Antennal sensilla in an anophthalmic wood‐dwelling species, Clinidium canaliculatum, Costa 1839 (Coleoptera, Rhysodidae)

Abstract The habit of feeding on slime moulds (Myxomycetes) commonly present in litter or dead wood requires specific morphological adaptations of the mouthparts and sensory structures involved in the search for habitat and food. In this study, the external morphology of antenna and its sensilla were studied using scanning electron microscopy in the saproxylic beetle, Clinidium canaliculatum, Costa 1839 (Coleoptera, Rhysodidae). Their moniliform antennae consist of a scape, pedicel, and nine flagellomeres. We identified seven different types of sensilla, according to their morphological characteristics: two types of sensilla chaetica (sc1 and 2), two types of sensilla basiconica (sb1 and 2), one type of sensilla campaniformia, one type of sensilla coeloconica, and Böhm sensilla. No sexual dimorphism was found regarding antennal morphology and sensilla type and distribution, except for the sensilla coeloconica. The functional role of these sensilla was discussed in relation to their external structure and distribution, and compared with the current knowledge on coleopteran sense organs. Results are basic information for further physiological and behavioral studies to identify their role in the selection of habitat, food, mates and oviposition sites.

This study was designed to describe the antennal sensory equipment of females and males in C. canaliculatum. Shape, size, number, and distribution of the sensilla were studied using scanning electron microscopy. The function of the sensilla found in C. canalicutatum was discussed comparatively with current knowledge on coleopteran sense organs. This research provides basic information for further ultrastructural, behavioral, and ecological studies.

| Insect collection
Adults of C. canaliculatum ( Figure 1a) were hand-collected under rotten pine bark in the Sila National Park (39 21 0 16.79 00 N, 16 37 0 57.64 00 E, Monte Spina 1,550 m a.s.l. San Giovanni in Fiore, Calabria, Southern Italy) in May 2021. In the laboratory, beetles were identified by using a dichotomous key and separate by gender.

| Scanning electron microscopy
Beetles (three males and three females) were anesthetized in a cold chamber at 4 C for 3 min and beheaded under a stereomicroscope.
Heads were fixed in 2.5% glutaraldehyde and 1% paraformaldehyde in phosphate buffer (PBS, 10 mM pH 7.4; Electron Microscopy Sciences) over night at 4 C. They were then washed with PBS (Sigma-Aldrich), dehydrated in an ethanol (Sigma-Aldrich) series and finally specimens were immersed in hexamethyldisilazane (HMDS, (Sigma-Aldrich) to remove liquids. Heads of males and females were mounted on aluminum stubs with double-sided sticky tapes with ventral or dorsal sides.
Immediately before the observation, they were graphite coated in a

| Data analyses
Measurements were taken with ImageJ open source software on digitized images and processed as means ± standard error. The morphofunctional types of the sensilla were described and classified according to Schneider (1964) and Zacharuk (1985).
The differences between the sexes for both antenna and numbers of sensilla were assessed by the nonparametric Mann-Whitney

| Böhm sensilla
Böhm sensilla (bs) were short, smooth, sharp-tipped, thorn-like bristles located at the base (condyle) of the scape (Figure 4d). They had a length of 2.7 μm and a diameter at the base of 1.4 μm.

| Glandular pores
In all specimens examined, pores were present on the antennomeres.
They were usually associated with sb1 and sco from fifth to eighth flagellomeres in the ventral sensorial field (Figure 3c,d) Note: Data are presented as mean ± SE, (n = 3). Data are not significantly different between sexes (Mann-Whitney U test, p >.05. glandular pores were located on ninth antennomere (Figure 4c). They were round or oval and measured about 500 nm. The distribution of the glandular pores showed no sexual dimorphism.
We also identified large areas of sensilla dorsally in the frontal and antennal grooves and ventrally on the mentum and gular grooves (Figure 1b-g). sc2 This is the first study on antennal sense organs of males and females in C. canaliculatum. We identified and measured seven types of sensilla that show differences in morphology, abundance and, distribution on the antenna of C. canaliculatum as an adaptation to improve their efficiency and sensitivity in perceiving chemical and physical stimuli.
The most abundant type of sensilla was sc1 on the antenna and occurred in association with sc2 from fifth to eighth flagellomere. The wide articular socket, the pointed tip of the long bristles, and the longitudinal grooves as well as ultrastructural evidences in previously described species (Altner, Schaller-Selzer, Stetter, & Wohlrab, 1983), suggested that the mechanoreception is the most likely function of sc1. In addition, mainly those present on scape and pedicel probably have a proprioceptive function, giving information about the position of antennae in relation to the body (Schneider, 1964). Sc2 without an articular socket and blunt-tipped likely acts as a chemoreceptor (Altner & Prillinger, 1980). Sensilla, showing the external morphology of sc2 in C. canaliculatum, have already been described in other coleopteran species Hao et al., 2020;Seada & Hamza, 2018;Shao et al., 2019), including ground beetles, for example, Paussus favieri Fairmaire, 1851 (Di Giulio et al., 2012), Bembidion lampros Hdst (Merivee et al., 2002), even though in some case they are named as sensilla trichodea. In electrophysiological studies on Pterostichus aethiops (Panzer, 1796), a species that lives mainly in forest habitats, sc2 are referred to as contact chemoreceptors that can detect changes in salinity and pH (Merivee et al., 2004). The porous structures found mainly at the base of sb1 and sco in C. canaliculatum are part of glands associated with sensilla and are likely responsible for secretion of mucous-like substances for lubrication of the antennal surface (Giglio, Ferrero, & Brandmayr, 2005).
They are located on the antennal ventral site of C. canaliculatum from fifth to eighth and on ninth flagellomere in association with sco. Further ultrastructural analyses may be elucidate their possible function.
Sensilla basiconica type 2 were found from the scape to the sixth flagellomere of C. canaliculatum. Their morphology correspond to those of contact mechanoreceptors likely involved in the perception of antennal distortions caused by external stimuli (Giglio, Perrotta, & Zetto Brandmayr, 2010;Keil, 1997;Zacharuk, 1985).
Sensilla campaniformia (sca), described as no-pore sensilla with a cupola or dome-shaped external apparatus (Altner, 1977), have been found on mouthparts, antenna, wing bases, and legs to be involved in exteroception and proprioception (Chapman, 2012;Keil & Steinbrecht, 1984;Kim & Yamasaki, 1996;McIver, 1975McIver, , 1985Schneider, 1964). Ultrastructural analyses of cerci in crickets indicated that they function as mechanoreceptors (Keil, 1997). The position on the antennal tip may allow it to detect environmental mechanical stimuli and regulate movement and locomotion of C. canaliculatum within the dead wood, replacing the scanning function of the eyes.
In Rhysodidae, morphology and distribution of antennal sensilla have so far been described only in males and females of Omoglymmius americanus (Baker, 2001 Moreover, distribution pattern, number and types of sensilla found in this species differ to C. canaliculatum because the evolutionary adaptation to different biotic and abiotic factors of its ecological niche.

| CONCLUSION
This preliminary study described the sensory equipment for the first time in C. canaliculatum, a species belonging to Rhysodidae. We found no significant sexual differences in types, numbers, and distribution of sensilla in the antennae, except for the sensilla coeloconica. The distribution pattern of sensilla suggested that the antennae are involved in the scanning surrounding area for habitat selection to compensate for the absence of eyes (anophthalmic species). Furthermore, we assessed that the ability to detect temperature and humidity variation is crucial for identifying habitats where the amoeboid stage of Myxomycetes on which it feeds are present and for avoid overheating and dehydration. In addition, chemoreception can be useful in locating rotting trees suitable for laying eggs and providing food for their larval stages.
Further ultrastructural and electrophysiological studies of antennae are needed to clarify our hypotheses on the functional role of these sensilla in the behavioral process of habitat selection, localization, and recognition. The results are a contribution to the knowledge and conservation of these species.

ACKNOWLEDGMENTS
The Italian Ministry of Education, University and Research (MIUR) (grant n UA.00.2014.EX60) supported this research. Open Access Funding provided by Universita della Calabria within the CRUI-CARE Agreement.

CONFLICT OF INTEREST
The authors declare that they have no conflict of interests.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.