that were phagocytosed by the somatic cells of the cyst. Breland et al. (1966) claimed that the sperm tail of the North American Hylobittacus apicalis has a 9 + 9 + 2 pattern of axoneme microtubules, but their published figures are not convincing as was pointed out by Jamieson et al. (1999). Similarly, in a few spermatid axonemes of P. germanica, Dallai et al. (2003) also observed two microtubules outside the nine axonemal doublets, speculating that the two additional microtubules as the result of an abortive formation of accessory tubules. This seems to present a reasonable explanation to the claim of 9 + 9 + 2 axoneme pattern in H. apicalis (Breland et al., 1966). In the European P. annexa and P. germanica, the outer chromatin consists of tubular filaments (Baccetti et al., 1969) or orthogonally arrayed parallel nuclear fibres (Dallai et al., 2003). Similar tubular filaments were not observed in our investigation on the Chinese P. liui and P. longihypovalva, as well as in the North American P. nuptialis (Gassner et al., 1972). A deep fossa was observed at the posterior end of the nucleus both in P. liui and P. longihypovalva. Moreover, P. liui also has a postnuclear rather than prenuclear location of the centriole and two mitochondrial derivatives, a plesiomorphy character present in P. nuptialis but not in P. annexa and P. germanica (Dallai et al., 2003; Gassner et al., 1972; Jamieson, 1987; Jamieson et al., 1999). This indicates that P. liui is close to P. nuptialis, both being at the basal position in the phylogeny of Panorpa. Significant features of the spermatozoon that P. longihypovalva differ from P. liui lie in the presence of the lateral lamellae extending from the anterior region of the nucleus to the flagellum and the possession of only one accessory body. These two features also exist in P. germanica (Dallai et al., 2003) and P. nuptialis (Gassner et al., 1972), though the accessory body was called fibrous rod by Gassner et al. (1972). All the data discussed above on the sperm ultrastructure further confirm the paraphyly of the genus Panorpa reached by external morphology (Cai et al., 2008; Willmann, 1989)
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and molecular data (Misof et al., 2000), as well as the eggs (Ma et al., 2009) and the female reproductive system (Hou and Hua, 2008). The spermatozoon of B. planus is peculiar by the presence of the globular units from the acrosome through the nucleus to the flagellum. The globular structure has not been described previously, and their function, if any, is unknown. Another peculiarity in B. planus mature spermatozoon lies in the presence of the Golgi complex-derived membrane, which was argued to be involved in the crystallization of insect mitochondria during a complicated process of rearrangement and fusion among spermatid mitochondria (André, 1962). The occurrence of Golgi complex-derived membrane has not been described previously in the spermatozoa of Mecoptera. Although some fascinating discoveries were found on the spermatozoal ultrastructure of Mecoptera in our present study and some early researches (Baccetti et al., 1969; Dallai et al., 2003; Gassner et al., 1972), only eight species in three families have been examined in this interesting order so far. More work on the spermatological research of Mecoptera is desperately needed.
Acknowledgements We thank Dr. Yankai Li for providing the spermatid light micrograph of P. liui. Our thanks are also due to Guoyun Zhang and Qingmei Han for their technical assistance in transmission electron microscopy observation. Special thanks are due to two anonymous reviewers for their helpful and valuable comments on the revision of our manuscript. This research was financially supported by the National Natural Science Foundation of China (grant nos. 30670255 and 309703863) and the Research Fund for the Doctoral Program of Higher Education (grant no. 20090204110004).
References André, J., 1962. Contribution à la connaissance du chondriome: étude de ses modifications ultrastructurales pendant la spermatogénèse. J. Ultrastruct. Res. Suppl. 3, 1–185. Baccetti, B., 1972. Insect sperm cells. Adv. Insect Physiol. 9, 315–397. Baccetti, B., 1987. Spermatozoa and phylogeny in orthopteroid insects. In: Baccetti, B. (Ed.), Evolutionary Biology of Orthopteroid Insects. Ellis Horwood Limited, Chichester, UK, pp. 12–112. Baccetti, B., 1998. Spermatozoa. In: Harrison, F.W., Locke, M. (Eds.), Microscopic Anatomy of Invertebrates. Vol. 11C. Insecta. Wiley-Liss, New York, pp. 843–894. Baccetti, B., Dallai, R., Rosati, F., 1969. The spermatozoon of Arthropoda. III. The lowest holometabolic insects. J. Micros. 8, 233–248. Breland, O.P., Gassner, G., Riess, R.W., Biesele, J.J., 1966. Certain aspects of the centriole adjunct, spermiogenesis, and the mature sperm of insects. Can. J. Genet. Cyt. 8, 759–773. Byers, G.W., 1979. Hylobittacus, a new genus of North American Bittacidae (Mecoptera). J. Kans. Entomol. Soc. 52, 402–404. Byers, G.W., Thornhill, R., 1983. Biology of the Mecoptera. Annu. Rev. Entomol. 28, 203–228. Cai, L.J., Huang, P.Y., Hua, B.Z., 2008. Sinopanorpa, a new genus of Panorpidae (Mecoptera) from the Oriental China with descriptions of two new species. Zootaxa 1941, 43–54. Cheng, F.Y., 1949. New species of Mecoptera from northwest China. Psyche 56, 139–173. Dallai, R., Lupetti, P., Afzelius, B.A., Frati, F., 2003. Sperm structure of Mecoptera and Siphonaptera (Insecta) and the phylogenetic position of Boreus hyemalis. Zoomorphology 122, 211–220. Gassner, G., Breland, O.P., Biesile, J.J., 1972. The spermatozoa of the scorpionfly Panorpa nuptialis: a transmission electron microscope study. Ann. Entomol. Soc. Am. 65, 1302–1309. Hou, X.Y., Hua, B.Z., 2008. Structures of the female reproductive systems in Panorpidae (Mecoptera) with remarks on their taxonomic significance. Acta Zootaxon. Sin. 33, 427–434. Hua, B.Z., 1997. A new species of the genus Panorpa (Mecoptera: Panorpidae) from northeast China. Entomotaxonomia 19, 213–215. Hua, B.Z., Cai, L.J., 2009. A new species of the genus Panorpa (Mecoptera: Panorpidae) from China with notes on its biology. J. Nat. Hist. 43, 545–552. Jamieson, B.G.M., 1987. The Ultrastructure and Phylogeny of Insect Spermatozoa. Cambridge University Press, Cambridge, pp. 205–208. Jamieson, B.G.M., Ausio, J., Justine, J.L., 1995. Advances in spermatozoal phylogeny and taxonomy. Mém. Mus. natn. Hist. nat. 166, 1–565.
632
S. Xie, B. Hua / Micron 41 (2010) 622–632
Jamieson, B.G.M., Dallai, R., Afzelius, B.A., 1999. Insects: Their Spermatozoa and Phylogeny. Science Publishers, Enfield, NH, pp. 313–320. Kaltenbach, A., 1978. Mecoptera (Schnabelhafte, Schnabelfliegen). Handbuch. Zool. 4, 1–111. Lambkin, K.J., 1988. An Australian species of the genus Bittacus Latreille (Mecoptera: Bittacidae). Mem. Queensland Mus. 25, 439–444. Ma, N., Cai, L.J., Hua, B.Z., 2009. Comparative morphology of the eggs in some Panorpidae (Mecoptera) and their systematic implication. Syst. Biodivers. 7, 403–417. Misof, B., Erpenbeck, D., Sauer, K.P., 2000. Mitochondrial gene fragments suggest paraphyly of the genus Panorpa (Mecoptera, Panorpidae). Mol. Phylogenet. Evol. 17, 76–84.
Penny, N.D., Byers, G.W., 1979. A check-list of the Mecoptera of the world. Acta Amazon. 9, 365–388. Tan, J.L., Hua, B.Z., 2009a. Bicaubittacus, a new genus of the Oriental Bittacidae (Mecoptera) with descriptions of two new species. Zootaxa 2221, 27–40. Tan, J.L., Hua, B.Z., 2009b. Terrobittacus, a new genus of the Chinese Bittacidae (Mecoptera) with descriptions of two new species. J. Nat. Hist. 43, 2937–2954. Whiting, M.F., 2002. Mecoptera is paraphyletic: multiple genes and phylogeny of Mecoptera and Siphonaptera. Zool. Scr. 31, 93–104. Willmann, R., 1989. Evolution und Phylogenetisches System der Mecoptera (Insecta: Holometabola). Abh. Senckenberg. Naturforsch. Ges. 544, 1–153.