Two new species of parasitic demodecid mites in the European polecat Mustela putorius and their co-infestation with Miridex putorii (Acariformes: Demodecidae)

Abstract The biodiversity of parasitic mites of the Demodecidae, infesting mammalian carnivorans, is poorly understood. To date, 18 host-specific species have been described, including four each from domestic dogs and cats, and only 10 from wild carnivores, known from single or duplicate reports. No data is available on the level of infestation of wild populations, or the co-occurrence of different demodecids in the same host, as only single species have been identified in individual hosts. A convenient model for such studies turned out to be the European polecat Mustela putorius, in which a new genus and species, Miridex putorii, was recently described in the vibrissae region of the skin of the head. Our study reveals that M. putorii co-occur with other species: Demodex putorii sp. nov. (associated with the hairless skin of the head) and Demodex foetorii sp. nov. (associated with hairy skin). Thus, the present study provides descriptions of species new to science. It also provides the first analysis of the occurrence of Demodecidae in wild mammalian carnivorans at the species and individual animal level based on the co-occurrence of three demodecid mite species in the skin of the head in M. putorius. Demodecid mites were found in 75.7% of 37 polecats; M. putorii showed the highest prevalence (56.8%), and D. putorii showed the highest abundance in the skin (mean 10.8 mites in 9 cm2). The three species co-occurred in 5.4% of the polecats, and two species in 27.0%. Despite the very high abundances, infestation was not associated with gross skin pathology (a feature of stable parasite–host systems formed by long-term co-evolution). The co-occurrence of several species and the separation of microhabitats are also typical of Demodecidae of other mammalian groups and illustrate the optimal use of the host body as a habitat and food source with limited impact. urn:lsid:zoobank.org:act:4D12FF6B-5A9E-4346-A496-52AC8448E7FE https://zoobank.org/NomenclaturalActs/4D12FF6B-5A9E-4346-A496-52AC8448E7FE urn:lsid:zoobank.org:act:07C533F5-9392-4FD3-BF9A-E1058C0FC1D6 https://zoobank.org/NomenclaturalActs/07C533F5-9392-4FD3-BF9A-E1058C0FC1D6


Introduction
The Demodecidae (Acariformes: Prostigmata) are among the smallest parasitic arthropods; however, they are also among the least understood due to their lifestyle hidden in the skin or in other mammalian tissues and structures.They are likely to be common in host populations, exhibiting high host specificity (Izdebska & Rolbiecki 2020;Cierocka et al. 2022).In individual host species, there may be several particular species of these mites, associated with different microhabitats: they have been found in various skin structures, tissues and organs, including normal and sensory hair follicles, various glands, ear canals, eyeball, tongue and gums (Izdebska & Rolbiecki 2020).At the same time, each species shows a clear preference regarding their location, inhabiting different regions of hairy skin (e.g.only the head, or only the limbs, abdomen, or back), sparsely hairy or hairless skin (e.g. the region of the lips or nose, hairless skin of the feet, the flight membranes of bats, genital or anal areas).Topical (related to the specific microhabitat) and topographic (distribution in various regions of the body) preference and the co-occurrence of synhospital Demodecidae species are mostly observed in rodents, especially cosmopolitan and synanthropic species like the house mouse Mus musculus Linnaeus, 1758 and the brown rat Rattus norvegicus (Berkenhout, 1769), or the widely distributed Apodemus spp.(Izdebska & Rolbiecki 2013a, 2013b, 2014b).There is little information on their occurrence in other mammals, and in the case of carnivorans, they refer only to the domestic dog Canis lupus familiaris Linnaeus, 1758 and the domestic cat Felis catus Linnaeus, 1758, in which four different species each have been described, showing different location preferences (Desch & Nutting 1979;Desch & Hillier 2003;Izdebska 2010;Izdebska & Rolbiecki 2018).
However, data from domestic animals do not necessarily provide an appropriate model for parasitological analyses for most members of the Demodecidae that are associated with wild mammals.In domestic mammals, infestation often has a different course, associated with the appearance of the disease symptoms of demodecosis (demodicosis), resulting from excessive multiplication of a particular demodecid species or, less frequently, from infestation by two or more species.Domestic animals live under different (artificial) environmental conditions, and such host-parasite systems probably have a much shorter evolutionary pedigree than in wild mammals; hence, this affects its instability − a greater chance of crossing the tolerance threshold of the host and the subsequent development of parasitosis (Izdebska et al. 2023).
However, in wild mammals, this type of analysis is hampered by a very poor understanding of the biodiversity of their Demodecidae.In individual hosts, only single representatives, usually of the genus Demodex, have so far been described based on isolated or sparse findings (Izdebska & Rolbiecki 2020).In the light of current research, a suitable example for such study has proved to be the European polecat Mustela putorius Linnaeus, 1758 (Carnivora: Mustelidae), in which a new species and genus Miridex putorii Izdebska, Rolbiecki et Rehbein, 2022 has recently been described residing in the vibrissae (Izdebska et al. 2022).The species is characterized by unusual structural features, making it one of the more mysterious and exceptional representatives of mites (Izdebska et al. 2022).
The aim of the current study was to describe two new species of Demodecidae which are typical and probably specific parasites of M. putorius and have been discovered in further research on the skin samples that resulted previously in the discovery of Miridex putorii (Izdebska et al. 2022).This study also provides the first analysis of the occurrence of Demodecidae in a mammalian carnivore based on the co-occurrence of three mite species from two genera in the skin of European polecat, both at the mite species level and at the individual host level.In addition, data on host associations and the global distribution of carnivore demodecid mites are updated.

Material and methods
Skin from the head and adjacent neck area of 37 European polecats was collected during a survey of the animals' parasitic fauna in Germany from October 2013 to August 2015 (Table I) and preserved in 70% ethanol.The animals were traphunted according to the hunting regulations in Germany, then placed in separate plastic bags and frozen (Kretschmar 2016).
Nine ~1 cm 2 fragments were taken from each skin sample representing eight head regions, viz. the area around the eyes, nose, area of vibrissae, lips, chin, cheeks, ear pinnae and vertex, plus the adjacent neck area (total ~9 cm 2 per animal).For the recovery of demodecid mites, skin fragments were individually digested in 10% potassium hydroxide solution as described previously (Izdebska 2004).The digest material was decanted and examined under phase-contrast microscopy (Nikon Eclipse 50i) with 1 cm 2 of skin sample yielding approximately 100 wet preparations.The mites were placed in polyvinyl-lactophenol solution.The following measurements were taken: total body length = length of gnathosoma, podosoma and opisthosoma; gnathosomal width (at base); podosomal and opisthosomal width (maximum width).All measurements are given in micrometers (µm).
The number of eggs and mite stages (larva, protonymph, deutonymph, adult male, adult female) per 1 cm 2 for each mite species were identified.The total number of specimens was counted to estimate the abundance of infestation as the number of mites (larvae + nymphs + adult mites) per examined host/skin samples.
To assess the relationship between presence of mite infestation (interpreted as prevalence -percentage of hosts infested with mites) and host demographic factors, all 37 polecats were considered, which New species of demodecid mites in Mustela putorius comprised 14 "juvenile" animals (≤1 year; nine male, five female), 18 "young adult" animals (~2-3 years old; 13 male, five female) and five "old" animals (>3 years old; three male, two female) (see Table I).
Associations between mite presence and variables representing host demographic factors (age group, sex) were assessed using contingency tables and Fisher's exact test.Abundance of infestation (total demodicid mite count between sexes of polecats per age group) was analyzed using the Mann Whitney U-test.All testing was two-sided, and the level of significance for all analyses was set at p < 0.05.(Zhang 2018).
The description of the species adopted the nomenclature commonly used for the family Demodecidae (Nutting 1976) and was completed with the nomenclature proposed by Bochkov (2008) for the superfamily Cheyletoidea (Acariformes: Prostigmata) and by Izdebska and Rolbiecki (2016).The scientific and common names of the hosts follow Wilson and Reeder (2005) and the Integrated Taxonomic Information System (2022).
Species descriptions were prepared including all specimens, and morphometric features were analyzed based on all (14 females and 10 males) D. foetorii specimens and 140 selected D. putorii specimens (100 females and 40 males) from different locations and different host individuals.

Infestation, and demodecid mites found in the European polecats
A total of 629 specimens (including eggs) of Demodecidae were recovered from the examined skin samples, including 204 Miridex putorii, 401 Demodex putorii sp.nov.and 24 D. foetorii sp.nov.The overall prevalence of infestation (including animals infested with at least one of the three mite species) was 75.7% with the abundance (mean, median, range) of mites 16.6, 4.0 (1-366) (Table II).The highest prevalence was exhibited by M. putorii (56.8%), with the highest abundance by D. putorii sp.nov.(10.8, 0.0, 1-360), where the most specimens (360) were found in one "old" male polecat from Northrhine Westfalia/county Borken; however, no skin lesions related to the infestation were observed.
While all ontogenetic stages were recovered for M. putorii and D. putorii sp.nov., only adult mites were isolated for D. foetorii sp.nov.Some variation in the adult male to female ratio was observed for the three mite species in individual hosts; in particular, a female predominated ratio was observed for D. putorii sp.nov.(1:5.5)(Table III, Figure 1)., 83.3, 19.0, 1-366) 28 (75.7%, 16.6, 4.0, 1-366) N mite+: number of mite-positive animals."Total demodicid mites" refers to animals infested with at least one of the three mite species.
For total demodecid mite infestation (including animals infested with at least one of the three mite species) with respect to host age group, prevalence of infestation was higher among combined "young adult" plus "old" animals (i.e.sexually mature, capable of reproducing) than for "juvenile" polecats (sexually immature) (20/23 vs. 8/14, p = 0.0569).In addition, male polecats were significantly more likely to be infested than female polecats: all animals − 22/25 (male) vs. 6/12 (female), p = 0.0355; combined "young adult" plus "old" animals − 16/16 (male) vs. 4/7 (female), p = 0.0198.Pairwise comparison of the total mite abundance by sex of the polecats demonstrated that "young adult" male animals harbored significantly more mites than "young adult" females (p = 0.02), and that the total male polecats harbored significantly more mites than total females (p = 0.0063).No significant difference in the abundance of demodecid mite infestation was noted between "juvenile" male and "juvenile" female animals (p > 0.1); counts of "old" animals were not analyzed separately because of small sample size (Table IV).
Male (n = 40).Distinctly smaller than female, 203 μm (177−229 μm) long and 31 μm (27−35 μm) wide, but of similar shape.Gnathosoma shape similar to female, but smaller.Pharyngeal bulb and morphological details of gnathosoma similar to those in female.Shape of podosoma and legs also similar to those in female, but epimeral plates I connect at one point, pairs II−IV separated.On dorsal side of podosoma, podosomal shield with distinctly vertical striation, reaches level of legs III.Opisthosoma relatively long, smaller than females, constitutes 64% (60−68%) of body length; distinctly annulated; annuli relatively wide at ca.

Etymology
The specific epithet putorii refers to the specific name of the host.

Location in the host
Demodex putorii sp.nov.was found in the skin around the eyes, nose, area of vibrissae, lips, chin, cheek, and neck (Figure 2).The observed mites did not cause any skin lesions in the examined European polecats.
Differential diagnosis of Demodex putorii sp.nov.Demodex putorii sp.nov. is similar in shape and some features to other Demodecidae described from mustelid mammals, especially D. lutrae Izdebska et Rolbiecki, 2014 (Table VII).However, both the female and male specimens of Demodex putorii sp.nov.are noticeably larger.The gnathosoma of both species is trapezoidal, but in D. lutrae it is elongated (length exceeds width at the base), while in D. putorii sp.nov., the length and width at the base are similar.The supracoxal spines on the gnathosoma are hammer-shaped and slightly larger in D. lutrae (1.5-2.0 μm in length), directed medially; in D. putorii sp.nov.they are smaller (1.0-1.5 μm in length), directed toward the outer edges of the gnathosoma.There are three spines each on the terminal segments of the palpi in both species, but they are conical and of different sizes in D. lutrae, and comprise one small conical, two much larger (one bifurcated) in D. putorii sp.nov.In addition, v"F setae are present on the palpi of D. putorii sp.nov., whereas in D. lutrae these setae are absent.The subgnathosomal setae in D. lutrae are located on both sides at the level of the posterior edge of the pharyngeal bulb; in contrast, they are located at the level of the anterior edge of the pharyngeal bulb in D. putorii sp.nov.The posterior edges of the epimeral plates IV of D. lutrae females form a triangular, distinct incision, and a slightly arched incision in D. putorii sp.nov.; in addition, the epimeral plates I−IV connect medially in D. lutrae males, while they are slightly separated in D. putorii sp.nov.males.The leg claws differ mainly in the shape of the spur (straight in D. lutrae, curved in D. putorii sp.nov.); in addition, no solenidia were observed in D. lutrae, but they are present in D. putorii sp.nov.Furthermore, the aedeagus of males of the two species are in similar locations but it is slightly longer in D. lutrae.The opisthosoma has a similar shape in the two species but is relatively shorter compared to body length in D. lutrae (i.e.59% in the male and 61% in the female), and is more elongated (64% ) ± 3 Body length to width ratio 6.5:1 (5.4-8.1:1)± 0.5:1 8.0:1 (6.8-9.6:1)± 0.6:1 5.0:1 (4.4-6.0:1)± 0.4:1 6.0:1 (4.9-7.1:1)± 0.5:1 Opisthosoma length to body length ratio (%)

Etymology
The specific epithet foetorii is derived from one of the Latin names for the host, Foetorius putorius.nov.has the characteristic shape of a scorpion's telson.The opisthosoma is conical and slender in D. putorii sp.nov., while it is short and oval in D. foetorii sp.nov.The microhabitat preferences are also a little different: D. putorii sp.nov.was mainly found in the sparsely hairy skin of the head (lips, chins), and mainly in the in the hairy skin of the head in D. foetorii sp.nov.Demodex foetorii sp.nov.generally differs in its shape and features from known Demodecidae, including species described from mammal carnivorans.It only resembles D. erminae Hirst 1919, described on the basis of two males specimens found in Mustela erminea Linnaeus, 1758, with regard to its habit and the presence of large supracoxal spines.This species, however, requires re-description, as its description lacks many of the features currently considered important in the taxonomy of Demodecidae, and no description exists of the females.Based on the laconic description, measurements and figures, it can be concluded that the males of D. erminae are smaller than those of D. foetorii sp.nov., but have slightly different body proportions and features of the gnathosoma (including supracoxal spines) New species of demodecid mites in Mustela putorius and a shorter (higher located) and differentshaped aedeagus.It is likely that this is a morphologically similar species from a host belonging to the same genus, which is a phenomenon typical of Demodecidae.

Distribution and associations of demodecid mites with families and species of carnivoran hosts
Taking into account the currently described species of demodecid mites from the European polecat and the latest literature data on the occurrence of these mites in carnivores, the list of Demodecidae species associated with Carnivora and their distribution in the world has been updated (Table X).

Discussion
Although the Demodecidae have been studied for more than 180 years, knowledge of their biodiversity remains scant.Most of the data concern their occurrence in domestic mammals and humans, and knowledge of the Demodecidae fauna of wild animals is fragmentary.This is also true for the order Carnivora, a widely distributed and otherwise relatively well-studied group of mammals (Izdebska & Rolbiecki 2020;Izdebska et al. 2023).Of the 10 known species of Demodecidae of wild carnivorans, six have been described from single hosts kept in zoos (Table X), of which only Demodex phocidi has also been confirmed in a host from a natural population (Izdebska et al. 2020).Of the remainder, two (D.erminae Hirst, 1919, D. lutrae) are known from single findings (Hirst 1919;Izdebska & Rolbiecki 2014a), one (D.melesinus Hirst, 1921) has been found worldwide twice, in single hosts in the UK and Poland (Hirst 1921;Izdebska et al. 2018); only the recently described Miridex putorii was found in a sample of a dozen host individuals (Izdebska et al. 2022).Thus, data are lacking on not only the species diversity of Demodecidae of individual wild carnivores, but also the most basic information on their occurrence in host populations including the level of infestation.The current study, which resulted in the discovery of two more demodecid mite species in Mustela putorius, has made it possible to carry out such an analysis, taking into account the co-occurrence of these mites.
The European polecat is a common mammal carnivoran inhabiting Europe and North Africa.It also appears to be a good model for studying the occurrence of demodecid mites -it is less territorial than other mustelids, and is also polygamous, which undoubtedly promotes inter-individual contact and transmission of skin mites between individuals within a population.Despite having a relatively wellrecognized parasitofauna (Kretschmar 2016), data on the occurrence of so-called skin and tissue mites of the order Prostigmata (Demodecidae, Psorergatidae, Epimyodecidae) in this mammal was lacking until recently.Unidentified Demodex sp. have only been recorded in the ferret Mustela furo (= M. putorius furo Linnaeus, 1758), the domesticated form of the European polecat, in the Netherlands and New Zealand (Nutting et al. 1975;Noli et al. 1996).However, no descriptions of specimens obtained from ferrets have been published, only metric data (Noli et al. 1996), which differ (length of adults in the range of about 60-70 µm) significantly from the dimensions of specimens found in the present study.
In the light of the discovery of the new species, the question of co-occurrence of several species of Demodecidae in the same host seems interesting.Such synhospital species are known in mammals from other groups, especially rodents; for example, in the house mouse, seven species from two Demodecidae genera have been described so far.They inhabit different regions of the skin, in different areas of the body, thus making optimal use of the host's body (Izdebska & Rolbiecki 2015a, 2015b, 2016;Izdebska et al. 2016), while not exceeding its tolerance for the presence of skin parasites, resulting in infestations which are not associated with skin lesions.However, in the case of the European polecat, the described species inhabit the same region, i.e. the skin of the head, without gross pathology.In this case, what may be the correlation of the occurrence of synhospital species, allowing for optimal use of environmental resources?According to the analyses, all species can occur simultaneously in the same host, although relatively rarely, at different abundances and within different microhabitats.Thus, M. putorii prefers the vibrissae region, D. putorii the hairless or sparsely hairy parts of the head, and D. foetorii the adjacent but more intensely hairy regions.In general, a higher abundance was noted on the anterior (facial) part of the head, which is characterized by a greater diversity of potential microhabitats.An analogous diversity of species associated with adjacent head microhabitats was described in rodents, such as the field mouse Apodemus agrarius (Pallas, 1771), in which demodecid mites specific to the vibrissae region (D.gracilentus Izdebska et Rolbiecki, 2013), eye area (D. huttereri Mertens, Lukoschus et Nutting, 1983), ear canals (D. agrarii Bukva, 1994), or the rest of the hairy skin on the head (D. apodemi Hirst, 1918) were found; however, the possibility of co-occurrence at the individual level was not analyzed (Izdebska & Rolbiecki 2013b).
While Miridex putorii and D. putorii sp.nov.were more numerous and observed in all life stages, only adults were recorded for D. foetorii sp.nov.It is possible that these species engage in reproduction and population development during different periods or seasons, which prevents the simultaneous overgrowth of Demodecidae.Such seasonality has been observed in other Demodecidae, such as D. plecoti Izdebska, Rolbiecki, Mierzyński et Bidziński, 2019 of the brown bat Plecotus auratus (Linnaeus, 1758) (Izdebska et al. 2019).Miridex putorii and D. putorii, although more numerous and represented by all life stages, also demonstrated differing dynamics of infestation: the former showed high prevalence, with a lower level of abundance in the skin, while the latter was much less frequent, but with a higher abundance.It appears that the host habitat can be optimized, not only by the separation of microhabitats (e.g., different species may use sensory hair follicles, normal hair follicles, glands, or other structures) but also by seasonal fluctuations in abundance.This may occur especially for longestablished host-parasite relationships.In general, the stability of host-parasite relationships is related to the adaptation of parasites to function in the host, optimizing its use as a habitat and food source, and limiting nuisance to the host and thus pathogenicity; this is a feature of the old host-parasite relationships, developed as a result of long-term evolution.
Demodecidae were noted in nearly 76% of the studied European polecats.However, a higher level of infestation was observed in males; this may be a result of polygamy and, at the same time, greater activity of males in inter-individual contacts.This has been confirmed by studies of, for example, Demodecidae in rodents (Izdebska 2012;Izdebska et al. 2017).In addition, higher levels of infestation were observed in older individuals, which is also a typical phenomenon in Demodecidae infestations and is due to a greater number of contacts over a longer life span, i.e. greater opportunities to acquire parasites with age.The phenomenon also applies to demodecid mites of other mammals, including, for example, humans (Izdebska & Jankowski 2006).
Previous studies of infestation levels of Demodecidae in mammalian carnivorans have focused only on domestic dogs and cats, although even here, few analyses of the typical non-clinical infestation associated with these mites have been carried out.Studies on domestic dogs and cats from Poland confirm frequent and even widespread occurrence of these mites in their hosts (Izdebska 2010;Izdebska & Rolbiecki 2018;Izdebska et al. 2023).However, in the case of pets from animal shelters or areas where they live in high densities (high possibility of contact between individuals), the transfer of parasites is easier, which determines a high prevalence of infestation.In the case of wild animals, which exhibit territorialism and where contacts between individuals mainly occur during the breeding season, the level of infestation observed in the present population can be considered very high.Interestingly, despite the high level of abundance of mites in the skin of some hosts, no signs of demodecosis were observed in them.Similar observations apply to D. melesinus from badgers, D. lutrae from European otters and D. phocidi from common seals, where even very high abundances did not generate the occurrence of gross pathology (Izdebska & Rolbiecki 2014a;Izdebska et al. 2018Izdebska et al. , 2020)).It is likely that the level of host tolerance, formed as a result of long-term co-evolution of the parasite and host, is much higher here than in the case of analogous relationships in domestic mammals or humans, where the abundance of even a few individuals per 1 cm 2 of skin determines clinical demodecosis (Forton & De Maertelaer 2017).
In summary, our findings extend the current knowledge of the biodiversity of the Demodecidae in wild mammalian carnivorans from 10 to 12 species.They also provide new data on the cooccurrence of different species of this family in the same host, as well as on level of infestation among a larger number of individuals than in previous studies.They also confirm data obtained from studies of other host groups indicating high topical and topographic specificity associated with optimal use of available microhabitats within the host.

%
Area around the eyes Nose Area of vibrissae Lips Chin Cheek Ear pinnae Vertex Neck

December 2013 ,
February 2014, November 2014, January 2015, and December 2014; parasites coll.J. N. Izdebska and L. Rolbiecki; host coll.S. Rehbein; the whole-type material (mounted microscope slides with the demodecid mites) deposited within the framework of the Collection of Extant Invertebrates in Department of Invertebrate Zoology and Parasitology, University of Gdańsk, Poland.

Table I .
Sampling details for the European polecats examined for demodecid mites.

Table III .
Recovery of demodecid mite eggs and ontogenetic stages (larvae, nymphs, adults) in the areas of head/adjacent skin (~1 cm 2 skin fragments each) of Mustela putorius.

Table V .
Body size (µm) for adults of Demodex putorii sp.nov.
and 67%, respectively) in D. putorii sp.nov.The typical microhabitat is also different: D. lutrae was found mainly in the hairy skin of the head, and D. putorii sp.nov. in the sparsely hairy skin of the head (lips, chins).
The supracoxal spines on the gnathosoma in D. putorii sp.nov.are small (1.0−1.5 μm in length), conical, and directed outwardly, while they are larger (5.0−6.0 μm in length), wedgeshaped, and directed to the center of the gnathosoma in D. foetorii sp.nov.There are three spines on the terminal segments of the palpi in both species, but one small, conical and two large, including one bifurcated in D. putorii sp.nov., compared to one small, conical and two large, curved, in D. foetorii sp.nov.In addition, a v"F seta is present on the palpi of D. putorii sp.nov., which was not found in D. foetorii sp.nov.The subgnathosomal setae are located at the level of the anterior edge of the pharyngeal bulb in D. putorii sp.nov., but they are located clearly below the anterior edge in D. foetorii sp.nov.Moreover, the posterior edge of epimeral plate IV has a slight arched shape in D. putorii sp.nov.females but a triangular shape in D. foetorii sp.nov.females; in addition, epimeral plates I −IV are slightly separated in D. putorii males, while only epimeral plates II and III are slightly separated, additionally epimeral plate IV is weakly sclerotized in D. foetorii sp.nov.males.The leg claws differ mainly in the shape of the spur, which is larger and more curved in D. putorii sp.nov.The solenidia are present in D. putorii sp.nov., but absent in D. foetorii sp.nov.Furthermore, the aedeagus in D. foetorii sp.
sp. nov. is short, broad, with an oval opisthosoma.The gnathosoma of both species is trapezoidal, but the length is close to the width at the base in D. putorii sp.nov., and the length is less than the width in D. foetorii sp.nov.

Table X .
Carnivoran host associations and distribution of demodecidae of the world.