Ultrastructure of spermatozoa in three cicada species from China (Hemiptera, Cicadomorpha, Cicadidae)

Abstract The ultrastructure of mature spermatozoa of three cicada species, Subpsaltria yangi, Karenia caelatata, and Platypleura kaempferi, was investigated using epifluorescence and transmission electron microscopies. This is the first investigation of the sperm ultrastructure of species in the subfamily Tibicininae and the tribe Sinosenini, represented by S. yangi and K. caelatata, respectively. The three species all produce two or three types of spermatozoa with various lengths, viz., polymegaly. The centriolar adjunct of spermatozoa in S. yangi shows a granular substructure, which is different from that of other cicada species, suggesting that spermatozoa in Tibicininae may have their own characteristics in comparison with other cicadas. The centriolar adjunct of spermatozoa of K. caelatata displays characteristics similar to that of the Cicadinae. Combined with other morphological characters, it is reasonable to remove K. caelatata and its allies (i.e., Sinosenini) from Cicadettinae to Cicadinae. The study of sperm ultrastructure, particularly in the species of Tibicininae and Sinosenini, expands the spermatological research of Cicadidae and provides more information for phylogenetic analysis of Cicadidae.


Introduction
As germ cells, sperm are evolving at the fastest speed and are among the most diverse cell types with the highest degree of variation in insect growth , Jamieson 1987, Joly et al. 1989). Spermatological characteristics have been used for distinguishing taxa and for clarifying phylogenetic relationships of related taxa (Jamieson 1987, 1991, Jamieson et al. 1999, Lino-Neto and Dolder 2001, Zhang and Hua 2017. The sperm structure of insects, similar to those of other metazoans, is broadly the same, but each group has unique characteristics. Most of these generally tiny, motile spermatozoa are slender with a single flagellum, but some have more than one flagellum (Chawanji et al. 2005(Chawanji et al. , 2006. The flagellum is the power source for sperm mobility (Ciolfi et al. 2016).
There are two aspects of insect sperm, the length and structure, together revealing any sperm polymorphism. The sperm length in Diptera extends across a great range (Joly et al. 1991, Joly et al. 1995. It has been reported that two types of nuclei of distinctly discrete lengths are produced in some species of Drosophilidae and Diopsidae (Diptera) (Snook et al. 1994, Pasini et al. 1996, Presgraves et al. 1999). Another aspect is nucleation. Males in butterflies and moths (Lepidoptera) can produce nucleated eupyrene sperm and non-nucleated apyrene sperm; the former has the ability to fertilize eggs, while the exact function of the latter is still uncertain (Katsuno 1977, Silberglied et al. 1984, Osanai et al. 1989, Friedländer 1997, Yamashiki and Kawamura 1997, Kubo-Irie et al. 1998, Watanabe and Bon'no 2001, Friedländer et al. 2005, Hayakawa 2007). There exists an extreme case in Dahlbominus fuscipennis (Hymenoptera, Eulophidae) which has at least five different types of spermatozoa with diverse appearances (Lee and Wilkes 1965). Although the function of polymorphic sperm in insects remains uncertain, some authors suggest that it may be related to sperm competition (Snook 1998, Swallow andWilkinson 2002).
The family Cicadidae of the order Hemiptera includes approximately 3,000 extant species worldwide, and about 210 extant species distributed in China (Sanborn 2013, Chou et al. 1997. This family includes four subfamilies, i.e., Cicadinae, Cicadettinae, Tibicininae and Tettigomyiinae (Marshall et al. 2018). Up to the present, studies on the sperm ultrastructure of Cicadidae have addressed 13 species (Snook et al. 1994, Pasini et al. 1996, Presgraves et al. 1999, Kubo-Irie et al. 2003, Chawanji et al. 2005, 2006, Chawanji et al. 2007). It was found that Graptopsaltria nigrofuscata (Cicadinae) can produce two types of spermatozoa, but only the longer spermatozoa have fertility (Kubo-Irie et al. 2003). Chawanji et al. (2005) revealed polymegaly in spermatozoa of all four investigated species of African platypleurine cicadas of the subfamily Cicadinae. The sperm ultrastructure of five cicadas, currently belonging to Cicadettinae and Tettigomyiinae, respectively, has also been studied (Chawanji et al. 2006). Such scant information is applicable to phylogenetic study of Cicadidae from the point of view of spermatology. However, studies on sperm structure in Cicadidae are still insufficient, particularly for some taxa whose systematic status remains controversial, and for the subfamily Tibicininae, of which the sperm structure has never been investigated in any species.
Herein, the sperm ultrastructure of three cicada species were observed using both epifluorescence and transmission electron microscopies (TEM). The sperm ultrastructure of Subpsaltria yangi is the first detailed description of spermatozoa investigated in the subfamily Tibicininae. We also address the systematic placement of the genus Karenia based on a comparison of the sperm ultrastructure of this species and other species. In addition, coupled with previous studies, we discuss the similarities and differences in sperm ultrastructure among different subfamilies of Cicadidae, aiming to provide useful clues for taxonomic and phylogenetic studies of the Cicadoidea.

Materials and methods
Male adult cicadas were collected using a net. Their identities and detailed collecting information are shown in Table 1. The higher classification of Cicadidae follows that of Marshall et al. (2018).

Sample preparation and epifluorescence microscopy observation
Samples (at least five individuals of each species) were anesthetized with alcohol at a concentration of 75%, and dissected with a fine scalpel blade under a binocular microscope (Olympus SZX16, Olympus Corporation, Tokyo, Japan) to obtain the seminal vesicles from which spermatozoa were recovered. For measuring the sperm total length, sperm samples were placed in 1% bisbenzimidazole Hoechst 33258, a cellpermeable adenine-cytosine binding epifluorescent dye used to stain DNA (Sakaluk and O'Day 1984), for 1 min, then rinsed in three changes of 0.1 M phosphate buffered saline (PBS, pH 7.2). Spermatozoa were evenly spread on a microscope slide and covered with a coverslip. Slides were examined with an Olympus BX-51 epifluorescence microscope (Olympus Corporation, Tokyo, Japan) at a wavelength of 343 nm. Digital images of 50-100 spermatozoa were randomly captured from each species under the same microscope with an Olympus DP72 camera (Olympus Corporation, Tokyo, Japan). Sperm lengths were measured using the Olympus DP2-BSW software version 2.1.

Sample preparation and transmission electron microscopy observation
Sperm samples were fixed in 2.5% glutaraldehyde (0.1 M PBS, pH 7.2) for 12 h at 4 °C, and the materials were rinsed with 0.1 M phosphate buffered saline (PBS, pH 7.2), then fixed in 1% osmium tetroxide for 2 h at room temperature. Alcohol dehydration with a concentration gradient was performed after rinsing with the same PBS. Treated samples were then embedded in Epon 812 resin. A diamond knife was used to obtain ultrathin sections which were collected on 300 mesh copper grids before staining with uranyl acetate and lead citrate. Sections were examined and photographed with a HT7700 transmission electron microscope (HITACHI, Tokyo, Japan) at 80 kV.

Data analysis
The measured data were recorded using Microsoft Excel version 2010. Then an analysis of variance was conducted to verify the mathematically significant differences in sperm length between different sperm types within species using SPASS version 19. Correlation analysis between nucleus length and tail length was performed using R version 3.3.2. Measurements are reported as mean ± standard error.

Epifluorescence Morphology
Mature spermatozoa of the three species are all linear with needle-like heads and long tails that tapered posteriorly ( Fig. 1A-C). The heads of the spermatozoa are aggregated into bundles, with the thread-like tails scattered freely (Fig. 1D). Sperm morphologies of the three species are similar, but the spermatozoa vary in length. Based on their remarkably different total length, spermatozoa within a species are divided into disparate types (Table 2). In K. caelatata, the spermatozoa can be divided into three types: long spermatozoa, medium spermatozoa, and short spermatozoa. The spermatozoa of the other two species, P. kaempferi and S. yangi, can be classified into two types: long spermatozoa and short spermatozoa. Within each species, the total lengths of different types of spermatozoa are significantly different (P < 0.01; one-way ANOVA). Among the long spermatozoa of these three species, the spermatozoa of K. caelatata is the longest (178.45 ± 10.82 μm); among the short spermatozoa, the spermatozoa of S. yangi is the shortest (64.36 ± 5.13 μm) (Table 2).
Additionally, the differences in total length of spermatozoa and the sizes of sperm nuclei and tails both within and between species are also significantly different. In S. yangi, the lengths of nuclei fall into two classes, and the lengths of tails present  three classes. There is a weak correlation between the nucleus and tail lengths in S. yangi (Table 3). In K. caelatata, the lengths of nuclei fall into two classes; the lengths of tails fall into two clear classes. There is a moderately correlation between the nucleus and tail lengths in K. caelatata (Table 3). Sperm nuclei of P. kaempferi can be classified into two classes according to their length; the tail lengths fall into three classes. The nucleus length is moderately correlated to the tail length in this species (Table 3).

Ultrastructure
Subpsaltria yangi Chen, 1943 The head region that is embedded into a homogenous matrix consists of an acrosome and a nucleus, and the anterior section of nucleus intrudes into an invagination of the acrosome as shown in longitudinal section ( Fig. 2A). The acrosomal contents are differentiated internally with tubular substructures (Fig. 2B-F). The acrosome has two processes with an extension on both sides of the anterior section of the nucleus, and gradually widens in diameter in cross-sections ( Fig. 2D and E). The nucleus appears to have a cylindrical profile ( Fig. 2A). The nucleus accommodates to the processes of the acrosome, and is bilaterally concave ( Fig. 2D and E), finally becoming circular shape in a cross-section (Fig. 2F). There is a shallow invagination in the post-lateral part of the nucleus, where the granular centriolar adjunct is located in longitudinal sections (Fig. 3A, G). The centriolar adjunct is limited by the invagination of the nucleus, and its shape changes in different cross sections ( Fig. 3B-E). The diameter of nucleus decreases towards the base of nucleus ( Fig. 3B-F). A centriole runs from the flat base of the nucleus, connecting the nucleus and the axoneme (Fig. 3A). The flagellum is formed by a 9 + 9 + 2 axoneme (i.e., nine accessory microtubules, nine double microtubules, and two central microtubules) flanked by a pair of equal mitochondrial derivatives with crystalline regions (Fig. 4B, C). The derivatives are composed of cristae arranged in an orderly array in longitudinal section (Fig. 4A). At the end of the tail, axonemal microtubules appear less well organized and disappeared gradually (Fig. 4D, E).

Karenia caelatata Distant, 1890
Spermatozoa are all gathered together, with their conical acrosomes and part of the electron-dense nuclei inserted into a homogenous matrix forming a spermatodesm ( Fig. 5A). In cross-section, the acrosome is conical, and a sub-acrosomal invagination is eccentric in position anteriorly (Fig. 5B). The acrosome forms two processes posteriorly, which flank the anterior part of nucleus ( Fig. 5C-F). The acrosomal contents are not homogenous in appearance, which are filled with numerous tubular substructures as shown in cross sections (Fig. 5B-F). The diameters of the two tubular acrosomal processes increase towards the base of the acrosome, and the nucleus becomes mushroom-shaped in cross sections (Fig. 5B-G). The electrondense centriolar adjunct forms a sheath shape and runs parallel to the posterior part of nucleus (Fig. 6A). The posterior segment of the nucleus develops a lateral invagination; the centriolar adjunct is confined within the invagination in cross sections ( Fig. 6B-E). The centriole is attached to the base of the nucleus (Fig. 6A).The flagellum is composed of an axoneme with a typical 9 + 9 + 2 microtubular pattern and a pair of mitochondria derivatives (Fig. 6F, G). The mitochondrial derivates are composed of numerous cristae, which can be seen from the longitudinal section of the tail (Fig. 6G). All of derivates with different diameters have a crystalline region in cross-section of tail (Fig. 6F).

Platypleura kaempferi (Fabricius, 1794)
Spermatozoa aggregate together with their heads inserted into a homogenous matrix to form a spermatodesm. The head region consists of an acrosome and a compact and homogeneous nucleus (Figs 7A and 8A). The acrosomal contents have tubular substructures (Fig. 7B-G). The acrosome is laterally flattened, and an electron-lucent space (viz., subacrosomal space) lies in an eccentric position anteriorly (Fig. 7A). The acrosome gradually widens posteriorly, and forms two processes that flank the anterior part of the nucleus in cross-sections (Fig. 7C-G). The centriolar adjunct lies beneath the posterior of the nucleus and is parallel to it. The diameter and shape of the centriolar adjunct vary, adapting to the lateral invagination of the nucleus (Fig. 8E-G). With the extension of the nucleus, the diameter of nucleus gradually decreases (Fig. 8C-H). The centriolar adjunct gradually vanishes where the mitochondrial derivatives emerge (Fig. 8H).
The centriole emerges from the base of the nucleus, connecting the nucleus and the axoneme (Fig. 8B). In the tail region, the paired mitochondrial derivatives are formed by cristae at the periphery, and a typical axonemal arrangement of 9 + 9 + 2 microtubules is present ( Fig. 9B and C). Each derivative is positioned laterally to the axoneme and contains an elliptical crystalline region (Fig. 9C). Cross-sections through the end of the tail show a progressive loss of microtubules: the 9 accessory microtubules disappear first (Fig. 9D), followed by the two central microtubules (Fig. 9E). The axoneme extends to almost the end of the sperm tail (Fig. 9A).

Discussion
In this study, a number of similarities are revealed in the mature spermatozoa of S. yangi, K. caelatata and P. kaempferi. The motile spermatozoa, all aggregated into bundles, intrude into a homogenous matrix to form a spermatodesm. The spermatozoa of each species can be divided into two or three types based on their total length, nucleus length, and tail lengths (viz., polymegaly). There is a conical acrosome with a subacrosomal space in an eccentric position, and the acrosome sits above the anterior part of the nucleus. The centriolar adjunct is located at the postero-lateral invagination of the nucleus and is parallel to it. In the tail region, two equal mitochondrial derivatives with electron-dense crystalline regions comprise cristae, which are arranged in an orderly array. A single axoneme displays a 9 + 9 + 2 microtubule arrangement. The mitochondrial derivatives and the axoneme both extend to almost the end of the tail. There is no accessory body in the sperm tail. These features are all likely common to spermatozoa of other investigated cicadas (e.g., Folliot and Maillet 1970, Kubo-Irie et al. 2003, Chawanji et al. 2005, 2006. Although the sperm ultrastructures of these three cicada species have some similarities, the centriolar adjunct of S. yangi presents a different appearance, i.e., with a granular substructure. In many insects, the centriolar adjunct has been identified as derived from additional pericentriolar material (PCM) deposited beneath the nucleus at the end of spermiogenesis (Dallai et al. 2016a). The centriolar adjunct is apparently an apomorphy of Insecta (Dallai et al. 2016b). In Dicondylia of the Insecta the centriolar adjunct and the accessory bodies may be variably developed, and their size and shape are important characteristics for taxonomy (Dallai et al. 2016a). Cicadas are classified into two families, Cicadidae and Tettigarctidae (Moulds 2005, Marshall et al. 2018, with the former being divided into four subfamilies (Cicadinae, Cicadettinae, Tibicininae, and Tettigomyiinae) (Marshall et al. 2018). So far, the centriolar adjunct of spermatozoa in Cicadinae has been found to be composed of homogenous, moderately electron-dense material (Folliot and Maillet 1970, Kubo-Irie et al. 2003, Chawanji et al. 2005. In our present study, the structure of the centriolar adjunct in spermatozoa of P. kaempferi and K. caelatata is consistent with previous descriptions for the Cicadinae. In the Cicadettinae and Tettigomyiinae, the centriolar adjunct of the spermatozoa is lamellar (Chawanji et al. 2006). In our study, we found that the centriolar adjunct of S. yangi is granular, which is different from that of other cicada species, indicating that spermatozoa in Tibicininae may have their own characteristics in comparison with other cicadas. Chawanji et al. (2005) described vesicular-like structures associated with the centriolar adjunct in some sections of spermatozoa of the cicada P. hirtipennis (Germar, 1834), but the centriolar adjunct itself is not granular, which is different to the granular centriolar adjunct of S. yangi. This characteristic may add more information for spermatology of Tibicininae and, together with other results, may inform future studies on the phylogeny of Cicadoidea.
Cicadas of the genus Karenia, remarkably without timbals, are currently placed in the Cicadinae. This is the only genus of the tribe Sinosenini. The systematic placement of this tribe remains controversial (Boulard 1973, 1988, 2008, 2001, Chou et al. 1997, Moulds 2005, Wei et al. 2009, Marshall et al. 2018. Species of this group are restricted to southwestern China, Myanmar and Vietnam (Wei et al. 2009, Pham andYang 2012). Moulds (2005) attributed Karenia to the subfamily Cicadettinae, but this genus and other probably related taxa were not included in his morphological phylogenetic analyses. Boulard (2008), Wei et al. (2009) and Pham and Yang (2012) followed Moulds (2005), attributing this genus into the Cicadettinae. However, Boulard (2013) and Marshall et al. (2018) put this genus in the Cicadinae. Morphologically, the metanotum of Karenia is distinctly concealed by the cruciform elevation at the dorsal midline, which is the same as species in Cicadinae, but is different from members of Cicadettinae whose metanotum is partly visible at dorsal midline (Moulds 2005(Moulds , 2012. Furthermore, in Cicadettinae the uncus is duck-bill shaped and undeveloped, and the pair of claspers are well developed; while in Cicadinae the uncus is well developed with uncal lobes much swollen and elongated, and the claspers are usually degenerate or even disappeared (Moulds 2005(Moulds , 2012. The strongly swollen uncus in Karenia is similar to that in the Cicadinae. In some species of Cicadettinae, the centriolar adjunct presents as a lamellate substructure (Chawanji et al. 2006). However, in our study, there is no such substructure in the centriolar adjunct of the spermatozoa of K. caelatata. Coupled with the above-mentioned morphological characters and the morphology of ovipositors (Zhong et al. 2017), antennae (Wang et al. 2018, in press) and Malpighian tubules (Li et al. 2015), our results confirm that it is reasonable to place this genus in Cicadinae, which is consistent with the results of Marshall et al. (2018) based on molecular data.
Spermatozoa possess more than one size type (viz., polymegaly), which has been described widely within the Insecta. For example, some species of vinegar flies (Diptera, Drosophilidae) and stalk-eyed flies (Diptera, Diopsidae) produce two discrete lengths of nucleated sperms (Snook et al. 1994, Pasini et al. 1996, Presgraves et al. 1999). In the Cicadomorpha, all of the 13 previously investigated species of Cicadoidea can produce more than one size type of spermatozoa reflected in nuclear length and total length within and between species (Kubo-Irie et al. 2003, Chawanji et al. 2005, 2006. In addition, a distinct correlation between nuclear length and total length was found in the spermatozoa of cicada G. nigrofuscata (Kubo-Irie et al. 2003). However, Chawanji et al. (2005Chawanji et al. ( , 2006 found the nucleus length and total length of spermatozoa have no significant correlations in their examined species. In our study, the three examined species also produce two or three distinct size types of spermatozoa, and there is a weak (in S. yangi) or modest (in K. caelatata and P. kaempferi) correlation between the nuclear length and tail length within a species (Table 3). In contrast, polymegaly of spermatozoa does not appear in the Cicadellidae and Cercopoidea within Cicadomorpha. The production of only one size type of spermatozoa has been revealed in investigated species of Membracoidea (Cruz-Landim and Kitajima 1972, Araújo et al. 2010, Zhang and Dai 2012, Su et al. 2014. Although the sperm length varies within individual males of Locris transversa (Cercopidae), this variation has no statistical significance, which was also observed in other two cercopids (Folliot and Maillet 1970). Hodgson et al. (2016) presumed that polymegaly may be an apomorphy of Cicadoidea within Cicadomorpha based on the study of Locris transversa (Cercopidae). Therefore, our results, coupled with other previously related studies, suggest that polymegaly is a narrow occurrence in the Cicadomorpha.
The results of our study provide more clues for further studies of classification and phylogeny of the Cicadoidea. There may also be some ultrastructural features that can be used as morphological evidence for the phylogeny of the Cicadomorpha.