Antennal sensilla of two species of Gymnetis MacLeay, 1819 (Coleoptera: Scarabaeidae: Cetoniinae)

ABSTRACT The present study describes the morphology of the antennal sensilla of adults of Gymnetis holosericea (Voet, 1779) and Gymnetis rufilateris (Illiger, 1800). The adults of Gymnetis spp. were sexed and antenna of males and females were dissected. Both species have sensilla chaetica, trichodea, placodea (type I and II), coeloconica (type I and II), and ampullacea (or pore). Females of G. holosericea have a total of about 19,995 sensilla and males have about 23,273 sensilla, and females of G. rufilateris have about 16,633 sensilla and males have about 21,184 sensilla. Sensilla placodea are the predominant type of sensilla in males and females of G. holosericea and G. rufilateris.


A B S T R A C T
The present study describes the morphology of the antennal sensilla of adults of Gymnetis holosericea (Voet, 1779) and Gymnetis rufilateris (Illiger, 1800).The adults of Gymnetis spp.were sexed and antenna of males and females were dissected.Both species have sensilla chaetica, trichodea, placodea (type I and II), coeloconica (type I and II), and ampullacea (or pore).Females of G. holosericea have a total of about 19,995 sensilla and males have about 23,273 sensilla, and females of G. rufilateris have about 16,633 sensilla and males have about 21,184 sensilla.Sensilla placodea are the predominant type of sensilla in males and females of G. holosericea and G. rufilateris.
The antennal sensilla of the genus Gymnetis is still undescribed until now and the present work aim to provide the first sensilla characterization of the genus based in the study of two species: G. holosericea and G. rufilateris.The morphological description provided here adds new characteristic to differentiate species and new information about to Scarabaeidae antennal structure.
Antennal sensilla were studied by scanning of antennal clubs taken using an electron microscope in the Departamento de Física e Química, Universidade Estadual Paulista (UNESP), Campus of Ilha Solteira, SP (a method by Tanaka et al., 2006).Antennae of six males and six females of G. holosericea and four males and six females of G. rufilateris were dissected and maintained in 70% ethanol.Afterwards, appendages were successively washed in 80% and 90% ethanol for 15 minutes each, and then 100% ethanol for 20 minutes.The pieces were dried in a CO 2 critical point dryer (model Leica® CPD300).Subsequently, they were coated with gold using a Quorum® Q150TE turbo molecular pump.Images were obtained using a Zeiss® EVO LS15 scanning electron microscope (SEM).The images obtained in SEM were subjected to image enhancement filters available in the software Image-Pro Plus 6.0.The sensilla were quantified in the images obtained by SEM of the coverslips of six males and six females.The Student's t-test was used to compare the distribution of sensilla.
Sensilla terminology follows Keil (1999).The term sensilla trichodea is here used as a very comprehensive term, to quite variable hair-like sensilla (Snodgrass, 1935;Keil, 1999).And the term sensilla chaetica is here used as a restricted term to a hair-like sensilla that are distinguished from other hair-like sensilla by their distribution or form (usually as a stout or spine-like setae Snodgrass, 1935;Keil, 1999).

Results
The general structure of the antenna of G. holosericea and G. rufilateris is typic to Scarabaeoidea and comprises (basis to apex): a scape, a pedicel, funicle with 5 antennomeres, and the distal clava or club with three lamellate antennomeres (Fig. 1).The clava of both species have sensilla trichodea, chaetica, placodea, coeloconica, and ampullacea (pores).Sensilla trichodea are hair-like long setae distributed mainly on edges of lamellae (Fig. 4F).Sensilla chaetica are also hair-like, but are shorter than sensilla trichodea, and are mainly found grouped in the outer surface of the proximal lamella (Fig. 4F).
Sensilla placodea are distributed in inner side (the distal surface) of proximal lamella, inner and outer sides of medial lamella, inner side of distal lamella (the proximal surface), and in the posterior area of outer side of distal lamella (Figs.4-7).Two types of sensilla placodea are found, type I and II (Figs. 2 and 3).The sensilla placodea type I are rounded plates surrounded by a peripherical ditch or furrow and   have a diameter of 7.1 ± 2.1 µm in G. holosericea and 5.8 ± 2.1 µm in G. rufilateris.The sensilla placodea type II are rounded plates without peripherical furrow and have a diameter of 8.1 ± 2.1 µm in G. holosericea and 10.7 ± 2.1 µm in G. rufilateris.Few observed sensilla placodea type I show some minor punctures (Figs.2B and 3).
The sensilla placodea type I are mainly arranged at medial and anterior area of inner side of proximal lamellae, inner and outer sides of medial lamella, inner side of distal lamella (Figs.4B, 5B, 6B and 7B).Sensilla placodea type II are mainly arranged at posterior area of: inner side of proximal lamellae and inner and outer sides of medial and distal lamellae (Figs.4A, 5A, 6A and 7A).
Sensilla coeloconica are small projections inside a pit, and are divided into type I, with sharped apex, and type II, with blunt apex (Figs. 2 and 3).These sensilla are distributed in anterior and medial areas of inner side of proximal lamella, inner and outer sides of medial lamella, inner side of distal lamella.Both types can present some grooves in their surface or the grooves are indistinct and the surface seems smooth.
Sensilla ampullacea are visible as pores and they are scarcely distributed in all sides of lamellae (Figs 2A and C).
When comparing the amounts of sensilla between the two species of Gymnetis, it was verified that the amounts of placodea sensilla between females of G. holosericea and G. rufilateris differ significantly from each other (t=44.95;df=10; P<0.001), among males, significant differences were also observed for the amount of placodea sensilla (t=17.62;df=10; P<0.001).The amounts of coeloconica sensilla between G. holosericea and G. rufilateris females do not differ from each other (t=1.63;df=10; P<0.14), however, they differ significantly between males (t=11.22;df=10; P<0.001).For the total amount of sensilla among females of   G. holosericea and G. rufilateris, significant differences were observed (t=41.71;df=10; P<0.001), with significant differences also for the total amount of sensilla among males (t=18.45;df=10; P<0.001).
Within the subfamily, Valgus hemipterus (Valgini) is separated from all other flower chafers, or even from other scarabaeoid beetle, by the presence of sensilla placode bearing invaginations like pores or points, those pores could by irregularly distributed or have a concentric distribution (Bohacz et al., 2020: sensilla 1E;Meinecke, 1975: E1, E2, E3).Some sensilla placodea type I of Gymnetis has some minor punctures and these punctures are quite distinctive from the invaginations above discussed to Valgus.The above differentiation of the puncture of Gymnetis and Valgus is given to show that both structures are possibly not homologous.
All Cetoniinae have the outer side of proximal lamella bearing a group of setae.These setae are designated here with sensilla chaetica, but other studies termed these setae as sensilla trichodea (as Bohacz et al., 2020).Despite the term used (see terminology in Material and Methods), the presence of this group of setae is a conspicuous character of Cetoniinae and part of Dynastinae (Bohacz et al., 2020;Costa et al., 2021), is inconspicuous to Rutelinae and part of Dynastinae (Cyclocephalini; Rodrigues et al., 2019;Saldanha et al., 2020;Nagamine et al., 2022), and is indistinct in Melolonthinae (Bohacz et al., 2020;Menis et al., 2021).In Rutelinae and Cyclocephalini, the outer side of proximal lamella has a sparse group of setae, and these setae are not evidently different from other setae present on lamellar edges (Rodrigues et al., 2019;Nagamine et al., 2022).To Melolonthinae, the outer side of proximal lamella has several setae that are not grouped (Menis et al., 2021).The presence of this grouped setae is here discussed to encourage future works investigate the evolution of this character, checking its homology, or even the possibility of usage of it as a diagnosis to family-group names.
Some here termed sensilla coeloconica type I and II have shallow striae on its surface (Bohacz et al., 2020: sensilla GSC).Sensilla coeloconica variations are sometimes difficult to clarify and intermediate types are common.Present study tried to classify these variations based on its apical shape, type I and II with blunt or acute tip, respectively.Despite the apex shape, the surface of sensilla coeloconica is smooth or grooved.The grooved sensilla coeloconica are common to all Scarabaeoidea and those with smooth surface is common to Scarabaeidae subfamilies: Cetoniinae, Rutelinae, Dynastinae, and part of Melolonthinae (Diplotaxini, Melolonthini) (Bohacz et al., 2020).To melolonthine species, Shao et al. (2019) proposed that the sensilla coeloconica are sensible to humidity and temperature, and Romero-López et al. (2004) suggested that these sensilla detect plant volatiles.
The inner side of proximal and distal lamella and both sides of medial lamella have two well defined area, a posterior homogeneous area mainly formed by sensilla placodea type II, and an anterior heterogeneous area with sensilla coeloconica (type I and II), and sensilla placodea (mostly type I).These areas are present in Cetoniinae, Dynastinae, Rutelinae, and Melolonthinae (Rodrigues et al., 2019;Bohacz et al., 2020;Costa et al., 2021;Menis et al., 2021;Nagamine et al., 2022).Larsson et al. (2001) noted that the sensilla of homogeneous area is responsible to the sexual pheromone detection, and those of heterogeneous area are responsible to other volatiles detection in the ruteline Anomala cuprea (Hope, 1839).
The number of sensilla are higher in males than in females of Gymnetis, and the sexual dimorphism is more evident in G. rufilateris than in G. holosericea.The intrageneric sexual variation of number of sensilla as similarly noted to Hoplopyga species, and the sexual dimorphism is more evident in H. albiventris than in H. liturata (Costa et al., 2021).Males of G. rufilateris have almost 24.4% more sensilla than females, and males of G. holosericea have about 14.1% more sensilla than females.And males of H. albiventris have about 34.5% more sensilla than females, males of H. liturata have almost 14.8% more sensilla than females.
Both Gymnetis and Hoplopyga have the anterior area of outer side of distal lamella bearing sensilla placodea type II homogeneously distributed (Costa et al., 2021).
The distribution and structure of the antennal sensilla are almost the same to G. holosericea and G. rufilateris, except the diameter of sensilla placodea that are slightly larger in G. rufilateris than in G. holosericea (see above).The main difference of antennal sensilla to both species is relative to the number of elements, especially regarding placodea sensilla present in posterior homogeneous area, and this area is relatively bigger in G. rufilateris than in G. holosericea.
Present study reveals that the distribution (into a well delimited anterior posterior areas), shape and size of antennal sensilla are variable between congeneric species.This variability may indicate different patterns of volatile detection and chemical communication.Despite the functional importance, sensilla interspecific variations are a rich data source for phylogenetic studies and also add new morphological descriptions to formerly described species.Some differences and similarities regarding the structure and vestiture of the antennal lamella were discussed above.The significance of these comparative effort is to highlight the variation observed in the antennal sensilla of phytophagous scarab beetles, encourage future descriptive and comparative studies to found new differences and similarities, and also support future cladistic studies by adding new primary homology hypothesis to by checked.

Figure 5
Figure 5 Gymnetis holosericea, male antenna.Distal lamella, outer and inner sides (A, B), medial lamella, outer and inner sides (C, D), proximal lamella, outer and inner sides (E, F). White dotted line showing the posterior homogeneous area, black dotted line showing the anterior heterogeneous area.Scale = 200 µm.

Figure 6
Figure 6 Gymnetis rufilateris, female antenna.Distal lamella, outer and inner sides (A, B), medial lamella, outer and inner sides (C, D), proximal lamella, outer and inner sides (E, F). White dotted line showing the posterior homogeneous area, black dotted line showing the anterior heterogeneous area.Scale = 200 µm.

Figure 7
Figure 7 Gymnetis rufilateris, male antenna.Distal lamella, outer and inner sides (A, B), medial lamella, outer and inner sides (C, D), proximal lamella, outer and inner sides (E, F). White dotted line showing the posterior homogeneous area, black dotted line showing the anterior heterogeneous area.Scale = 200 µm.