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Arthropod Structure & Development 40 (2011) 77e92

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Arthropod Structure & Development

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Sperm accessory microtubules suggest the placement of Dipluraas the sister-group of Insecta s.s.

Romano Dallai a,*, David Mercati a, Antonio Carapelli a, Francesco Nardi a, Ryuichiro Machida b,Kaoru Sekiya b, Francesco Frati a

aDepartment of Evolutionary Biology, University of Siena, I-53100 Siena, Italyb Sugadaira Montane Research Center, University of Tsukuba, Ueda, Nagano, Japan

a r t i c l e i n f o

Article history:Received 20 May 2010Accepted 11 August 2010

Keywords:Hexapod sperm structureSperm phylogenySperm ultrastructure

* Corresponding author. Tel.: þ39 0577 234 412; faE-mail address: dallai@unisi.it (R. Dallai).

1467-8039/$ e see front matter � 2010 Published bydoi:10.1016/j.asd.2010.08.001

a b s t r a c t

Sperm ultrastructure and spermiogenesis of the dipluran Japygidae (Japyx solifugus, Metajapyx braueriand Occasjapyx japonicus) and Campodeidae (Campodea sp.) were studied with the aim of looking forpotential characters for the reconstruction of the phylogenetic relationships of basal hexapods. BothJapygidae and Campodeidae share a common sperm axonemal model 9þ 9þ 2, provided with nineaccessory microtubules. These microtubules, however, after their formation lose the usual positionaround the 9þ 2 and migrate between the twomitochondria. In Japygidae, four of these microtubules arevery short and were observed beneath the nucleus after negative staining and serial sections. Accessorymicrotubules have 13 protofilaments in their tubular wall. Diplura have a sperm morphology which isvery different from that of the remaining Entognatha (Proturaþ Collembola). On the basis of the presentresults, the presence of accessory microtubules suggests that Diplura are the sister-group of the Insectas.s.. Moreover, Japygidae and Campodeidae differ with regards to the relative position of the spermcomponents, the former having the axoneme starting from beneath the nucleus (above which sits theshort acrosome), while the latter having a long apical acrosome and a nucleus running parallel with theproximal part of the axoneme. The present study also allowed to redescribe the male genital system ofJapyx.

� 2010 Published by Elsevier Ltd.

1. Introduction

The ultrastructure of spermatozoa has been extensively studiedin a vast number of species (Gagnon, 1999; Birkhead et al., 2009),including insects (Jamieson,1987; Jamieson et al., 1999). In additionto their intrinsic value for the understanding of the functionalmorphology of such fundamental cells, details of sperm ultra-structure may serve as useful characters to assess evolutionaryrelationships and, in a cladistic framework, reconstruct phyloge-netic relationships (Jamieson et al., 1995; Dallai et al., 2003, 2007).Albeit with a variable degree of utility in different taxa, spermcharacters have provided interesting information in a number ofcases, including the relationships of pentastomids with Branchiura(Wingstrand, 1972; Giribet et al., 2005), of Myzostomida withRotifera and Acanthocephala (Zrzavý et al., 2001), and of Solifugaearachnids with actinotrichid mites (Alberti, 1980; Klann et al.,2009). It appears that, in many instances, sperm morphology may

x: þ39 0577 234 476.

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be more conservative than external morphology with respect tofunctional adaptations to specific lifestyles.

One of the peculiar features of insect spermatozoa is the presenceof 9 accessory microtubules to the flagellar axoneme. This set of 9microtubules, with a variable number (13e20) of protofilaments,surrounds the 9þ 2 doublet microtubules of the axoneme. Severalhypotheses have been formulated on their function, includingmechanical reinforcement, contribution to motility, and storage ofpolysaccharides (Dallai and Afzelius, 1999). During spermiogenesis,accessory microtubules originate from the proliferation of theB-subtubule of each axonemal doublet, starting from the fourth pro-tofilament and growing up to form a complete microtubule (Dallaiand Afzelius, 1999). At the end of this growth, each accessory micro-tubule loses contact with the corresponding doublet, but remains inclose association with it often surrounded by electrondense materialcalled “intertubular material” (Dallai and Afzelius, 1992).

Accessory microtubules are found in all ectognathan insects(Insecta s.s.), from the basal Microcoryphia (¼Archaeognatha) andZygentoma (Dallai et al., 1992; Dallai, 1994), to the most derivedHolometabola, and are therefore considered as a synapomorphicfeature for the whole group (Kristensen, 1981). They are primarily

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present in nearly all insect orders except Phthiraptera, Mecoptera,and Siphonaptera and are also secondarily lost in subgroups of theDiptera, Trichoptera, and Lepidoptera (Dallai and Afzelius, 1999).

Diplura is a small group of basal hexapods which live in the soil.Three main lineages of Diplura are distinguished on the basis of theform of the cerci. Campodeoidea (also referred to as Campodeina)exhibit filiform cerci; Japygoidea (also referred to Japygina) possessforceps-like cerci; Projapygoidea have short and stout terminalappendages. The classification of Pagés (1997) and Dunger (2003)recognizes the suborders Rhabdura (Projapygoidea and Campo-deoidea) and Dicellurata (Japygoidea), while the classificationbased on ovary structure and molecular data unites Projapygoideawith Japygoidea. With the presumably allied taxa Protura andCollembola, they compose a group of primitively wingless hexa-pods, exhibiting enclosed mouthparts (Entognatha), that arecommonly classified as the basal lineages of Hexapoda, or as thesister-taxa of the Insecta (Kristensen, 1981). Nevertheless,a remarkable set of peculiar characters possessed by these organ-isms has stimulated the reconsideration of their correct phyloge-netic relationships (reviewed by Grimaldi, 2010). With respect tothe position of Diplura, apart from the controversial hypothesis ofa semi-entognathan fossil (Kukalova-Peck, 1987), the monophyly ofEntognatha has been questioned on the basis of ovary structure byBüning (1998), who suggested the Japygina as the sister-group ofEctognatha, thereby implying the paraphyly of Diplura. Differencesin the structure and development of the female reproductivesystem (�Stys and Bilinski, 1990; Bilinski, 1994) have also suggestedthe possible paraphyly of Diplura, with the lineage of Campodeinabeing the sister taxon of Ellipura (Proturaþ Collembola), and theJapygina being the basal lineage of all Entognatha. The interpreta-tion of the morphological characters potentially useful for thereconstruction of the phylogenetic relationships of basal hexapods(Protura, Collembola and Diplura) has also been the subject ofextensive revision (Koch, 1997; Bitsch and Bitsch, 2004; Klass andKristensen, 2001; Grimaldi, 2010), and new data are now alsobeing accumulated on the phylogenetic relevance of embryologicalaspects (Machida, 2006). Recently, a wealth of molecular data havebeen collected, based on bothmitochondrial and nuclear sequences(reviewed in Carapelli et al., 2005). Mitochondrial genome datainvariably suggest the possible paraphyly of the Hexapoda astraditionally defined, with Collembola (Nardi et al., 2003a,b;Negrisolo et al., 2004; Cook et al., 2005), as well as Diplura(Carapelli et al., 2006, 2007), not sharing a recent common ancestorwith Pterygota, but branching off the tree of Pancrustacea in a basalposition. This hypothesis, if confirmed, would implicate thathexapody, terrestrialization and other putative hexapod apomor-phies (Edgecombe 2010) have been acquired several times inde-pendently (Carapelli et al., 2005, 2007). However, this hypothesiscould be potentially affected by peculiarities of the molecularevolution of the mitochondrial genome (Delsuc et al., 2003;Cameron et al., 2004; Giribet et al., 2004; Hassanin et al., 2005).On the other hand, molecular analyses based on nuclear genesstrongly support the monophyly of the Hexapoda (Regier et al.,2004, 2010; Kjer et al., 2006), although the position and recip-rocal relationships of basal lineages remain controversial. Oneunexpected outcome of these analyses is the presumed relation-ship between Protura and Diplura (with the exclusion of Collem-bola), for which the new taxon Nonoculata has been proposed(Luan et al., 2005; Dell’Ampio et al., 2009; von Reumont et al.,2009). In all these analyses, the monophyly of Diplura isconfirmed, and morphological data in support of the Nonoculatahave also subsequently accumulated (Szucsich and Pass, 2008;Koch, 2009).

Preliminary data on the presence and position of the accessorymicrotubules in the diplurans Japyx and Campodeawere presented

in Dallai and Afzelius (1999), showing that Diplura, likewise allInsecta s.s., possess 9 accessory microtubules. However, theirposition and dynamics of assemblage in the flagellar axoneme issomewhat peculiar, and, in this work, we aim at showing the detailsof this process. We also provide a detailed description of the malegenital system of Japyx, thereby revising the original description byGrassi (1888) which is always reported in the specialized literature(see Berlese, 1909; Grassé, 1949; Imms,1973 for relevant examples).

2. Materials and methods

The following material was used for the present work:

Japygidae- Occasjapyx japonicus (Enderlein, 1907) (R. Machida &K. Sekiya leg.), Tsukuba, Japan

- Japyx solifugus Holiday, 1864 (A. Carapelli leg.), Siena, Italy- Metajapyx braueri (Verhoeff, 1904) (E. Dell’Ampio leg.), WienAustria

Campodeidae- Campodea sp., (R.Dallai leg.), Siena, Italy

After collection, the specimens of the four species were kept insmall containers in a climate chamber at 20 �C and 80% of humidityand fed with small fragments of dried fish food and of barks ofdifferent plants. For transmission electron microscope (TEM)preparations, the whole male genital organs were dissected in0.1 M pH 7.2 buffer to which 3% of sucrose was added (PB). Thematerial was fixed overnight in 2.5% glutaraldehyde in PB at 4 �C,then rinsed in PB and post-fixed in 1% OsO4 for 1 h, after rinsing inPB. The material was dehydrated in a series of graded alcohol andembedded in a mixture of Epon-Araldite resin. Part of the materialwas treated with 2.5 glutaraldehyde in PB to which 1% of tannicacid was added, according to Dallai and Afzelius (1990). Thinsections, obtained with a Reichert ultramicrotome, were routinelystained with uranyl acetate and lead citrate and observed witha Philips CM 10 electron microscope operating at 80 kV.

For light microscopical observations, the dissected male genitalsystem was photographed with a Olympus SZX12 stereo lightmicroscope equipped with a Zeiss Axiocam MRC5 digital camera.

Negative staining preparations were performed on free sperm ofJ. solifugus, taken from deferent ducts, suspended on a drop ofdistilled water, then transferred on a grid and stained with 1%uranyl acetate.

Freeze fracture replicas were obtained on previously fixeddeferent ducts of J. solifugus. The material was cryoprotected byimmersion in PB buffer with increasing glycerol concentration (10%,20%, 30% glycerol), then it was frozen by plunging it into Freon�22,refrigerated with nitrogen liquid, and mounted on a freeze BalzersBAF 400 fracturing device. Fractured pellets were shadowed with2.5 nm of platinumecarbon and backed with 20 nm of carbon.Finally, tissues were removed from replica with sodium hypo-chlorite, rinsed in water, and mounted on rhodiumecopper 50meshes grid for TEM observations.

For scanning electron microscope (SEM) observations, sperma-todesms taken from deferent ducts were suspended in a drop of PBplaced on coverslips previously treated with 1% poly-L-lysine.A drop of fixative (2.5% glutaraldeyde in PB) was added to thesuspension. After several rinsings, coverslips with adherent spermwere treated according to the critical point drying method ina Balzers CDP 030. The coverslips, coated with about 20 nm goldmetal in a Balzers MED 010 sputtering device, were attached ona mounting base and observed with a Philips XL 20 scanningelectron microscope operating at 10 kV.

Fig. 1. (A) Light microscopic view of one of the branches of the male genital system of Japyx solifugus. Note the apical masses of fat body (Fb), which extend also along the testes (T),the deferent duct (D), and the accessory gland (ag). The arrow indicates the insertion of the accessory gland. Details of testes (upper-left) and of the transition region between testesand the deferent duct (bottom-right) are shown in the insets. (B) Drawing of the male genital system of Japyx solifugus compared to that by Grassi (1888) (C).

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3. Results

3.1. The general anatomy of the male genital system of J. solifugus

In all examined specimens (of different ages) of J. solifugus, aswell as in the several specimens of the other two species studied in

the present work (M. braueri and O. japonicus), the organization ofthe male genital system is quite similar. It consists of an apical,elongated (about 1 mm long) mass of fat body, connected througha thin peduncle to the anterior end of testes. These are tubularstructures, 70e80 mm in diameter, which extend in a sinuous wayfor 1.5e1.7 mm; along their bends, large amounts of fat body are

Fig. 2. (A) Three living spermatodesms of Japyx solifugus at the interference light microscope. (B) SEM detail of the anterior part of a J. solifugus spermatodesm showing thehelicoidal array of the sperm heads (H). (C) SEM micrograph. Three sperm of J. solifugus isolated from the spermatodesm; sperm head (H) and tail (T) are visible. (D) SEMmicrograph. An isolate spermatozoon of J. solifugus; H, head; T, sperm tail. (E) Freeze fracture replicas of a J. solifugus spermatodesm. Note that a cluster of intramembrane particlesare associated with the outer flagellar membrane E-face (arrowheads).

Fig. 3. (A) Longitudinal section through a Japyx solifugus spermatodesm showing two sperm cells. a, acrosome; ax, flagellar axoneme; N, nucleus. (B,C) Longitudinal sectionsthrough the sperm cells in a J. solifugus spermatodesm. Note the short bi-layered acrosome (a) and the compact nucleus (N) beneath which the flagellar axoneme (ax) is visible. Notethat a dense long structure (P) is visible attached to the acrosome. (D) Cross section through a sperm bundle in the J. solifugus spermatodesm. Ten sperm cells are sectioned at theflagellar level and each of them exhibits a 9þ 2 axoneme, 5 accessory microtubules (at) and 2 mitochondria (m). Note that in the axial part of the bundle the amount of the stickymaterial is visible (Sm).

Fig. 4. (A,B) Negative stainings of Japyx solifugus sperm to show the nucleus (N) and, beneath it, four short (arrows) and five long accessory microtubules (numbers 1e5).

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present, sometimes hiding the testes. The deferent ducts are long,tubular ducts about 3e4 mm long, the diameter of which is greatlyvariable, depending on the amount of spermatodesms they contain.Usually, they are larger than testes, about 150 mm in diameter, butwhen they are filled with sperm they become very large, up to250 mm in diameter. Close to the point where the two deferentducts fuse to give origin to the short ejaculatory duct, an accessorygland is evident, branching from the inner side of each deferentduct. These glands, often tightly adhering to the deferent duct, havethe same diameter of testes (about 70 mm) and are about 1.5e2 mmlong (Fig. 1A,B).

3.2. The sperm structure of Japygida

The ultrastructural study performed on the three genera ofJapygidae (Japyx, Metajapyx, Occasjapyx) gave similar results andthus their sperm structure will be described together and onlywhen important different details occur in a certain species theywillbe mentioned.

The sperm length is not easy to establish, as sperm is in bundlesto form spermatodesms. Approximately, it measures about 150 mmin J. solifugus, when a sperm cell is detached from the spermato-desms (Fig. 2D). The length of each spermatodesm in the threespecies examined is 180e200 mm (Fig. 2A), thus a little longer thanthe sperm length, while its width, in J. solifugus, is of about1.1�0.83 mm or 1.5� 0.74 mm (Fig. 3B,C); spermatodesm widthvaries according to the shape acquired in the deferent duct, wherethey can be compressed by closely assembled bundles. What hasbeen observed by the comparison of the spermatodesms in thethree species is that, while a cross section of J. solifugus and O.japonicus spermatodesms shows 10 sperm in each of them, in

M. braueri the cross section of spermatodesm exhibits 8 sperm cells(compare Fig. 3D with Figs. 7C,D and 9C).

The sperm in each bundle is stuck together by a secretionpresumably produced by the deferent epithelial wall, whichextends along the axial part of the bundle (Figs. 3D, 7BeD, 9C). Thisstructure is analogous to the “axial filament” described by Bareth(1968) in Campodea. The anterior tract of the sperm flagellumadheres to this structure. The sperm are aligned along the sper-matodesm in such a way that two nuclei are often at the same leveland then the cells prolong with their flagella in helicoidal fashionaround two other sperm cells positioned at a short distance fromthe previous ones. The same is repeated for all sperm cells along thewhole length of the spermatodesm (Figs. 2B, 6B). During thepreparation of material for SEM observations, some sperm cells candetach from the spermatodesms (Fig. 2C) and also isolated spermcells can be observed (Fig. 2D). After freeze fracture replicas, theplasma membrane of each sperm flagellum of the spermatodesmbundle shows a series of intramembrane particles (IMPs), associ-ated with the E-face of the plasma membrane, thus towards thematerial in the spermatodesm axis (Fig. 2E). The secretion containsfinely fibrous material and small elliptical dense bodies (Figs. 3B,C,7BeD). Only in O. japonicus the material seems to be less organizedthan in the other species (Fig. 9C). The membrane specializationreflects of the intimate contact of the plasma membrane of eachsperm in the bundle with the material present in the axial part ofthe spermatodesms.

The most intriguing point in the study of spermatogenesis andspermiogenesis in the three species is the lack of evidence of themeiotic process, as well as of sperm differentiation. Many dissec-tions of J. solifugus performed throughout several years have failedin giving information on this process; thus, only spermatogonialcells were found in the testes of all the species studied. In

Fig. 5. (A,B,C) Cross section through the basal body region of J. solifugus to show the progressive appearance of the 9 accessory microtubules (1e9) on a side of the 9þ 2 axoneme,between the two mitochondria (m). N, nucleus; P, sections through the prolongation adherent to the acrosome. (D,E) Cross section through the sperm flagellum at two differentlevels. Note that in (D) the accessory tubules are still present, while in (E) they are missing.

Fig. 6. Metajapyx braueri. SEM micrograph showing a spermatodesm. (B) Detail of the apical region of the spermatodesm to show the nucleus (N) and the tail (T).

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O. japonicus these cells are interconnected by cytoplasmic bridgesand are provided with a large nucleus; in the cytoplasm, a Golgiapparatus and a few mitochondria are visible. The centriole hasdoublets rather than triplet microtubules. In some sections, cells inmitotic division are also visible (Fig. 9A,B). In J. solifugus the sper-matogonial cells are intermingled with others, which are invadedby large masses of microorganisms. The whole testes are filled withdegenerating cells, presumably spermatogonial cells (not shown).

The sperm of the three species is filiform, with an apical small bi-layered acrosome consistingof an acrosome vesicle, that in J. solifugusis 34 nmthick andonly 0.12 mmhigh,with the shapeof a convex vaultbeneathwhich somedensematerial is present (Fig. 3AeC). The spermprolongs anteriorly in an elongated structure (1.3e1.4 mm long),triangular in cross section in its basal part andflattened apically (Figs.3A, 5B), throughwhich it adheres to the stickymaterial present in theaxial part of the spermatodesm. The acrosome is more expanded(0.4 mm long) in M. braueri and O. japonicus; in cross section theyclearly exhibit the axial perforatorium surrounded by the acrosomevesicle filled with dense material (Fig. 7A).

The nucleus has a compact chromatin; it has a cylindrical shapeand is about 3.5 mm in J. solifugus (Fig. 3AeC), 6 mm long inM. braueri (Fig. 7A) and 4.2 mm in O. japonicus. Beneath the nucleus,the region of the basal body occurs, with 9 microtubular doubletsdevoid of dynein arms. These doublets are almost embedded in 9dense amounts of material of the centriole adjunct which, ina centrifugal way, detach from the axial part (Fig. 5A). In M. brauerithe centriole adjunct material is more expanded and obscures thestructure of the basal body (Fig. 8AeC). Microtubular doublets havethe subtubules A provided with the usual 13 protofilaments in theirtubular wall and are filled with dense material, while subtubules B,which have 11 protofilaments, are not (Figs. 5A,B, 8B,C). In the spacebetween the axoneme and the plasma membrane, on the sidewhere the two mitochondria will be also located, 9 additionalaccessory microtubules are present (Figs. 5A,B, 8AeC). Shortlybehind the basal body region, only five of these nine microtubulesremain and they accompany the axoneme up to almost the tail end.These five microtubules, located among the two mitochondria,have 13 protofilaments in their tubular wall and contain densematerial (Figs. 3D, 5D, 7C,D).

While in J. solifugus the presence of the 9 singlet microtubules isnot always easy to detect beneath the nucleus and serial sectionsare needed to count all of them (Fig. 5AeC), in M. braueri theirpresence is easy to observe, as they are positioned at the same level,just beneath the basal body in the space between the beginning ofthe two mitochondria and the plasma membrane (Fig. 8B,C). Theoccurrence of the nine microtubules just beneath the basal bodyregion and the shortness of four of them is clearly evident afternegative staining preparations of J. solifugus sperm. Beneath thenucleus, four short microtubules, only 0.14 mm long, are visible,while the other five microtubules extend up to almost the tail end(Fig. 4A,B). Towards the flagellar end, in all three species examined,the single 5 microtubules end and they are no longer visible (Figs.5E, 7D, 9C). At the tail end, only a few doublets are still visible, eventhough they have lost the usual position and their dynein arms.Moreover, other singlet microtubules are visible, possibly bya modification of some doublets, and the flagellar diameter isreduced in size to only 0.12e0.15 mm (Fig. 7D). The sperm bundlesare actively motile in the common buffer; the sperm beating issynchronous in all the sperm cells of a bundle, but soon somesperm detach from the bundle and continue the beating as isolatedsperm cells.

3.3. The sperm structure and the spermiogenesis of Campodea sp.(Campodeida)

Contrary to the above mentioned species of Japygidae, thedifferent steps of sperm maturation can be easily followed in thegonad of Campodea. During spermiogenesis, the sperm cells ofCampodeidae, about 300e350 mm long, cluster to form large spermbundles. A series of axial filaments of 1.5e2 mm of diameter,produced by the epithelial wall and consisting of a compacthomogeneous secretion, contact the sperm bundles. The axonemeof each sperm in the bundle adheres to the periphery of the fila-ment which, for this reason, exhibits several indentations (Fig.10A).At the same time, the winding up of the sperm bundle occurs. Thus,in a cross section, close to the axial filament, acrosomes will beevident, while the nuclei and the flagellar axonemes will be visiblefar from it (Fig. 10A). In a cross section through one of these sperm

Fig. 7. (A) Metajapyx braueri. Longitudinal section through a spermatodesm showing the acrosome (a), the nucleus (N), and the prolongation (P) adherent to the acrosome. (B)Longitudinal section through a spermatodesm showing thematerial constituting the axial part (asterisks) towhich the sperm adhere. (C,D) Cross section of two spermatodesms. In (C)the section passes through the anterior sperm region and each flagellar axoneme contains also 5 accessorymicrotubules; in (D) the section is through the posterior flagellar region andthe accessory microtubules are missing (asterisks). At the tail end (arrowheads) the structure of the flagellum is reduced to few microtubule doublets. N, nucleus; m, mitochondria.

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bundles we can estimate that the filament forms 5e6 turns. Lateron, two sperm bundles will be commonly used for the building ofthe spermatophore droplet, used in Campodeidae for reproductionby indirect sperm transfer (Schaller, 1952, 1954; Bareth, 1974).

The acrosome of Campodea sp. is about 8 mm long and, in a crosssection, it measures 0.3e0.35 mm; it consists of a thin acrosomalvesicle and an inner cylindrical perforatorium, which is constitutedby a crystalline material (Fig. 11A). Beneath the acrosome, theregion of basal body occurs. The basal body consists of 9 doublets,with the usual configuration and protofilament composition, whichare devoid of arms and are embedded in a double dense ring ofcentriole adjunct material (Fig. 11B). From this region, the nucleusand two small mitochondria are visible running in parallel with theaxoneme 9þ 2. On the same region, also 9 singlet microtubules,each one with 13 protofilaments in their tubular wall, are visible: 5microtubules are in a linear array close to one mitochondrion andthe other 4 are close to the opposite mitochondrion. Sometimes 1or 2 microtubules are intermingled with the mitochondria and thenucleus and the remnant microtubules are close to the plasmamembrane (Fig. 10B). The origin of this peculiar array of microtu-bules can be well understood studying the process of spermio-genesis. In the late spermatids, an acrosome and a stilluncondensed nucleus are visible, both surrounded by a layer ofmicrotubules, which are connected to the outer membrane of thevesicle by short bridges (Fig. 11A,C). Beneath the acrosome, thenucleus, the two mitochondria and the axoneme run in parallel(Fig. 11C). Microtubules also surround the two mitochondria. Fromthe basal body, a 9þ 2 axoneme is found. Doublet microtubuleshave two dynein arms and radial spokes ending in quite expandedspoke-heads. As it occurs in pterygotan insects, each doubletmicrotubule gives origin to a centrifugal projection starting fromB-subtubule which, at the end of the process, originates 9 accessorymicrotubules provided with 13 protofilaments in their tubular wall(Fig. 11D). These accessory microtubules maintain their contactwith the doublets until spermiogenesis is near to the end. Whenspermiogenesis is finished, however, the accessory microtubulesdetach from the doublets and migrate to the flagellar periphery tobe placed close to the mitochondria and to the surrounding plasmamembrane (Fig. 10B).

When transferred in the buffer solution, the spermatodesmsshow a progressive motility, with sperm beating synchronously.Contrary to the previous species of Japygidae, we were unable tosee free sperm cells.

4. Discussion

4.1. Japygidae and Campodeidae have a 9þ 2 axoneme with 9accessory microtubules

Our data show that the three species of Japygidae and thesingle species of Campodeidae studied here share a commonsperm flagellar pattern. In particular, in both major lineages ofDiplura, nine accessory microtubules in the sperm axoneme areformed during spermiogenesis. The evidence presented heredefinitely corrects the wrong conclusions drawn by Baccetti andDallai (1973), who suggested the five microtubules observed

Fig. 8. Metajapyx braueri. (A,B,C) Serial cross sections through the basal body region. In(A) the basal body (bb) with microtubule doublets embedded in the dense centrioleadjunct material is visible. a, acrosome; P, peduncle. In (B), a shortly more posteriorsection through the basal body (bb); the 9 accessory microtubules (arrowheads) areevident between the two small mitochondria. a, acrosome; P, peduncle. In (C), tran-sition zone between the basal body and the initial part of the 9þ 2 axoneme withdoublets still devoid of arms. Note the 9 accessory microtubules (1e9) between thetwo mitochondria (m); a, acrosome.

Fig. 9. Occasjapyx japonicus. (A) Cross section through a group of spermatogonial cells at the end of the mitotic division. (B) Cross section through a spermatogonial cell with nucleus(N) and a cluster of mitochondria (m). (C) Cross section through a deferent duct lumen to show sperm cells in spermatodesm bundles. Note that 10 sperm cells surround the axialregion where the sticky material (asterisks) is present. The bundle on the left upper corner consists of sperm cells sectioned through their posterior ends, where the 5 accessorymicrotubules are missing (arrowheads).

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Fig. 10. (A) Cross section through a deferent duct of Campodea sp. In the duct lumen, sections of the spiral axial filament (fi) are visible; several acrosomes (a) adhere to the filament,while sections of the nucleus (N) and of the flagella (F) are at a distance from the filament due to the winding up of the sperm cells in the spermatodesm. (B) Detail of the sperm tailshowing the parallel position of the nucleus (N), of the mitochondria (m), and of the axoneme (ax). Note the position of the 9 accessory microtubules (arrowheads) lateral to theaxoneme and close to the mitochondria.

Fig. 11. (A) Campodea sp. Cross section through the acrosome of a mature spermatid. Note the acrosome vesicle (a), the crystallized material of the perforatorium (p), and the crownof microtubules (mt) surrounding the structure. Note the short bridges anchoring the microtubules to the acrosomal membrane. (B) Cross section through a spermatid basal body. Itconsists of 9 doublet microtubules embedded in the dense material of the centriole adjunct. (C) Cross section through three spermatids. The acrosome (a) is visible on the bottom;the two other spermatids show the nucleus (N) parallel to the axoneme (ax) and to the two mitochondria (m). Note that the microtubule doublets of the axoneme are forming the 9accessory microtubules. (D) Detail of the formation of the accessory microtubules from the axonemal doublets (arrowheads). m, mitochondria.

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along the most part of the sperm flagellum of Japyx to be theremnants of the microtubular “manchette” which helps theshaping of the sperm components during spermiogenesis. On theother hand, these microtubules unequivocally correspond toaccessory microtubules, homologous to those described in pter-ygotan insects (Dallai and Afzelius, 1990). Nevertheless, while theevidence is quite clear and convincing in Campodea, the identi-fication of the 9 accessory microtubules in Japygidae is madedifficult by the fact that four microtubules are very short andplaced right beneath the nucleus. Here, negative stainingprovides the necessary evidence to identify them as accessorymicrotubules. In addition, it has proven impossible to study theorigin of the accessory microtubules in Japygidae, a fact poten-tially associated with the habit of these organisms that movefrom the lower strata to the soil surface only for reproduction.Therefore, immature stages (where initial stages of spermiogen-esis are likely to be found) are difficult to collect, thereby pre-venting the description of the full process. Indeed, many aspectsof the reproductive biology of Japygidae need further clarification,including the presumed presence of spermatophores (Pagés,1967) and the mode of fertilization.

4.2. Phylogenetic implications of the presence of accessorymicrotubules

The presence of accessory microtubules in Diplura bearsremarkable implications in terms of the evolution and phylogenyof basal Hexapoda. Accessory microtubules are considered as oneof the crucial synapomorphies of Insecta s.s. (Kristensen, 1981).The classical framework of Diplura, as part of the Entognatha,would imply an independent acquisition of accessory microtu-bules. On the other hand, the presence of accessory microtubulesplaces the Diplura as the sister-group of the Insecta (Dallai andAfzelius, 1999; Giribet et al., 2005; Dallai et al., 2006; Beuteland Gorb, 2006). This is also supported by the presumeddifferent type of entognathy shown by Diplura (Manton, 1977;Koch, 2001). The position of Diplura as the sister-taxon (or thebasal lineage) of the Ectognatha (¼Insecta) is also at odds with allmolecular reconstructions that either support Entognatha (Regieret al., 2004, 2010; Kjer et al., 2006), and sometimes Nonoculata(Dell’Ampio et al., 2009), or even the paraphyly of Hexapoda(Carapelli et al., 2007). If considered the synapomorphy ofDipluraþ Ectognatha, the presence of accessory microtubulesdoes not say much about the monophyly or paraphyly of Diplura.Indeed, accessory microtubules of Campodeidae and Japygidaehave similar features (including the number of protofilaments, 13,and their unstable position) that might indicate monophyly of thetaxon Diplura. It should also be mentioned that paraphyly of thetaxon was only considered in the framework of the Entognatha(�Stys and Bilinski, 1990). Nevertheless, Japygidae and Campodei-dae sperm differ regarding the arrangement of the spermcomponents. Species of Campodeidae, in fact, show a long bi-layered acrosome, beneath which the elongated nucleus andmitochondria run parallel to the proximal part of the axoneme.The presence of a long acrosome was also found in machilidMicrocoryphia (Jamieson et al., 1999), while the parallelarrangement of the nucleus and the axoneme was found also insome lepismatids (Zygentoma). On the other hand, species ofJapygidae exhibit a more traditional sperm cell, with the short bi-layered acrosome sitting above the nucleus, and the axonemestarting right beneath the nucleus.

The other two entognathan orders, Collembola and Protura, lackaccessory microtubules. They also show other peculiarities insperm morphology, although these differences are irrelevant forphylogenetic reconstruction in a cladistic framework. Collembolan

spermatozoa are uniform throughout the group, with a 9þ 2axoneme, and the flagellum rolled-up around an extracellularmaterial (Dallai, 1994; Dallai et al., 2003). The encysted sperm ofProtura have an axoneme without central complex and a variablenumber of doublet microtubules; the encysted sperm are formedby a mechanism reminiscent of that occurring in some Arachnida(Dallai et al., 2010).

In the next basal ectognathan lineage, the Microcoryphia,accessorymicrotubules have a different protofilament number (16),and their position is also unusual. However, while accessorymicrotubules in Campodea and Japyx are clustered on one side ofthe plane of flagellar beating, in Microcoryphia they are arranged intwo groups (of 5 and 4 microtubules) placed on the opposite sidesof such a plane (Dallai, 1994). The loss of the connection of acces-sory microtubules with the axonemal doublets observed inMicrocoryphia and Diplura may also be explained by the lack ofintertubular material. From a functional point of view, the asym-metrical position of accessory microtubules of Diplura and Micro-coryphia, and their short length in Japygidae, prevent them to beuseful to stabilize the flagellar beating, as suggested for pterygotaninsects (Dallai and Afzelius, 1990). Nevertheless, the reduction inlength of the sperm flagellum and the association of sperm cells inspermatodesms may render this function less important in theseorganisms.

Another peculiar feature of the sperm of Diplura is the presenceof the “axial filament”, produced by the epithelial cells of the testesand responsible of anchoring the sperm cells in a bundle (Bareth,1968; Pagés, 1997).

The sperm of the members of the Japygidae exhibits an apicalpeduncle which is reminiscent of that described in the sperm ofCollembola (Dallai, 1970; Dallai et al., 2008a,b, 2009), although thesimilarity is only superficial. In Collembola, in fact, the peduncleends up in being modified in the female spermatheca and origi-nates many vesicles, whose function may be related with theunwinding and the activation of sperm motility (Dallai et al.,2008a,b).

4.3. The redescritpion of the male genital system of Japyx

According to Grassi (1888), the male genital system of J. sol-ifugus consisted of one pair of large testes with long, convolutedvasa deferentia with two accessory glands closely adhering tothem. A short ductus ejaculatorius opened on the 8th sternum(Fig. 1C). This has remained for many years the only informationavailable on the structure of the male genital system of Japyx, andhas been used in most books and papers dealing with the subject.The data collected here show that that description is wrong. Thetestes are not as large as in Grassi’s description, but rather thin,elongated and sinuous. We argue that what Grassi described asthe testes is in fact a large mass of fat body which can beobserved at the distal end of the Japygidae testes. The fat bodiesthen extend for a short distance along the true testes. The mostremarkable feature of Japyx genital system are the deferent ducts,which are large cylindrical canals of variable diameter (presum-ably depending on the amount of sperm contained). Deferentducts, in fact, are filled with mature sperm, organized in sper-matodesms. The two tubular structures detaching from thedeferent ducts are indeed accessory glands, as they never containsperm cells in their lumen. By the time sperm cells are stored inthe deferent ducts, spermatogenesis is stopped, and no meioticdivisions, nor spermatids, are observable in the testes. Only in theJapyx population from Siena, degenerating testes are invaded bylarge aggregates of bacteria.

R. Dallai et al. / Arthropod Structure & Development 40 (2011) 77e92 91

Acknowledgements

The authors wish to thank Dr. Emiliano Dell’Ampio for providingspecimens ofMetajapyx braueri. This work was supported by a PRINgrant from the Italian Ministery of Research to R.D.

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