Morphology of the antennal sensilla of two species of Hoplopyga Thomson, 1880 (Coleoptera, Scarabaeidae, Cetoniinae)

Antennal sensilla are important functional elements of sensory systems in insects. This study aimed to determine the morphology and structure of the sensilla of two species of the genus Hoplopyga. Adults of Hoplopyga liturata (Olivier, 1789) were collected in traps with sugarcane juice as an attractant. Thereafter, larvae of Hoplopyga albiventris (Gory and Percheron, 1833) were collected in mounds of termites (Cornitermes cumulans (Kollar, 1832) Isoptera). Then, they were reared in the laboratory for adult observations. Antennae of H. liturata and H. albiventris have sensilla chaetica, trichodea, placodea (type I and II), coeloconica (type I and II), and ampullacea (or pore). Females of H. liturata have a total of about 10657 sensilla and males have about 12512, whereas females of H. albiventris have about 16490 sensilla and the males 24565 sensilla. Sensilla placodea are predominant in the antenna of males and females of both species.

Insect antennae are important sensory structures involved in the detection of odorants, and temperature, besides being tactile and gustative organs (Schneider, 1964;Hansson and Stensmyr, 2011). Antennal sensilla are responsible for reception of semiochemicals (or infochemicals), such as sexual or aggregative pheromones, and of other volatiles, helping insects to locate plants used as food and mating sites (Visser, 1986;Tegoni et al., 2004;Wee et al., 2016).
The study of sensorial organs of insects is important to clarify questions about fine morphology, the process of communication, and habitat perception by these animals. The present paper aimed to describe the morphology of antennal sensilla of males and females of two species of phytophagous scarab beetles included in the genus Hoplopyga: H. albiventris and H. liturata. Larvae of H. liturata were collected from decaying woods, pupal instar lasted about 18 days (Garcia et al., 2013), and immatures were described by Morón e Arce (2002). In Brazil, the species prefer cerrado landscapes (Brazilian savanna) to urban parks (Correa et al., 2020). Hedström and Elmqvist (1985) observed H. liturata damaging stems of the vine species Gouania polygama (Jacq.) Urb. (Rhamnaceae) in Costa Rica to feed on its exudates. Such an interaction attracted the butterfly species Prepona laertes (Hübner, 1811) (Nymphalidae, Lepidoptera), which also fed on the vine exudates and directly on beetle excreta. The authors suggested that this interaction between the beetle and the butterfly could be a case of commensalism. Occasional copresence of cetoniine beetles and butterflies around sources of plant exudates have been sometimes observed (Di Iorio, 2014). Luederwaldt (1911) found larvae of H. albiventris in termite mounds of Cornitermes sp. Also, Puker et al. (2014) noted the preference of this species for pastures due to the presence of mounds of Cornitermes in these agroecosystems regularly.

Material and methods
The study was conducted at the Universidade Estadual de Mato Grosso do Sul (UEMS), Campus of Cassilândia, Mato Grosso do Sul, Brazil.
Larvae of H. albiventris were collected from mounds of termites (Cornitermes cumulans (Kollar, 1832) (Isoptera: Termitidae)) between August 2017 and September 2018 in a pasture area (Urochloa decumbens Stapf, Poaceae). These mounds were open in the field. After collection, larvae were reared in the laboratory. Each larva was placed into plastic containers (500 mL) filled with the substrate used by immatures.
Adults of H. liturata were collected using traps baited with a banana and sugarcane juice mixture as an attractant. The species had been collected in recent studies using the same method as in Rondônia State (Puker et al., 2020a(Puker et al., , 2020b. Each trap was constructed from a PET bottle (2 L) with two 8 × 5 cm side openings. Three traps were used between November and December 2018, two in a "Cerrado" (Brazilian savanna) fragment and one in a pasture area. They were installed about 1.5 m high from the soil. The attractant was replaced two times per week (methodology modified by Rodrigues et al., 2013).
The reared adults of H. albiventris and those of H. liturata collected were preserved in 70% alcohol. The adults of H. albiventris were identified by comparison with the material of the Museu de Zoologia da Universidade de São Paulo, while those of H. liturata by comparison with the material of the UEMS, Campus of Cassilândia. The revision of the genus Hoplopyga was also used for identifications (Shaughney and Ratcliffe, 2015).
Antennal sensilla were studied by scanning of antennal clubs taken using an electron microscope (SEM, model Zeiss EVO LS15) in the Departamento de Física e Química, Universidade Estadual Paulista (UNESP), Campus of Ilha Solteira, São Paulo State (a method by Tanaka et al., 2006). Antennae of 10 males and 10 females were dissected and maintained in 70% alcohol. Afterwards, appendages were successively washed in 80% and 90% ethanol for 15 minutes each, and then 100% ethanol for 20 minutes. The pieces were taken to dry in a CO 2 critical point dryer (model Leica® CPD300).
Antennal lamellae were also mounted on slides for optical microscope observation (model Nikon E200), following the method of Romero-López et al. (2004).
Sensilla terminology follows Keil (1999). Meinecke (1975) was used as an alternative sensilla terminology (in brackets) to easily term comparison. The Student's t-test was used to compare the distribution of sensilla.

Results
Adults of H. liturata and H. albiventris have antennae with ten antennomeres, and the antennal club has three antennomeres ( Fig. 1).

Hoplopyga liturata
Antennae of male and females are quite similar to each other, but males have medial lamella longer than to females (Table 1 e Fig. 2).
Lamellae have sensilla trichodea, chaetica, placodea, coeloconica and ampullacea. Sensilla trichodea are hair-like, thin, and long, and found on edges of lamellae in both genders. Sensilla chaetica are like sensilla trichodea but short and grouped all together in a brushlike structure in the outer surface of the proximal lamella. Sensilla trichodea and sensilla chaetica have been sometimes termed together as sensilla chaetica (e.g., Zauli et al., 2016) or as sensilla trichodea (e.g., Bohacz et al., 2020). Hair-like sensilla cannot be easily separated into two categories (as used here and by the generalization for insects by Keil, 1999). They are quite variable in length and shape (with acute or blunt apex, simple or serrate, smooth or with perforations or striae; see Meinecke (1975) and Bohacz et al. (2020) for sensilla comparison between scarab beetles). The present study considered sensilla trichodea to be long setae distributed in all antennomeres and sensilla chaetica the minor setae grouped in a brush-like structure (Fig. 3).
The sensilla placodea found are type I (Meinecke, 1975: sensilla B2; Fig. 4A) and type II (Meinecke, 1975: sensilla B3; Fig. 4B). These are the predominant types of antennal sensilla found along lamellae in both sexes except on the outer side of the proximal lamella.
The sensilla placodea type I are somewhat rounded and have a diameter of 8.2 ± 2.1 μm (n= 50; Fig. 4A). These sensilla are predominant in the posterior third of the outer side of proximal lamella (Fig. 4C).
The sensilla placodea type II are rounded and have diameter of 9.8 ± 3.1 (n= 50; Fig. 4D). These sensilla are predominant in the anterior half of the inner side of the medial lamella.
The sensilla coeloconica found are type I (short with acute apex; Meinecke, 1975: sensilla L1; Fig. 5B) and type II (short with blunt apex; Meinecke, 1975: sensilla L3; Fig. 5C). Both are found in the inner side of proximal and distal lamellae and inner and outer sides of medial lamella (sides of lamellae that can be closed). They are distributed mainly in the central areas of lamellae (Fig. 5A).

Hoplopyga albiventris
The proximal and distal lamellae of male antennae are longer than those of females (Table 3 and Fig. 7).
The antennae of H. albiventris are like those of H. liturata (Fig. 3) and both species have sensilla of the same types and with similar distribution. The differences of antennal structure of H. albiventris (regarding H. liturata described before) are listed below.
Sensilla placodea of type I have a diameter of 8.8 ± 2.1 μm (n = 50) and those of type II have diameter of 10.1 ± 2.1 (n = 50).

Discussion
Adults of H. liturata and H. albiventris have similar antennae structure, shape, and size, but the amount of sensilla is quite different between both species. Male antennae are slightly longer than those of females and have more sensilla than female antennae. Sexual dimorphism in scarab beetle antennae have been frequently reported (e.g., Allsopp, 1990;Romero-López et al., 2004;Mutis et al., 2014) and it could be related to how species find sexual partners or food sources by detecting volatiles with antennae.
In Cetoniinae, plant volatiles are important for aggregation of the genus Pachnoda (Cetoniini; Larsson et al. 2003a;Bengtsson et al., 2011). For Cotinis nitida (Linnaeus, 1758) (Gymnetini), Domek and Johnson (1987, 1988, 1990 found evidence of females releasing sexual attractant  scarab beetles have been reported with both behaviors: females directly attract males and females are attracted to damaged plants by males (e.g., Popillia japonica Newman, 1841, Rutelinae, studied by Smith 1923). However, it is still not clear whether Hoplopyga males are directly attracted to females or they firstly find a food source and then attract females. It is also unclear whether these are behaviors related to antennal sexual dimorphism.
Both H. albiventris and H. liturata have large amounts of sensilla placodea, which are more abundant in males than in females of both species. This sort of sensillum was considered the main sexual pheromone receptor, as in Anomala cuprea (Leal and Mochizuki, 1993), Phyllopertha diversa (Nikonov et al., 2001), and Popillia japonica (Kim and Leal, 2000). Larsson et al. (2001) found that the sensilla placodea of A. cuprea antennae are both sexual pheromones and plant volatile receptors and that where they are is decisive for their function.
Sensilla coeloconica are arranged mainly in the central area of lamellae. In antennal pedicel, Shao et al. (2019) suggested that this kind of sensilla respond to humidity and temperature (hygro-receptors and thermo-receptors). For lamellae, Kim and Leal (2000) and Romero-López et al. (2004) considered sensilla coeloconica as receptors to plant volatiles.
Hoplopyga albiventris adults have more antennal sensilla (males: 24565, females 16490) than H. liturata (males: 12512, females 10657). Perhaps such an abundance of antennal sensilla in H. albiventris is related to the detection of termite mounds or food sources in open areas. However, the number of antennal sensilla in other Cetoniinae species (found or not associated with termites) are scarce for such an assumption.
The inner parts of Hoplopyga lamellae have two main areas, one homogeneous with sensilla placodea type I and another heterogeneous with placodea (type I and II) and some coeloconica (Fig. 4). In ruteline beetles (A. cuprea), Larsson et al. (2001) observed that the homogeneous area is responsible for the detection of sexual pheromones, while the heterogeneous is related to the detection of other volatiles.  and males and females releasing aggregation volatiles related to the beetle diet (sites with fruits and feeding males attract more males and females than fruit alone or females alone). Therefore, both aggregation and sexual infochemicals could work together in the subfamily. Another possibility is that males attract females with sexual pheromones, as in Osmoderma eremita (Scopoli, 1763) (Cetoniinae, Osmodermatini, studied by Larsson et al., 2003b), perhaps to guide females to a suitable habitat. In some phytophagous scarab beetles (e.g., Maladera matrida Argaman, 1986, Melolonthinae, studied by Harari et al., 1994), males first find and damage a host plant and then attract females. Other The kind of sensilla found in Hoplopyga is like that found in other Old-World Cetoniinae genera (Meinecke, 1975;Baker and Monroe, 2005;Zauli et al., 2016;Bohacz et al., 2020). More data on quantity and distribution of antennal sensilla are needed to further comparison between different Cetoniinae species.  Total  outer  inner  outer  inner  outer  inner   Female   Placodea  0  3569  2686  3379  2801  3344  15779   Coeloconica  0  201  136  144  0  230  711   Total  0  3770  2822  3523  2801  3574  16490   Male   Placodea  0  5041  5516  5706  3035  5267  24565   Coeloconica  0  142  81  110  0  251  584   Total  0  5183  5597  5816  3035  5518  25149