1 Luca Dorigo, Museo Friulano di Storia Naturale, via Sabbadini 32, 33100 Udine, Italy, e-mail: luca.dorigo1979@gmail.com 2 Andrea Squartini, Dipartimento di Biotecnologie Agrarie, Università di Padova, Legnaro (PD), Italy, e-mail: squart@unipd.it 3 Vladimiro Toniello, Gruppo Speleologico C.A.I. di Vittorio Veneto (TV), Italy, e-mail: vladimiro.toniello@virgilio.it 4 Angelo Leandro Dreon, Dipartimento di Biologia, Università di Padova, Agroecology and Ethnobiology, Padova, Italy, e-mail: leandro.dreon@libero.it 5 Alberto Pamio, Dipartimento di Biologia, Università di Padova, Agroecology and Ethnobiology, Padova, Italy, e-mail: albertopamio@gmail.com 6 Giorgio Concina, Gruppo Speleologico Pradis, Clauzetto (PN), Italy, e-mail: g.concina@libero.it 7 Venicio Simonutti, Gruppo Speleologico Pradis, Clauzetto (PN), Italy, e-mail: veniciosimonutti@alice.it 8 Enrico Ruzzier, The Natural History Museum, London, UK, e-mail: symphyla@gmail.com 9 Michel Perreau, IUT Paris Diderot, Université Paris Diderot, Sorbonne Paris Cité, France, e-mail: michel.perreau@univ-paris- diderot.fr 10 Annette Summers Engel, Department of Earth and Planetary Sciences, University of Tennessee, Knoxville, Tennessee, USA, e-mail: aengel1@utk.edu 11 Federico Gavinelli, Dipartimento di Biologia, Università di Padova, Agroecology and Ethnobiology, Padova, Italy, e-mail: federico.gavinelli@gmail.com 12 Isabel Martinez-Sañudo, Dipartimento di Agronomia Animali Alimenti Risorse Naturali e Ambiente, Università di Padova – Agripolis, Legnaro (PD), Italy, e-mail: isabel.martinez@unipd.it 13 Luca Mazzon, Dipartimento di Agronomia Animali Alimenti Risorse Naturali e Ambiente, Università di Padova – Agripolis, Legnaro (PD), Italy, e-mail: lmazzon@unipd.it 14* Maurizio G. Paoletti, Dipartimento di Biologia, Università di Padova, Agroecology and Ethnobiology, Padova, Italy, e-mail: paoletti@bio.unipd.it * Corresponding Author Received/Prejeto: 08.02.2017 COBISS: 1.01 ACTA CARSOLOGICA 46/2−3, 317–328, POSTOJNA 2017 Abstract UDC 595.76:591.5 Luca Dorigo, Andrea Squartini, Vladimiro Toniello, Angelo Leandro Dreon, Alberto Pamio, Giorgio Concina, Venicio Si- monutti, Enrico Ruzzier, Michel Perreau, Annette Summers Engel, Federico Gavinelli, Isabel Martinez-Sañudo, Luca Mazzon & Maurizio G. Paoletti: Cave hygropetric beetles and their feeding behaviour, a comparative study of Cansiliella servadeii AND Hadesia asamo (Coleoptera, Leiodidae, Cholevinae, Leptodirini) Several Leptodirini beetles (Leiodidae) are known to dwell in hygropetric habitats where films of water run down the cave walls, but observations of their behaviour are lacking. The ul- tra-specialised hygropetricolous beetles belonging to the gen- era Cansiliella and hadesia are biogeographically and phylo- genetically unrelated leptodirines. As the species of the former CAVE HyGROPETRIC BEETLES AND THEIR FEEDING BEHAVIOUR, A COMPARATIVE STUDy OF CANSILIELLA SERVADEII AND hADESIA ASAMO (COLEOPTERA, LEIODIDAE, CHOLEVINAE, LEPTODIRINI) PRIMERJAVA JAMSKIH HIGROPETRIčNIH HROŠčEV IN NJIHOVEGA PREHRANJEVANJA; CANSILIELLA SERVADEII IN hADESIA ASAMO (COLEOPTERA, LEIODIDAE, CHOLEVINAE, LEPTODIRINI) Luca DORIGO1,*, Andrea SqUARTINI2, Vladimiro TONIELLO3, Angelo Leandro DREON4, Alberto PAMIO5, Giorgio CONCINA6, Venicio SIMONUTTI7, Enrico RUZZIER8, Michel PERREAU9, Annette Summers ENGEL10, Federico GAVINELLI11, Isabel MARTINEZ-SAÑUDO12, Luca MAZZON13 & Maurizio G. PAOLETTI14 Izvleček UDK 595.76:591.5 Luca Dorigo, Andrea Squartini, Vladimiro Toniello, Angelo Leandro Dreon, Alberto Pamio, Giorgio Concina, Venicio Simonutti, Enrico Ruzzier, Michel Perreau, Annette Sum- mers Engel, Federico Gavinelli, Isabel Martinez-Sañudo, Luca Mazzon & Maurizio G. Paoletti: Primerjava jamskih higropetričnih hroščev in njihovega prehranjevanja; Can- siliella servadeii in Hadesia asamo (Coleoptera, Leiodidae, Cholevinae, Leptodirini) Številni hrošči iz poddružine Leptodirinae (Leiodidae) so pri- lagojeni na življenje v posebnem habitatu »jamskem higro- petriku«, t.j. tankem sloju tekoče vode, ki teče po sigi. Zaenkrat je njihovo vedenje še precejšnja neznanka. Visoko speciali- zirani higropetrični vrsti Cansiliella servadeii in hadesia asamo sta geografsko ločeni in filogenetsko nesorodni. Ker je prva ACTA CARSOLOGICA 46/2–3 – 2017318 L. DORIGO, A. SqUARTINI, V. TONIELLO, A. L. DREON, A. PAMIO, G. CONCINA, V. SIMONUTTI, E. RUZZIER, M. PERREAU, ... genus are known to be associated with the moonmilk deposits our study aimed to obtain data on their foraging behaviour, as well as to compare the feeding strategies of both genera. In situ monitoring of C. servadeii from the cave Grotta della Foos (Italy) and h. asamo from Bravenik Cave (Bosnia and Herzegovina), was complemented by video recordings to en- sure accurate results. Mouthparts and tarsi of both species were examined using scanning electron microscopy and compared with h. weiratheri from Montenegro to evaluate potential morphological adaptations to the hygropetricolous ecological niches. The three species had significantly different mouthpart morphologies, likely due to differences in semi-aquatic feeding strategies and overall ecology. A series of new observations on site movement and feeding behaviour are presented, compared and discussed. Key words: Coleoptera, ecology, behaviour, moonmilk, cave hygropetric. očitno vezana na depozite mehke sige (t.i. jamsko mleko), smo želeli raziskati iskanje hrane pri tej vrsti ter primerjati strate- gijo hranjenja obeh vrst. Da bi povečali zanesljivost rezultatov, smo poleg in situ opazovanja C. servadeii iz jame Grotta della Foos (Italija) in h. asamo iz jame Bravenik (Bosna in Herce- govina), njuno vedenje tudi posneli. Z vrstičnim elektronskim mikroskopom smo raziskali obustne okončine in stopalca obeh vrst in jih primerjali s h. weiratheri iz črne gore, da bi preve- rili potencialne morfološke prilagoditve na specifično ekološko nišo. Bistvene razlike v zgradbi ustnega aparata so najverje- tneje posledica razlik v strategiji hranjenja, kot tudi različne splošne ekologije omenjenih vrst. V prispevku predstavljamo, pri merjamo in razpravljamo o novih opažanjih povezanih z gibanjem in prehranjevanjem higropetričnih hroščev. Ključne besede: Coleoptera, ekologija, vedenje, jamsko mleko, jamski higropetrik. INTRODUCTION The buccal apparatus (mouthparts) of some highly spe- cialised subterranean Leptodirini differs considerably in shape and structure with respect to what is observed in other tribe members. In some species, this fact has been interpreted as a result of an adaptation to a semi- aquatic trophic niche (Paoletti 1972; Casale et al. 2000a; Moldovan et al. 2004). In several unrelated species from eight genera (out of 235 globally described, unpublished data), ultra-specialized ecomorphological forms have evolved, including in hadesia Müller, 1911; Croatodirus Casale, Giachino & Jalžić, 2000; Nauticiella Moravec & Mlejnek, 2002; Velebitodromus Casale, Giachino & Jalžić, 2004; kircheria Giachino & Vailati, 2006 (subtribe An- throherponina Jeannel); Radziella Casale & Jalžić, 1988 (subtribe Leptodirina Lacordaire); Cansiliella Paoletti, 1972 and tartariella Nonveiller & Pavićević, 1999 (sub- tribe Bathysciina Horn). All of these leptodirines are dis- tributed in the North-Eastern sector of the Italian Alps and in the Dinaric Alps, and share a similar semi-aquatic cave habitat, described originally by Remy (1940) and formally defined by Sket (1979, 2004) as “cave hygropet- ric”. As such, the distribution of the hygropetricolous lep- todirines has been linked to areas with high annual pre- cipitation at high altitudes (see, i.e., Fig. 33 in Giachino & Vailati 2006). Some species have been observed only in periods of considerable rainfall (Perreau & Pavićević 2008). Leptodirini can show highly modified mouth- parts. The mandibles are hoe-shaped and the galeae have a spoon shape (e.g., Fig. 4, 4 in Paoletti et al. 2011). The galeae are also covered by dense, bristly structures on the dorsal side (Paoletti 1972, 1973; Casale & Jalžić 1988; Moldovan et al. 2004; Paoletti et al. 2011, 2013; Lo- haj et al. 2012; Engel et al. 2013). Modified mouthparts (i.e., the presence of setae, the rotation of the apex of the mandibles), described by Jeannel (1924), Remy (1940), Nonveiller and Pavićević (1999), Moravec and Mlejnek (2002), Perreau and Pavićević (2008) and Polak et al. (2016), have been referred to as “filtering mouthparts” (Casale et al. 2000a; Casale et al. 2000b; Casale et al. 2004; Moldovan et al. 2004) or “brushing mouthparts” (Perreau & Pavićević 2008). The physiological and behavioural adaptations, as well as migration through the subterranean habitat, have been studied in only a few obligate cave inhabitants (Kane & Poulson 1976; Culver 1982; Lamprecht & Weber 1992; Rétaux & Elipota 2013). In general, the activities of cave organisms, predominantly invertebrates, are scarce- ly known, except for cases that appear as unique within cave fauna. For example, the feeding and foraging behav- iour of Cansiliella servadeii Paoletti, 1980 was notewor- thy because this species is not attracted by baits, nor has it been observed on deposits of guano or organic matter, but the species is found in association with moonmilk deposits (Paoletti et al. 2011). Moonmilk is a whitish, porous speleothem characterized by a soft texture (Hill & Forti 1997) that is usually permanently covered by percolating water and has a rich microbial biomass. The moonmilk microbiology, the digestive tract contents of several beetle individuals, and mouthpart morphology have been previously studied (Paoletti et al. 2011, 2012, 2013; Engel et al. 2013). For this study, our aims were to uncover the trophic ecology of C. servadeii by obtaining accurate tempo- ral data on behavioural activities and to compare the ACTA CARSOLOGICA 46/2–3 – 2017 319 CAVE HyGROPETRIC BEETLES AND THEIR FEEDING BEHAVIOUR, A COMPARATIVE STUDy OF CANSILIELLA SERVADEII ... MATERIALS AND METHODS The is only known locality for C. servadeii is the cave Grotta della Foos (507/229 FR) (total length 2625 m, total depth 161 m), in Tramonti di Sotto (PN), Carnic Prealps, northeastern Italy. Observation sites were cho- sen where at least two to three individuals per square meter were found. Sites were marked with flags and photographed. Each observation session for each beetle lasted six hours per researcher. Individual beetles under observa- tion were pointed at to the next-shift researcher at the end of an observation period to guarantee continued data recording of the same beetle during the long-term tracking. Using this approach, the same individuals were monitored by different observers in 24-h sessions. Indi- viduals were observed at close distance and were illumi- nated by a cold LED light to avoid heat-related distur- bance. In order to appreciate the details of the trophic and cleaning activities, and to identify the sex of the bee- tles, a 30X magnifying lens was used. Sex determination in C. servadeii was possible due to external morphologi- cal sexual dimorphism. Beetle behaviour was recorded on a standardized data collection form prepared before sampling and based on prior observations (Paoletti et al. 2011). Photographs and video were also taken with a 550D Canon camera. Each behavioural activity lasting more than 1 min, as well as shorter activities that were considered significant, were recorded. To simplify the recording process, five different be- havioural categories were distinguished: trophic activity, cleaning or preening, site movement, interaction with other individuals, and other activities. As a trophic ac- tivity, the action of a beetle, with head facing down, in contact with the substrate, moving jaws and galeae and seemingly browsing the moonmilk surface was consid- ered. Cleaning was denoted by a repeated preening ac- tion, or rubbing legs and antennae against mouthparts. This behaviour is always accompanied by movement of jaws and galeae. For every movement of the individual on the substrate, we noted moving and recorded direc- tion, distance travelled, and any other possible type of movements. Interaction was recorded in the presence of conspecifics. Lastly, other activities were considered as any behaviour that did not fall under the prior four categories. Anomalous behaviours were recorded for further investigation. The amount of time spent within and outside of the water film during activities was also recorded. hadesia asamo is known from a single pit called Bravenik Cave (total length 225 m, total depth 200 m) at 800 m a.s.l. in the Zubačko Polje, about 10 km from Trebinje (Perreau & Pavićević 2008), and h. weiratheri was collected from an unnamed pit near Ledenice, Ka- meno more, Montenegro. Behaviour observations of three h. asamo individuals were done, one of them was filmed with a 550D Canon camera for 40 min with cold LED light to avoid any heat-related stress. No behav- ioural observations were possible in h. weiratheri, it was only used for morphological comparisons. As already reported in Perreau and Pavićević (2008), the presence of hadesia spp. has been strongly correlated to the water flow rate and, in general, only a few individuals can usu- ally be seen during cave expeditions. In the caves, conductivity, temperature, pH, and oxygen level in the water, were recorded in situ with a standard HACH portable sensIONTM 156 Multiparam- eter Meter calibrated at 20 °C. Electrical conductivity was measured following APAT CNR IRSA 2030 Man 29 2003 method, total hardness according to APAT CNR IRSA 2040 A Man 29 2003 method, and ammonia-nitro- gen following APAT CNR IRSA 4030 A2 Man 29 2003 method. Anions were detected according to EPA 9056 A 2007 method and cations according to EPA 6020 B 2014, with the chromatographic instruments CS-1600+ HPLC Dionex and CS-2000+ HPLC Dionex from Thermo Sci- feeding and foraging strategy of the species with those evolved in other cave hygropetricolous leptodirines, in- cluding hadesia asamo Perreau & Pavićević, 2008, and hadesia weiratheri Zariquiey, 1927. In addition to behav- ioural observations, microscopic examination of mouth- parts and other body traits was done in C. servadeii, h. asamo and h. weiratheri. Collectively, these results pro- vide important information on the behavioural biology of biogeographically and phylogenetically unrelated hy- gropetricolous beetles. The implications of this research also extend broadly to beetle ecology, particularly with respect to the relationship between morphological ad- aptation and occupation of different ecological niches in a similar habitat. Although additional observational data will be needed for verification, our findings may be extrapolated to other species of both genera, i.e., Can- siliella tonielloi Paoletti, 1972, C. montisceseni Piva, 2000, and hadesia vasiceki Müller, 1911, h. lakotai Perreau & Pavićević, 2008, and h. zetae Delić, Polak & Trontelj, 2016. ACTA CARSOLOGICA 46/2–3 – 2017320 entific. The metal composition of substrate, including moonmilk, was detected with atomic absorption spec- trophotometry using a graphite cooker SMEWW 3113 B (APHA 2012). Mouthparts and tarsal segments of C. servadeii, h. asamo, and h. weiratheri were prepared and compared using a scanning electron microscope (SEM), using methods as described by Paoletti et al. (2011). RESULTS OBSERVATIONS OF BEHAVIOUR Video footage for Cansiliella and hadesia is available at https://www.youtube.com/watch?v=hWNkAiArSwk&fe ature=youtu.be. Cansiliella. The population size of C. servadeii is currently unknown. All individuals were observed in a part of the cave named "Cavernone" (sensu Gasparo 1971) where extensive moonmilk deposits exist and water flows over the deposits (Paoletti et al. 2011). In a single observation session, a maximum of 83 individu- als were counted, with a maximum density of 8 indi- viduals per m2 of moonmilk deposit. During the year, the amount of water flowing at the sampling site did not significantly change and aqueous conditions were con- stant. The air temperature of the “Cavernone” was 8.6 °C, humidity reached saturation, and the water temperature was 8.8 °C. Tab. 1 lists the water and moonmilk physi- cochemical properties in the caves where Cansiliella and hadesia were present. The behaviour and activities of eight C. servadeii in- dividuals were continuously monitored for 1826 minutes (Tab. 2; Fig. 1). Individuals, which were 2.6 to 3.0 mm long, were mainly observed in areas covered by moon- milk and not on moist, bare rock. Although there was behavioural variability among individuals, beetles spent as much as 75 % of their time on trophic activities (i.e., feeding and foraging) and preening legs and antennae. For all C. servadeii except individual “E,” which did not exhibit any feeding or foraging trophic activi- ties (Tab. 2), trophic activity lasted on average 39 % of the observation time, and was performed in and out of water (57 % and 43 %, respectively). During immersion, a bubble of air was constantly present and covered the beetle (Fig. 2, top row), as previously reported (Paoletti et al. 2011). Often, individuals moved from moist surfac- es to areas with flowing water that was up to 4 cm deep. Sloped moonmilk surfaces appeared to be preferred to flat surfaces. In overall, movement represented just un- der a quarter (23.6 %) of the observation time, with dis- tances ranging from less than 1 cm/min to 11 cm/min, with individuals often moving within the water film. All individuals were observed on moonmilk, inside or out- side the water film. Only individual “D” moved over bare rock without any trophic or preening activities for eleven minutes before returning to moonmilk. Preening activ- ity varied by individual, representing (on average) up to 36 % of the observation time. Individual “N” did not show any cleaning activity, whereas individual “V” spent 78 % of the time preening (Tab. 2). A case of conspe- cific interaction was also observed: a female (individual “D”) approached another female (“E”), and when they touched with antennae, “D” immediately turned around and left the area. hadesia. Behaviour of three h. asamo individu- als, from 7.3 to 8.0 mm long, was observed and video- recorded for about 40 minutes, while the assessment of the activity changes over time (as in C. servadeii) has not been possible. The cave inhabited by h. asamo mostly lacks moonmilk deposits, and where they are present, deposits only constitute a very thin layer. h. asamo in- dividuals moved actively and quickly on bare rock or/ and on compact carbonate concretions. When they ap- proached water, they turned their abdomen against the water flow (Fig. 2, bottom row), as if to collect organic particles transported by water. An individual was ob- served standing with body trunk above a stream of water fig. 1: Proportions of the activity types for all observed Cansiliel- la servadeii in 24-hour observation. Activities are annotated as: At, trophic activity; S, site movement; Pz, cleaning of legs (and antennae); O, other activities; I, interaction with other individu- als; IW, activity performed in water; OW, activity performed out of water. L. DORIGO, A. SqUARTINI, V. TONIELLO, A. L. DREON, A. PAMIO, G. CONCINA, V. SIMONUTTI, E. RUZZIER, M. PERREAU, ... ACTA CARSOLOGICA 46/2–3 – 2017 321 for about 20 minutes, moving only for a few cm. Of the three individuals, none was seen with fully submersed whole body; only their heads were underwater. The three individuals were also not seen scraping the rocky sur- face, with their mouthparts in contact with the substrate. No preening activity was observed in h. asamo. tab. 1: Chemical and physical characteristics of percolating waters in karst caves hosting Cansiliella spp. and hadesia asamo. Locality GROTTA DELLA FOOS CAVE Friuli, Italy BUS DELLA GENZIANA CAVE Friuli, Italy BUS DELLA GENZIANA CAVE Friuli, Italy BRAVENIK CAVE Bosnia and Herzegovine VECCHIA DIGA CAVE Friuli, Italy MCL CAVE Friuli, Italy Cave entrance (m a.s.l.) 422 1020 1020 800 483 970 Date of Measurement 2008–2010 Average values 01/01/2009 Measured Value 01/06/2010 Measured Value 03/07/2011 Measured Value 01/10/2009 Measured Value 24/10/2015 Measured Value Mesured Parameters Air temperature (°C) 8.60 6..2 7.3 6.4 Water temperature (°C) 8.80 7.0 8.8 6.6 Relative Humidity 100 % 100 % 100 % 100 % Oxygen dissolved (mg/L) 10.27 7.87 10.8 8.08 11.56 pH (in cave) 8.15 7.9 8.21 8.10 Electric conductance (μS/cm) 233.50 390 216 201.00 Redox Potential (mV) 231.50 220 220.00 236 pH (laboratory) 7.98 7.8 7.45 7.87 8.0 8.10 Electric conductance (μS/cm) 210.50 339 175.30 315 194.7 201.00 Total hardness (°F) 11.48 11.15 9.53 18.3 10.1 11.90 Bicarbonate (mg/L) 149.27 107.55 124.89 228.8 137.0 136.00 Carbonate (mg/L) absent absent absent absent absent absent Silica (SiO2) not detected not detectable not detected not detectable not detected 1.40 Water NH4 + (mg/L) 0.13 <0.01 <0.01 <0.01 <0.05 <0.05 Water PO4 3– (mg/L) 0.10 <0.05 <0.05 <0.05 <0.05 <0.05 Water NO2– (mg/L) 0.15 <0.05 <0.05 <0.01 <0.05 <0.05 Water Cl– (mg/L) 0.83 47.15 1.27 2.7 0.80 0.60 Water NO3– (mg/L) 3.18 6.1 2.43 0.9 5.60 2.80 Water SO42– (mg/L) 2.58 2.55 1.37 2.9 1.80 2.20 Water Na+ (mg/L) 0.35 19.5 0.73 2 0.60 0.50 Water K+ (mg/L) 0.03 0.35 0.20 0.4 <0.1 <0.1 Water Mg2+ (mg/L) 0.45 0.5 0.23 19.8 1.6 0.50 Water Ca2+ (mg/L) 45.50 43.8 37.77 40.7 37.8 46.80 Water F– (mg/L) not detected not detected not detectable not detectable not detectable <0.1 Iron (Fe3+ μg/L) 5.45 not detected not detected 17 not detected not detectable Manganese (Mn2+ μg/L) 0.19 not detected not detected 3.95 not detected not detectable Strontium (Sr3+ μg/L) 21.78 not detected not detected 40 not detected not detectable Barium (Ba3+ μg/L) 17.60 not detected not detected 4.46 not detected not detectable Iodine (I– μg/L) not detected not detected not detected <0.05 not detected not detectable Aluminum (Al3+ μg/L) 14.11 not detected not detected 732.9 not detected not detectable MOUTHPART AND TARSAL MORPHOLOGy In C. servadeii, mandibles have a characteristic asymmet- ric shape (Fig. 3) that is clearly distinct from a typical (i.e., non-hygropetric) Leptodirini beetle, such as Orosty- gia pretneri Müller, 1931 (Fig. 4). Mandibles in all hadesia species are symmetric and have more teeth (Fig. 5), especially when compared to C. CAVE HyGROPETRIC BEETLES AND THEIR FEEDING BEHAVIOUR, A COMPARATIVE STUDy OF CANSILIELLA SERVADEII ... ACTA CARSOLOGICA 46/2–3 – 2017322 servadeii mandibles (Fig. 7: panels 4–9). Differences be- tween the two genera can also be seen from their mouth- part setal pattern; while dorsal side of galeae are covered by long and filamentous setae in hadesia (Fig. 6), short spines sorted in dense rows are present in C. servadeii (Fig. 7: panel 3 with permission from Paoletti et al. 2011). fig. 2: Cansiliella servadeii (top row) and hadesia asamo (bottom row) exhibit different behaviours while moving on the hygropetric surface. Specifically, C. servadeii moves in all directions, also with its body perpendicular to the water flow, but h. asamo keeps its body parallel to the water flow, with the keel-shaped abdomen facing upwards (Upper photo: A. Pamio, lower photo: M. Perreau). tab. 2: 24-hour behaviour and movement activities of Cansiliella servadeii in the cave grotta della foos. Activities are annotated as: At, trophic activity; S, site movement; Pz, cleaning of legs (and antennae); O, other activities; I, interaction with other individuals; IW, activity performed in water; OW, activity performed out of water. Individual; sex Observer Date (2010) Time Behaviour and notes C; indeterminate Simonutti 13.06 00:40-01:08 (28 min) AT: 21 % (100 % IW); PZ: 25% (100 % IW); S: 54 % (100 % IW) D; female Dorigo, Simonutti 12-13.06 17:30-04:17 (515 min) AT: 23 % (22 % IW; 78 % OW); PZ: 33.8 % (18 % IW; 82 % OW); S: 39.8 % (31 % IW; 69 % OW); O: 2.5 % (100 % OW); I: 0.2 % (100% OW) While out of water, beetle floated on a film of water while remaining anchored to the rock with one claw. Attempt to cross running water failed due to strong water flow. Touched another animals with its antennae, followed by immediate turning and changing direction. E; female Dreon 12.06 17:40-22:05 (210 min) PZ: 72 % (100 % OW); S: 28 % (100 % OW) G; male Dreon 12.06 22:35-23:23 (48 min) AT: 31 % (100 % OW); PZ: 10 % (100 % OW); S: 59 % (36 % IW; 64 % OW) H; female Dorigo 13.06 05:15-11:15 (360 min) AT: 56.7 % (14 % IW; 86 % OW); PZ: 27.2 % (10 % IW; 90 % OW); S: 15.8 % (63 % IW; 37 % OW); O: 0.3 % 100 % OW) M; indeterminate Dreon 13.06 05:15-06:53 (70 min) AT: 23 % (62,5 % IW; 37,5 % OW); PZ: 64 % (100 % OW); S: 13 % (100 % OW) N; indeterminate Concina 13.06 05:23-11:15 (352 min) AT: 91 % (97 % IW; 3 % OW); S: 9 % (100 % IW) For a few minutes, this beetle was a few centimetres away from another individual, but ignored it. V; female Dreon 13.06 07:00-11:15 (243 min) AT: 12 % (100 % IW); PZ: 78 % (100 % IW); S: 24 % (100 % IW) In Cansiliella, different degrees of wear, likely due to abrasion, are seen on the mandibular apex (Fig. 7: panels 4−9), but abrasion is not present in hadesia. Fur- thermore, the mouthparts of h. asamo and h. weiratheri show no presence of debris, which contrasts Cansiliella (Fig. 7, with permission from Paoletti et al. 2011). L. DORIGO, A. SqUARTINI, V. TONIELLO, A. L. DREON, A. PAMIO, G. CONCINA, V. SIMONUTTI, E. RUZZIER, M. PERREAU, ... ACTA CARSOLOGICA 46/2–3 – 2017 323 fig. 3: Cansiliella servadeii: mouthparts, in dorsal view (1); left (2) and right (3) mandibles (Photo 1: E. Ruzzier, photos 2 & 3: M. Beggio). fig. 4: Orostygia pretneri: mouthparts, in dorsal view (1); right mandible (2). Beetle was collected in the cave grotta della foos, near the entrance. (Photo 1: E. Ruzzier, photos 2 & 3: M. Beg- gio). fig. 5: Mouthparts of hadesia asamo (Photo: by E. Ruzzier). CAVE HyGROPETRIC BEETLES AND THEIR FEEDING BEHAVIOUR, A COMPARATIVE STUDy OF CANSILIELLA SERVADEII ... fig. 6: Mouthparts of hadesia asamo, dorsal setation of the galeae consisting of long setae. Upper row: h. weiratheri, male. Lower row: h. asamo, female (Photo: E. Ruzzier). ACTA CARSOLOGICA 46/2–3 – 2017324 fig. 7: hoe-shaped mandibles, spoon-shaped galeae, and labial palps of Cansiliella servadeii.1, a moonmilk particle (m) is visible within mouth- parts; 2, magnified view of labial palp (p), left galea (g) and 3, galea setation. 4-6, 3-dentate right mandibles and 7-9, left mandibles. Different degree of apical abrasion, caused by scraping of the substrate, is visible (Photos1 and 3: Paoletti et al., 2011; other photos: M. Beggio). fig. 8: tarsi of hadesia spp. with characteristic bent-blade bristles, more prominent on the right side. Upper row: h. weiratheri, male. Lower row: h. asamo, female (Photo: E. Ruzzier). L. DORIGO, A. SqUARTINI, V. TONIELLO, A. L. DREON, A. PAMIO, G. CONCINA, V. SIMONUTTI, E. RUZZIER, M. PERREAU, ... Tarsi (pro-, meso- and metatarsus) of h. asamo and h. weiratheri have characteristically shaped setae, i.e., bent-blade bristles, that are up to 300 µm long and pres- ent on the lateroventral surface of tarsomeres 2-3-4, of both males and females (Fig. 8). ACTA CARSOLOGICA 46/2–3 – 2017 325 During the observation period Cansiliella servadeii and hadesia asamo displayed different strategies of mov- ing in water flow and in feeding behaviour. C. servadeii moved trough the running water, completely submerged and protected by an air bubble, movements were irregu- lar and in all possible directions. h. asamo, however, was never completely submerged but merely lapped by the flowing water surface; when entering the water film, this species oriented its abdomen upstream, facing the water flow, and then moved laterally. Also morphological comparison between the mouthparts of both species showed important dissimi- larities. It is uncertain whether mouthpart structural differences between C. servadeii and h. asamo are relat- ed to their different trophic habits. In C. servadeii, piec- es of moonmilk between the mandibles had previously been observed by using SEM; the different degrees of mandibular abrasion were interpreted as a result of the browsing of moonmilk and the shape and distance of (1–1.5 µm) the upper galeae spines, as the means for brushing bacteria (Fig. 7; Paoletti et al. 2011). Nothing of the kind was noticed in hadesia. Moreover, the pres- ence of long bristles on the dorsal galeae suggests that h. asamo and h. weiratheri may perform water filtra- tion rather than scraping of rock surfaces like C. ser- vadeii. The above listed differences possibly indicate that C. servadeii interacts with the moonmilk surface while h. asamo, and probably also its congeners, interact ex- clusively with the water film. It may be possible that the evolution of two different feeding behaviours was pro- moted by different body sizes of both genera. C. serva- deii is small and able to move freely inside the water by creating a bubble of air; a reduced body size combined with the air bubble probably help the animal to easier resist the water flow, allowing a free set of movements in the water film. In contrast, h. asamo is larger and potentially more subjected to water flow; consequently the particular movement strategy may help prevent its drowning. Furthermore the orientation of the elytra apex against the water flow could help breaking the flow, thereby decreasing the body’s resistance against it. Anchorage to the substrate by h. asamo and probably by other species of this genus may likely be facilitated not only through long claws, but also by characteris- tic bent-blade bristles, present on the ventral surface of tarsi. These bristles are present also in h. weirath- eri (Fig. 8) and in h. zetae (Polak et al. 2016), as well as in some other Leptodirini. However, they have been observed neither in Cansiliella (Paoletti et al. 2013: Fig. 2b), nor in other tribes of Cholevinae for which the structure of tarsi have been investigated in detail (Antunes-Carvalho & Gnaspini 2015). The true func- tion of the bristles has not yet been verified and must be tested. In C. servadeii the most commonly observed in situ behaviours were trophic and preening activities. In particular, C. servadeii dedicated 36 % of its time to preening while in h. asamo such behaviour has never been observed. Self-preening activity in insects is a multipurpose behaviour whose functions could range from the collection of food (Rath 1999), removal of objects or material from the body surface, including parasites (Peng et al. 1987; Božič & Valentinčič 1995), distribution of substances across the body surface, as a displacement behaviour in stressful conditions (Howse 1974; Golenda & Forgash 1986), grooming of tarsi to maintain adhesion of attachment pads (Hosoda & Gorb 2011), or spontaneous action with no apparent external stimuli (Spruijt et al. 1992). Preening activity can be performed in different forms, such as by using mouthparts to remove material or parasites (e.g., in Or- thoptera and Coleoptera) or by depositing debris onto a substrate (e.g., in Diptera and Lepidoptera) (Zhuk- ovskaya et al. 2013). Debris can be either manipulated into the hypopharynx and ingested (Robinson 1996) or not (Božič & Valentinčič 1995). We hypothesize that preening in Cansiliella is con- nected with trophic activity because, to date, parasites have not been observed in the moonmilk or biofilm above the moonmilk (Engel et al. 2013; Paoletti et al. 2013), and all individuals were observed in situ, under natural conditions and free of stress sources. More- over, as abundant microbial cells have been observed on Cansiliella appendages (Paoletti et al. 2011, 2013) we consider plausible that microorganisms picked up during browsing are thereby removed from legs and antennae. This could suggest that preening behaviour is closely connected with the trophic activity and poten- tially related to animal-bacterial symbioses. Another plausible hypothesis is that the action of brushing an- tennae could have the function of keeping the sensory appendages clean (Böröczky et al. 2013). In Leptodirini this function may be important to ensure the function- ality of hygroreceptors (Hamann’s organ), which are fundamental devices for finding wet habitats (Lucarelli & Sbordoni 1977). The fact that preening has not been observed in hadesia asamo so far – despite comparable observation conditions – seems to disregard the latter hypothesis and suggest that for hadesia this activity is not as rel- evant as it is for C. servadeii. CAVE HyGROPETRIC BEETLES AND THEIR FEEDING BEHAVIOUR, A COMPARATIVE STUDy OF CANSILIELLA SERVADEII ... DISCUSSION ACTA CARSOLOGICA 46/2–3 – 2017326 An evaluation of behavioural activities and morphologi- cal features of two Leptodirini beetles living in hygro- petric habitats, hadesia asamo and Cansiliella servadeii, revealed distinct differences in their feeding, movement, and preening activities. At the same, time differences in the microstructure of the mouthparts were discovered, specifically in the presence and configuration of the spe- cialised setae on the galeae. Together with the observed differences in the foraging behaviour, these results sug- gest that these hygropetricolous beetles have the ability to exploit different resources in their habitat, which po- tentially emphasizes the ecological principle of finding "a niche within a niche." Body size may influence the way of beetle movement in the semi-aquatic habitat, likely af- fecting also their feeding strategies. The observed differences suggest that Cansiliella interacts with the moonmilk surface while hadesia in- teracts with the water film, not with the rock surface. To date, the association of C. servadeii with a moonmilk habitat still represent a peculiar animal-bacterial system. Additional behavioural observations of other Lep- todirini with “hadesian habits” are needed to improve the knowledge of the activities, feeding patterns, and other trophic associations in hygropetricolous beetles. More research on the physicochemistry, organic carbon concentrations, and overall nutrient conditions of hy- gropetric habitats is also needed, serving as a basis for understanding how these habitats fit into the overall cave ecosystem and how resources are used by the cave fauna. ACKNOWLEDGEMENTS We thank all members of the "Gruppo Speleologico Pradis" and members of the "Circolo Speleologico Id- rologico Friulano "(CSIF) for their help during in cave field activities in Italy. We also thank the speleologists of Zelena Brda speleological group in Trebinje for their col- laboration for field work in Bosnia and Herzegovina. The research and previous manuscript drafts benefited from ideas and contributions from R. Caicci, O. Coppellotti, L. Deharveng, A. Faille, B. Jalžić, L. 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