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Arthropod Structure & Development 40 (2011) 531e547

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Arthropod Structure & Development

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The male reproductive system of Zorotypus caudelli Karny (Zoraptera):Sperm structure and spermiogenesis

R. Dallaia,*, D. Mercatia, M. Gottardoa, R. Machidab, Y. Mashimob, R.G. Beutelc

aDepartment of Evolutionary Biology, Via A. Moro 2, I-53100 Siena, Italyb Sugadaira Montane Research Center, University of Tsukuba, Ueda, Nagano 386-2204, Japanc Institut für Spezielle Zoologie und Evolutionsbiologie mit Phyletischem Museum, Friedrich-Schiller-Universität Jena, Ebertstrasse 1, D-07743 Jena, Germany

a r t i c l e i n f o

Article history:Received 18 April 2011Accepted 13 July 2011

Keywords:Insect reproductionInsect spermiogenesisSperm structureInsect phylogenyElectron microscopy

* Corresponding author. Tel.: þ39 577 234412; fax:E-mail address: romano.dallai@unisi.it (R. Dallai).

1467-8039/$ e see front matter � 2011 Elsevier Ltd.doi:10.1016/j.asd.2011.07.001

a b s t r a c t

Considering the overall uniformity of the morphology of Zoraptera, the structural diversity of the malegenital system is remarkable. Structures related to the male reproductive system of Zorotypus caudellidiffer profoundly from those of Zorotypus hubbardi. The testes are elongated rather than spherical, theseminal vesicle is apparently absent, and the deferent ducts are very long. A feature shared by these twospecies and other zorapterans examined is that the two accessory glands are closely adherent to eachother and form a single large structure, from which the ejaculatory duct originates. This is a potentialzorapteran autapomorphy. Another feature possibly present in the groundplan of the order is the strongelongation of the sperm cells. This may be connected with a reproductive strategy of males trying toavoid re-mating of females with other males after the first copulation. The extremely long and coiledspermathecal duct of Z. caudelli and other zorapteran species is possibly correlated with the spermelongation, and both features combined may result in a sexual isolating mechanism. The short durationof mating of Zorotypus barberi and Zorotypus gurneyi suggests that the male introduces sperm into thefemale tract up to the opening of the spermathecal duct using their long coiled aedeagus. A thick gly-cocalyx around the sperm in the distal part of the deferent ducts probably protects the sperm cells duringtheir forward progression towards the long spermathecal duct, and is removed when they reach theapical receptacle. The spermatogenesis of Z. caudelli follows a pattern commonly found in insects, butdiffers distinctly from that of Z. hubbardi in the number of spermatids in each sperm cyst. An unusual andpossibly autapomorphic feature of Z. caudelli is a disconnection of sub-tubules A and B at the level ofmicrotubule doublets 1 and 6 of the mature sperm cells. It is conceivable that this results in a shorterperiod of sperm motility. The character combination found in different zorapteran species supports theview that the sperm, a very compact functional unit, does not evolve as a unit, but like in other morecomplex body regions, sperm components can also be modified independently from each other. Thisresults in different mosaic patterns of plesiomorphic and derived features in a very compact entity indifferent species of the very small and otherwise uniform order Zoraptera. In Z. caudelli, for instance, thebi-layered acrosome and small accessory bodies are plesiomorphic states among several others, whereasthe mitochondrial derivatives and the elongate nucleus are apparently derived conditions. Othercombinations likely occur in other zorapteran species. Only few but noteworthy sperm charactersindicate possible phylogenetic affinities of Zoraptera. A possible synapomorphic feature, the presence ofdense laminae radiating in a cartwheel array between neighbouring centriolar triplets, is shared withPhasmatodea and Embioptera. Another potential synapomorphy shared with Phasmatodea is the pres-ence of 17 protofilaments in the tubular wall of the outer accessory microtubules.

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1. Introduction

The placement of Zoraptera, the insect order described bySilvestri (1913), represents one of the most intriguing phylogenetic

þ39 577 234476.

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problems among pterygote insects. The different hypothesesdealing with the position of the taxon have been carefully exam-ined (Engel and Grimaldi, 2002) and discussed (Yoshizawa, 2007).According to these authors, four hypothetical sister groups of Zo-raptera are considered at the present state of knowledge: Derma-ptera (Jarvis et al., 2005; Terry and Whiting, 2005), Dictyoptera(Boudreaux, 1979; Wheeler et al., 2001), Embioptera (Minet and

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Bourgoin, 1986; Engel and Grimaldi, 2000, 2002; Grimaldi andEngel, 2005; Yoshizawa, 2007) and Acercaria (Hennig, 1953;Beutel and Weide, 2005).

Yoshizawa (2007) investigated the wing base structure of repre-sentatives of different hemimetabolous lineages and some holome-tabolous taxa and reached the conclusion that structural affinities inthis character system support a close phylogenetic relationshipbetween Zoraptera and Embioptera. He pointed out that the rate ofevolution of this character complex is low due to functional restric-tions related to flight and wing folding mechanisms.

The sperm structure of Zorapterawas still an unexplored sourceof phylogenetic information. Sperm structure has been

Fig. 1. Schematic drawing of the male genital system of Z. caudelli, A. Dorsal view. B. Ventr

successfully used to infer the phylogenetic position of taxa(Jamieson et al., 1999). There are several examples supporting thevalidity of the sperm structure to establish, often unexpected, closerelationships between groups. The best example is that describedby Wingstrand (1972) who, on the basis of a quite similar spermstructure, suggested a close relationship between Pentastomidaand the crustacean Branchiura, rather than with Tardigrada andOnychophora. Wingstrand’s hypothesis appeared rather unlikelyconsidering structural features of the worm-like parasitic penta-stomids, which differ radically from “typical” crustaceans in all lifestages. However, it was further supported from sperm ultrastruc-ture by Storch and Jamieson (1992) and was later unequivocally

al view. AG, accessory glands; D, deferent ducts; E, ejaculatory duct; S, spiral; T, testes.

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confirmed by single gene molecular analyses (e.g., Abele et al.,1989; Lavrov et al., 2004; Giribet al., 2005) and also in a recentstudy with a phylogenomic approach (Regier et al., 2010). Analo-gously, Myzostomida, highly aberrant ectoparasites of echino-derms, were placed with Rotifera and Acanthocephala(Gnathifera), rather than with Annelida, based on their spermstructure and molecular data (Zrzavý et al., 2001). Among Arach-nida, Solifugae display a sperm structure more similar to

Fig. 2. A. General organization of the male genital system of Z. caudelli, after dissection. Noaccessory glands (AG) with a spiral structure (S) in the anteriormost region. Accessory glacopulatory organ (C). B. Detail of the testes (T), deferent ducts (D) opening into the symmeplaced anteriorly in the complex. Posteroventral part removed during dissection. C. Detail as icuticular spiral forming the aedeagus (E). The structure ends with two cuticular branches.posteroventral region showing the cuticular spiral of the long aedeagus and the two cuticulNote the cuticular spiral of the aedeagus (E).

actinotrichid mites rather than to that of pseudoscorpions (Alberti,1980; Alberti and Peretti, 2002; Klann et al., 2009). A well knownexample of an apparently important feature in insects is thepresence of an additional outer ring of 9 single microtubules in thesperm axoneme, which supports a clade Diplura þ Insecta (e.g.,Klass, 2007; Dallai et al., 2011a).

In general, and for insects in particular, it is clear that externalmorphological characters are often affected by strong selective

te the testes (T), the long deferent ducts (D), the symmetrical structures forming thends connected to the posteroventral part (V) crossed by the ejaculatory duct and thetrical complex forming the accessory glands (AG). Note the typical spiral (S) structuren B, but also showing the posteroventral region containing the ejaculatory duct and theT, testes; D, deferent ducts; AG, accessory glands; S, spiral structure. D. Detail of thear branches (asterisks in E). E. Copulatory organ after potassium hydroxide treatment.

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pressure. For example, the bug Coptosoma scutellatum looks likea beetle at first glance, but its sperm structure is unequivocally ofthe heteropteran type (Dallai and Afzelius, 1980; Mercati et al.,2009). Likewise the sperm structure of Braula coeca, a morpho-logically aberrant wingless bee parasite, immediately indicates itsposition within Brachycera (Dallai, personal observation).

In combination with parallel investigations on the egg struc-ture and the female genital apparatus, using Zorotypus caudelli asmaterial, the aim of the present work is to contribute to a step-wise solution of the persistent Zorapteran problem by comparingtheir sperm structure with those of representatives of suppos-edly related insect orders. The general structure of the malegenital system will also be described and compared withconditions found in the related species Zorotypus hubbardirecently studied by Hünefeld (2007) and also in members ofother insect lineages.

2. Materials and methods

Six females and 12 males of Z. caudelli collected at Ulu Gombak,Selangor, Malaysia, by Y. Mashimo and R. Machida were used asmaterials. After dissection in 0.1 M phosphate buffer pH 7.2, towhich 3% of sucrose was added (PB), the male and female genitalsystems were isolated and photographed with a light microscope.The material was then transferred in 2.5% glutaraldehyde in PB

Fig. 3. A. Cross section through early spermatids with a spheroidal nucleus (N) and the largeB. Cross section through an early spermatid showing the nucleus (N) and the flagellar axonespermatid with the roundish homogenously dense nucleus (N), with a flattened acrosome (giving rise to the perforatorium space (p). Beneath the nucleus, the centriole adjunct materiasection through flagella of early spermatids. Axoneme (ax) provided with complete set of m

overnight at 4 �C. After careful rinsing in PB, the material was post-fixed in 1% osmium tetroxide for 1e2 h. After rinsing, the materialwas dehydrated in a graded series of ethyl alcohol (50�e100�), thentransferred in propylene oxide and finally embedded in an Epon-Araldite resin. Part of the material was treated with the tannicacid method following Dallai and Afzelius (1990a). Ultrathinsections were obtained with a Reichert ultramicrotome, routinelystained with uranyl acetate and lead citrate, and observed witha CM10 Philips transmission electron microscope operating at80 kV. Semithin sections were stained with 1% toluidine blue andphotographedwith a Leica DMRB interference-contrast microscopeequipped with an AxioCam HR camera (Carl Zeiss).

Free spermatozoa were obtained by dissecting testes in 0.1Mphosphate-buffered saline (PBS), pH 7.2. They were observed andphotographed using interference-contrast and epifluorescentmicroscopy with a Leica DMRB light microscope equipped withan AxioCam HR camera (Carl Zeiss). DNA was stained by incu-bating the samples for 3e4 min in Hoechst 33258 1 mg/ml(Sigma). The material was then mounted in a drop of 90%glycerol.

A suspension of sperm in PB was spread on a coverslip previ-ously treated with a 1% of poly-L-lysine, then fixed in 2.5% glutar-aldehyde for 30 min at 4 �C. Samples were then rinsed in buffer andpost-fixed in 1% osmium tetroxide, dehydrated in ethanol, driedafter terbutilic alcohol sublimation, and coated with gold in

“Nebenkern” (m). Note the huge Golgi complex (G) with the pre-acrosomal vesicle (av).me (ax) with the two lateral mitochondria (m). C. Longitudinal section through an earlyA) on top. At half length of the acrosome, the nuclear envelope shows a small infoldingl (arrowheads), the flagellar axoneme (ax) and a mitochondrion (m), are visible. D. Crossicrotubular structures, flanked by two mitochondria (m) still showing inner cristae.

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a Balzers MED 10 sputtering device. Specimens were finallyexamined and photographed in a Philips XL20 scanning electronmicroscope operating at 10 kV.

3. Results

Themale reproductive system of Z. caudelli consists of two testesoccupying the dorsal region of the abdominal cavity (Figs. 1 and2AeC). Each testis is formed by two elongate, banana-shaped folli-cles (ca. 500 mm long, ca. 10 mm wide). They open into two longdeferent ducts (ca. 1 mm) directed towards the anteroventral bodyregion (Fig. 2A, B). Their diameter is slightly larger at the beginning,and in some specimens they are dilated to form a small roundishstructure fromwhich the deferent ducts originate. The diameter ofthe ducts is constantly about 15 mm for most of their length;however, close to a conspicuous elliptical symmetrical heart-shapedstructure, it is increased to up to 40 mm. They penetrate into this

Fig. 4. A. Cross section through a spermatid surrounded by a layer of microtubules (arrowcondensed in thin threads forming a meshwork. B. Cross section through the nucleus (N) ofby a layer of microtubules (arrowhead). C. Cross section through centriolar region of a spderivatives (m), both embedded in centriole adjunct material (ca). Nucleus (N) and centriolarcentriolar region of an early spermatid showing the centriole structure (c) consisting of 9 mithe periphery. Centriole surrounded by granular centriole adjunct material (ca). N, nucleus. Ederived from 25 spermatocytes. Cyst enveloped by a cyst cell (Cc).

structure upon reaching half length of its dorsal part and then bendtowards the posterior region. The heart-shaped structure consists oftwo closely adhering accessory glands (Fig. 2AeC); it is 560 mmwideat its middle region and it continues anteriorly as a spiral-shapedappendage with 5e7 loops (ca. 250 mm long) (Fig. 2AeC), which isthe first structure to appear when the abdomen of a specimen isdissected starting from the anteriormost part of the abdomen. Theheart-shaped structure is continuous with an elongated region (ca.250 mmlong and 150e200 mmwide) containing the ejaculatory duct(Fig. 2AeC), which is prolonged as a long chitinized spiral-shapedaedeagus (Fig. 2CeE). Posteriorly, a cuticular fork is visible, withtwo branches ending with short appendages (Fig. 2CeE).

3.1. The testes and the early spermiogenesis

The two banana-shaped follicles forming the testes are con-nected to the deferent duct by a short canal. Each follicle is filled

heads), showing the flattened acrosome (A). In the nucleus (N) note the chromatina spermatid showing a different stage of chromatin condensation. Nucleus surroundedermatid. Note the initial part of the axoneme (ax) flanked by the two mitochondrialregion surrounded by layer of microtubules (arrowheads). D. Cross section through thecrotubular triplets in the typical cartwheel array and the “L” dense laminae (arrows) at. Cross section through a germ cell cyst containing about 120 almost mature spermatids

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with elongate cysts with germinal cells at different stages ofspermiogenesis. In the apical region, early spermatids arepresent. They are characterized by a large “Nebenkern” close toa spherical nucleus, 4e4.5 mm in diameter (Fig. 3A). A conspic-uous Golgi system is present in the cytoplasm; it forms a largepro-acrosome vesicle (Fig. 3A). On top of the nucleus a flattenedacrosome is formed at a later stage (Fig. 3C). At the middle part ofthe acrosome, the nuclear envelope forms a small invaginationwithin which filamentous material is visible. This is the earlyperforatorium (Fig. 3C). The nuclear chromatin in the earlyspermatids is often distributed in small masses, often adhering tothe inner face of the nuclear envelope. However, sometimes itconsists of a homogenously granular material (Figs. 3C and 4D).Later, the chromatin condenses as a meshwork and then intoa series of dense patches progressively filling the entire nucleus(Fig. 4AeC).

The dense granular material of the centriole adjunct liesbeneath the nucleus; it surrounds both the centriole and the twomitochondria (Fig. 3C). The centriole consists of 9 triplets ofmicrotubules, which exhibit the conventional cartwheel arrayexhibiting dense “L” laminae. The inner region of the centriole isfilled with dense material (Fig. 4D). The two elliptic mitochondriaare placed beside the axoneme and still have long, easily recog-nisable cristae (Fig. 3B, D). At a slightly later stage, with theelongation of the spermatids, a process starts in the mitochon-drial matrix with dense material deposited on the inner mito-chondrial membrane on the side facing the axoneme.

The axoneme has the typical microtubule organization9þ 9þ 2; thus the 9 accessory microtubules, external to the central9 þ 2, are formed very early during spermiogenesis. The accessorymicrotubules contain moderately electron-dense material (Fig. 3D).Between adjacent accessory microtubules, intertubular materialwith an elliptical shape is visible.

3.2. Late spermiogenesis

With the elongation of the spermatids, the components, as forinstance the nucleus, modify their shape and appearance. A crosssection through a spermatid cyst shows about 120 germ cells, as theresults of themeiotic division of primary 25 spermatocytes (Fig. 4E).Each cyst is surrounded by a somatic cyst cell. In the apical region ofeach spermatid, the acrosome strictly adheres to the nucleus asa flattened structure with a dense content. The cylindrical nucleusis filled with a homogenous and dense material; together with theacrosome it is delimited by fine, filamentous material withinwhichthe longitudinal microtubule of the “manchette” is visible (Fig. 5).The diameter of the nucleus is reduced progressively from 0.6 to0.35 mmbefore its apex is reached (Fig. 5CeD). The acrosome is bell-shaped in cross section and becomes irregularly elliptic at theanteriormost spermatid region (0.5 mm in diameter) where thenucleus ends; it contains a small hole (0.2 mm in diameter) as in thetypical configuration of a bi-layered acrosomewith a perforatorium(Fig. 5AeB). The dense granular material of the centriole adjunct isvisible beneath the nucleus, where the axoneme originatesaccompanied by two elliptic mitochondrial derivatives (Fig. 4C);these derivatives usually are similar in shape and size and containparacrystalline material along the region towards the axoneme(Fig. 6AeB). At a later stage, the densematerial expands and fills the

Fig. 5. A. Cross section through the anterior region of aged spermatids showing theacrosome (A) with the perforatorium (arrow). B. The acrosome (A) still showing theperforatorium (arrow) is located lateral to the nucleus (N) and it has a bell-shaped incross section. CeD. Cross sections of the nucleus (N) and of the flattened acrosome (A).The layer of microtubules (arrowhead) is still present.

Fig. 6. A. Cross section through a maturing spermatid. The spermatid components are enclosed by a cistern (arrowheads) and the axoneme consists of a complex of 9 þ 9 þ 2microtubules. The intertubular material is regularly arranged between the accessory microtubules. Lateral to the axoneme two relatively small dense dots (arrows) are visible.Beneath the axoneme two mitochondrial derivatives (m), with advanced crystallization of the inner material, are evident. A cistern (cs) is visible between the axoneme and the twomitochondrial derivatives. Glutaraldeyde-tannic acid fixation. B. Cross section through two spermatids at the same stage as in A, after conventional double glutaraldehyde-osmiumfixation. C. Cross section through a nearly mature spermatid. The axoneme has not changed its structure, but the intertubular material between doublets 1 and 6 appears twice aslong as between the others and the doublets are broken into A and B sub-tubules (arrows). Accessory microtubules with 17 protofilaments in their tubular wall. Lateral to theaxoneme, two characteristic dense arches (arrowheads) are visible, while dense material of centriole adjunct (ca) is visible beneath the axoneme. Between the axoneme and the twomitochondrial derivatives (m) a cistern filled with dense material is present (cs). Glutaraldehyde-tannic acid fixation. D. Cross section through three nearly mature spermatidssimilar to that in C, after conventional double glutaraldehyde-osmium fixation.

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Fig. 8. A. Light microscope view of the Z. caudelli sperm. Note the undulated nucleus (N) and the long flagellum (F) running in helicoidal fashion. B. Hoechst staining of the spermobserved with fluorescent microscope. The acrosome (A) and the nucleus (N) are clearly visible. C. SEM micrograph of the apical region showing the acrosome (A) and the nucleus(N).

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entire mitochondrial matrix (Fig. 6CeD). Laterally on both sides ofthe axoneme a small dot is visible (Fig. 6AeB). This structureprogressively extends and assumes the shape of an arch in crosssection, 0.15 mm long and only 25 nm thick (Fig. 6CeD). Moreover,a dot of about 40 nm surrounded by a dense circumference is visible(Fig. 6CeD) in the axial part of the spermatid cross section. This dotand the longitudinal expansions of the centriole adjunct materialgive rise to accessory bodies between the axoneme and the twomitochondrial derivatives (Fig. 6CeD). A peripheral cisternsurrounds all the sperm components; a minimal distance of 60 nmseparates it from the plasma membrane. A shorter cistern is placedabove the two mitochondrial derivatives; this is the place of originof the mitochondrial cisterns at the end of the spermiogenesis(Fig. 6A, C).

At this stage of spermatid maturation a peculiar featurebecomes visible in the cross sectioned axoneme: the intertubularmaterial around microtubules 1 and 6 is about twice as long as thatof the others (Fig. 6CeD); moreover, the microtubule doublets 1and 6 are divided into two subunits A and B (Fig. 6C). As a conse-quence, the appearance of the whole axoneme appears notperfectly circular but slightly flattened. The sub-tubules A and B areformed by 13 and 9 protofilaments, respectively, and both containa dense material in their lumen (Fig. 6C).

The axoneme is still accompanied by the two mitochondrialderivatives for most of the flagellar length. Nevertheless, they

Fig. 7. A. Cross section through the posterior end of two maturing spermatids. Note thatsurrounds the axoneme (arrowheads). The axonemal doublets apparently lost their subtubusection through the flagellar end of a maturing spermatid. The axoneme exhibits disorganisemature spermatids showing the degeneration of the axoneme. The peculiar arches above(arrowheads). The accessory bodies have disappeared. When the two mitochondrial derivatitwo central microtubules and the accessory microtubules maintain their typical structure. Fdisappear. Finally, the flagellar end shows a reduced diameter (4).

reduce their size towards the posterior end when the two lateralarches disappear, and a diffuse, dense material surrounds theaxoneme (Fig. 7AeB). A short distance behind this level, the twomitochondrial derivatives end, and only the axoneme is visible. Ithas an unusual appearance with the microtubule doublets dis-appearing and empty singlet microtubules becoming visible intheir place; only the accessory microtubules filled with densematerial maintain their usual position (Fig. 7B). The cytoplasmaround the spermatid components in the posterior flagellar end ischaracterized by a parallel series of cisterns alternating with a layerof microtubules.

When the spermatid maturation is almost completed, theposterior region of the axoneme is modified; at this stage theaxonemal doublets cannot be distinguished as they fuse to forma dense mass around the two central microtubules. The tail end hasa smaller diameter in cross section (Fig. 7C).

3.3. The mature sperm

The sperm cell of Z. caudelli is long and thin (about 770e800 mmlong). It contains a long cylindrical homogenously dense nucleus(ca. 40 mm) and a bi-layered acrosome (2.5 mm long) with a shortperforatorium on the nuclear tip (Fig. 8AeC). Behind the nucleusthe centriole adjunct material forms a compact ring around theanteriormost part of the axoneme (Fig. 9B). Further behind lies the

the two arches above the axoneme are no longer present and that fibrous materialle B (left bottom). Layers of microtubules surround the spermatid components. B. Crossd microtubular doublets. C. Cross section through the posterior flagellar end of almostthe axoneme reduce their size and the axoneme is surrounded by fibrous material

ves (m) are greatly reduced (1) the axonemal doublets become a dense mass, while theurther behind, the mitochondrial derivatives (2) and also the central microtubules (3)

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origin to the long helicoidal flagellum (Fig. 9A). A cross sectionthrough the main part of the flagellum shows that it consists of anaxoneme with two dense arches above it (Fig. 9C). Beneath theaxoneme three small triangular accessory dense bodies are present(Fig. 9C). Two mitochondrial derivatives of the same size and filledwith compact dense material occupy half of the flagellar sectionbeneath the axoneme. Mitochondrial cristae are not recognisable incross and longitudinal sections, but an axial hole of less densematerial is often visible (Fig. 9B, C). A small cistern adheres to eachmitochondrial membrane facing the axoneme (Fig. 9C).

The axoneme maintains the same pattern described previouslyfor the late spermatids. The accessory tubules contain 17 proto-filaments in their microtubular wall, and their lumen containsglobular units (Fig. 9D). The dense intertubular material is elliptic inshape and those close to microtubular doublets 1 and 6 are twice aslong as the others; the doublets are disconnected at the connectionbetween subtubule A and B, so that the former is a singlet with 13protofilaments in the tubular wall, two dynein arms, and a radialspoke, whilst the subtubule B is a groove with 9 protofilaments(Fig. 9CeD).

Towards the flagellar apex the size of one of the mitochondrialderivatives is reduced and the axoneme becomes surrounded bya thick layer (50 nm of thickness) of dense material (Fig. 9E). Theaxoneme begins to degenerate and the two central microtubulesare apparently displaced from the axonemal central axis; themicrotubular doublets are no longer visible in their normalconfiguration, but are transformed into a single coarse pear-shapedstructure in which 9 protofilaments are only visible along the smallprotuberances of the structure corresponding to the B subtubule(Fig. 9E); the dynein arms and radial spokes are lost. While themicrotubular doublets and central tubules do not maintain theiroriginal position in the axoneme, the accessory tubules are stillregularly arranged at the periphery beneath the dense layer.Further towards the posterior end, the two mitochondrial deriva-tives end and the 9 degenerated axonemal doublets are still visible;sometimes one of them is shifted to the central axonemal region(Fig. 9E); only few (2e3) accessory microtubules are still visible inthe distal part of the axoneme. The anteriormost region is elliptic incross section (0.17 � 0.25 mm) and contains only 9 degeneratedaxonemal doublets surrounded by a thin layer of dense material.

3.4. The deferent ducts

As mentioned above, the long deferent ducts have a smallerdiameter in the proximal part close to the testes (15e20 mm) andwiden (ca. 40 mm) close to the heart-shaped structure of theaccessory glands (Figs 1 and 2AeB). Their ultrastructure is quiteuniform. A longitudinal section through the deferent duct proximalto the testes shows cubic cells with sinuous cell membranes withapical zonulae adherentes and septate junctions. In the cytoplasma few organelles and some microtubules are present and few smalldroplets of secretion are visible in the apical cell region. Beneaththe epithelial cells a very thin basal lamina and a layer of 1.5 mm ofmuscle fibers are present. In the duct lumen many sperm cells arevisible or the lumen can appear reduced and empty depending on

Fig. 9. A. Longitudinal section through a sperm showing the helicoidal array of mitochondriaa sperm. Note the compact appearance of the centriole adjunct material (ca) and the twothrough the sperm flagellum showing the axoneme (ax), the two arches (arrowheads) abov(m) with the flattened cistern (arrows). Glutaraldehyde-tannic acid fixation. D. Detail of the fl

A and B sub-tubules; moreover, the intertubular material at this level is longer than thosefilaments. Glutaraldehyde-tannic acid fixation. E. Cross section through the flagellar end shoand one of them has shifted to the central part. Radial spokes disappear and only one centraenlarged, while the B sub-tubules show the protofilaments in their tubular wall. Accessory mmaterial. m, mitochondrial derivatives.

the time of the reproductive cycle (Fig. 10A). Towards the accessoryglands, the number of apical dense droplets of secretion increasesin the epithelial cells. The thickness of the basal lamina increases to0.25 mm and a layer of muscle fibers is visible beneath it. The lumenis filled with numerous sperm cells (Fig. 10B). The deferent ductspenetrate into the glands after having covered about half the lengthof the accessory gland complex (Fig. 2B). At this level the twostructures run parallel; the diameter of the ducts is about60e70 mm. A cross section through them shows that the epitheliummaintains the organization of the proximal part of the tract, andnumerous dense droplets of secretion are visible in the cytoplasm(Fig. 10B).

Numerous sperm cells are visible in the lumen of both closelyadjacent deferent ducts (Fig. 10C, E). They show the typical ultra-structure described previously, but a thick glycocalyx of 85e90 mmis recognisable outside the plasma membrane of many sperm cells(Fig. 10E, F). Some sperm cells are also visible in the cytoplasm ofthe epithelial cells, which are apparently involved in a spermio-phagic activity (Fig. 10E). After a 180� bend within the accessoryglands the two ducts are directed towards the posterior part of theglands before reaching the ventral region, where they are contin-uous with the ejaculatory duct (45 mm in diameter) (Fig. 12BeC, G).

3.5. The accessory glands

As described above a large heart-shaped structure is present inthe ventral abdominal region. It receives the terminal part of thedeferent ducts and after that continues with the initial part of theregion containing the ejaculatory duct (Figs 1 and 2BeC). Theheart-shaped structure consists of a large (0.4� 0.33mm) perfectlysymmetrical body constituted by two paired glands, which areseparated by a narrow space (2e5 mmwide) where elongate musclefibers and tracheoles are visible (Fig. 10D). Cross sections throughthe anterior region show that the upper part is occupied by the twoparallel deferent ducts, while the bottom region contains a large(0.2 � 0.12 mm) structure which results from the adhesion of twodistinct glandular districts.

Anteriorly, the two glands narrow and form a long (ca. 0.6 mm)spiral structure (Fig. 2AeC). The posterior region of the accessoryglands is crossed by the proximal section of the ejaculatory duct.The two accessory glands consist of a complex of secretory cellswhich delimit the central areas where the secretion is stored. Threecenters of secretion were identified in each gland (Fig. 11A), eventhough only a single large storage area is visible at the end of thecell secretory activity; it extends over most of the length of theglands (Fig. 11D). The secretory cells are flat (only 0.2e0.3 mmwide)and thin microvilli are visible in the apical region; zonulae adher-entes and gap junctions are present along the cell contacts; theirsmall elliptic nuclei are about 5 mm long and form a compact layerat the periphery of the gland; their cytoplasm is rich in roughendoplasmic reticulum cisternae (Fig. 11B). Cells are involved in thesecretion of dense small droplets but progressively their cytoplasmbecomes charged with vesicles of various density. The final result isthe production of large amounts of spongy structures (Fig. 11C) thatare then discharged into the central part of the gland. At the end of

l derivatives (m) and the axoneme (ax). B. Cross section through the centriolar region ofmitochondrial derivatives (m). Glutaraldehyde-tannic acid fixation. C. Cross section

e the axoneme, the three accessory bodies (ab) and the two mitochondrial derivativesagellar axoneme. Note that the microtubules doublets 1 and 6 (arrows) are broken intobetween the other accessory microtubules. The tubular wall is formed by 17 proto-wing the axoneme degeneration. The position of the microtubule doublets is changedl tubule is visible. The microtubule doublets have the A sub-tubules (asterisks) greatlyicrotubules maintain their position and structure. The axoneme is surrounded by dense

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the secretory process the region is filled with many of thesestructures which fuse to form a compact mass of dense material(Fig. 11D).

The anteriormost region of each gland is prolonged as a cylin-drical part which runs closely adherent in a characteristic spiralwith 5e6 loops and a total length of 0.5e0.6 mm (Fig. 2AeC). Theultrastructure of the epithelium of this region does not differ formthat of the main part of the glands; it surrounds a narrow lumen(Fig. 12AeD) where a dense secretion is stored. The epithelial cellsshow the same appearance as those in the region of the mainglands and a few dense droplets are visible in their cytoplasm(Fig. 12EeF). Beneath the epithelium of the two accessory glandsand of the spiral structure a thin basal lamina is visible and beneathit a thin layer of muscle fibers (Figs. 11A, D and 12D).

The posteriormost region of the male genital system consists ofan approximately 200 mm long and roughly cylindrical part, whichis connected with the accessory glands by a narrowed region of100e200 mm length; it ends with two cuticular appendages(Fig. 2A, CeE). This region contains the terminal part of the ejacu-latory duct and the long cuticular spiral constituting the aedeagus.The structure is formed by several longitudinal muscle fibers whichsurround the long branches of a thin epithelium lined by cuticle(Fig. 12B, C, G). This corresponds to the ejaculatory duct which runsthroughout the middle part of the structure (Fig. 12G). At theperiphery a series of long folds with a crenulated cuticle is present.The ejaculatory duct ends with the long aedeagus (Fig. 2DeE).

4. Discussion

The male genital system of Z. caudelli is structurally differentfrom that of Z. hubbardi (Gurney,1938; Kuznetsova et al., 2002). Thetestes are elongated rather than spherical, the seminal vesicle isabsent, and the deferent ducts are very long. Considering the greatmorphological differences between the species so far examined indetail, a careful analysis of the internal anatomy of other membersof the order should have high priority, as already underlined byEngel (2000).

A feature occurring in Z. caudelli and Z. hubbardi, and also inother zorapterans, is the unusual elongation of the sperm cells, asalready described by Silvestri (1913) in his study on Zorotypusguineensis. Another feature which possibly belongs to the ground-plan of the order is the presence of paired accessory glands closelyadherent to each other and forming a compact and large structurefromwhich the ejaculatory duct originates (Fig. 2C). Both characterstates are potential autapomorphies of Zoraptera.

The presence of long sperm is not an uncommon trait amonginsects. Sperm cells measuring more than 0.5 mm or even up to6 cm (Drosophila bifurca) have been described (Pitnick et al., 1995;Jamieson et al., 1999). The greatly increased length is likely relatedwith a reproduction strategy of males, which may avoid re-matingof females with other males after the first copulation. In manyspecies the sperm length increases when an intense spermcompetition occurs (Gage, 1994; Montgomerie and Fitzpatrick,2009). There is tentative evidence, at least in Drosophila, that theincreased length of sperm is correlated with an elongation of thefemale tract (Miller and Pitnick, 2002). A similar coadaptation wasalso demonstrated by Dybas and Dybas (1981) for Ptiliidae. Both

Fig. 10. A. Longitudinal section through the proximal region of a deferent duct. Note the sinunucleus. B. Cross section through the epithelial cells of distal region of a deferent duct show(dr) in their cytoplasm. C. Cross section through the two adherent deferent ducts above the aD. Cross section through the two accessory glands (ag) beneath the deferent ducts (d) showithe two accessory glands tracheoles (t) and muscle fibers are visible. E. Detail of the epith(arrowheads), indicating spermiophagic activity. L, duct lumen; sp, sperm. F. Cross sectionSperm structure unchanged, two arches (arrows) above axoneme, two mitochondrial derivadoublets 1 and 6 broken (arrowheads), the intertubular material at their level is longer tha

features combined may be linked with a sexual isolating mecha-nism. An increased efficiency of sperm propulsion caused by theelongation appears less likely (Pitnick and Markow, 1994). Thisinterpretation fits with our observation of the spermathecal duct ofZ. caudelli, which indeed is extremely long, coiled and adherent tothe common oviduct, as already observed by Gurney (1938) andKuznetsova et al. (2002) in Z. hubbardi. How the sperm reach theapical receptacle of the spermatheca is still unclear.

Considering the short duration of mating of Zorotypus barberiand Zorotypus gurneyi (Choe, 1997), we tentatively assume that themale introduces sperm into the female tract up to the opening ofthe spermathecal duct using its long coiled aedeagus. There is nodirect evidence for this, but it is likely that the secretions of theaccessory glands facilitate the penetration into the female tract andalso enable the sperm to reach the apical receptacle of the sper-matheca. The presence of few free sperm cells in the lumen of thecalyx could be interpreted as remnants of sperm escaped duringmating and transfer of sperm fluid into the spermathecal duct.Alternatively, these isolated cells may be remnants of the fertil-ization, when sperm is squeezed from the spermatheca. In thiscontext, it is also interesting that a thick glycocalyx around thesperm is present in the terminal part of the deferent ducts, close tothe entrance into the accessory glands. This glycocalyx could beinterpreted as a device to protect the sperm cells during theirforward progression towards the long spermathecal duct; theprotective cover is removed when they reach the apical receptacle.The presence of some sperm cells in the cytoplasm of the deferentduct epithelial cells suggest a spermiophagic activity performed atthis level (Dallai et al., 2011b).

The spermatogenesis of Z. caudelli follows a pattern commonlyfound in insects. The number of spermatids and of sperm cells ineach sperm cyst is 128 as the consequence of the number of sper-matocytes before the meiotic process corresponding to 25. Inter-estingly, this seems to be quite different from observations inZ. hubbardi, which according to Kuznetsova et al. (2002) has 32spermatids in each sperm cyst, thus originated by 23 spermato-cytes. The early spermatids show a bi-layered acrosome on top ofthe nucleus with a small perforatorium. It is formed by a large Golgicomplex, as it is commonly found in insects and other groups ofanimals. An elliptic nucleus and a very long flagellum are visible atthis stage. Beneath the nucleus, a typical centriole provided withmicrotubular triplets is embedded in the dense material of thecentriole adjunct. This is the usual condition shared with membersof other polyneopteran lineages (Dallai et al., 2003a, 2005).

Uncommon feature in these orders is the presence of denselaminae radiating in a cartwheel array between neighbouringtriplets. However, this condition was found in Phasmatodea (stickinsects) sperm (Baccetti et al., 1973) and in the embiopteran Hap-loembia solieri (Baccetti et al., 1974), even though with thickerlaminae in the latter. This unusual feature is a potential synapo-morphy of Zoraptera, Phasmatodea and Embioptera. However,a similar condition has also been observed in the odonatan Calo-pteryx sp. (R. Dallai, pers. obs.).

The shaping of the acrosome and of the nucleus proceeds asusual among insects involving the microtubular “manchette”surrounding the sperm components. After the differentiation of theaxoneme two dense spots appear, which then transform into

ate limiting plasma membrane with the long apical zonulae adherentes (arrowheads). N,ing numerous sperm cells in the lumen (L). The epithelial cells contain dense dropletsccessory glands (ag). Note numerous sperm (sp) in the duct lumen (L), and (s) secretion.ng the secretory cells surrounding the lumen where the secretion (s) is stored. Betweenelial cell cytoplasm of the two adherent deferent ducts with some sperm cells visiblethrough sperm flagellum in the apical deferent duct. Note the thick glycocalyx (gly).tives (m), 3 accessory bodies and mitochondrial cisternae (ci) still visible. Microtubulen between the other accessory microtubules.

Fig. 11. A. General view of a cross section through an accessory gland. Three foci of secretion are visible (asterisks). Secretory cells (sc) surround the lumen where the secretion isstored. The gland is surrounded by a basal lamina and a thin muscle layer (arrowheads). B. Detail showing the density of the rough endoplasmic reticulum (rer) in the secretory cells.Note the microvilli (mv) at the cell apices. s, secretion. C. Cross section through a secretory cell showing the secretion of the spongy structures (ss) then discharged in the glandlumen (L). D. At the end of the secretory process, a large compact mass of secretion (s) extends along the entire gland. In the inset note the symmetric appearance of the twoaccessory glands with the large extension of the secretion (s) in the central part (semithin section). Dorsal to the glands the two deferent ducts (d) are visible.

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elongate, curved structures. Their function is still unclear but it is anevolutionary novelty apparently not shared with other insect taxa.The distribution within Zoraptera still needs to be clarified.

The axoneme at an early stage is of the conventional type, with9 þ 9 þ 2 microtubular elements. However, a very unusual featureis the presence of 17 protofilaments in the tubular wall of the outeraccessory microtubules, which are separated from neighbouringones by dots of dense intertubular material. This structural featureis shared only by Phasmatodea among the polyneopteran orders(Dallai and Afzelius, 1999), even though the same number of

Fig. 12. A. Semithin section through accessory glands with the anterior spiral structure. Notesections of two successive levels of the ejaculatory duct (E). Note the large mass of muscleaccessory glands with the cylindrical lumen filled with dense secretion (s). E. Cross section th(region without dense compact secretion in the lumen). F. Detail of elongated epithelial cells(s). G. Cross section through ejaculatory duct. Note the branched epithelium (ep) lined byepithelial folds.

protofilaments has also been found in a few basal representatives ofTrichoptera (Dallai et al., 1995) and in the strepsipteran Xenos(Dallai et al., 2003b).

An unusual and thus autapomorphic feature of the flagellaraxoneme of Z. caudelli is the disconnection of sub-tubules A and B atthe level of microtubule doublets 1 and 6 of a mature sperm. Alonger area with intertubular material is also visible at these twolevels. An ad hoc explanation of this unusual phenomenon wouldbe a correlation with the helical array of the long sperm flagellum.However, this condition does not occur in other insects showing

the secretion (s) stored in the lumen surrounded by the epithelium. BeC. Two semithinfibers (ms) surrounding the epithelium. D. Cross section through the spiral region ofrough epithelial cells of spiral gland with dense droplets of secretion in their cytoplasmof the spiral gland showing apical microvilli and the lumen filled with dense secretiona cuticle, limiting the duct. Duct lumen is not evident due to the close apposition of

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a similar sperm array (Jamieson et al., 1999). It is conceivable thatthis organization has serious effects on the sperm motility as thetwo doublets involved in the modifications are those located alongthe plane of stroke of the sperm flagellum (Tokuyasu, 1974). Thetemporarily limited sperm motility of Z. caudelli is possiblya consequence of the observed modifications. However, in othercases of a discontinuity between axonemal doublets, as for instancein thrips (Paccagnini et al., 2007), or in the giant sperm axoneme ofsome gall-midge flies (Dallai, 1988; Mencarelli et al., 2001, 2008),the sperm motility is not affected.

Another unusual feature observed in Z. caudelli is the axonemedegeneration in the posterior end of the sperm tail. A similarcondition has apparently independently evolved in some brachy-ceran taxa including Drosophila (Perotti, 1969; Dallai and Afzelius,1991) and also in the mole cricket Gryllotalpa (Baccetti et al.,1971; Dallai and Afzelius, 1990b).

Considering the overall uniformity of the morphology of Zora-ptera the structural diversity of the male genital system isremarkable. The character combination found in Z. caudelli andother zorapteran species supports the view that the sperm, a verycompact functional unit, does not evolve as an entity. The evidencepresented here suggests that, like elements of more complex bodyregions (e.g., head, thorax), sperm components can be modifiedindependently from each other, as already suggested by Dallai et al.(2006). This results in varying mosaic patterns of ancestral andderived features. In Z. caudelli, for instance, the bi-layered acrosomeand small accessory bodies are plesiomorphic states, whereas themitochondria modified into mitochondrial derivatives and theaxoneme degeneration in the posterior end of the flagellum areapparently derived conditions. Other combinations occur in otherinsect species (Jamieson et al., 1999).

Apparently a combination of highly conserved elements of thereproductive system (e.g., sperm axoneme pattern) with otherparts evolving very rapidly (e.g., short and simple versus long andcoiled aedeagus in species of Zorotypus) results in missing orsecondarily obscured phylogenetic signals and impedes phyloge-netic and evolutionary interpretations. Nevertheless, the presentstudy has revealed some characters potentially relevant for theplacement of the enigmatic Zoraptera. Whereas no affinities withmembers of acercarian lineages could be identified, unusual char-acteristics of the reproductive system are shared with two poly-neopteran groups, Phasmatodea and Embioptera. Considering thewell supported monophyly of Phasmatodea, a possible sistergrouprelationship between this order and Embioptera (Jintsu et al., 2010;Wipfler et al., 2011; Friedemann et al., 2011), and recently sug-gested affinities between Embioptera and Zoraptera (Minet andBourgoin, 1986; Engel and Grimaldi, 2000, 2002; Grimaldi andEngel, 2005; Yoshizawa, 2007), it is conceivable that derivedfeatures of the egg (Mashimo et al., 2011) and sperm axonemewerepresent in a common ancestor of Zoraptera, Phasmatodea andEmbioptera, with secondary modifications in Euphasmatodea andEmbioptera. Considering the apparent key role of Zoraptera, andthe persistent difficulties to assess its phylogenetic position basedon other features, a broad and formal evaluation of charactersrelated to the reproductive system appears highly desirable, incombination with a broad spectrum of other character complexesand also molecular data.

Acknowledgements

For financial support of this project we want to thank the MIUR(PRIN 2008/FL2237 to RD), the VolkswagenStiftung (to RGB) andthe German Science Foundation (DFG, grant BE 1789/10-1 to RGB),and the Japan Society for the Promotion of Science (Grant-in-AidScientific Research C, 21570089 to RM).

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