Contribution to the fauna of chiggers (Acariformes: Trombiculidae) parasitizing bats in Spain

Four chigger mite species were collected from different bat hosts in several provinces of Spain. Leptotrombidium europaeum (Daniel & Brelih, 1959) and Ascoschoengastia latyshevi (Schluger, 1955) has been recorded for the first time on bats; Leptotrombidium russicum (Oudemans, 1902), A. latyshevi and Willmannium cavus moldaviensis Kudryashova, 1992 have been recorded for the first time from Spain. The latter record represents also the first finding of the genus Willmannium Vercammen-Grandjean & Langston, 1976 in Western Europe.

Our collections added four Chiroptera parasitizing chiggers to the Spanish fauna.
(Kuhl) -1.One of the two adult males of E. isabellinus carried ca. 10 very small chiggers in plagiopatagium and uropatagium on both sides.One of the two adult males of H. savii was heavily infested with conspicuous reddish chiggers all over the patagium, whereas second male carried only a few chiggers in plagiopatagium and uropatagium.
• 2 -Vicinity of Palacio de Doñana, Doñana National Park, Almonte, Huelva province, 36.9907°N,6.4435°W.In July 2019, we examined an entire maternity colony of N. lasiopterus (40 adult females, 10 adult males and 19 juveniles) occupying five bat boxes.Whitish chiggers were recorded on the face, eyelids and ears of only one adult female.All bats of the colony were tagged with rings and transponders; the infested female, ringed that day as K03283, could be identified as new in the colony.
Thus, we collected trombiculids in summer months of 2019-2021 from four species of bats belonging to the family Vespertilionidae, E. isabellinus, E. serotinus, H. savii, and N. lasiopterus.Chiggers were attached to head and wing membrane of the bats.The mites were removed using O'TOM tick twisters (H3D, Lavancia, France) or spraying TABERNIL [Tetrametine + Piperony butoxide] (Divasa-Farmavic, S.A., Barcelona, Spain) on a swab and applying it to the chiggers, and after some time removing them by pushing gently with the forceps, without need for grabbing, avoiding the possibility of damaging the mites.Samples were stored in Eppendorf tubes with 70% ethanol at room temperature.Chiggers from different bat individuals of the same species from La Rioja were combined.
Mites were mounted on microscope slides using Berlese's medium.Specimens were examined and photographed under a Leica DM2500B microscope (Leica Microsystems GmbH, Wetzlar, Germany) with a Leica DMC 4500 digital camera using differential interference contrast.Measurements necessary for identification were made using a microscope MBI-3 (LOMO plc, St. Petersburg, Russia) with phase contrast optics and ocular micrometer.We used monographs of Kudryashova (1998) and Vercammen-Grandjean & Langston (1976), a revision of the genus Willmannium Vercammen-Grandjean & Langston, 1976 (Kudryashova 1992), and other referenced sources for identification of chiggers.
All chigger specimens are deposited in the collection of the Zoological Institute of the Russian Academy of Sciences (ZIN).
Our specimens depart from the original description in having a concave (vs.almost straight) posterior margin of scutum and by shorter legs -Ip = 815 -887 vs. 952 in the holotype (Table 1).However, the length of leg III tarsus in our material is almost the same as in the holotype -TaIIIL = 76 -86 (mean 81) vs. 83.In general, we do not support the practice of subspecies descriptions in the chigger taxonomy.Revision of the genus, with the possibility of raising some subspecies to the species level and synonymizing of others, is needed.
According to Kudryashova (1992), species of the genus Willmannium are known from Asia, Africa, and America.In Europe, three taxa belonging to this genus were recorded -Willmannium bulgaricum (Dusbabek, 1964) from Bulgaria, W. cavus moldaviensis from Moldova and Albania (Kudryashova 1992, 1998, 2004; Benda et al. 2019), and Willmannium cavus cavus Kudryashova, 1992 from Albania (Benda et al. 2019).Here, we record a representative of this genus in the Western Europe for the first time.

Discussion
Leptotrombidium russicum was described by a single specimen collected from an unidentified bat near Kiew (currently Kyiv, Ukraine) (Kudryashova 1998).The holotype is deposited in Rijksmuseum van Natuurlijke Historie (RMNH; currently, Naturalis Biodiversity Center, Leiden, Netherlands).Fuller (1952) noted that this specimen is nearly fully engorged, its scutum is tipped forward, and the posterior scutal margin is hidden under a fold of the cuticle.Vercammen-Grandjean & Langston (1976) provided a complete re-description of L. russicum on the base of the holotype, one specimen from Maastricht (Netherlands) deposited in the Oudemans collection (RMNH), and literature data.Kudryashova (1998) re-described this species on the base of new materials from Moldova, Kyrgyzstan, and European Russia (Moscow, Tver, and Tula Oblast); her measurements and morphological drawings were in good agreement with those of Vercammen-Grandjean & Langston (1976).Fernandes & Kulkarni (2003) also gave full morphological and morphometric data for an Indian sample of L. russicum; these data are consistent with previous descriptions, except for the barbs of scutal setae, which look fairly long on their drawing of the scutum.
Many records of this species from hosts other than bats were misidentifications (Vercammen-Grandjean & Langston 1976; Kudryashova 1998.In part, the species Leptotrombidium subrussicum Kolebinova, 1970 is morphometrically similar to L. russicum (Stekolnikov 2013) and could be misidentified.This species was described from rodents on Corsica (Kolebinova 1970) and later recorded from rodents, soricomorphs, and one bird in Bulgaria (Vercammen-Grandjean & Langston 1976; Kolebinova 1992).We also found a series of specimens belonging to L. subrussicum in the collection of ZIN collected from rodents in North and Western Caucasus (Russia) and in Transcaucasia (Armenia) and originally identified as L. russicum or L. fulleri (Ewing, 1945).
There are several species of Leptotrombidium that may be specific to bats, including four described from Romania and one from the UK (Stekolnikov 2013).Leptotrombidium russicum differs from all of them in having fewer idiosomal setae and fD = 2H-8-6-6-4-2 vs. 2H-10-8-8-… or 2H-8-6-6-6-….One larva from France (Ariège) identified by Kolebinova (1970) as L. russicum, with fD = 2H-9-8-8-8-6-4-2, probably belongs to another species.However, our finding of L. europaeum on E. serotinus from La Rioja demonstrates that some European species of Leptotrombidium, usually parasitizing rodents and soricomorphs, can also infest bats.Therefore, we may expect the joint occurrence of morphologically similar species, such as L. subrussicum and L. russicum, which can be represented by different intraspecific forms, on same individuals of bats in Europe.Two size forms of L. russicum were described in the present work; L. subrussicum also shows a wide range of size variations, probably depending on altitude (Vercammen-Grandjean & Langston 1976).
A recent work on bat-infesting chiggers in Poland reported the presence of four different Leptotrombidium identified by mitochondrial cytochrome c oxidase subunit I gene (COI) sequences.One was identified morphologically as L. russicum, whereas the identity of other three forms remain unclear (Zajkowska & Mąkol 2022).Interestingly the genetic distance between the form designated as Leptotrombidium sp. 2 and L. russicum was only 5.6 -5.9%.This distance is rather close to the threshold of the intraspecific variation in Trombiculidae, which attained 4.6% by the COI sequence (Zajkowska & Mąkol 2022).Further morphological examination of these samples is required to establish the nature of the observed molecular  Kudryashova (1998).Abbreviations as in Table 1.
forms more definitely.
Noteworthy is that our material on L. russicum was collected from N. lasiopterus, a bat usually inhabiting tree cavities (Ibáñez et al. 2004).This allows us to conclude that hollow trees can be at least one of possible habitats where the life cycle of L. russicum takes place.Stekolnikov et al. (2014) presented the list of 19 chigger mite species known from Spain at that time, but omitted Ericotrombidium ibericense (Vercammen-Grandjean & Langston, 1976) from this list.However, we suppose that the previous record of E. hasei (Feider, 1958) could have been E. ibericense.Later, Stekolnikov & Quetglas (2019) added two more species to the chigger fauna of Spain.Currently, 25 chigger species are recorded from Spain, including the results of the present study.Six species were collected from bats and three -L.russicum, O. komareki, and T. knighti -only parasitize bats.