Insects have highly variable reproductive systems, reflecting a diversity of reproductive strategies and adaptations. Such variation has been widely used to classify and estimate phylogenetic relationships. Here, the morphology and ultrastructure of the internal reproductive systems of two leafhopper species are described and illustrated, using both light and transmission electron microscopy, and representing two tribes of Deltocephalinae: in Chiasmini, Nephotettix cincticeps (Uhler, 1896), and in Deltocephalini, Deltocephalus vulgaris (Dash & Viraktamath, 1998). Tables comparing the morphology of male and female internal reproductive structures of these studied species are provided and indicate that the main differences are in the relative shapes, sizes, and colors of these structures. The overall structure and organization, including details of the ultrastructure, of these two leafhopper species’ male and female internal reproductive systems are very similar to those of previously studied leafhoppers. The main differences observed among species include the number of testicular follicles, the relative position of seminal vesicles and the degree of development of the accessory glands in the male, the number of ovaries, and the shape and color of the vagina and spermatheca in the female.

Auchenorrhyncha, comparative study, Memberacoidea, transmission electron microscope

Insects are the dominant component of terrestrial biodiversity. Their ability to survive across complex and varied natural environments is closely related to their strong reproductive capacity. The structures of the insect reproductive system are complex and varied, reflecting different reproductive strategies (Song 2011). The reproductive system of insects consists of multiple organs in the abdominal cavity that work together to produce sperm or ova and facilitate copulation. A study of the insect reproductive system is needed to improve the understanding of insect reproductive potential and oviposition mode, which may ultimately facilitate pest management (Lei and Rong 2003). Comparative study of the reproductive structures of different insects also increases our understanding of the evolution and phylogeny of insects.

Despite leafhoppers’ high diversity and economic importance and the widespread use of the external sclerotized structures of their genitalia for taxonomy and phylogenetics, their internal reproductive organs remain little studied. Helms (1968) described in detail the morphology of the leafhopper Empoasca fabae (Harris, 1841) male reproductive system. The adult male consists of two testes (each composed of four follicles), vasa deferentia with seminal vesicles, paired accessory glands, paired ejaculatory ducts, and a median ejaculatory bulb. Moreover, Mishra (1979) concluded in the comparison of male leafhoppers Cofana spectra (Distant, 1908), C. unimaculata (Signoret, 1854), and Amritodus atkinsoni (Lethierry, 1889) that the number of testicular follicles is different, and the corresponding accessory glands also differ in shape. Similarly, Tsai and Perrier (1996) studied the morphology of male reproductive systems of the two deltocephaline leafhoppers representing different tribes, Dalbulus maidis (DeLong, 1923) and Graminella nigrifrons (Forbes, 1885). They found that the males of these species have two lateral testes (each with six follicles). A pair of accessory glands arise at the posterior of each seminal vesicle and open into a lateral ejaculatory duct. Hayashi and Kamimura (2002) conducted a specific study on the leafhopper Bothrogonia ferruginea Fabricius, 1787 and found that during the process from production to entry into the female body, male sperm first transported to the female’s bursa copulatrix through a spermatophore. After successful mating, the sperm enters the spermatheca for fertilization. At this stage, the sperm-binding material (trypsin degradable proteins) and the spermatophore disappear in the bursa and an enlarged portion of the genital duct. They suggested that females could incorporate proteinaceous material derived from male spermatophores and/or sperm-binding material into their oocytes. Tian et al. (2006) compared the male reproductive systems of 39 species of Cicadellidae and focused on spermatogenesis, classifying the testes into two types according to the presence or absence of the sheath. Their results revealed that the testicular follicles of Cicadellidae are spherical, while testicular follicles in Heteroptera are lamellar and sheathed. Su et al. (2014) compared the ultrastructure of the male reproductive systems of Psammotettix striatus (Linnaeus, 1758) and Exitianus indicus (Distant, 1908) (Deltocephalinae) and described the morphology and ultrastructure of the sperm of these two species. The sperm of both species have a 9+9+2 axonemal pattern, as in other Auchenorrhyncha, and provide a reference for the comparative study and phylogeny of other groups. Vitale et al. (2015) analyzed the morphology of the male reproductive system of Balclutha brevis (Lindberg, 1954). Their results showed that secretory activity occurring mainly in the lateral ejaculatory ducts and the accessory glands. The ultrastructural features of the seminal vesicle differed from those of the lateral ejaculatory duct, suggesting that these structures play distinct roles in the organization of the sperm bundles. Recently, Zhang et al. (2016) found differences in the organizational structure of the male reproductive systems of the closely related cicadelline leafhoppers Cicadella viridis (Linnaeus, 1758) and Kolla paulula (Walker, 1858).

Similarly, Helms (1968) found that the female reproductive system of E. fabae (Harris, 1841) consists of two ovaries (each composed of four telotrophic ovarioles), lateral oviducts, a median oviduct, a spermatheca, a genital chamber with a pair of spermatozoal pouches, and a median accessory gland. Additionally, Cheung (1994, 1995) studied the female reproductive system of Euscelidium variegatus Kirschbaum, 1858; it was clear that the ovary is endotrophic, and the main source of nutrition for growth and development is also completed by trophoblasts. Studies on the ultrastructure of the cicada reproductive system have found ovarian sheaths and tracheae on the periphery of the ovarian tubes, which are called perimetrium. Similarly, Tsai and Perrier (1996) also compared the morphology of the female reproductive systems of D. maidis (DeLong, 1923) and G. nigrifrons (Forbes, 1885) and found that ovaries of these species contain six ovarioles, each with six follicles. In G. nigrifrons each ovariole usually contains only one egg within the last follicle, whereas the ovarioles of D. maidis usually contain two eggs. The ovarioles open into the lateral oviduct, common oviduct, and the vagina. Besides, Hummel et al. (2006) studied the development of the ovaries in Homalodisca vitripennis (Germar, 1821) and found that a single ovary is composed of 10 ovarioles, and the development of an ovary occurred in seven stages. Recently, Pappalardo et al. (2016) studied the ultrastructure of the female reproductive system of Balclutha brevis (Lindberg, 1954). Their results showed that the female reproductive system has a morphological configuration comparable to most species of Auchenorrhyncha.

These previous studies indicate that characteristics of the reproductive system may provide a basis for phylogenetic analysis and classification. However, the available data are scattered, and studies have focused on different reproductive characteristics. Thus, additional comparative analyses are needed.

In this study, the overall morphology and ultrastructure of the male and female internal reproductive systems of two species of Deltocephalinae are described and illustrated for the first time. The overall composition and structure of the internal genitalia of these species are similar to those of other studied leafhoppers. Although we observed some differences between N. cincticeps and D. vulgaris, further study will be needed to determine whether such differences, e.g., in the color of the seminal vesicles of the males and degree of development of the spermatheca and accessory glands of the females, are consistent or whether they may reflect different ages or physiological stages of the studied individuals.

Comparing our observations to the few published observations of the male internal reproductive organs of other leafhopper species (Table 1), we note that, in contrast to the two species of Deltocephalinae studied here, in which males have the seminal vesicles well separated from each other, as in the megophthalmine leafhopper Agallia constricta (Gil-Fernandez & Black, 1965), another deltocephaline species, B. brevis (tribe Macrostelini) has the seminal vesicles joined to each other medially (Vitale et al. 2015). Two previously studied species of sharpshooters (subfamily Cicadellinae) also have paired seminal vesicles joined medially (Tian et al. 2006; Zhang et al. 2016). Vitale et al. (2015) also noted that the male accessory glands of B. brevis are divided into distinct proximal and distal sections distinguishable by color. Such sections are evident in the accessory glands of K. paulula but not in C. viridis, based on illustrations by Zhang et al. (2016), and we also did not observe distinct regions in the two species examined here. However, the distal section of the accessory gland of D. vulgaris is somewhat paler than the darker proximal section. In other respects, leafhoppers’ male internal reproductive structures appear to be highly conservative, although differences among species in the number of testicular follicles have been reported (Vitale et al. 2015), as observed among the two species studied here.

Comparing our observations to the few published observations of the female internal reproductive organs of other leafhopper species (Table 2), we note that, among female leafhoppers, the most obvious differences among species in the internal reproductive structures seem to be the number of ovarioles. The two studied species each had six ovarioles per ovary, as in most previously studied leafhopper species. However, the number of ovarioles may be much larger and vary within a species, e.g., 8–10 in H. vitripennis (Hummel et al. 2006). The structure of individual ovarioles may also vary among species, which may reflect differences in fecundity (Tsai and Perrier 1996), although such variation also occurs within individuals at different stages of development and vitellogenesis (Hummel et al. 2006). Much more comparative study, including the variation among individuals of the same species at different stages of development, is needed to elucidate further the morphological variability of leafhoppers’ male and female internal reproductive structures.

The overall structure and organization, including details of the ultrastructure, of the male and female reproductive systems of Nephotettix cincticeps (Uhler, 1896) and Deltocephalus vulgaris (Dash & Viraktamath, 1998) are very similar to those of previously studied leafhoppers. The main differences observed among species include the number of testicular follicles, the relative position of seminal vesicles, the degree of development of the accessory glands in the male, the number of ovarioles, and the shape and color of the vagina and spermatheca in the female. This suggests that, compared to the external genitalia, which highly varies among species and is often used in taxonomy, the internal reproductive structures of leafhoppers offer relatively few characters useful for classification and phylogenetics. Nevertheless, relatively few species have so far been studied in detail. Future work should focus on representatives of the many additional leafhopper subfamilies that have not yet been studied in detail.

We sincerely thank J.R. Schrock, Emporia State University, USA, for commenting on an earlier draft of this paper and anonymous referees and the subject editor for constructive criticism.