INTRODUCTION

The species of Scarabaeidae exhibit great morphological and ecological diversity, which is reflected in the anatomy of their reproductive system. However, the anatomy of the male reproductive system in this family has received very little study, except for the aedeagus, which is often used for species identification (^{Sharp & Muir, 1912}). Unfortunately, only some isolated descriptions of the male internal genitalia in representatives of different Scarabaeidae subfamilies are available.

In Melolonthinae, there are morphological descriptions of the male reproductive system for Melolontha melolontha (Linnaeus) (^{Dufour, 1825}; ^{Straus-Dürckheim, 1828}; ^{Bordas, 1900}), Anoxia villosa (Fabricius) and Hymenoplia strigosa (Illiger) (^{Bordas, 1900}), Phyllophaga sp. (^{Williams, 1945}), Amphimallon majale (Razoumowsky) (^{Menees, 1963}), Costelytra zealandica (White) (^{Fenemore, 1971}; ^{Stringer, 1990}), Phyllophaga anxia (LeConte) (^{Berberet & Helms, 1972}), Phyllophaga sanctipauli (Blanchard) (^{Diefenbach et al., 1998}), Prodontria lewisi Broun (^{Ferreira & McKinlay, 2001}), Macrodactylus mexicanus Burmeister (^{Benítez-Herrera et al., 2015}) Hoplia argentea (Poda) (^{Bordas, 1900}), H. subcostata Bates and H. squamifera Burmeister (^{Carrillo-Ruiz et al., 2008}).

In Dynastinae the male reproductive system has been studied for Oryctes nasicornis (Linnaeus) (^{Straus-Dürckheim, 1828}), Oryctes rhinoceros (Linnaeus) (^{Mathur et al., 1960}; ^{Jacob, 1989}) and Adoryphorus couloni (Burmeister) (^{Hardy, 1981}).

In Rutelinae it has been studied for Anisoplia agricola (Poda) (^{Bordas, 1900}), Anomala dubia (Scopoli) and Phyllopertha horticola (Linnaeus) (^{Rittershaus, 1927}), Popillia japonica Newman (^{Williams, 1945}; ^{Anderson, 1950}), Anomala ausonia Erichson (^{Lupo, 1947}), and Chrysina costata (Blanchard) (^{Morón, 2010}).

Due to the limited knowledge of the internal genitalia of males in Scarabaeidae, here is described and illustrated for 21 Mexican June bug species. The systems of these species are compared with those reported in other published studies, differentiating between some subfamilies of Scarabaeidae and other families of Scarabaeoidea. The external genitalia were not studied because it is well known and used for taxonomic purposes (^{Sharp & Muir, 1912}).

This study is a contribution to the line of research Biología, Sistemática e Importancia de los Coleópteros Lamelicornios en América Latina, established by the late Miguel Ángel Morón^† several years ago.

MATERIALS AND METHODS

The adults of most of the species studied were collected in May and June over several years, from different places where it was known that they could be found. Beetles were collected manually, at random, mainly near Xalapa City, Veracruz, Mexico. A total of 350 males of the different species were collected. The species were determined by Miguel Angel Morón†.

The species studied were the following:

Melolonthinae. Phyllophaga testaceipennis (Blanchard) (El Sumidero, Xalapa, Veracruz), Phyllophaga (Phytalus) obsoleta (Blanchard) (El Sumidero, Xalapa, Veracruz), Phyllophaga (s. str.) opaca (Moser) (Valle del Carrizo, Ahome, Sinaloa), Phyllophaga (Phytalus) pruinosa (Blanchard) (Coatepec, Veracruz), Phyllophaga (s. str.) ravida (Blanchard) (El Haya, Xalapa, Veracruz), Phyllophaga (s. str.) rugipennis (Schauffus) (Briones, Coatepec, Veracruz), Phyllophaga (s. str.) setifera (Burmeister) (La Higuera, Xalapa, Veracruz), Phyllophaga (s. str.) subrugosa (Moser) (Briones, Coatepec, Veracruz), Phyllophaga (s. str.) tenuipilis (Bates) (Las Ánimas, Xalapa, Veracruz), Chlaenobia latipes Bates (Las Ánimas, Xalapa, Veracruz), Macrodactylus nigripes Bates (Las Vigas, Veracruz) and Isonychus neglectus Moser (Briones, Coatepec, Veracruz).

Dynastinae. Cyclocephala jalapensis Casey (Rancho Guadalupe, Xalapa, Veracruz), Cyclocephala picta Burmeister (Briones, Coatepec, Veracruz), Cyclocephala sexpunctata Castelnau (Briones, Coatepec, Veracruz), Cyclocephala mafaffa Burmeister (Briones, Coatepec, Veracruz), Cyclocephala lunulata Burmeister (Briones, Coatepec, Veracruz), and Cyclocephala weidneri Endrödi (Briones, Coatepec, Veracruz).

Rutelinae. Paranomala marginicollis (Bates) (Briones, Coatepec, Veracruz). Paranomala semicincta (Bates) (Briones, Coatepec, Veracruz), and Paranomala cupricollis (Chevrolat) (Briones, Coatepec, Veracruz).

The suprageneric classification used follows the proposal of ^{Smith (2006}). All examined specimens were deposited in the collection of Miguel Ángel Morón, Xalapa, Veracruz, Mexico (MXAL).

For each species two to ten live males were selected and dissected in Ringer’s saline solution to obtain their reproductive system. The complete system from each male was placed and extended in AFATD fixer (96% ethanol-formaldehyde-trichloroacetic acid-dimethylsulfoxide), and later completely stained using the Feulgen light green and chlorazol black staining techniques (^{Martoja & Martoja, 1967}; ^{Gabe, 1968}; ^{Carayon, 1969}; ^{Martínez, 2002}). The internal genitalia of several specimens of each species were initially observed and drawn to scale using a stereomicroscope with a camera lucida (Zeiss® SV11). For each species Chinese ink was used for the line drawings (unpublished techniques, learned by the first author directly from J. Carayon and Dominique Pluot-Sigwalt). The figures were edited into plates using Corel Photo-Paint® software. The descriptions use the terminology of ^{Snodgrass (1935)} and ^{Matzuda (1976)} for the general morphology of the internal genitalia.

RESULTS

In general terms, the males of these three subfamilies have similar reproductive systems, though there are some differences between them.

Melolonthinae (Fig. 1-12). The male reproductive system of P. testaceipennis (Fig. 1) is typical of all of the Melolonthinae species examined. It consists of two testes, each with six lobed and dorsoventrally flattened testicular follicles, without an outer membrane to group them together. Each testicular follicle has a long and very visible vas efferens, which emerges from the ventral region. The six vas efferens flow into the vas deferens of each testis.

Figures 1-12 Male reproductive system of 12 species of Melolonthinae. 1. Phyllophaga testaceipennis 2. Ph. obsoleta 3. Phy opaca 4. Ph. pruinosa 5. Ph. ravida 6. Ph. rugipennis 7. Ph. setifera 8. Ph. subrugosa 9. Ph. tenuipilis 10. Chlaenobia latipes 11. Macrodactylus nigripes 12. Isonychus neglectus. Only in P. testaceipennis is the complete reproductive system shown, in the other figures only half is shown. (ae= aedeagus, ag= accessory glands, eb= ejaculatory bulb, ed= ejaculatory duct, gd= glandular duct, gr=glandular reservoir, te= testis, vd= vas deferens). Scale= 4 mm. 

Each vas deferens has an anterior region that is smaller in diameter than the posterior region, and both ducts flow into the anterior region of the ejaculatory duct. The ejaculatory duct is a very elongated organ, with a thick muscular wall that opens to the inner sac, inside the aedeagus (Fig. 1).

There are two accessory glands, each with the anterior glandular region and a posterior glandular duct greater in diameter than the previous one. The glandular region is filamentous, small in diameter, which rolls over on itself and measures approximately 42 mm long. The glandular duct, larger in diameter than the glandular region, measures approximately 34 mm. Both glandular ducts open into the anterior part of the ejaculatory duct, next to the vas deferens (Fig. 1).

The males of all the Phyllophaga species examined have two testes with six testicular follicles and their respective vasa deferens, and two accessory glands open to the ejaculatory duct, which continue with the inner sac inside the aedeagus. Only in P. rugipennis (Fig. 6), M. nigripes (Fig. 11) and I. neglectus (Fig. 12) is the ejaculatory duct shorter, and it opens to an ejaculatory bulb with thick muscular walls.

Testes. In all species of Melolonthinae there are six follicles per testis (Fig. 1-12). In Phyllophaga, the six testicular follicles are free, lobed, and dorsoventrally flattened. In I. neglectus they are free, flattened, and lobed as in the species of Phyllophaga, while in M. nigripes (Fig. 11) the testicular follicles are spherical and grouped within an outer membrane. The vas efferens are long and thin, and the vas deferens is long, with thicker walls, as in all species. Likewise, the anterior part of the vas deferens is smaller in diameter than the posterior part.

Accessory glands. The two accessory glands in all species have an anterior region that is glandular, filamentous, long, and small in diameter, but their length varies among species (Table 1). The longest glands, more than 300 mm long, were found in Ph. tenuipilis (Fig. 9). Glands of 100 to 164 mm in length were found in Ph. obsoleta (Fig. 2), Ph. ravida (Fig. 5), Ph. rugipennis (Fig. 6), and Ph. setifera (Fig. 7), and glands 41 to 86 mm long were found in Ph. testaceipennis (Fig. 1), Ph. opaca (Fig. 3), Ph. pruinosa (Fig. 4), Ph. subrugosa (Fig. 8) and Ch. latipes (Fig. 10). The glandular duct varies in size between nine and 34 mm, depending on the species (Table 1). The smallest accessory glands were found in M. nigripes (Fig. 11). Only in this species was a widening observed in the anterior part of the glandular duct, as a glandular reservoir, but it does not occur in any other of the species studied in Melolonthinae.

Ejaculatory duct. This duct is very elongated and occurs in all species of Phyllophaga, though in some species it is slightly thicker towards the back (Table 1), as in Ph. testaceipennis (Fig. 1), Ph. pruinosa (Fig. 4) and Ph. rugipennis (Fig. 6).

Ejaculatory bulb. Only Ph. rugipennis (Fig. 6), M. nigripes (Fig. 11), and I. neglectus (Fig. 12) have an ejaculatory bulb (Table 1). This organ was not seen in all the species of Phyllophaga examined, though in Ph. pruinosa (Fig. 4) and Ph. setifera (Fig. 7) the muscular walls of the ejaculatory duct are thickened and are wider than long, which would represent an incipient ejaculatory bulb.

Dynastinae (Fig. 13-18). The male reproductive system in C. jalapensis (Fig. 13) exemplifies the system seen in all the species examined of this subfamily. It consists of two testicles, each with six dorsoventrally flattened free testicular follicles (Table 1), two vas deferens, two accessory glands with their respective glandular duct, and an ejaculatory bulb that opens into the aedeagus.

Figures 13-18 Male reproductive system of six species of Dynastinae species. 13. Cyclocephala jalapensis 14. C. picta Burmeister 15. C. sexpunctata 16. C. mafaffa 17. C. lunulata 18. C. weidneri. Only in C. jalapensis is the complete reproductive system shown, in the other figures only half is shown. (ae= aedeagus, ag= accessory glands, eb= ejaculatory bulb, ed= ejaculatory duct, gd= glandular duct, gr=glandular reservoir, te= testis, vd= vas deferens). Scale= 4 mm. 

Testes. The six species studied have two testicles, each with six flattened and lobed testicular follicles, and the vas efferens are long. The vas deferens are small in diameter in their anterior region and much larger diameter, with thick walls in the posterior region (Fig. 13-18).

Accessory glands. The longest-almost 349 mm long-were found in C. mafaffa (Fig. 16), and the smallest (44 mm) in C. jalapensis (Fig. 13) (Table 1). In all species, these glands are small in diameter. The glandular duct is long, with thick walls.

Ejaculatory duct. This duct is present in all six species (Table 1), although it is very small in C. jalapensis (Fig. 13), C. sexpunctata (Fig. 15), and C. weidneri (Fig. 18). In the other three species, it is elongated (Fig. 14, 16, 17).

Ejaculatory bulb. In the six species studied, there is a well-developed ejaculatory bulb (Table 1), with thick muscular walls (Fig. 13-18).

Rutelinae (Fig. 19-21). The male reproductive system of Paranomala marginicollis (Fig. 19) is typical of all the Rutelinae species examined. It consists of two testicles, each with six testicular follicles (Table 1), with their respective vas efferens, and a vas deferens. There are two very long accessory glands, with their respective glandular duct, the ejaculatory duct, and the ejaculatory bulb. The organs of the reproductive system of these three species have the same distribution plane.

Figures 19-21 Male reproductive system of three species of Rutelinae. 19. Paranomala marginicollis 20. Paranomala cupricollis 21. Paranomala semicincta. Only in P. marginicollis is the complete reproductive system shown, in the other figures only half is shown. (ae= aedeagus, ag= accessory glands, eb= ejaculatory bulb, ed= ejaculatory duct, gd= glandular duct, gr=glandular reservoir, te= testis, vd= vas deferens). Scale= 4 mm. 

Testes. The three species studied have two testicles, each with six-lobed and dorsoventrally flattened testicular follicles. The vas deferens are large in diameter.

Accessory glands. The accessory glands are 144 mm long in P. cupricollis (Fig. 20), but only 17.9 mm in P. semicincta (Fig. 21) (Table 1).

Ejaculatory duct. The ejaculatory duct is short and is present in all species.

Ejaculatory bulb. In the three species studied, there is a well-developed ejaculatory bulb.

DISCUSSION

Testes and vasa deferens. In all species studied there are six testicular follicles as occurs in the species of other Scarabaeoidea subfamilies (Table 2). However, in species of Passalidae there are two testicular follicles (^{Krause, 1946}; ^{Cruz & Castillo, 2008}; ^{Salazar et al., 2016}), whereas species of Aphodiinae have six to seven testicular follicles, while some species of Eupariini have two testicular follicles (^{Martínez et al., 2001}) and Lucanidae have twelve (^{Reyes-Castillo et al., 2004}).

The vas deferens are similar in all the species of the three subfamilies studied and in none of them was a seminal vesicle observed. Nevertheless, the presence of a seminal vesicle has been observed in other Melolonthinae species: Prodontria lewisii Broun, 1904 and Costelytra zealandica (White, 1846) (^{Ferreira & McKinlay, 2001}; ^{Stringer, 1990}). But, in two species of Hoplia (^{Carrillo-Ruiz et al., 2008}) in reality the seminal vesicle is only a dilation of the final part of the vas deferens. In one species of Dynastinae, Oryctes rhinoceros (Linnaeus, 1758) (^{Jacob, 1989}; ^{Mathur et al., 1960}), the seminal vesicle appears as an elongated dilation in the middle of the vas deferens. And in one species of Rutelinae, Plusiotis costata (Blanchard, 1851), it appears as two independent hemispherical dilations between the anterior and posterior vas deferens (^{Morón, 2010}).

Accessory gland. These glands are very long in Melolonthinae, Dynastinae, and Rutelinae. In fact, these glands are much longer than those of the other scarab species that have been studied (^{Berberet & Helms, 1972}; ^{Jacob, 1989}; ^{Diefenbach et al., 1998}; ^{Stringer, 1990}; ^{Carrillo-Ruiz et al., 2008}; ^{Morón 2010}). The glandular reservoir, which is located between the gland and the glandular duct, was only observed in M. nigripes (Fig. 11). This organ was also observed in M. mexicanus and O. rhinoceros (^{Mathur et al., 1960}; ^{Benítez-Herrera et al., 2015}). Some authors call the final part of the glandular duct the “glandular reservoir”, perhaps because that region is very wide, but it is not a glandular reservoir.

Ejaculatory duct. This duct is straight and long in the Melolonthinae species studied (Fig. 1-5, 7-10) except in Ph. rugipennis, M. nigripes and I. neglectus, in which the back of the ejaculatory duct thickens to form an ejaculatory bulb (Fig. 6, 11, 12). A long duct is also reported for other Melolonthinae such as C. zealandica and M. mexicanus (^{Stringer, 1990}; ^{Benítez-Herrera et al., 2015}). It has also been described as a short duct in P. anxia and P. sanctipauli (^{Berberet & Helms, 1972}; ^{Diefenbach et al., 1998}). In Dynastinae and Rutelinae, the ejaculatory duct is short before entering the ejaculatory bulb in all the species examined, as observed in O. rhinoceros and Ch. costata (^{Mathur et al., 1960}; ^{Jacob, 1989}; ^{Morón, 2010}).

Ejaculatory bulb. This organ occurs in three species of Melolonthinae, Ph. rugipennis, M. nigripes, and I. neglectus, and in all species of Dynastinae and Rutelinae (Table 1). It has also been observed in Ph. anxia (^{Berberet & Helms, 1972}), though these authors refer to it as the "erection fluid pump". It is also present in Ph. sanctipauli (^{Diefenbach et al., 1998}) and C. zealandica (^{Stringer 1990}), for which the latter author calls it the “muscular sheath of ductus ejaculatorius”, and in M. mexicanus (^{Benítez-Herrera et al., 2015}). In the genus Hoplia, the ejaculatory bulb is found in H. squamifera and H. subcostata (^{Carrillo-Ruiz et al., 2008}), and these authors referred to it as the “ejaculatory duct”. In Dynastinae there is an ejaculatory bulb in O. rhinoceros (^{Mathur et al., 1960}; ^{Jacob, 1989}), which the latter author refers to as the “spermatophore sac”. In Rutelinae it occurs in C. costata (^{Morón, 2010}).

In species of Aphodiinae and Scarabaeinae, it is known that the ejaculatory bulb, a complex muscular structure that contains the ejaculatory duct of ectodermal origin, is well developed. In Aphodius pseudolividus (Aphodiinae), the ejaculatory bulb is formed by a thick (50 µm) muscular wall, inside of which is the ejaculatory duct and a part of the internal sac. In Canthon indigaceus (Scarabaeinae) this structure is more complicated, with a 100 µm thick muscular wall and, in addition to the ejaculatory duct, there is a third gland that is ectodermal in origin (an ectadenia). Its structure indicates that it has a pumping function; the muscular contractions probably ensure the emission of the secretions of the accessory glands and the seminal fluid with the spermatozoa, which participate in the formation of the spermatophore (^{Pluot-Sigwalt & Martínez, 1998}). Perhaps the complexity of the ejaculatory bulb is related to the shape of the spermatophore. The spermatophore of several Scarabaeinae species is more elaborate than that of Aphodiinae species (Cruz & Martínez, 1992; Martínez, 2003) and in other Scarabaeoidea species its shape is very simple, including Melolontha melolontha (^{Landa, 1960}), Costelytra zealandica (Striger, 1988) and Phyllopertha horticola (^{Rittershaus, 1927}). In the species studied, the spermatophore is still unknown.