Larvae of the family Passalidae occur with relative abundance in tropical forest and they are associated with adults in fallen trunks. Only a few studies on the larvae of Neotropical Pas&salidae have been published (Costa & Fonseca, 1986; Costa, Vanin, & Casari-Chen, 1988; Schuster & Reyes-Castillo, 1981, 1990; Schuster, 1992), and up to date, the most important work on Brazilian species (Costa & Fonseca, 1986) has described the larvae of 21 species.
All known Passalidae are subsocial, with parental adults, immature adults and larvae, all living in a family group in a gallery system constructed by the adults, almost exclusively within or beneath decaying wood (trunks, limbs, roots, and wood particles). This is asso&ciated with interesting behaviors including cooperation, parental care, defense, hierar&chic systems and communication, including a complex acoustical repertoire (Reyes-Castillo, 1970; Schuster, 1983; Reyes-Castillo & Halff-ter, 1984).
The subgenus Pertinax in Passalus comprises 40 species, of which 23 have been recorded in Brazil (Hincks & Dibb, 1935; Fonseca & Reyes-Castillo, 2004). Passalus (Pertinax) gravelyi Moreira, 1922 is an endem&ic species from the Atlantic Forest (Mata Atlantica) and known only from the states of Rio de Janeiro and Minas Gerais, and from elevations above 1 000 m.
Our current research includes a systematic study of the larvae of Neotropical Passalidae. While preparing the description of the 3rd instar larva of Passalus (Pertinax) gravelyi Moreira, 1922, we noted the occurrence of a sexual dimorphism in the 2nd and 3rd instar larvae of this species; the male larvae has a terminal ampulla on the margin of the 9th sternite, and investigated the occurrence of this character in the species from all of supra-generic taxa of Passalidae.
The terminal ampulla was first described by Herold (1815) in the larvae of Lepidoptera and defined more specifically by Snodgrass (1941) in Hymenoptera. In Coleoptera the terminal ampulla was described in Scarabaei-dae by Hurpin (1953), and subsequently the presence of sexual dimorphism in Passali-dae, Lucanidae, Scarabaeidae and Melolon-thidae larvae has been reported in literature of Scarabaeoidea (Menees, 1957; Onore, 1994; Martínez & Lumaret, 2003, 2005; Ramírez-Salinas, Pacheco-Flores, Castro-Ramírez, & Moròn, 2010). The sexual dimorphism in Pas-salidae has never been studied in detail, espe&cially in larvae and pupae. The goal of our study was to describe the larvae of Passalus (Pertinax) gravelyi Moreira, 1922, and to begin the systematic study of larvae with the knowl&edge about the sexual dimorphism in larvae of the Neotropical Passalidae.
Material and Methods
The samples were collected in decayed wood in the Parque Nacional do Itatiaia (Itati-aia, Rio de Janeiro, Brazil); a total of 10 specimens of larvae were collected from the same tunnel system as three adults, and all were preserved in ethanol 96 % until their evaluation.
At least 300 specimens of 62 species (Table 1) were comparatively studied about the terminal ampulla in the following Institutions: Colecào José Alfredo Pinheiro Dutra, Departamento de Zoologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro (DZRJ) and Colección Entomológica del Instituto de Ecología Xalapa (IEXA).
The morphological characters and the illustrations were made with the aid of a Leica MZ 7.5 stereomicroscope with a drawing tube and integrated digital camera Leica 3.0 CMOS. The larvae terminology followed the primary setal patterns proposed by Schuster & Reyes-Castillo (1981), the general terms of structures as Costa et al. (1988), and the general taxo-nomic classification followed Boucher (2006).
The electronic photographs were made after the dehydration of some larval structures in a graded ethanol series (70-100 %), air dried, and mounted on aluminum stubs and palladium gold covered. Subsequently, the samples were photographed in a JEOL model JSM 5600 LV Scanning electron Microscope.
In order to study the terminal ampulla, the larvae were fixed following the techniques described by Martínez (1999, 2002) using Black of chlorazol and Feulgen-woven green. We investigated the occurrence of the sexual dimorphism in passalid larvae, with the dimor&phism present in the structure of the terminal ampulla. We observed the terminal ampulla in male larvae of the 62 passalid species across the two subfamilies and six tribes.
Results
Passalus (Pertinax) gravelyi Moreira, 1922, larva
Passalus (Pertinax) gravelyi Moreira, 1922:276-278; Luederwaldt, 1931:116; Fon-seca & Reyes-Castillo 2004:15 (cat.) (Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7, Fig.
8, Fig. 9).
Description: Larva of third instar (n = 5). Length = 8.09-15.99 mm; head width = 1.89&2.66 mm; prothorax width = 2.98-3.83 mm. Head, with short and sparse pubescence (Fig. 2 and Fig. 3); two or three post-antennal setae (2-3 HPA), elongate, reaching antennal tip when extended and folded forward. Epicranial suture present with distinct frontal stem in V shaped. Coronal suture almost half of head capsule length. Clypeus distinctly transverse and pubescent. Labrum transverse, with anterior margin rounded and with moderately elongate and sparse setae. Epipharynx (Fig. 4) with corypha rounded and with setose, clithrum small and pigmented, acanthoparia with elon&gate setae, acroparia smooth, chaetoparia with long and sparse setae, pedium marked by the presence of many setae in the central area, laeotorma and dexiotorma strongly pigmented. Mouthparts protracted (Fig. 5, Fig. 6 and Fig. 7): mandibles (Fig. 5) symmetrical, movable, triangular, tridentate at apex, external margin distinctly angulate, with many sparse setae (except in apical part), mesal surface curved and smooth, inner cutting edge with a blunt tooth, mola developed, baso-lateral area, near the mola, with a declivity and with one tuft of short and dense setae near the condilun, and brustia developed with a dense, compact tuft of elongate setae. Maxillae (Fig. 6) mobiles and elongate, with galea elongate, almost as long as apical segment of maxillary palp, falcate at apex, with elongate setae at inner edge; lacinia entire, reaching the apical third of galea, with single apex, inner margin with fringe of elongate setae; palpifer present with six basal setae; maxillary palp with two segments, the apical segment slightly longer than basal segment and narrowing near the apex, basal segment with four setae. Labium with prementum, mentum and submentum; ligula (Fig. 7) rounded at apex and dense and short setae; prementum with a single fringe of setae near base; mentum short, smooth, and posterior margin converging back in V-shaped; submentum with one pair of elongate setae at the sides the suture between men-tum and submentum; submentum fused at the gula; gula laterally with many setae, medially glabrous. Thorax. Segments glabrous, with prothoracic spiracles larger than and directed opposite to mesothoracic, metathoracic and abdominal spiracles. Pronotum entirely pubescent and with two lateral sclerotized areas, each with 3-5 long lateral setae (3-5 PSL); median area between sclerotized pronotal areas distinct glabrous. Mesonotum no dorsal mesonotal setae (MSD) and one pair of lateral mesono-tal setae (0MSD; 2 MSL). Metanotum with one pair dorsal metanotal setae and two lateral metanotal setae (1 MTD; 2 MTL). Pro and mesothoracic legs well developed (with coxa, trochanter, femur, tibia and tarsungulus) and pubescent. Metathoracic legs reduced (Fig. 8) with only one segment bearing six small, lateral teeth basal to the apex. Abdomen. Ten segments visible when viewed dorsally or laterally. Abdominal segments each with one pair of medial tergal setae and one pair of lateral tergal setae (1 TM; 1 TL). Anal opening (Fig. 9) V-shaped; anal ring with 16-20 setae (18-20 AR); IX abdominal ventrite with one pair of setae in medial area (AV9).
Setal Pattern 2° and 3° instars: 2-3 HPA; 3-4 PSL; 0 MSD; 2 MSL; 1 MTD; 2 MTL; 1 TM; 1 TL; 1 AV9; 16-18 AR.
Setal Pattern 1° instar (Fig. 1): 3 HPA; 4 PSL; 1 MSD; 2 MSL; 1 MTD; 2 MTL; 1 TM; 1 TL; 1 AV9; 18 AR. Measurements of first instar - Length = 7.49 mm; head width = 1.49 mm; prothorax width = 2.66 mm.
Material examined: BRAZIL. Rio de Janeiro: Itatiaia (Parque Nacional do Itatiaia,
Estrada para Agulhas Negras, 44°36'25.3" W - 22°26'3.3" S, 2.091 m), 20.VII.2012, 1 larva (1° instar), 4 larvae (2º instar), 5 larvae (3º instar), 3 adults, Mermudes et al. leg. (DZRJ).
In this study sexual dimorphism in pas-salid larvae is characterized in detail for the first time, with the dimorphism present in the structure of the terminal ampulla (sometimes referred to as "genital ampula"). The termi&nal ampulla has been reported and described in various holometabolous insects (Herold, 1815; Snodgrass, 1941; Hurpin, 1953; Menees, 1957; Matsuda, 1976; Onore, 1994; Martínez & Lumaret, 2005) as the cuticular structure in medial-ventral area of the 9th abdominal sternite in males (Fig. 10, Fig. 11, Fig. 12, Fig. 13, Fig. 14 and Fig. 15). Ramírez-Salinas et al. (2010) reported that the terminal ampulla (referred to as "genital ámpula") was present in the last (tenth) abdominal sternite of the pupae of the dynastine scarab, Ligyrus nasutus (Burmeister) but that it was less prominent than the terminal ampula in male pupae.
In other Scarabaeoidea, the terminal ampulla is evident in all larval instars and it is observed at apex of the aedeagus at the last abdominal ventrite in male pupae (Martínez & Lumaret, 2005). We were unable to detect the terminal ampulla in the first instar passalid larvae, but in the second and third instar larvae and in pupae, the sex of the immatures can be determined. We observed the terminal ampulla only in male larvae of the 62 passalid species across the two subfamilies and six tribes (sensu Boucher, 2006) of Passalidae (Table 1), using the techniques of Martínez (1999) for dissecting and staining the terminal ampulla in the Scarabaeinae.
The terminal ampulla of late-instar passa-lid male larvae is visible ventrally as a distinct patch of cuticular tissue of the 9th sternite, between and internal to the pair of ventral abdominal setae (AV) of this segment (Fig. 10, Fig. 11, Fig. 12, Fig. 13, Fig. 14 and Fig. 15). This structure, terminal ampulla, it is a very thin layer, with no apparent differences among species.
Discussion
Our current examination included a sys&tematic study of the larvae of Neotropical Passalidae, with the description of the larva of Passalus (Pertinax) gravelyi Moreira, 1922. Based on the three larval instars studied and the absence of some primary setae in the later instars (2° and 3°), the following general setal pattern is proposed for Passalus (Pertinax) gravelyi: 2-3 HPA; 3-5 PSL, 0-1 MSD; 2 MSL; 1 MTD; 2 MTL; 1 TM; 1 TL 1 AV9; 16-18 AR. The larvae of Passalus gravelyi has the general setal 'Pertinax' pattern (Schuster & Reyes-Castillo, 1981) and differ from others of Pertinax subgenus larvae by the high number of setae in the anal ring (16-18 AR). Passalus (Pertinax) affinis Percheron, 1835, P. (Perti-nax) anguliferus Percheron, 1835, P. (Pertinax) mancus Burmeister, 1847, P. (Pertinax) lati-frons Percheron, 1841, and also P. (Pertinax) dubitans Kuwert, 1891 present the typical anal ring with 10 setae (10AR). While, P. (Perti-nax) convexus Dalman, 1817 reveal variation between 10-12 AR.
The descriptions of Passalidae larvae were based on ornamentations or setae for chae-totaxy analysis (see Costa & Fonseca, 1986; Costa et al., 1988; Schuster & Reyes-Castillo, 1981, 1990; Schuster, 1992). Nevertheless, in all previous descriptions of Passalidae larvae, the authors did not report the presence of a terminal ampulla (sensu Snodgrass, 1941; Mat-suda, 1976). The terminal ampulla structure was originally described on Lepidoptera larva by Herold (1815), it was described in Hyme-noptera by Snodgrass (1941), and in Coleop&tera: Scarabaeidae larvae by Hurpin (1953) and it was reported at three instars larvae of Canthon c. cyanellus LeConte by Martínez and Lumaret (2003, 2005); it was cited from immature stages of six species of Sphaenogna-thus (Lucanidae) by Onore (1994).
The reports to larval species of the fami&lies assemblage in Scarabaeoidea published by Menees (1957), cited the Passalidae Popilius disjunctus (Illiger) transferred to Odontotae-nius by Reyes-Castillo (1970), and by Martínez and Lumaret (2003, 2005), that mentioned 26 species from Scarabaeidae, Aphodiidae, Trogi-dae, Orphnidae, Dynastidae, and two species of Passalidae: Passalus unicornis Saint-Fargeau (a species from Lesser Antilles endemic to Guadeloupe) and P. alticola Kirsch (a spe&cies described from Ecuador), which have not been described.
The occurrence of the sexual dimorphism in passalid larvae was characterized in detail for the first time based in the structure of this terminal ampulla. This structure was local&ized on the margin of the 9th sternite in male larvae, in species of the supra-generic taxa of Passalidae: all subfamilies and tribes, except the Leptaulacini, sensu Boucher (2006). In this study, we were unable to detect the ter&minal ampulla in first instar passalid larvae, but in second and third instar male larvae, and pupae (only clearly evaginated aedeagus in males), the sex of immatures was determined for species of subfamilies Aulacocyclinae and Passalinae. Data from this study included the largest world range of information to Passali-dae, comprising 62 passalid species, 29 genera in six tribes -Aulacocyclini, two species from Brunei & Australia, and Ceracupini, two species from Japan & Taiwan; Solenocyclini, two species from Ivory Coast; Macrolinini, one species from Brunei; Passalini, 19 species from Mexico, Guatemala, Colombia, Ecuador, Jamaica, Guadeloupe Lesser Antilles & Brazil; and Proculini, 36 species from United States of America, Mexico, Guatemala, Colombia & Brazil)-. Obviously is easier to determine the sexual dimorphism in larvae than in adults, for which the recent studies pointed specific areas of the adult pubescent body (e. g., spe&cies of Paxillus, see Mattos & Mermudes, 2013). Finally, the testicle, deferent ducts and other structures observed in Scarabaeidae by Martínez and Lumaret (2005), were not seen in Passalidae species, possibly because sex&ual maturation occurs later, which can be further investigated.