Male reproductive system and spermatogenesis of Limodromus assimilis (Paykull 1790)

Based on advanced light and electron microscopy, we describe the male reproductive system and sperm development of Limodromus assimilis. The genital tract consists of pairs of uni-follicular testes, spermatic ducts with diverticula regions, seminal vesicles, accessory glands, an unpaired ejaculatory duct and an aedeagus containing an internal sac equipped with sclerotic scales. Based on their morphology, we draw conclusions about their functions. After spermatogenesis within the follicle, the spermatozoa become released from the sperm cysts. The single spermatozoa move into the diverticula of the vasa deferentia I. Here, they become attached to central rods (spermatostyles), forming secondary conjugates (spermiozeugmata). The coordinated flagella movement of the conjugates possibly improves sperm velocity. Using super-resolution microscopy, we identified highly condensed reticulate chromatin in the lancet-shaped spermatozoa heads and the mitochondrial derivates of the flagella, likely formed by genomic and mitochondrial DNA, respectively. The results show, for the first time, sperm bundle formation in a Platynini species mainly corresponding to that found in Pterostichini species.

Previously, the morphology and formation of Carabidae spermiozeugmata were mainly described within the tribe Pterostichini originating from East Asia [17]. It was not yet clear whether the formation of spermiozeugmata, as described for Pterostichini, is also true for other Carabidae species. Therefore, we analysed Limodromus assimilis (Paykull 1790), which belongs to the Platynini tribe also containing sperm bundles [18]. Due to its eurytopicity, the black 10-to 12-mm-long ground beetle (Fig 1) is common, in addition to many other Palaearctic areas, in riparian forests of Middle Europe [32]. The propagation of hibernated imagos occurs during spring.
In the present work, we describe the morphology of the male reproductive system, spermiozeugmata formation and the spermatogenesis of L. assimilis. Based on these observations and simple staining procedures delivering first rough hints on the chemical components of the reproductive organs and tissues, we conclude their functions.
Until preparation, the beetles were kept in a climate chamber (Sanyo) under long-day conditions (16 h light, 8 h dark, 20000 Lux) at 23˚C in order to positively influence the maturation of the genital organs. Tenebrio molitor Linnaeus 1758 larvae served as food.

Sample preparation and staining procedures
The beetles were killed with chloroform, and the dissected organs were put into Ringer's solution to remove the trachea, Malpighian tubules and fat bodies. Spermiozeugmata were obtained by scratching out the seminal vesicles in Ringer's solution. The genital organs were fixed in Carnoy's solution, treated in an ascending ethanol series (70%, 80%, 96%) and isopropanol, and imbued with paraffin at 65˚C. Using a Leica (SM 2010 R) microtome, the sample blocks were cut in 5-or 8-μm thin sections and then placed onto protein-glycerine treated slides. The deparaffinised samples were stained with haematoxylin and eosin (HE) after Romeis [33].
The aldehyde fuchsin-alcian blue (AF-AB) staining was applied after Spicer and Meyer [34]. For the alcian blue and periodic acid-Schiff (AB-PAS) staining, the samples were first treated with alcian blue, followed by periodic acid, Schiff's reagent and haematoxylin. Then, the sections were embedded in Canada balsam. For DAPI staining the spermiozeugmata were placed onto polysine coated slides (Thermo Scientific) into a drop of Ringer's solution. Then, 8 μl of 0.005% DAPI in Antifade (Vectashield) was added, and the sample was covered by an 18×18 mm coverslip.

Light microscopy
The genital organ sections and the DAPI-stained spermiozeugmata were analysed under a Leica microscope (Leitz DMRBE) applying Differential Interference Contrast (DIC) and fluorescence microscopy, respectively. To analyse the structure of chromatin in spermatozoa heads and the mitochondrial derivates above the diffraction limit of light (super-resolution), spatial Structured Illumination Microscopy (3D-SIM) was applied using a C-Apo 63×/1.2W Korr objective of an Elyra PS.1 microscope system and the software ZEN (Carl Zeiss GmbH) to achieve a lateral resolution of~120 nm and an axial resolution of~250 nm. Images were captured using a 405-nm laser for excitation and the appropriate emission filter to identify DAPI [35]. SIM image stacks were used to produce 3D movies by Imaris 8.0 (Bitplane) and ZEN software.

Scanning electron microscopy
The evaginated aedeagus was fixed in Carnoy's solution, dehydrated with ethanol (70%, 80%, 96%) and isopropanol and then fixed in hexamethyldisilazane according to Bock [36]. The dry samples, glued on a plate, were coated with gold and then investigated with a Hitachi (S-2004) scanning electron microscope at 18 kV.  Table summarizes size measurement data of its main parts. Testes, spermatic ducts and accessory glands are present in pairs. Testes are localized lateral-dorsal and the accessory glands median-ventral in the abdomen. The globular and unifollicular testes have a size of~3.8 x 1.9 mm comprising one follicle, the spermatic duct and the vas deferens-region I. First, the follicle leads into a short vas efferens (defined by Snodgrass [24]) followed by the three parts of the vas deferens. The first spiral part (vas deferens I) contains several diverticula. The tube is twisted either left-handed, right-handed or alternating left-and right-handed, at which the loops are extended in the posterior direction. The second part (vas deferens II) does not contain diverticula, and the third part, known as seminal vesicle (defined by Kaulenas [25] and Snodgrass [24]), represents a part of the spermatic duct with an increased diameter. Eventually, the spermatic duct leads into the posterior third part of the accessory glands. The measured length of the whole spermatic duct from the follicle to its outlet averages~118 mm. The curved accessory glands re-join posterior into the ejaculatory duct which leads into the median lobe of the aedeagus. The invaginated median lobe lies within the abdomen at the side, in which its tip points to the left when regarded dorsal-caudally. In addition, the aedeagus has two lateral lobi, called parameres.

Spermatogenesis
The formation of spermatozoa occurs in the testis follicles, which are divided into different parts of progressive spermatozoa development within sperm cysts (Fig 3A). The follicle is surrounded by a thin epithelial layer and the diameter of its distal part reaches~170 μm. In this region, the spermatogonia are localized and originated from primordial germ cells via mitosis.  Depending on the different number of already-performed somatic divisions, a different number of these are enclosed within a sperm cyst (Fig 3a 1 ). A thin epithelial cell layer surrounds the sperm cyst. The continuous spermatogonia generation induces their proximal movement. After mitosis completion, the spermatogonia nearly double in size and then perform meiosis as indicated by different stages (Fig 3a 2 ). The resulting globular spermatids elongate during spermatogenesis starting with flagella formation, in which for now the chromatin (shown in violet by HE staining) remains in the globular sperm head (Fig 3a 3 ). Afterwards, the chromatin becomes elongated, indicating the mitochondrial derivate formation along the flagella. The spermatid heads are disposed to the cyst membrane, where they seem to be attached (Fig 3a 4 ). Later, in the head, the nucleus containing condensed chromatin becomes visible ( Fig 3C). In this state the flagella are arranged nearly straight to each other. Afterwards, they show a synchronous wave-like behaviour suggesting that they have reached their functionality. In this spermatogenesis state, the spermatozoa reached their final lengths and lie tightly within the sperm cysts. Via HE staining, the mitochondrial derivates in the flagella become visible (Fig 3a 5-1 and 3a 5-2 ). Here, the follicle diameter amounts to 160 μm and then narrows in the region ahead of the vas efferens where the sperm cyst membranes dissolve, and the spermatozoa are released ( Fig 3B). The vas efferens, showing a diameter of~50 μm, exhibits a muscle layer and a broad layer of epithelial cells. The separated spermatozoa, showing a mean length of~310 nm, are transported via this short spermatic duct region into the following vas deferens I.
To analyse the distribution and structure of chromatin within spermatozoa, they were labelled by the DNA-specific stain DAPI followed by super-resolution microscopy (SIM). The highly condensed genomic chromatin was identified in the spearhead-like shaped sperm heads. In addition, chromatin-free regions are evident in the sperm head nucleus (Fig 3c 3-4 , S1 and S2 Movies). The mitochondrial DNA of mature spermatozoa forming two parallel connected mitochondrial derivates of the same shape and size was present posteriorly within the second half of the flagella region and extended~80-100 μm. At a distance of~5 μm, the derivates exhibited narrow constrictions of~300 nm in diameter (Fig 3c 1-2 , S1 Fig, S3 and S4 Movies).

Sperm bundle formation and transport
The first (I) region of the vasa deferentia appears as an~20-mm long spiral containing more than 400 diverticula. All these wall protrusions are outside of the spiral and their tips are oriented towards the vas efferens (Fig 4A and 4B, S5 Movie). The anterior part of vas deferens I close to vas efferens (S2 Fig) has a diameter of~28 μm and comprises smaller ovoid-like diverticula (~27 x 22 μm) than in the posterior part where the diameter increases up to 60 μm, and the thumb-shaped diverticula reach a length and width of~70 μm and~50 μm, respectively. The vas deferens I wall consists of a cuboidal epithelium surrounded by a muscular layer. The HE staining indicates DNA (Fig 4C). In this part of the spermatic duct, spemiozeugmata appear first. The lumen of each diverticulum contains a single central rod and the anterior parts of the spermatozoa (Fig 4D). Life observations indicated that the rhythmic contractions of the diverticula are accompanied by a lateral wall constriction and consequently by a lumen reduction (S5 Movie). The diverticula opening orients towards the seminal vesicle (Fig 4B),  The spermiozeugmata comprise a central rod where up to~200 spermatozoa are attached. Their mean length is~335 μm (Fig 4J-4L, S1 Table). The slightly bent central rod has a length of~160 μm and can be subdivided into a head, middle and tail part (Fig 4K). The convex head region showing a rounded apex~23 μm long. The middle piece (width:~13-15 μm; height: 24 μm) narrows towards the short filamentary tail. The red central rod stained by AB-PAS reagent indicates the presence of polysaccharides (Fig 4H). The spermatozoa attach to the front two-thirds of the central rod via their heads (Fig 4L). Living spermatozoa, isolated from the seminal vesicle, show a coordinated sinusoidal cilium-like flagella movement. This results in a spiral movement allowing the sperm conjugates to move in Ringer's solution~200 μm/ min (S6 Movie).
The second vas deferens region (II) does not contain diverticula, has a diameter of~70 μm and is only slightly contorted. HE staining indicated that it consists of a single-layer columnar epithelium surrounded by a thin muscle layer (Fig 4F). Similar to vas deferens I, the sperm bundles are transported via propulsive peristaltic movements of the spermatic duct.
The seminal vesicle is an~4-6 mm long part of the vasa deferentia and has, depending on its filling state, a variable diameter of~150-250 μm. Similar to vas deferens II, the wall consists of a single-layer columnar epithelium surrounded by a thin muscular layer. The seminal vesicle accumulates and stores a high amount of spermiozeugmata (Fig 4G). After AB-PAS staining, the spermatozoa show a mixed colour of magenta and blue, indicating the presence of acid and neutral mucosubstances (Fig 4H). An AF-AB staining leading to an exclusive magenta labelling of the spermiozeugmata suggests the presence of sulphured mucosubstances (Fig 4I). In contrast, spermatozoa present in the spermatic duct region between the proximal follicle and vas deferens I show both magenta and blue. Thus, it seems that they contain in addition carboxylated glycoproteins (Fig 4E).
From the seminal vesicle, the sperm bundles are transported to the accessory glands via a tight corridor which has a strong circular muscular layer. The accessory glands (length:~4-6 mm, width:~350-400 μm) are filled with a white viscous secretion. The inner wall epithelium (~12 μm thick) contains secretory adenocytes. This columnar gland wall epithelium is surrounded by circular and longitudinal muscle layers~30 μm thick (Fig 5A-5C). An AB-PAS and AF-AB staining of the glandular lumen indicates acid and neutral glycoproteins, polysaccharides, and glyco-and phospho-lipids. The blue colour of the secretion specifies that the acid glycoproteins yield carboxyl groups (Fig 5D and 5E).
Sperm bundles present in the accessory glands are transported via contraction of their double-layered musculature (Fig 5A) into the ejaculatory duct. The area where the accessory glands fuse (diameter:~230 μm) contains a sclerotic structure dividing them (S3 Fig). The ejaculatory duct is also surrounded by muscle cells mediating the sperm bundle transport via contraction. Its end flows into the median foramen of the median lobe of the aedeagus. The median lobe (length: 2.5 mm) is a curved sclerotic tube containing at its convex side an invaginated internal sac (Fig 6A and 6B).
During copulation, this sac becomes evaginated, and the ejaculate is released through the gonopore. The internal sac surface has sclerotic scales which are oriented basally. They are flat at the basal region and become spicule-like towards the gonopore (Fig 6C-6E). Scale shape and orientation are possibly helpful to fix the aedeagus in the female genital tract during copulation. At the concave side of the median lobe small and large parameres are situated. They are surrounded by muscle cells (Fig 6A and 6B).

Discussion
Testes Adephaga (Coleoptera) testes consist of a single tube-like coiled follicle (Lawrence & Britton 1994). Similar to the globular testes of two Sardinian Carabids (Pterostichini) (Carcupino et al. 2002, Cadeddu et al. 2008, L. assimilis testes contain, in addition, a part of the vas deferens. As by all other insects [37], the complete spermatogenesis proceeds in the follicle. Similar to P. nigrita [29] and A. parallelpipidus [19], cyst resolution and transport of single spermatozoa towards the vas deferens appears when spermatogenesis is finished.

Vasa deferentia
Similar as the spermatic duct of other Carabidae [19] the vas deferens I of L. assimilis contains diverticula. However, they vary significantly in size and number. L. assimilis has four times more (~400) diverticula than Calathus fuscipes Goeze 1777 (Carabidae: Pterostichini), but they are ca. twice (~160 μm) as long [13]. Similar size ratios are evident when compared to A. parallelpipidus, but these diverticula appear croissant-like [19]. Similar to P. nigrita [26], diverticula size increases along the spermatic duct of L. assimilis, and their ovoid shape is similar to the globular protrusions of Percus strictus strictus Dejean 1828 [15].
Probably, similar as that described for other Pterostichini, A. parallelpipidus [19], P. strictus strictus [15], Speomolops sardous Patrizi 1955 [16], P. nigrita [14], and Calathus species [13], the diverticula containing vas deferens region of L. assimilis is responsible for sperm bundle formation. Each diverticulum contains one central rod and spermatozoa. We suggest that spermatozoa move from the anterior tract into the diverticulum where they attach at the central rod via their heads. Ferenz [14] suggests that there is an active movement mediated by attractants in P. nigrita. In addition, it could be that the diverticula contractions absorb the spermatozoa of L. assimilis.
The origin of the substances forming the spermatostyle is not yet clear. Indicated by the HE staining, they are possibly secreted from the diverticulum epithelia cells. The secretory function of diverticulum epithelial cells to produce central rod substances has been described for some Carabidae species [15,16,19,26]. The rhythmic diverticulum contractions could be required to form spermatostyles, as suggested by [26] for P. nigrita.
The sperm bundle apex of L. assimilis is not attached by sperm heads, probably because it is not accessible due to its embedding in the apical diverticulum epithelium when spermatozoa attach. Similarly, the spermiozeugmata heads of P. nigrita are fixed in a microvilli-rich impression of the epithelium in the diverticulum apex [26]. Because sperm bundle formation proceeds after spermatogenesis is completed, L. assimilis contains secondary sperm bundles, which should be called spermiozeugmata according to Higginson and Pitnick [21]. These spermiozeugmata become released from the diverticula and are then transported into the seminal vesicle where an AF-AB staining of spermatozoa indicates the presence of sulphated glycoproteins. In contrast, spermatozoa evident in the spermatic duct region between the proximal follicle and vas deferens I contain, in addition, carboxylated glycoproteins. This indicates a chemical turnover of the structural glycosides in the seminal vesicle whose physiological function is not yet clear.

Sperm and conjugate morphology
Analysing L. assimilis, we confirm the spermiozeugmata variability present in the family Carabidae, and for the first time, also within the tribe Platynini. J. estriata, belonging to the same tribe, shows a similar sperm bundle structure as L. assimilis, but its spermatostyle is filamentary and 15-times (2.4 mm) longer [18].
The AB-PAS staining of the L. assimilis spermatostyles indicates neutral glycosylated substances. Chemical analyses of Carabidae sperm bundles identified the presence of polysaccharides and proteins [27,28], suggesting that the central rod of L. assimilis is also formed by neutral glycoproteins.
Similar to that described for Membranipora membranacea Linnaeus 1767 [38], the spiral spermiozeugmata movement of L. assimilis indicates a coordinated spermatozoa behaviour probably important to improve sperm movability. The number of spermatozoa per conjugate may be positively correlated with swimming velocity, in which a synchronous flagella movement avoids interference [21]. Thus, the~200 spermatozoa of L. assimilis arranged in a conjugate seem to improve the velocity compared to that of a single spermatozoon. Due to different osmolarities and the presence of more viscous fluids, the velocity of~200 μm/min we identified in Ringer solution may be slower than that in the male and female genital tract.
Similar to other Pterygota species, based on SIM imaging we found that the spermatozoa nuclei of L. assimilis, likely achieved by the substitution of nuclear histones by protamines [39], contain highly condensed chromatin. It is distributed in a lancet-like manner, a spermatozoon head shape common in this insect subclass [1]. The high degree of nuclear chromatin compaction seems to be required to transport effectively the transcriptionally silenced male genetic material into the female egg cells. In addition, the lancet-like head shape may improve the swimming efficiency of the spermatozoa. The variability of sperm nuclei shapes is extraordinary between species [40]. E.g. the worm Tubiluchus troglodytes has a double-helical sperm nucleus as identified by electron microscopy [41]. Similarly, the sperm nuclei of L. assimilis contain a clearly structured, highly condensed reticulate chromatin.
At the end of meiosis, each spermatozoon receives an equal set of mitochondria. They aggregate and form the "nebenkern", from which two mitochondrial derivates originate [42]. In several hymenopteran species, the two mitochondrial derivates can strongly differ in shape and size [1,2,22]. As is typical for Pterygota species [1], and Cicindela campestris Linnaeus 1758 (Carabidae: Cicindelinae), [9] and Pasimachus subsulcatus Say 1823 (Carabidae: Scaritinae) [10], two parallel mitochondrial derivates occur along nearly the whole flagellum. Interestingly, during the condensation of nuclear chromatin, in Dendroctonus armandi Tsai and Li (Coleoptera: Scolytinae), these derivatives acquire different sizes and become laterally located in relation to the nucleus [43]. Similarly, in Tribolium castaneum (Herbst, 1797) (Coleoptera: Tenebrionidae) also two asymmetric mitochondrial derivatives were observed [44]. Mature spermatozoa of Rhynchophorus ferrugineus Oliv. (Coleoptera: Dryophthoridae) contain mitochondrial derivates of different size, in which the large derivative is embedded into an infolding of the nucleus [45]. In contrast, in L. assimilis the DNAspecific DAPI staining identified two identical parallel, tightly connected, and recessed mitochondrial derivates, which were found only along the posterior flagellum region. They show a finer reticulated structure, likely representing cristae, than the highly condensed head nucleus chromatin. Similar shaped mitochondrial derivates have not yet been found in other insects. Nevertheless, in despite of the different shape we suggest that they are required for sperm motility as has been found in Drosophila melanogaster Meigen, 1830 (Diptera) [46].

Accessory glands and ejaculatory duct
The accessory glands (a pair of tubes) of L. assimilis belong to the simplest type known among Coleopterans [25]. This type has also been found in Leptinotarsa decemlineata Say 1824 (Coleoptera: Chrysomelidae), Popillia japonica Newman 1841 (Coleoptera: Scarabaeidae) and in Cleridae and Carabidae species [30,[47][48][49]. The accessory glands of L. assimilis show a curved shape, confirming the finding of Will et al. [30]. The gland structure consisting of lumen, columnar epithelium and surrounding muscular layer is typical for many insects [25].
The secretion excreted by the epithelium of many insects mainly contains polysaccharides, proteins and mucosaccharides [31]. The AB-PAS and AF-AB staining suggest that these substances (with exception of non-glycosinolated proteins) also occur in L. assimilis. Contrary to L. decemlineata and P. nigrita, which exclusively include proteins and neutral mucines [48,50], the gland secretion of L. assimilis additionally contains acid (carboxylated) mucosubstances.
The main accessory gland function of many insects is the formation of spermatophores [25,51]. They consist of secretion and spermiozeugmata as found in the bursa copulatrix of female L. assimilis individuals after copulation [52]. Possibly, in L. assimilis, the gland lumen secretion is responsible for the formation of spermatophores, similar as is described for T. molitor (Coleoptera: Tenebrionidae) [53]. However, it is not yet clear where exactly the association of spermiozeugmata and secretion appears because in L. assimilis glands they were identified only separately. The spermatophore formation could proceed in the posterior (glandular) parts of the vas deferens, similar as has been found by Krüger et al. [50] in P. nigrita.
The circular and longitudinal gland muscle layers of L. assimilis, present also in P. nigrita [50] and S. sardous [16], allow via directed contraction the transport of secretion and spermiozeugmata into the posterior regions of the genital organs. According to Snodgrass [24] the region where both accessory glands fuse belongs to the ejaculatory duct. Here, a sclerotic structure is evident, possibly required to stabilize the tract during the transport of lumen content. The sclerotic structure also hints on the ectodermal origin of the ejaculatory duct of L. assimilis as is present in all other insects [25]. It seems that the L. assimilis ejaculate contains complete spermiozeugmata because they are still present in the ejaculatory duct, and according to Fritzsch [52], they have also been found in the female bursa copulatrix.