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Sperm ultrastructure of the European hornet Vespa crabro (Linnaeus, 1758) (Hymenoptera: Vespidae)
Arthropod Structure & Development 38 (2009) 54–59
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Arthropod Structure & Development journal homepage: www.elsevier.com/locate/asd
Sperm ultrastructure of the European hornet Vespa crabro (Linnaeus, 1758) (Hymenoptera: Vespidae) Karina Mancini a, *, Jose´ Lino-Neto b, Heidi Dolder a, Romano Dallai c a
Departamento de Biologia Celular, CP 6106, Universidade Estadual de Campinas, 13084-971 Campinas, SP, Brazil Departamento de Biologia Geral, Universidade Federal de Viçosa, 36570-000 Viçosa, MG, Brazil c ` di Siena, 53100 Siena, Italy Dipartimento di Biologia Evolutiva, Universita b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 March 2008 Accepted 2 July 2008
This study represents the first sperm description of a Vespinae species (Vespa crabro). The acrosome consists of an acrosomal vesicle and a perforatorium. The nucleus has compact chromatin and shows lenticular structures on the nuclear envelope. These structures, which have never been observed in a hymenopteran sperm, could be clusters of nuclear pores. The centriolar adjunct has an asymmetric pattern and shows a structured periphery. The centriole consists of 9 accessory microtubules and 9 doublet microtubules devoid of arms and spokes. The axoneme has a 9 þ 9 þ 2 microtubule pattern and the accessory microtubules have 16 protofilaments. The mitochondrial derivatives differ in length and diameter. The larger one is adjacent to the nuclear base, while the smaller one begins below the centriolar adjunct. They possess three distinct areas and a large paracrystalline region, which occurs only in the large one. The large mitochondrial derivative ends first, followed by the small one. The axoneme gradually disorganizes: first the central microtubules disappear, then the doublets, which show opened B-tubules, and finally the accessory microtubules. The sperm morphology of V. crabro is very similar to that of the polistine wasp, Agelaia vicina. This can indicate that, in Vespidae, sperm morphology is maintained without important variations among subfamilies and/or that this similarity indicates close phylogenetic relationship between these two subfamilies. Although Vespidae phylogenetically related to Formicidae, these data suggest that the former more closely related to Apoidea than to Formicidae. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Electron microscopy Insect phylogeny Insect spermatozoa
1. Introduction The order Hymenoptera is composed of the taxa ‘‘symphyta’’ and Apocrita and the latter is still divided into Aculeata and Parasitic wasps (LaSalle and Gauld, 1992). Vespidae belong to Aculeata and include about 4200 species divided into six subfamilies (Carpenter, 1982). Euparagiinae and Masarinae display only solitary behavior; Eumeninae exhibit both solitary and presocial behavior; Stenogastrinae are facultatively eusocial, and Polistinae and Vespinae are all eusocial (Cowan, 1991; Ross and Matthews, 1991; Crespi and Yanega, 1995). The subfamily Vespinae (hornets and yellowjacket wasps) consists of about 60 species divided into four genera, which are predominantly found in northern regions of the world (Carpenter and Kojima, 1997).
* Corresponding author. Tel.: þ55 19 3521 6116; fax: þ55 19 3521 6111. E-mail address: [email protected] (K. Mancini). 1467-8039/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.asd.2008.07.001
In Hymenoptera, as in many other insect groups, the sperm structure has become an important source of characters for phylogenetic studies (Baccetti, 1970; Dallai, 1974; Jamieson, 1987; Wheeler et al., 1990; Quicke et al., 1992; Dallai and Afzelius, 1993; Jamieson et al., 1999). The sperm morphology of Hymenoptera is known in several groups (Quicke et al., 1992; Newman and Quicke, 1999; Lino-Neto et al., 1999; Lino-Neto and Dolder, 2001a,b) including the Aculeata (Caetano, 1980; Wheeler et al., 1990; Peng et al., 1992, 1993; Lino-Neto et al., 2000b; Lino-Neto and Dolder, 2002; Zama et al., 2001, 2004, 2005a; Ba´o et al., 2004; Arau´jo et al., 2005; Fiorillo et al., 2005; Moya et al., 2007). However, in Vespidae, the sperm morphological data are limited to only one ultrastructural study dealing with the polistine wasp, Agelaia vicina (Mancini et al., 2006). All these studies have demonstrated that the hymenopteran sperm structure is very diverse among the taxon, but within different species, or even genera, it is quite similar and this allows these data to be used for taxonomy and phylogeny. The present study describes the sperm ultrastructure of the largest European eusocial wasp, Vespa crabro, with the aim of contributing to the knowledge of Vespidae sperm and to the systematics of Hymenoptera.
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2. Materials and methods Male specimens of Vespa crabro (Linnaeus 1758) were captured in the neighborhoods of Siena (Italy). After dissection, the seminal vesicles were processed according to Dallai and Afzelius (1990). Ultrathin sections obtained with a Reichert Ultracut 2E ultramicrotome were routinely stained with 3% uranyl acetate and 3% lead citrate and observed with a Philips CM 10 transmission electron microscope at 80 kV. 3. Results Spermatozoa of Vespa crabro consist of head and flagellar regions. The head contains an acrosome and a nucleus, while the flagellum has an axoneme, two mitochondrial derivatives and two accessory bodies. Between the two regions, a centriolar region with a centriolar adjunct is evident. The acrosome is 2.5 mm long and consists of an acrosomal vesicle and a perforatorium. The acrosomal vesicle is very thin and its axis is occupied by the perforatorium, which extends from just below the acrosome tip to the nuclear tip (Fig. 1A–D). In cross sections, it has a flattened profile at the tip (Fig. 1A), and an oval profile at the mid and posterior portions (Fig. 1B–D). The perforatorium is a long, dense, crystalline structure (Fig. 1B–D), cone shaped, with its base inserted into the anterior nuclear cavity (Fig. 1D). The nucleus shows a very homogenously compact chromatin (Fig. 1E,F,I) and appears oval shaped in cross sections (Fig. 1F). Along
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its whole length and preferentially in the posterior nuclear region several dense lenticular structures, similar to nuclear pores, about 80–90 nm, can be observed (Fig. 1E,G), which are located at irregular intervals along the nuclear envelope. The nuclear base is thin, shows an oblique profile, in close association with the centriolar region (Figs. 1H,I and 2A–C). The nucleus–flagellum transition region is composed of the nuclear base, the centriolar adjunct, the anterior portions of the large mitochondrial derivative and the axoneme (Figs. 1H,I and 2A–C). The centriolar adjunct has an asymmetric pattern; it begins as a thin flattened structure located between the nuclear base and the large mitochondrial derivative (Fig. 2A). A little posteriorly, it acquires a lateral location, between the centriolar region and the large mitochondrial derivative (Fig. 2B–C). Its flattened anterior portion displays an amorphous appearance (Fig. 2A), while the mid portion has a triangular profile in cross sections and shows a structured periphery (Fig. 3A). The centriolar adjunct extends up to the initial axoneme portion (Fig. 2A–C) and it ends at the small mitochondrial derivative tip. The centriolar region is located below the nucleus and laterally to the centriolar adjunct and the large mitochondrial derivative; it consists of 9 accessory microtubules surrounding nine doublets (Fig. 2C). Considering the head–tail direction, the accessory microtubules are the first ones to appear (Fig. 2B), followed by the doublets, which are devoid of dynein arms and spokes. No central tubules are as yet present. At this level, the nuclear posterior tip, surrounded by the two parallel membranes of the nuclear envelope, is evident (Fig. 2C). The axoneme has a 9 þ 9 þ 2 microtubule
Fig. 1. A–I. Anterior region of the Vespa crabro sperm. (A) Cross section of the acrosome tip, showing the flattened acrosomal vesicle (a). (B, C) Cross sections of the acrosome mid portion, showing the oval acrosomal vesicle (a) and the perforatorium (p). (D) anterior tip of the nucleus (n) with the perforatorium (p) in the nuclear cavity. (E) Longitudinal section of the nucleus (n) showing an area with lenticular structures (arrows) along the nuclear envelope. (F) Cross section of the nucleus, to show its oval shape. (G) Longitudinal section of the posterior region of the nucleus showing the presence of the lenticular structures (arrows) in this portion. Centriolar region (c) with the beginning of the axoneme (ax). (H) Longitudinal section of the posterior region of the nucleus (n) showing the large mitochondrial derivative (md1) and the centriolar region (c). Note the presence of the thick nuclear envelope (white arrow) in the posterior tip. (I) Longitudinal section of the posterior portion of the nucleus (n). Axoneme (ax) and nuclear envelope (white arrow) in the posterior tip. Scale bars: 0.5 mm; (F) 0.2 mm.
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Fig. 2. A–C. Nucleus flagellum transition region of the Vespa crabro sperm. (A) Cross section of the posterior region of the nucleus (n) showing the flattened centriolar adjunct (ca) and the large mitochondrial derivative (md1) with a large paracrystalline region (pc). Note the nuclear envelope (white arrow) and two lenticular structures (arrows) in the nucleus. (B) Cross section of the nucleus flagellum region showing the nuclear posterior tip (n) with two parallel membranes of the nuclear envelope (arrow) surrounded by some accessory microtubules (am). Amorphous material is intermingled between the different structures. The centriolar adjunct (ca) is located laterally to the large mitochondrial derivative (md1) that has a paracrystalline core (pc). (C) Cross section of the nucleus flagellum region showing the nuclear posterior tip region (n) with the two membranes of the nuclear envelope (arrow) surrounded by 9 accessory microtubules (am) and 9 doublets devoid of dynein arms and spokes (dm). Triangular centriolar adjunct (ca) laterally to the large mitochondrial derivative with its paracrystalline region (pc) is visible. Scale bars: 0.25 mm.
pattern, with 9 external single accessory microtubules provided with 16 protofilaments in their tubular wall, 9 microtubule doublets showing dynein arms and spokes and two central microtubules (Fig. 3A,B). The mitochondrial derivatives differ in length and diameter. The larger one appears first, adjacent to the nuclear base (Figs. 1H and 2A–C), and extends parallel along the axonemal length (Fig. 3A–E). Its anterior portion, at the centriolar level, is approximately circular in cross section, and has a large paracrystalline organization (Fig. 2A–C). Its mid portion is pear shaped in cross section, and its area is twice as large as its anterior tip (Fig. 3A,B). This portion comprises a large paracrystalline region (distal in relation to the axoneme) (Fig. 3A,B,D) and an amorphous region (proximal in relation to the axoneme). The amorphous region shows a central
clear area (Fig. 3A,B), cristae located at the periphery close to the plasma membrane (Fig. 3A–D) and a dense area located at the peripheral portion, close to the axoneme (Fig. 3A–C). The paracrystalline region occupies more than half of the mitochondrial derivative area (Fig. 3A,B,D). The small mitochondrial derivative begins below the centriolar adjunct. It shows, approximately, a circular profile in cross section, and is half the size of the large mitochondrial derivative (Fig. 3B). Differently from the large one, the small mitochondrial derivative does not display a paracrystalline region, but only amorphous material in three distinct areas (Fig. 3B). The accessory bodies are almost triangular in cross section, and are positioned between the axoneme and both mitochondrial derivatives (Fig. 3A,B,E).
Fig. 3. A–I. Flagellar region of the Vespa crabro sperm. (A) Beginning of the flagellar region, showing the complete axoneme, with 9 accessory microtubules (am), 9 doublets provided with dynein arms and spokes (dm) and 2 central microtubules (cm). The centriolar adjunct (ca) is triangular and shows a structured periphery (double arrow). The large mitochondrial derivative (md1) has a paracrystalline region (pc), and an amorphous region with a central clear area (arrowhead), cristae (arrows) and a dense area (asterisk). The accessory body (ab) is close to the mitochondrial derivative. (B) Flagellar region, showing the complete axoneme (ax); the large mitochondrial derivative (md1) and the small one (md2) which have a central clear area (arrowhead), cristae (arrow) and a dense area (asterisk). The large one has a paracrystalline region (pc). Accessory bodies (ab) are close to the mitochondrial derivatives. (C, D) Longitudinal sections of the flagellar region, showing the cristae of the mitochondrial derivatives (arrows). Axoneme (ax), paracrystalline region (pc) and the dense area (asterisk). (E) Flagellar posterior region, showing the axoneme (ax), the small mitochondrial derivative (md2), with cristae (arrow), and the posterior tip of the large one (md1). The accessory bodies (ab) are close to the derivatives. (F–I) Flagellar posterior tip, showing the axoneme disorganization. Accessory microtubules (am), doublets (dm) and central microtubules (cm). Scale bars: 0.25 mm.
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In the posterior sperm tip, the large mitochondrial derivative ends first (Fig. 3E), followed by the small one. Then, the axoneme begins to be disorganized; the central microtubules end first, followed by the doublets, which show opened B-tubules, and finally by the accessory microtubules (Fig. 3F–I).
4. Discussion This study represents the first sperm ultrastructural description of a Vespinae species. The other study on Vespidae sperm deals with the polistine wasp, Agelaia vicina (Mancini et al., 2006). The sperm morphology of Vespa crabro and A. vicina is very similar, since in both Vespidae: (a) the acrosome is composed of an acrosomal vesicle and a perforatorium; (b) the acrosomal vesicle is oval shaped in cross sections; (c) the nucleus has homogenous compact chromatin; (d) the centriolar adjunct is located between the nuclear base and one of the mitochondrial derivatives; (e) the nuclear posterior region is surrounded by the centriolar region; (f) the axoneme has a 9 þ 9 þ 2 microtubular pattern; (g) the mitochondrial derivatives begin at different levels and have different shapes and diameters; (h) a paracrystalline region is present only in the larger mitochondrial derivative; and (i) the accessory microtubules are the last ones to finish at the posterior axonemal tip. The acrosome, formed by an acrosomal vesicle and a perforatorium, is a common characteristic of Hymenoptera sperm, being described in ‘symphyta’ (Newman and Quicke, 1999a) and Aculeata (Wheeler et al., 1990; Peng et al., 1992, 1993; Zama et al., 2001, 2004, 2005a,b; Lino-Neto and Dolder, 2002). The acrosomal vesicle of V. crabro and A. vicina shows an oval shape in cross sections and a similar appearance was also observed in bee sperm (Cruz-Ho¨fling et al., 1970; Peng et al., 1992, 1993; Ba´o et al., 2004; Fiorillo et al., 2005; Zama et al., 2005a), while an acrosomal vesicle with a circular profile in the anterior region and triangular in the mid region has been observed in the sperm of the stingless bees (Zama et al., 2001, 2004; Arau´jo et al., 2005) and ants (Wheeler et al., 1990; Lino-Neto and Dolder, 2002; Moya et al., 2007). The conical shape of the perforatorium of V. crabro sperm is shared with the Euglossinae Eufriesea violacea (Zama et al., 2005a), as well as the Halictinae Pseudaugochlora graminea bees (Fiorillo et al., 2005). In the other hymenopteran sperm studied so far, including A. vicina, the perforatorium is, instead, a cylindrical structure. In general, the hymenopteran sperm nucleus shows homogenously compact chromatin, as was observed in V. crabro and A. vicina. However, in some Apidae (Zama et al., 2004; Fiorillo et al., 2005), and in the ant Pseudomyrmex (Moya et al., 2007), the nucleus has poorly condensed chromatin. In addition, some species contain, within the homogeneously compact chromatin, some electronlucent lacunae, as reported for the ant Solenopsis invicta (Lino-Neto and Dolder, 2002) and the Halictinae bees (Fiorillo et al., 2005). The dense lenticular structures observed along the posterior nuclear region of V. crabro have not been reported before in Hymenoptera spermatozoa. They are most likely modified nuclear pores as suggested by their dimensions, although subunits of the eight proteins that surround the nuclear pore cannot be identified in this dense structure. Such structures were also observed in the neck region of mammalian spermatozoa (Friend and Fawcett, 1974) and can be interpreted as a sign of transit of molecules from the nucleus to the flagellum or as merely the evidence of sperm immaturity. Probably, the presence of these nuclear pores could be associated with the uncommon appearance of the nuclear envelope at the nuclear posterior tip, where the two membranes of the nuclear envelope are parallel, but loose and distant from one another. The finding observed in V. crabro sperm is quite similar to the structure interpreted as dense or connective material and lamellar structure of the Sphecidae wasp (Zama et al., 2005b) and bee sperm (Lino-Neto
et al., 2000b; Zama et al., 2000, 2004, 2005a; Ba´o et al., 2004; Fiorillo et al., 2005). The centriolar region, although described in many Hymenoptera sperm, is poorly known. Many papers do not describe in detail this region or just show and mention ‘‘the centriolar region’’ (Lino-Neto et al., 1999; Lino-Neto and Dolder, 2001a; Zama et al., 2004; Arau´jo et al., 2005). Only in some bees (Zama et al., 2001; Fiorillo et al., 2005), and in the ants of the genus Pseudomyrmex (Moya et al., 2007) has the organization of accessory and doublet microtubules in the centriolar region been mentioned. In Vespa crabro it is clear that the centriolar region consists of microtubular doublets without arms and spokes encircled by the accessory microtubules, which are prolonged upwards to surround the nuclear base. The occurrence of a larger mitochondrial derivative located laterally to the nuclear base, reducing the centriolar adjunct anterior portion to a thin structure, is the most evident characteristic shared by the two Vespidae sperm studied so far, V. crabro and A. vicina (Mancini et al., 2006), and apid sperm (Peng et al., 1992, 1993; Lino-Neto et al., 2000b; Zama et al., 2001, 2004; Ba´o et al., 2004; Fiorillo et al., 2005). In V. crabro and A. vicina, as occurs in many Hymenoptera spermatozoa (Newman and Quicke, 1998, 1999a, 2000; Lino-Neto et al., 2000b; Zama et al., 2001, 2004, 2005a,b; Ba´o et al., 2004; Fiorillo et al., 2005; Lino-Neto et al., 2008), the centriolar adjunct is located between the nuclear base and one of the mitochondrial derivatives. On the contrary, in Formicidae (Wheeler et al., 1999; Lino-Neto and Dolder, 2002; Moya et al., 2007), Chalcidoidea (Lino-Neto et al., 2000a; Lino-Neto and Dolder, 2001b) and in the ‘‘symphyta’’, Tremex sp. (Newman and Quicke, 1999a), the centriolar adjunct is located between the nuclear base and both mitochondrial derivatives. The finding of a structured periphery in the centriolar adjunct of V. crabro was also observed only in the ‘‘symphyta’’ Arge pagana (Lino-Neto et al., 2008) and in the Chrysidoidea Trichopria sp. (Miranda, unpublished data). The presence, in the V. crabro sperm, of two mitochondrial derivatives with different shapes, a paracrystalline core only in the larger mitochondrial derivative (and the paracrystalline core occurring distally in relation to the axoneme) can also be found in Apidae (Lino-Neto et al., 2000b; Zama et al., 2001, 2004, 2005a; Ba´o et al., 2004; Fiorillo et al., 2005) and in the parasitic wasps Eucoilidae and Megalyroidea (Newman and Quicke, 1999b, 2000). On the contrary, in Formicidae the mitochondrial derivatives are equal in size, the paracrystalline core is located in both mitochondrial derivatives and the paracrystalline core is proximal in relation to the axoneme (Caetano, 1980; Wheeler et al., 1990; Lino-Neto and Dolder, 2002; Moya et al., 2007). Moreover, in all of the Hymenoptera sperm flagella described so far, the small mitochondrial derivative ends first, followed by the large one (Ba´o et al., 2004; Zama et al., 2004, 2005a,b; Arau´jo et al., 2005). Interestingly, the larger mitochondrial derivative terminates above the smaller one in V. crabro, as also occurs in A. vicina, new data which we wish emphasize in relation to the first publication of Vespidae (Mancini et al., 2006). With regard to the ending of the axonemal microtubules, V. crabro resembles what has been observed in Apidae (Peng et al., 1993; Zama et al., 2001, 2004, 2005a; Ba´o et al., 2004; Fiorillo et al., 2005); in both the groups the accessory microtubules are the last to end. In contrast, in Chalcidoidea (Lino-Neto et al., 1999, 2000a; Lino-Neto and Dolder, 2001b), the accessory microtubules end first. In conclusion, although Vespidae are phylogenetically closely related to Formicidae, the sperm morphology data suggest that the former are more closely related to Apoidea (especially Apidae) than to Formicidae. Thus, this study supports the validity of insect sperm morphology as a tool for phylogenetic analysis within Hymenoptera.
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Arthropod Structure & Development journal homepage: www.elsevier.com/locate/asd
Sperm ultrastructure of the European hornet Vespa crabro (Linnaeus, 1758) (Hymenoptera: Vespidae) Karina Mancini a, *, Jose´ Lino-Neto b, Heidi Dolder a, Romano Dallai c a
Departamento de Biologia Celular, CP 6106, Universidade Estadual de Campinas, 13084-971 Campinas, SP, Brazil Departamento de Biologia Geral, Universidade Federal de Viçosa, 36570-000 Viçosa, MG, Brazil c ` di Siena, 53100 Siena, Italy Dipartimento di Biologia Evolutiva, Universita b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 March 2008 Accepted 2 July 2008
This study represents the first sperm description of a Vespinae species (Vespa crabro). The acrosome consists of an acrosomal vesicle and a perforatorium. The nucleus has compact chromatin and shows lenticular structures on the nuclear envelope. These structures, which have never been observed in a hymenopteran sperm, could be clusters of nuclear pores. The centriolar adjunct has an asymmetric pattern and shows a structured periphery. The centriole consists of 9 accessory microtubules and 9 doublet microtubules devoid of arms and spokes. The axoneme has a 9 þ 9 þ 2 microtubule pattern and the accessory microtubules have 16 protofilaments. The mitochondrial derivatives differ in length and diameter. The larger one is adjacent to the nuclear base, while the smaller one begins below the centriolar adjunct. They possess three distinct areas and a large paracrystalline region, which occurs only in the large one. The large mitochondrial derivative ends first, followed by the small one. The axoneme gradually disorganizes: first the central microtubules disappear, then the doublets, which show opened B-tubules, and finally the accessory microtubules. The sperm morphology of V. crabro is very similar to that of the polistine wasp, Agelaia vicina. This can indicate that, in Vespidae, sperm morphology is maintained without important variations among subfamilies and/or that this similarity indicates close phylogenetic relationship between these two subfamilies. Although Vespidae phylogenetically related to Formicidae, these data suggest that the former more closely related to Apoidea than to Formicidae. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Electron microscopy Insect phylogeny Insect spermatozoa
1. Introduction The order Hymenoptera is composed of the taxa ‘‘symphyta’’ and Apocrita and the latter is still divided into Aculeata and Parasitic wasps (LaSalle and Gauld, 1992). Vespidae belong to Aculeata and include about 4200 species divided into six subfamilies (Carpenter, 1982). Euparagiinae and Masarinae display only solitary behavior; Eumeninae exhibit both solitary and presocial behavior; Stenogastrinae are facultatively eusocial, and Polistinae and Vespinae are all eusocial (Cowan, 1991; Ross and Matthews, 1991; Crespi and Yanega, 1995). The subfamily Vespinae (hornets and yellowjacket wasps) consists of about 60 species divided into four genera, which are predominantly found in northern regions of the world (Carpenter and Kojima, 1997).
* Corresponding author. Tel.: þ55 19 3521 6116; fax: þ55 19 3521 6111. E-mail address: [email protected] (K. Mancini). 1467-8039/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.asd.2008.07.001
In Hymenoptera, as in many other insect groups, the sperm structure has become an important source of characters for phylogenetic studies (Baccetti, 1970; Dallai, 1974; Jamieson, 1987; Wheeler et al., 1990; Quicke et al., 1992; Dallai and Afzelius, 1993; Jamieson et al., 1999). The sperm morphology of Hymenoptera is known in several groups (Quicke et al., 1992; Newman and Quicke, 1999; Lino-Neto et al., 1999; Lino-Neto and Dolder, 2001a,b) including the Aculeata (Caetano, 1980; Wheeler et al., 1990; Peng et al., 1992, 1993; Lino-Neto et al., 2000b; Lino-Neto and Dolder, 2002; Zama et al., 2001, 2004, 2005a; Ba´o et al., 2004; Arau´jo et al., 2005; Fiorillo et al., 2005; Moya et al., 2007). However, in Vespidae, the sperm morphological data are limited to only one ultrastructural study dealing with the polistine wasp, Agelaia vicina (Mancini et al., 2006). All these studies have demonstrated that the hymenopteran sperm structure is very diverse among the taxon, but within different species, or even genera, it is quite similar and this allows these data to be used for taxonomy and phylogeny. The present study describes the sperm ultrastructure of the largest European eusocial wasp, Vespa crabro, with the aim of contributing to the knowledge of Vespidae sperm and to the systematics of Hymenoptera.
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2. Materials and methods Male specimens of Vespa crabro (Linnaeus 1758) were captured in the neighborhoods of Siena (Italy). After dissection, the seminal vesicles were processed according to Dallai and Afzelius (1990). Ultrathin sections obtained with a Reichert Ultracut 2E ultramicrotome were routinely stained with 3% uranyl acetate and 3% lead citrate and observed with a Philips CM 10 transmission electron microscope at 80 kV. 3. Results Spermatozoa of Vespa crabro consist of head and flagellar regions. The head contains an acrosome and a nucleus, while the flagellum has an axoneme, two mitochondrial derivatives and two accessory bodies. Between the two regions, a centriolar region with a centriolar adjunct is evident. The acrosome is 2.5 mm long and consists of an acrosomal vesicle and a perforatorium. The acrosomal vesicle is very thin and its axis is occupied by the perforatorium, which extends from just below the acrosome tip to the nuclear tip (Fig. 1A–D). In cross sections, it has a flattened profile at the tip (Fig. 1A), and an oval profile at the mid and posterior portions (Fig. 1B–D). The perforatorium is a long, dense, crystalline structure (Fig. 1B–D), cone shaped, with its base inserted into the anterior nuclear cavity (Fig. 1D). The nucleus shows a very homogenously compact chromatin (Fig. 1E,F,I) and appears oval shaped in cross sections (Fig. 1F). Along
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its whole length and preferentially in the posterior nuclear region several dense lenticular structures, similar to nuclear pores, about 80–90 nm, can be observed (Fig. 1E,G), which are located at irregular intervals along the nuclear envelope. The nuclear base is thin, shows an oblique profile, in close association with the centriolar region (Figs. 1H,I and 2A–C). The nucleus–flagellum transition region is composed of the nuclear base, the centriolar adjunct, the anterior portions of the large mitochondrial derivative and the axoneme (Figs. 1H,I and 2A–C). The centriolar adjunct has an asymmetric pattern; it begins as a thin flattened structure located between the nuclear base and the large mitochondrial derivative (Fig. 2A). A little posteriorly, it acquires a lateral location, between the centriolar region and the large mitochondrial derivative (Fig. 2B–C). Its flattened anterior portion displays an amorphous appearance (Fig. 2A), while the mid portion has a triangular profile in cross sections and shows a structured periphery (Fig. 3A). The centriolar adjunct extends up to the initial axoneme portion (Fig. 2A–C) and it ends at the small mitochondrial derivative tip. The centriolar region is located below the nucleus and laterally to the centriolar adjunct and the large mitochondrial derivative; it consists of 9 accessory microtubules surrounding nine doublets (Fig. 2C). Considering the head–tail direction, the accessory microtubules are the first ones to appear (Fig. 2B), followed by the doublets, which are devoid of dynein arms and spokes. No central tubules are as yet present. At this level, the nuclear posterior tip, surrounded by the two parallel membranes of the nuclear envelope, is evident (Fig. 2C). The axoneme has a 9 þ 9 þ 2 microtubule
Fig. 1. A–I. Anterior region of the Vespa crabro sperm. (A) Cross section of the acrosome tip, showing the flattened acrosomal vesicle (a). (B, C) Cross sections of the acrosome mid portion, showing the oval acrosomal vesicle (a) and the perforatorium (p). (D) anterior tip of the nucleus (n) with the perforatorium (p) in the nuclear cavity. (E) Longitudinal section of the nucleus (n) showing an area with lenticular structures (arrows) along the nuclear envelope. (F) Cross section of the nucleus, to show its oval shape. (G) Longitudinal section of the posterior region of the nucleus showing the presence of the lenticular structures (arrows) in this portion. Centriolar region (c) with the beginning of the axoneme (ax). (H) Longitudinal section of the posterior region of the nucleus (n) showing the large mitochondrial derivative (md1) and the centriolar region (c). Note the presence of the thick nuclear envelope (white arrow) in the posterior tip. (I) Longitudinal section of the posterior portion of the nucleus (n). Axoneme (ax) and nuclear envelope (white arrow) in the posterior tip. Scale bars: 0.5 mm; (F) 0.2 mm.
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Fig. 2. A–C. Nucleus flagellum transition region of the Vespa crabro sperm. (A) Cross section of the posterior region of the nucleus (n) showing the flattened centriolar adjunct (ca) and the large mitochondrial derivative (md1) with a large paracrystalline region (pc). Note the nuclear envelope (white arrow) and two lenticular structures (arrows) in the nucleus. (B) Cross section of the nucleus flagellum region showing the nuclear posterior tip (n) with two parallel membranes of the nuclear envelope (arrow) surrounded by some accessory microtubules (am). Amorphous material is intermingled between the different structures. The centriolar adjunct (ca) is located laterally to the large mitochondrial derivative (md1) that has a paracrystalline core (pc). (C) Cross section of the nucleus flagellum region showing the nuclear posterior tip region (n) with the two membranes of the nuclear envelope (arrow) surrounded by 9 accessory microtubules (am) and 9 doublets devoid of dynein arms and spokes (dm). Triangular centriolar adjunct (ca) laterally to the large mitochondrial derivative with its paracrystalline region (pc) is visible. Scale bars: 0.25 mm.
pattern, with 9 external single accessory microtubules provided with 16 protofilaments in their tubular wall, 9 microtubule doublets showing dynein arms and spokes and two central microtubules (Fig. 3A,B). The mitochondrial derivatives differ in length and diameter. The larger one appears first, adjacent to the nuclear base (Figs. 1H and 2A–C), and extends parallel along the axonemal length (Fig. 3A–E). Its anterior portion, at the centriolar level, is approximately circular in cross section, and has a large paracrystalline organization (Fig. 2A–C). Its mid portion is pear shaped in cross section, and its area is twice as large as its anterior tip (Fig. 3A,B). This portion comprises a large paracrystalline region (distal in relation to the axoneme) (Fig. 3A,B,D) and an amorphous region (proximal in relation to the axoneme). The amorphous region shows a central
clear area (Fig. 3A,B), cristae located at the periphery close to the plasma membrane (Fig. 3A–D) and a dense area located at the peripheral portion, close to the axoneme (Fig. 3A–C). The paracrystalline region occupies more than half of the mitochondrial derivative area (Fig. 3A,B,D). The small mitochondrial derivative begins below the centriolar adjunct. It shows, approximately, a circular profile in cross section, and is half the size of the large mitochondrial derivative (Fig. 3B). Differently from the large one, the small mitochondrial derivative does not display a paracrystalline region, but only amorphous material in three distinct areas (Fig. 3B). The accessory bodies are almost triangular in cross section, and are positioned between the axoneme and both mitochondrial derivatives (Fig. 3A,B,E).
Fig. 3. A–I. Flagellar region of the Vespa crabro sperm. (A) Beginning of the flagellar region, showing the complete axoneme, with 9 accessory microtubules (am), 9 doublets provided with dynein arms and spokes (dm) and 2 central microtubules (cm). The centriolar adjunct (ca) is triangular and shows a structured periphery (double arrow). The large mitochondrial derivative (md1) has a paracrystalline region (pc), and an amorphous region with a central clear area (arrowhead), cristae (arrows) and a dense area (asterisk). The accessory body (ab) is close to the mitochondrial derivative. (B) Flagellar region, showing the complete axoneme (ax); the large mitochondrial derivative (md1) and the small one (md2) which have a central clear area (arrowhead), cristae (arrow) and a dense area (asterisk). The large one has a paracrystalline region (pc). Accessory bodies (ab) are close to the mitochondrial derivatives. (C, D) Longitudinal sections of the flagellar region, showing the cristae of the mitochondrial derivatives (arrows). Axoneme (ax), paracrystalline region (pc) and the dense area (asterisk). (E) Flagellar posterior region, showing the axoneme (ax), the small mitochondrial derivative (md2), with cristae (arrow), and the posterior tip of the large one (md1). The accessory bodies (ab) are close to the derivatives. (F–I) Flagellar posterior tip, showing the axoneme disorganization. Accessory microtubules (am), doublets (dm) and central microtubules (cm). Scale bars: 0.25 mm.
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In the posterior sperm tip, the large mitochondrial derivative ends first (Fig. 3E), followed by the small one. Then, the axoneme begins to be disorganized; the central microtubules end first, followed by the doublets, which show opened B-tubules, and finally by the accessory microtubules (Fig. 3F–I).
4. Discussion This study represents the first sperm ultrastructural description of a Vespinae species. The other study on Vespidae sperm deals with the polistine wasp, Agelaia vicina (Mancini et al., 2006). The sperm morphology of Vespa crabro and A. vicina is very similar, since in both Vespidae: (a) the acrosome is composed of an acrosomal vesicle and a perforatorium; (b) the acrosomal vesicle is oval shaped in cross sections; (c) the nucleus has homogenous compact chromatin; (d) the centriolar adjunct is located between the nuclear base and one of the mitochondrial derivatives; (e) the nuclear posterior region is surrounded by the centriolar region; (f) the axoneme has a 9 þ 9 þ 2 microtubular pattern; (g) the mitochondrial derivatives begin at different levels and have different shapes and diameters; (h) a paracrystalline region is present only in the larger mitochondrial derivative; and (i) the accessory microtubules are the last ones to finish at the posterior axonemal tip. The acrosome, formed by an acrosomal vesicle and a perforatorium, is a common characteristic of Hymenoptera sperm, being described in ‘symphyta’ (Newman and Quicke, 1999a) and Aculeata (Wheeler et al., 1990; Peng et al., 1992, 1993; Zama et al., 2001, 2004, 2005a,b; Lino-Neto and Dolder, 2002). The acrosomal vesicle of V. crabro and A. vicina shows an oval shape in cross sections and a similar appearance was also observed in bee sperm (Cruz-Ho¨fling et al., 1970; Peng et al., 1992, 1993; Ba´o et al., 2004; Fiorillo et al., 2005; Zama et al., 2005a), while an acrosomal vesicle with a circular profile in the anterior region and triangular in the mid region has been observed in the sperm of the stingless bees (Zama et al., 2001, 2004; Arau´jo et al., 2005) and ants (Wheeler et al., 1990; Lino-Neto and Dolder, 2002; Moya et al., 2007). The conical shape of the perforatorium of V. crabro sperm is shared with the Euglossinae Eufriesea violacea (Zama et al., 2005a), as well as the Halictinae Pseudaugochlora graminea bees (Fiorillo et al., 2005). In the other hymenopteran sperm studied so far, including A. vicina, the perforatorium is, instead, a cylindrical structure. In general, the hymenopteran sperm nucleus shows homogenously compact chromatin, as was observed in V. crabro and A. vicina. However, in some Apidae (Zama et al., 2004; Fiorillo et al., 2005), and in the ant Pseudomyrmex (Moya et al., 2007), the nucleus has poorly condensed chromatin. In addition, some species contain, within the homogeneously compact chromatin, some electronlucent lacunae, as reported for the ant Solenopsis invicta (Lino-Neto and Dolder, 2002) and the Halictinae bees (Fiorillo et al., 2005). The dense lenticular structures observed along the posterior nuclear region of V. crabro have not been reported before in Hymenoptera spermatozoa. They are most likely modified nuclear pores as suggested by their dimensions, although subunits of the eight proteins that surround the nuclear pore cannot be identified in this dense structure. Such structures were also observed in the neck region of mammalian spermatozoa (Friend and Fawcett, 1974) and can be interpreted as a sign of transit of molecules from the nucleus to the flagellum or as merely the evidence of sperm immaturity. Probably, the presence of these nuclear pores could be associated with the uncommon appearance of the nuclear envelope at the nuclear posterior tip, where the two membranes of the nuclear envelope are parallel, but loose and distant from one another. The finding observed in V. crabro sperm is quite similar to the structure interpreted as dense or connective material and lamellar structure of the Sphecidae wasp (Zama et al., 2005b) and bee sperm (Lino-Neto
et al., 2000b; Zama et al., 2000, 2004, 2005a; Ba´o et al., 2004; Fiorillo et al., 2005). The centriolar region, although described in many Hymenoptera sperm, is poorly known. Many papers do not describe in detail this region or just show and mention ‘‘the centriolar region’’ (Lino-Neto et al., 1999; Lino-Neto and Dolder, 2001a; Zama et al., 2004; Arau´jo et al., 2005). Only in some bees (Zama et al., 2001; Fiorillo et al., 2005), and in the ants of the genus Pseudomyrmex (Moya et al., 2007) has the organization of accessory and doublet microtubules in the centriolar region been mentioned. In Vespa crabro it is clear that the centriolar region consists of microtubular doublets without arms and spokes encircled by the accessory microtubules, which are prolonged upwards to surround the nuclear base. The occurrence of a larger mitochondrial derivative located laterally to the nuclear base, reducing the centriolar adjunct anterior portion to a thin structure, is the most evident characteristic shared by the two Vespidae sperm studied so far, V. crabro and A. vicina (Mancini et al., 2006), and apid sperm (Peng et al., 1992, 1993; Lino-Neto et al., 2000b; Zama et al., 2001, 2004; Ba´o et al., 2004; Fiorillo et al., 2005). In V. crabro and A. vicina, as occurs in many Hymenoptera spermatozoa (Newman and Quicke, 1998, 1999a, 2000; Lino-Neto et al., 2000b; Zama et al., 2001, 2004, 2005a,b; Ba´o et al., 2004; Fiorillo et al., 2005; Lino-Neto et al., 2008), the centriolar adjunct is located between the nuclear base and one of the mitochondrial derivatives. On the contrary, in Formicidae (Wheeler et al., 1999; Lino-Neto and Dolder, 2002; Moya et al., 2007), Chalcidoidea (Lino-Neto et al., 2000a; Lino-Neto and Dolder, 2001b) and in the ‘‘symphyta’’, Tremex sp. (Newman and Quicke, 1999a), the centriolar adjunct is located between the nuclear base and both mitochondrial derivatives. The finding of a structured periphery in the centriolar adjunct of V. crabro was also observed only in the ‘‘symphyta’’ Arge pagana (Lino-Neto et al., 2008) and in the Chrysidoidea Trichopria sp. (Miranda, unpublished data). The presence, in the V. crabro sperm, of two mitochondrial derivatives with different shapes, a paracrystalline core only in the larger mitochondrial derivative (and the paracrystalline core occurring distally in relation to the axoneme) can also be found in Apidae (Lino-Neto et al., 2000b; Zama et al., 2001, 2004, 2005a; Ba´o et al., 2004; Fiorillo et al., 2005) and in the parasitic wasps Eucoilidae and Megalyroidea (Newman and Quicke, 1999b, 2000). On the contrary, in Formicidae the mitochondrial derivatives are equal in size, the paracrystalline core is located in both mitochondrial derivatives and the paracrystalline core is proximal in relation to the axoneme (Caetano, 1980; Wheeler et al., 1990; Lino-Neto and Dolder, 2002; Moya et al., 2007). Moreover, in all of the Hymenoptera sperm flagella described so far, the small mitochondrial derivative ends first, followed by the large one (Ba´o et al., 2004; Zama et al., 2004, 2005a,b; Arau´jo et al., 2005). Interestingly, the larger mitochondrial derivative terminates above the smaller one in V. crabro, as also occurs in A. vicina, new data which we wish emphasize in relation to the first publication of Vespidae (Mancini et al., 2006). With regard to the ending of the axonemal microtubules, V. crabro resembles what has been observed in Apidae (Peng et al., 1993; Zama et al., 2001, 2004, 2005a; Ba´o et al., 2004; Fiorillo et al., 2005); in both the groups the accessory microtubules are the last to end. In contrast, in Chalcidoidea (Lino-Neto et al., 1999, 2000a; Lino-Neto and Dolder, 2001b), the accessory microtubules end first. In conclusion, although Vespidae are phylogenetically closely related to Formicidae, the sperm morphology data suggest that the former are more closely related to Apoidea (especially Apidae) than to Formicidae. Thus, this study supports the validity of insect sperm morphology as a tool for phylogenetic analysis within Hymenoptera.
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