Characteristics of the Male Reproductive System and Spermatozoa of Leptophlebiidae (Ephemeroptera)

SYSTEMATICS, MORPHOLOGY AND PHYSIOLOGY

Characteristics of the Male Reproductive System and Spermatozoa of Leptophlebiidae (Ephemeroptera)

P Brito^I; FF Salles^II; H Dolder^I

^IDepto de Anatomia, Biologia Celular e Fisiologia, Instituto de Biologia, UNICAMP, Campinas, SP, Brasil

^IIDepto de Ciências da Saúde, Biológicas e Agrárias, Centro Universitário Norte do Espírito Santo, Univ Federal do Espírito Santo, São Mateus, ES, Brasil

^{Correspondence}

ABSTRACT

This study describes morphological changes in the male reproductive system of Miroculis amazonicus (Savage & Peters) from mature nymphs to subimago stages. The sperm ultrastructure of Massartela brieni (Lestage), Farrodes carioca (Domínguez et al) and Miroculis mourei (Savage & Peters), as well as aspects of cell fragments observed in these species' subimagos deferent ducts were described. Sperm from the three species studied are aflagellated and immotile, while those from F. carioca and Ma. brieni are approximately spherical with a homogenous nucleus and acrosome. Sperm of F. carioca present two or three mitochondria located between the nucleus and the acrosome. In Ma. brieni, only one lateral mitochondria was found. Sperm from Mi. mourei are shaped as a number 'eight', with electron lucent spots inside the nucleus and two mitochondria above the acrosome. Large cell fragments containing degenerative vesicles and some sperm were observed in the deferent duct lumen of the three species. Testes of Mi. amazonicus are extremely reduced in the subimago stage, which suggests that these cell fragments originated from testes degeneration.

Keywords: Morphology, sperm, aflagellate, sexual maturation

Introduction

Species from Leptophlebiidae are distributed around the world and can be found in very diverse aquatic environments. Recent studies show approximately 130 genera and 610 species described in this family (Barber-James et al 2008). In South America, it represents one of the two major families of Ephemeroptera (Pescador et al 2001), being represented by 40 genera and 150 species (Domínguez et al 2006). Species of Leptophlebiidae are grouped in three subfamilies: Leptophlebiinae, Atalophlebiinae and Habrophlebiinae. Only species from Atalophlebiinae are found in South America.

Sperm from Leptophlebiidae males are notably aflagellate (Soldán 1979a, Grimm 1985, Gaino & Mazzini 1991a), which differentiates them from other Ephemeroptera. However, only four species have been ultrastructurally described: Habrophlebia lauta (Eaton) (Grimm 1985), Habroleptoides umbratilis (Eaton), Habrophlebia eldae (Jacob & Sartori) and Choroterpes picteti (Eaton) (Gaino & Mazzini 1991a). Furthermore, little is known of the morphology of male reproductive system in Ephemeroptera, but it is considered simple. Male reproductive system is composed of a pair of testes where the sperm cells develop and the seminal ducts which also store the spermatozoa, without accessory glands or other specializations (Soldán 1979b). Secretions produced by accessory glands are usually reported to play a role in the reproductive success of most insects (Chen 1984, Gillott 2003), but nothing is known about the possible consequences of the absence of these glands in the physiology of Ephemeroptera.

To provide new information on the Leptophlebiidae biology, this study describes morphological changes in the male reproductive system of Miroculis (Atroari) amazonicus (Savage & Peters) from their last instar to the subimago stage. The sperm ultrastructure of Massartela brieni (Lestage), Farrodes carioca (Domínguez et al) and Miroculis (Ommaethus) mourei (Savage & Peters) was also described, as well as aspects of cell fragments observed in these species' deferent ducts.

Material and Methods

Light microscopy

Miroculis amazonicus at the last nymph stage were collected from a river in Presidente Figueiredo, state of Amazonas, Brazil. Some specimens were dissected as nymphs and others were maintained until the first winged stage (subimago) and then dissected. Their reproductive systems were fixed in a 2.5% glutaraldehyde and 4% paraformaldehyde solution. They were placed on a histological slide and photographed unstained with an Olympus BX41 light microscope.

Transmission electron microscopy

Farrodes carioca, Ma. brieni and Mi. mourei male subimagos were collected near a river in Santa Teresa, state of Espirito Santo, Brazil. The deferent ducts of the specimens were dissected and fixed in a 2% glutaraldehyde and 1% tannic acid solution, and post-fixed in 1% uranyl acetate solution. The material was dehydrated and embedded in Epon resin; ultrathin sections were contrasted with 3% uranyl acetate and lead citrate and analyzed in a Zeiss Leo 906 transmission electron microscope.

Results

The spermatozoa of F. carioca and Ma. brieni are approximately spherical and consist of a nucleus with uniformly condensed chromatin and an acrosome with median electron density (Fig 1a, b). Two or three spherical mitochondria are observed between acrosome and nucleus in F. carioca sperm (Fig 1a). Massartela brieni sperm cells generally present one spherical mitochondrion laterally observed between the nucleus and the acrosome (Fig 1b). Spermatozoa of Mi. mourei consist of a nucleus and an acrosome, but their shape is similar to a number 'eight', since they have a constriction at the acrosome base, near the nucleus. The nucleus is filled with compact chromatin containing electron lucent regions (Fig 1c). In general, spermatozoa of Mi. mourei present two spherical mitochondria located above the acrosome (Fig 1c). The longer axis measured from the acrosome tip to the nuclear base of the sperm is approximately: 1.4 μm in F. carioca and 1.6 μm in Ma. brieni and Mi. mourei.

The sperm are stored for copula in the deferent duct lumens of these species. Many cellular fragments were observed filling a large part of the volume of these ducts. These fragments are different among the species studied, but all three species have vesicles or degenerative vacuoles in their cellular fragments (Fig 1d-f). The cellular fragments observed in F. carioca presented a large vesicle partially filled with electron dense material and many small clear vacuoles in their cytoplasm (Fig 1d). Nuclei with partially condensed chromatin can still be recognized among these fragments. The cellular fragments observed in Ma. brieni made up most of the content of the deferent ducts. They presented vesicles partially filled with electron dense material and some interspersed spermatozoa (Fig 1e). There are two types of cellular fragments in Mi. mourei: one with variable electron density (Fig 1f, lower part of the figure), while the other type contained complex membranous structures derived from partially reabsorbed organelles (Fig 1f).

The male reproductive system of Mi. amazonicus undergoes profound changes from mature nymphs to subimagos. In mature nymphs, the testes were well developed (Fig 2a) while the deferent ducts were thin, approximately 52 μm in diameter, with empty lumens (Fig 2b). In subimagos, the testes were degenerated and the deferent ducts were dilated to approximately 124 μm in diameter, and their lumens were completely filled with sperm and cell fragments, as described earlier (Fig 2c).

Discussion

Data presented in this manuscript confirm aflagellate and non-motile sperm as an autapomorphy of Leptophlebiidae in the Order Ephemeroptera. Despite the apparent simplicity, the sperm morphology in this family allows us to distinguish each of the genera analyzed.

The presence of a perforatorium in the acrosome is a plesiomorphic characteristic of insects (Baccetti 1972, Jamieson et al 1999), which was lost in the main Ephemeroptera lineages (Baccetti et al 1969, Fink & Yasui 1988). This fibrous structure was described in the acrosome of H. umbralitis (Habrophlebiinae) and C. picteti (Atalophlebiinae) (Gaino & Mazzini 1991a). In H. eldae (Habrophlebiinae) (Gaino & Mazzini 1991a). However, in F. carioca, Ma. brieni and Mi. mourei (Atalophlebiinae) sperm, no perforatorium was observed. Probably, the presence of a perforatorium is related to the thickness of the egg chorion for these different species as suggested by Gaino and Mazzini (1991b), and does not seem to have a phylogenetic significance in Leptophlebiidae, since this characteristic is not shared among species of the same subfamily.

Mitochondrion position is another variable characteristic among Leptophlebiidae sperm. A mitochondrion was observed beneath the nucleus in H. umbralitis (Gaino & Mazzini 1991a), laterally located between the nucleus and the acrosome in C. picteti (Gaino & Mazzini 1991a) as well as in F. carioca, but different from Ma. brieni, where it occurs laterally in the nucleus/acrosome contact region, and from Mi. mourei, where it is found above the acrosome. Interestingly, no visible mitochondrion was observed in H. eldae sperm (Gaino & Mazzini 1991a). Most Ephemeroptera sperm present a single, enlarged mitochondrial derivative with a separated paracrystalline body that extends along the tail (Baccetti et al 1969, Phillips 1969, Fink & Yasui 1988, Gaino & Mazzini 1991b).

The presence of small, simple mitochondria in Leptophlebiidae sperm is a derivation of the typical pattern described for this order. Since the mitochondrial derivative is associated with the flagellum and its movement, these immotile species would have very different energetic needs and therefore their mitochondria were not modified in the same manner. To confirm a probable phylogenetic significance of different localizations of the mitochondria in the spermatozoa of Leptophlebiidae, more species must be studied. A phylogenetic inference based on the available data would be premature.

The presence of large cell fragments in the deferent duct lumens has not been previously reported in Ephemeroptera. Since the testes of Miroculis amazonicus were greatly reduced and the enlarged seminal duct in the subimago stage, the cell fragments observed in this duct probably originated from the degenerated testes. The presence of some spermatozoa (as observed in Ma. brieni) and of degenerative vesicles inside these cell fragments seem to confirm this hypothesis. Some studies have reported the presence of holocrine and apocrine secretion in the ducts of male reproductive systems of different insects (Leopold 1970, Perotti 1971, Riemann 1973, Almadoss 1990, Brito et al 2010). Since Leptophlebiidae, as all other Ephemeroptera, do not present accessory glands in the male reproductive system, the cell fragments observed in this study could act as functional secretions inside the deferent ducts or in the female reproductive tract. They could also provide nutrients for sperm nutrition or function as a protective medium for sperm storage, or modulate post copula behavior in females. However, this hypothesis must be confirmed by future investigations.

Acknowledgments

The authors would like to acknowledge Dr Neusa Hamada and Paulo V Cruz for their support for specimen collection. Some specimen collection expeditions were supported by PRONEX/CNPq/FAPEAM. This research was funded by the Brazilian agencies Fapesp and Capes (Proex).

Received 24 February 2010 and accepted 23 August 2010

Edited by Fernando L Cônsoli - ESALQ/USP

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Publication Dates

• Publication in this collection
  14 Mar 2011
• Date of issue
  Feb 2011

History

• Received
  24 Feb 2010
• Accepted
  23 Aug 2010