The order Palpigradi includes species characterized by millimetric size and a characteristic flagellum with bristles at the end of the opisthosoma. They represent one of the less well-known and obscure arachnid orders. In this paper, observations were made on the ecology and feeding behavior of species belonging to the genus Eukoenenia Börner, 1901, from the Western Italian Alps. Direct observations and photographic documentation of 141 individuals in their cave habitat, allowed the recording of data on the physical and trophic conditions such as the presence/absence of trophic resources, temperature and relative humidity, of the underground environment in which they were found. Results showed that the species of this taxon are not as rare as previously reported and that their presence is mainly influenced by temperature, relative humidity, trophic resources and the presence of two speleothems: rimstone dams and rafts. The combination of our observations as well as data previously published highlights that the taxon can have predatory and saprophagous feeding behavior depending on the availability of the food resources. This work represents the starting point for a further investigation of the taxon.

Arachnida, microwhip scorpions, subterranean biology, caves

Palpigradi is the last described arachnid order and, to date, one of the lesser known and studied. The order includes small size species characterized by the whip-like flagellum with bristles at the end of the opisthosoma. The world fauna of living palpigrads consists of more than 100 described species divided in two families: Eukoeneniidae and Prokoeneniidae (Harvey 2002; Giribet et al. 2014).

The first scientific investigation of the species in this order began in 1885 in Sicily (Italy), where the zoologist Giovanni Battista Grassi (Grassi and Calandruccio 1885) discovered a “mysterious species” of arachnid that he described as Koenenia mirabilis Grassi & Calandruccio, 1885, now Eukoenenia mirabilis (Grassi & Calandruccio, 1885). Silvestri (1905) described for the first time the anatomy of the species and he was followed by other authors but many aspects of the biology of the order, including food selection and reproduction, are still unknown (Condé 1996; Smrž et al. 2013; Christian et al. 2014; Parimuchová et al. 2021). Molecular phylogeny of the taxon was performed only by Giribet et al. (2014) that demonstrated the monophily of the order and of the family of Eukoeneniidae.

The elective habitats of the species in the genus Eukoenenia are interstitials (Howarth 1983; Mammola 2019) and the species can be ecologically classified in soil- and cave-dwelling (Mammola et al. 2021b). The cave-dwelling species have been reported walking on the cave walls and ground (Condé 1996), speleothems (Souza and Ferreira 2010), decomposing wood (Balestra et al. 2019), sand banks near water stream (Souza et al. 2020) and on the surface of water pools (Christian et al. 2014; Balestra et al. 2019).

There is currently little known about the feeding of palpigrades. An observation of Eukoenenia hunting springtail provides evidence of a predatory habit (Lukić 2012), however, the presence of Cyanobacteria in their gut indicates an alternative food source (Smrž et al. 2013) and scavenging is also another possibility. A recent study based on molecular analysis (NGS) on the gut-content of Eukoenenia spelaea (Peyerimhoff, 1902) in Ardovská Cave, Slovakia, supports carnivory behaviour (Parimuchová et al. 2021).

Because there is minimal data available about the biology of the species of this genus, a continuous survey was carried out within 20 caves in Piedmont and Liguria regions (Northern Italy) in order to describe and better understand some aspects of their biology: nutrition, microhabitat and distribution. In particular, the following questions have been addressed: i) Is the paucity of records of this taxon related with bias in sampling or is it related with the low density of the populations? ii) What kind of environmental/ecological parameters affect the presence and the distribution of palpigrades in caves? iii) What is the trophic role of the taxon (potential predatory species, saprophagous species or both)?

In the last year, due to the high sampling effort, the level of knowledge about the distribution of the species of the genus Eukoenenia in Western Italian Alps has been exponentially increased: 1) more than 100 individuals were observed and documented in the last four years; 2) before 2016 palpigrade specimens were observed only in seven caves, whereas today Eukoenenia species are reported from 17 caves.

Therefore, it has been possible to increase the number of sites where Eukoenenia species is present, both in term of caves and in term of areas within caves. For example, Eukoenenia strinatii was considered endemic in the Bossea cave until 2016, now this species is reported from other four caves and from different areas within the Bossea cave (from seven in 2016 to 10 in 2021) (Balestra et al. 2019; Condé 1977; Morisi 1992).

The data reported here highlights the fact that palpigrades in Western Italian Alps seem to be not so rare: probably the paucity of data on these organisms was due to the difficult access to some habitats (Ficetola et al. 2019; Howarth 1983; Mammola 2019) and to the lack of knowledge of the environmental parameters that define the preferred habitat for this taxon. Moreover, in (Mammola et al. 2021b) a significant species-people correlation effect (number of species/number of researchers studying palpigrades) has been described, showing that the concentration of palpigrade records in some regions of the globe can be influenced by the presence of researchers interested in this group.

As for other species of invertebrates present in the Bossea cave, palpigrades do not seem to be particularly and negatively influenced by tourism. Despite their troglomorphism they tolerate the presence of lights, even if not direct, and probably only for brief periods. The presence of palpigrades in touristic cave was also observed in the Brazilian Maquiné cave, where E. maquinensis Souza & Ferreira 2010 is reported (Ferreira and Souza 2012).

Temperature and relative humidity values recorded in this study are typical of Alpine caves, however, the sampling data revealed the absence of Eukoenenia in cold caves (T < 8 °C). The most favorable caves for Eukoenenia genus seem to be those with mild temperatures and very high relative humidity, or higher temperatures and lower relative humidity. The parameters monitored in Maquiné cave, Brazil, showed higher temperature (23.6 °C to 24.5 °C) but similar RH% (89 to 95%) for E. maquinensis (Ferreira and Souza 2012).

Palpigradi living in the hypogean environment of the Western Italian Alps have usually been observed on the surface of pools of rimstone dams. They have rarely been observed on wet wood, near water, on wet stones or on the cave ground. This does not mean that the favored environment of these small arachnids is the water surface of the underground pools, on which they probably can easily float thanks to the surface tension of the liquid or with rafts, but that this possibly reflect higher detectability in this cave habitat. Their main habitats can be those interstitials (Howarth 1983; Mammola 2019) and probably they could be transported in underground pools by water flow during rainy periods, getting trapped on the rafts, or they could voluntarily go into this environment in search of food. In fact, relying on different observations on individuals on the water surface, the presence of trophic resources in 90% of cases makes it reasonable to think that food availability is the main factor influencing Eukoenenia specimens to venture on the rafts and in the gours.

In more than 100 direct observations in caves it was never possible to document an attack of palpigrades on a prey. An approach on dead springtails sucking their internal liquids have been observed. If the ingestion of liquid food is their feeding system, as observed in other arachnids, it could be explained why no solid remains were found in the digestive tract of the palpigrades (Condé 1984; Millot 1942). A direct attack on collembola in caves was documented (Lukić 2012) and a specimen of E. strinatii that carried a dead springtail, keeping it with chelicerae, was photographed in this work. Moreover, a recent study on gut content of E. spelaea has shown the presence of spiders, beetles, mites, springtails, and flies DNA (Parimuchová et al. 2021), supporting carnivory in palpigrades. Wheeler (1900) suggested that palpigrades probably feed on eggs or juvenile stages of bigger arthropods invertebrates, however, our observations on scavenging activity could explain the presence of genetic material of large arthropods in the intestinal tube of palpigrades.

The combination of our observations and the data from other authors support the idea that palpigrades are predator and scavengers depending on the food resources availability, due to the fact that caves are extreme environments with limited trophic resources. Cyanobacteria and Fungi could be an alternative food source or, probably, an accidental consumption due to the ingestion of contaminated prey, as suggested by Parimuchová et al. (2021).

In conclusion the results of this study highlights that:

1. The hypogean palpigrades in the Western Italian Alps seems to be anything but rare and the paucity of records of this taxon is related to sampling bias. A correct knowledge of the preferred habitat and the environmental conditions where they live allows the observation of a relevant number of specimens.
2. Hypogean palpigrades were observed in distinct regions inside caves, located at different altitudes, including records in areas with artificial lighting for tourist access. The presence of these animals is mainly influenced by temperature, relative humidity, trophic resources and the presence of two speleothems: rimstone dams and rafts. The most favorable cave habitats for Eukoenenia genus seem to be those with mild temperatures and very high relative humidity, or higher temperatures and lower relative humidity whereas the taxon was not recorded from cold caves.
3. Palpigrades are predators and scavengers depending on the food resources availability due to the fact that caves are extreme environment with limited trophic resources.

Caves are special environments that host unique creatures in a world still to be explored and documented. In this study, it was possible to provide a first knowledge on the environmental parameters that can influence the distribution of hypogean palpigrades in Western Italian Alps. Moreover, the feeding behaviour in palpigrades is directly observed and photographed for the first time. Direct observation and photographs can be very useful to elucidate biological aspects of fragile groups that are difficult to keep under laboratory conditions, such as palpigrades. This research can be considered the starting point for future and more detailed studies on this curious genus and other apparently rare hypogean taxa in Italian caves.

Authors are very grateful to Erhard Christian for the determination of the samples and useful information. We cordially thank Loris Galli, Massimo Meregalli and Maysa Sousa for helpful comments, Bartolomeo Vigna and Jo De Waele for geological suggestions, Michelangelo Chesta, Sara Longo, Marco Marovino, Nicoletta Pastorino, Fabrizio Falco, Marco Isaia, Mariagrazia Morando, Massimo Sciandra, Denise Trombin, Arianna Paschetto, Pier Mauro Giachino, Speleo Club Tanaro, Gruppo Speleologico Valli Pinerolesi, Gruppo Speleologico Savonese DLF and Gruppo Speleologico Piemontese for helping us. We acknowledge Liguria Region authorities for providing the necessary permits to collect hypogean Palpigradi and the DISTAV-University of Genova for the thermohygrometer use. Authors are grateful to Hanna Moore (Cranfield University) for the English revision of the manuscript and for helpful comments.

This work was possible with the contribution of the Scholarship of the National Congress of Biospeleology (Cagliari 7–9 April 2017), won for the project “Study of distribution and behavior of hypogean palpigrades (genus Eukoenenia) of Piedmont” by V.B. and E.L., and with the contribution of the association Biologia Sotterranea Piemonte – Gruppo di Ricerca.