Morphology of immature stages of Paraegidium costalimai (Coleoptera: Scarabaeidae: Orphninae) and remarks on egg-busters in Scarabaeidae first-instar larvae

. Studies about the immature stages of Orphninae (Coleoptera: Scarabaeidae) species are scarce. The subfamily includes 214 species, but only 5 have the immature stages described: Chaetonyx robustus liguricus Mariani, 1946, Hybalus benoiti Tournier, 1864, H. rotroui Petrovitz, 1964 and Triodontus nitidulus (Guérin, 1844) from Old World; and Aegidium cribratum Bates, 1887 from the New World. The Neotropical genus Paraegidium Vulcano, Pereira & Martinez, 1966 encompass five species, mainly recorded from Brazil. Herein, the immature stages of P. costalimai Vulcano, Pereira & Martinez, 1966 are described and illustrated, along with remarks on the presence of egg-buster in Scarabaeidae first-instar larvae. A key to the third-instar larvae of known Orphninae and a comparative study of chaetotaxy are also provided.

The subfamily is distinguished from the other Scarabaeidae by having (Frolov, 2012;Frolov & Vaz-de-Mello, 2015): mandibles and labrum exposed in dorsal view (hidden by clypeus in Onorius), antennae with 10 antennomeres and 3 lamellae, protibiae of males without spur, anterior area of procoxae with a transversal furrow, dorsal surface of metacoxae with stridulatory striae, spurs of metatibiae separated by tarsal insertion, lateral areas of abdominal sternite II (hidden by metacoxae in ventral view) with a conspicuous sclerotized and pigmented area (plectrum). Sexual dimorphism is usually evident, with males' head and pronotum bearing horns or other ornamentation.
The purpose of the present study is I) to describe the immature stages of Paraegidium costalimai, II) to discuss the egg-busters of first-instar larvae, and III) clarify the classification of immature stages of Orphninae.

MATERIAL AND METHODS
Larvae of P. costalimai were collected at two urban parks in São Paulo (state of São Paulo, SP): Instituto Biológico de São Paulo (IBSP) and Parque da Independência, both with a secondary woodland cover ( Fig. 1). Larvae were found at 10 cm depth in the soil.
The reared adult was identified in comparison with paratypes of P. costalimai (MZSP) and following Frolov et al. (2017a).
Immatures were killed in boiling water and preserved in 70% alcohol. Observations and drawings were carried out in a Zeiss Stemi SV6 stereomicroscope or Zeiss Axioscop microscope, both with a coupled light camera. Detached larval structures (e.g., mouthparts and legs) were provisionally mounted on glycerin jelly slides (see Burrows, 1965;Johansen, 1940;Perina & Camacho, 2016;Widden, 2001).
Photographs were taken with a Canon EOS 80D DSLR Camera with a Canon MP-E 65 mm f/2.8 lens and an LED illumination system (Kawada & Buffington, 2016). For small dissected a Lucky Zoom USB eyepiece camera attached to a microscope was used. Images were processed using Zerene stacker. The Adobe Photoshop CC 2018 software was used for image processing. Measurements were taken in millimeters using a micrometer.
The terminology used followed Böving (1936) and Lawrence (1991) with modifications by Sousa et al. (2018). Head chaetotaxy followed Ritcher (1966) and Sawada (1991), as summarized by Sousa et al. (2018). Lobes of thorax and abdomen followed Rodrigues et al. (2018). The hair-like setae were separated into the two well-defined groups: minute setae and short-long setae (modification of Šípek et al., 2008;see Rodrigues et al., 2018). The term phobae was used by Böving (1936) to the fleshy projections, even though in P. costalimai it is stiff and slightly sclerotized.

Remarks:
Including present contribution, six species of Orphninae have their third-instar larvae described, and all of them have a distinct epipharynx: pedium surrounded by heli, but Morón (1991) described A. cribratum larvae with pedium surrounded by pine-like setae. Some confusion and discordant interpretations were done regarding heli (fixed spine) and spine-like setae (see discussion in Albertoni et al., 2014) and the revision of material of A. cribratum is here emphasized to check the nature of the ornamentation that surround the pedium. Other characters shared by Orphninae larvae are: antennomere IV with a dorsal and a ventral sensorial spot; ligula somewhat rectangular; raster with septula oval and palidia and tegillar setae barely differentiated from each other.

Remarks:
The first and third-instar larvae are quite similar to each other as noted to other scarab beetles (e.g., Böving, 1936). However, the presence of egg-busters on the first instar can be useful to distinguish it from other instars as mentioned in some studies (e.g., Emden,  1941; Šípek et al., 2008). Orphninae first-instar larvae are known only for Paraegidium costalimai (here described) and Chaetonyx robustus (illustrated by Gradinarov et al., 2015). (Figs. 56-58) length about 9.1 mm; thorax width about 3.5 mm; white, integument macroscopically smooth and glabrous but covered by a thin and short microscopic pubescence, which gives a velvety appearance to the surface (magnification of 30x). Head (Fig. 59). Vertex is almost hidden under pronotum in dorsal view (Fig. 56). Epistomal suture and canthus indistinct. Clypeus trapezoid, male with lateral areas prominent (Fig. 59). Labrum transverse and triangular . Maxillary palps prominent and somewhat triangular. Labium with a deep medial groove (Fig. 57). Antenna with three defined regions: scape-pedicel, funicle and clava. Thorax: Pronotum wider than long, great- est width at posterior margin, lateral margins rounded, males with a pair of large semispherical tubercles. Prosternum with posterior process rounded. Pronotum seeming longer than mesonotum, and mesonotum longer than metanotum in dorsal view (Fig. 56). Elytra curved ventrally around the body and with raised longitudinal carinae. Pro-, meso-and metacoxa contiguous; femur-tibia slightly exposed in prothorax and exposed in metathorax; protibia with three outer tubercle-like teeth; meso-and metatibia with 2 tubercle-like spurs, posterior spur at least twice longer than anterior spur; metatarsus with tarsomere I as long as other tarsomeres combined, or almost so. Mesothoracic spiracle present in a cavity between the pronotum, elytron and anterior and medial legs. Abdomen: Gin traps (dioneiform organs sensu Costa et al., 1988) absent, tergites I-II with a dorsal spine, III-V with three spines (anterior, dorsal, posterior), VI with two spines (anterior, posterior); VII-VIII with a longitudinal carina; I-VII with laterodorsally transverse carina. Abdominal spiracles I-IV with peritreme, I hidden under the wings, V-VIII as cuticular invagination; urogomphi present. Male terminalia (Fig. 60). Proximal genital ampulla divided in a cylindrical anterior area (phallobasis) and a posterior area bearing two dorsal spines (parameres); posterior ampulla semispherical and with a distal impressed line; sternite X hidden in ventral view. Female terminalia (Fig. 61). Sternite IX with genital ampulla formed by two parts, an anteromedial pair of small tubercles, and a posterolateral pair of prominent processes (see remarks); tergite X ventrally exposed as a membranous lobe.

Remarks:
The pupae are known to Orphninae species: A. cribratum (described by Morón, 1991), C. robustus (figured and measured by Gradinarov et al., 2015), and P. costalimai (here described). All three species have urogomphi, abdominal tergites I-VI with medial projection bearing hook-like acute spines, abdominal tergites VII-VIII with longitudinal carina, and abdomen without gin traps. Each medial projection of abdominal tergite I-VI has one spine in C. robustus, two spines in A. cribratum, and one spine (abdominal segments I-II), two (VI), or three spines (III-V) in P. costalimai. Other diagnostic characters to P. costalimai are (opposition to A. cribratum in parentheses): abdomen without prominent pleural lobes (tergites II-VII with pleural lobes prominent and covered with conspicuous minute pubescence), longitudinal ca-rina of abdominal tergite VII smooth (carina serrate). The description of pupa of C. robustus is needed to clarify its morphology.

Key to third instar of Orphninae known larvae
1 Epipharynx without plegmatia and corypha not prominent; lacinia with 1 or 2 unci (never with 3 tips); posterior area of ligula with thin setae; anal slit sinuate (Aegidiini

Egg-buster or egg-hatching structure of the first-instar larvae
The embryo of many insects has spines which aid the rupture of the chorion during hatching. These have been called hatching spines, oviruptors, or egg-busters (Hayes, 1929). With the aid of muscular contractions, the egg-busters assist the embryo to break the chorion during the hatching process (Vitner & Král, 2009). Besides the function of egg-busters, some authors showed that the structure is also useful to identify some first-instar Coleoptera larvae (e.g., Scarabaeidae, Emden, 1941;Šípek et al., 2008).
Egg-hatching structure was first described in Scarabaeoidea by Rittershaus (1925: figs. 1-4;1927: figs. 133-143) in two European species of Rutelinae (Anomala aenea Della Beffa, 1910; Phyllopertha horticula (Linnaeus, 1758)). This author described the structure as a triangular and sclerotized spine present on each dorsolateral side of metathorax. Afterwards, Gray et al. (1947) studied the embryo and the hatching process of P. horticula, and noted the egg-busters. Hayes (1929) did not found egg-busters in the first-instar larvae of several Scarabaeidae species, and indicated that some stiff dorsal setae probably aid the insect somehow to burst the egg chorion. The author added that the mandibles possibly also aid the hatching process.
Some studies have been reporting the presence of egg-busters with variable shapes in some Scarabaeidae species. Gardner (1935) found egg-busters in Rutelinae (Anomala polita Blanchard, 1851) and Cetoniinae (Heterorrhina elegans (Fabricius, 1781) and Oxycetonia albopunctata (Fabricius, 1798)), but Fidler (1936) did not find this structure in Melolonthinae (Amphimallon solstitialis (Linnaeus, 1758), Melolontha melolontha (Linnaeus, 1758), and Serica brunnea (Linnaeus, 1758)). Vitner & Král (2009) in a study of immatures of Synapsis yunnanus Arrow, 1933 (Scarabaeinae) suggested that egg-busters may not always be responsible for the initial fracture of chorion and that these sclerites possibly have a supportive function. The authors noted that the egg-busters probably serve as mechanoreceptors, since the sclerotized insertions of setae of the egg-busters keep them in a constant position relative to the chorion.
The shape of egg busters in other scarab beetles can be different from the above-mentioned. The egg-buster of the dung beetle Synapsis yunnanus (Scarabaeinae, described by Vitner & Král, 2009) has three conical projections backward directed, each projection bearing one robust subdistal seta. However, the egg-busters of P. costalimai are composed of a backward directed spine and other two small conic upward-directed projection, and a separated sclerite bearing projections. The presence of a separated sclerite was not reported in any of the works cited above.
Studies on the egg-busters and their function are very scarce in most families of Scarabaeoidea. Additional data on the functional morphology of this structure can help us to understand better the behavior and biology of these animals. Also, it can provide important information for taxonomy, systematics and cladistics. Sousa, R.C.J. & Fuhrmann, J.: Larva and pupa of Paraegidium costalimai Pap. Avulsos Zool., 2020;v.60.special-issue: e202060(s.i.).13 14/16