First detailed records of water mite larvae (Hydrachnidia: Hydrovolzidae, Hydryphantidae) parasitizing empidid flies (Diptera: Empididae: Clinocerinae)

Five species of the subfamily Clinocerinae from the Tatra Mountains (S Poland) were observed to be parasitized by larval water mites. Two of them: Kowarzia plectrum Mik, 1880 and Clinocera storchi Mik, 1880 are recorded from this massif for the first time. In addition, C. storchi, is new for Polish fauna. The most infected species was Clinocera appendiculata Zetterstedt, 1838, following by Wiedemannia mikiana (Bezzi, 1899), Clinocera storchi Mik, 1880, Kowarzia plectrum Mik, 1880 and Wiedemannia jazdzewski Niesiołowski, 1987. The highest number of hosts occurred in the case of Panisopsis curvifrons (Walter, 1907) with five host species, following by both Hydrovolzia placophora (Monti, 1905) and Protzia eximia (Protz, 1896) with one species each. In the case of Clinocera appendiculata more parasites were recorded on males than on females and in C. storchi more parasites were recorded on females. The abdomen of the hosts was the most often chosen by water mites larvae.


Introduction
The subfamily Clinocerinae (Diptera: Empididae) is a large group of predacious, long-legged flies that exist in lotic freshwater habitats. It contains 18 genera and well over 360 described species worldwide (Yang et al., 2007;Sinclair and Shamshev, 2014;Palaczyk et al., 2015). Adults are small to medium flies, usually greyish or sometimes brownish in colour, with generally narrow wings. So far as it is known, the immature stages of Clinocerinae are aquatic. Larvae live below the water surface predominantly in the moss or algal mats covering stones in the streams where they prey on small insects larvae, mainly on chironomids and simuliids (Vaillant, 1952(Vaillant, , 1953(Vaillant, , 1967. Sometimes larvae invade the pupal cocoons and attack the pupae (Zwick et al., 2011).
Among clinocerine genera, members of the genus Clinocera Meigen, 1803 usually occur in seepage habitats and also on wet stones or rocks and moss in headwater streams (Sinclair, 2008). In some cases they may be found in larger streams or rivers. In contrast, adults of Wiedemannia Zetterstedt, 1838) are generally confined to large and relatively clean, cool streams and rivers. Members of this genus are common resting on emergent stones and boulders. Adults of both genera prey on larvae and adults of chironomids and simuliids. In addition, adults of Clinocera are able to prey on larvae of thaumaleids as Sinclair (2008) reported. They are good and active fliers. However, most often, during the day, they remain on stones or boulders, on which they exhibit various forms of activity. On the other hand, during the night, they can gather in large numbers in the "sleeping association", from where they can be easily caught using insect collecting nets (Wagner and Gathmann, 1996, first author observation). Members of the subfamily Clinocerinae are active mainly in spring and summer but they are often present during colder months.
It is well known that dipterans are one of the most important hosts for larvae of many families of water mites (Acariformes: Hydrachnidia). Members of 17 families of nematoceran and five brachyceran flies are known as hosts for water mites (Martin and Gerecke, 2009). The family Chironomidae is most frequently parasitized by larvae of water mites (Smith and Oliver, 1986;Smith et al., 2001). Information of the interactions between empidid flies and mites is sparse. In particular, there is little data available on parasitism on the adults of the subfamily Clinocerinae (Tuzovskij et al., 2001;Sinclair, 2008).
In Poland, the subfamily Clinocerinae has been relatively well studied, especially in mountainous areas (Niesiołowski, 1990(Niesiołowski, , 2005Klasa et al., 2000;Palaczyk and Klasa, 2003;Krysiak and Niesiołowski, 2004;Krysiak, 2005;Palaczyk and Słowińska-Krysiak, 2013;https://doi.org/10.1016/j.ijppaw.2020.06.001 Received 17 April 2020; Received in revised form 7 June 2020; Accepted 7 June 2020 T Krysiak, 2014a, b;Słowińska, 2017Słowińska, , 2019. It is represented by 38 species, including Bergenstammia glacialis Palaczyk et Słowińska (2015), the species described recently from the Tatra Mountains (Palaczyk et al., 2015). Most species belonging to this subfamily occur in the mountains, however, many areas in the Sudetes and the Polish Carpathians are scarcely investigated. Despite undertaking long-term field studies on the family Empididae by the abovementioned authors, no cases of mites parasitizing on the Clinocerinae species have been reported in the country. Our study provides the first detailed records of water mites parasitic on Clinocerinae species from Poland.

Material and methods
Material analysed in this paper was collected during the regular studies on the Clinecerinae and Hydrachnidia in the area of Tatra mountains, during the summer-autumn period (from May to November in 2015, 2017 and 2018 years). The research was carried out at 75 sampling sites in the High Tatras (built of granites with typical alpinetype glacial relief) and the Eastern Tatras (built of metamorphic, crystalline and sedimentary rocks). Infested clinocerine adults were caught with an insect net in only four of the 75 sampling sites ( Fig. 1 Flies were preserved in 95% ethanol on-site. Larvae of water mites were removed from clinocerines by using forceps after recording their number and attachment sites in different regions of the host body. The larvae were mounted by embedding in the Hoyers' medium, and identified by using the keys and original papers of Tuzovskij et al. (2001) and Martin (2000Martin ( , 2003. Adults of the genus Clinocera were determined by reference to the original description (Mik, 1880) and using a key by Sinclair (1999). Photographs were taken using a Leica M205C stereomicroscope. Material was deposited in the Department of Invertebrate Zoology and Hydrobiology of the University of Łódź (Łódź, Poland).
Prevalence and intensity of infestation (number parasites per infested host individual) relative to hosts, sexes of hosts and parts of host body were analysed. One-way ANOVA was used, based on the logtransformed dataset, to assess the statistical significance of differences between number of water mite larvae on different host's sexes and particular parts of host's body, their size of body and number of hosts, and Shapiro-Wilk test showed that all log-transformed dataset were normally distributed (Jayaraman, 1999). These analyses were performed using the Statistica 13.1 PL software.
Water mite larvae were more frequently attached to the host's abdomen than to the head, cervix, thorax or legs (F = 5.44608; df = 1; p < 0.05), but intensity of infestation was the same on each part of body (F = 0.54930; df = 1; p > 0.05). The situation was the same both for all species of hosts (Table 3), but larvae of Hydrovalzia placophora were more commonly found on the thorax and legs (Table 4).
Infected clinocerids were recorded in June, July, August, October and November, and the most number of parasites occurred in July (Table 5), differences between number of parasites was statistically significant (F = 2.83428; df = 4; p < 0.05). Larvae of Panisopsis curvifrons were most abundant in July, Hydrovolzia placophora in October and Protzia eximia in August. Larvae of Panisopsis curvifrons were recorded from June to October, Hydrovolzia placophora from June to November, and Protzia eximia only in August (Table 5, Fig. 1). The body

Discussion
Empididae have seldom been reported as hosts for water mite larvae (Smith and Oliver, 1986). Up to now only Hydrovolzia cancellata Walter, 1906 and H. placophora have been found on the family. Hydrovalzia placophora was found on Clinocera and Roederioides genera, whereas H. cancellata has been reported from Clinocera, Wiedemannia and Kowarzia (Tuzovskij et al., 2001). Our study adds two other water mite species (Panisopsis curvifrons and Protzia eximia) to the list of parasites of family Empididae. Hydrovolzia placophora and Protzia eximia are opportunistic and known as parasites of other hosts, while Panisopsis curvifrons was recorded only from Chironomidae (Smith and Oliver, 1986;Tuzovskij et al., 2001;Gerecke and Martin, 2006).
The most infected clinocerids belongs to the most abundant species (Clinocera appendiculata, C. storchi, Wiedemannia mikiana) which is common for other water mite species, and intensity of infestation is rather low, as usual in dipterans and in contrast with odonates, beetles and bugs. Earlier literature data pointed to a much broader list of host species for Hydrovolzia placophora and Protzia eximia than for Panisopsis curvifrons (Smith and Oliver, 1986;Tuzovskij et al., 2001;Gerecke and Martin, 2006). Our research does not confirm this pattern as Panisopsis curvifrons was recorded on five hosts species, and Hydrovolzia placophora and Protzia eximia only on one host species each. It seems that the later two species of parasites occur on more orders of insects while Panisopsis curvifrons attach only dipterans but at least of three families (Simulidae, Chironomidae and Empididae). Among of the three water mite species Hydrovolzia placophora and Panisopsis curvifrons are crenobionts, and Protzia eximia has a little wider distribution including springs and streams. This is reflected in the lists of host species, where Hydrovolzia placophora has a wide diversity of semiaquatic or semiterrestial host species, Panisopsis curvifrons parasitized on simuliids, chironomids and empidids occurred in springs, and Protzia eximia has the widest list of typically aquatic host species including Diptera and Trichoptera (Davids et al., 2007;Di Sabatino et al., 2010).
In water mite literature there are many reports assessing differential parasitism of host sexes. Some of them indicate differences in infestation of different sexes, others indicate the lack of such differences. For example Atwa et al. (2017), Kirkhoff et al. (2013) and Edwards and Smith (2003) show females of mosquitos and chironomids were more infected than males, but Martin and Stur (2006) did not see such differences between sexes in Limoniidae, Simuliidae, Dixidae and Chironomidae. Differing reports of sex-biased parasitism also apply to odonates. Zawal (2004), Baker et al. (2008) presented data suggesting clear preferences in relation to the infestation of females in Coenagrion puella and C. pulchellum. However, they were not confirmed in other papers (Zawal, 2006a, b). Ilvonen et al. (2016) found significant differences between prevalence and intensity of infestation the sexes within certain host species of Odonata, but on a general level of Odonata more parasitized sex does not exist in the order. It seems that the situation is quite complicated and the differences in sex infestation depends on the host species and the parasite species. In our research Panisopsis curvifrons preferred famales, and Hydrovolzia placophora preferred males as a host, but the reason are unknown.
Usually larvae of water mites attach to the thorax or abdomen of their hosts; this holds for most dipterans, odonates, water beetles and water bugs (Biesiadka and Cichocka, 1994;Zawal, 2002Zawal, , 2003Baker et al., 2007;Stryjecki et al., 2015). This is unsurprising given that the thorax and abdomen make up the majority of the host's body.   Słowińska, et al. IJP: Parasites and Wildlife 12 (2020) 165-171 Additionally the integument is soft on abdomen, and in the case of beetles and bugs, water mite larvae are protected by elytra and hemelytra (Biesiadka and Cichocka, 1994;Zawal, 2002Zawal, , 2003Arjomandi et al., 2019). Most probably legs play the same role as elytra and enclose the water mite larvae attached to the thorax (Odonata, Diptera) (Zawal and Buczyński, 2013). In a few cases water mite larvae attached to the legs of their hosts: Ranatra linearis (Linnaeus, 1758) parasitized by Hydrachna gallica Thor, 1916, and Clinocerinae parasitized by Hydrovolzia cancellata (Tuzovskij et al., 2001). It is not known whether they attack soft hosts shortly after the moulting, or whether they are hard integuments of fully mature forms.
The most numerous occurrence and the highest activity (including procreation activity) water mites of crenophilous and crenobionts species coincides with the later period of the year (late summer or autumn) than limnophilous species (Zawal, 2002(Zawal, , 2003Martin and Stur, 2006;Zawal and Szlauer-Łukaszewska, 2012). Among the investigated species Hydrovolzia placophora has the most activity period annually later than Panisopsis curvifrons.
The body size range of all three water mite species larvae was very wide in all months, that means the time of ovipositions is elongated. And body size of the larvae was much smaller than body size of the adults (Di Sabatino et al., 2010), which means that significant body gains were realized in the deutonymph stage like in other water mite larvae parasitizing on Diptera and Trichoptera (Martin and Stur, 2006;Buczyńska et al., 2015) unlike the water mite larvae parasitizing on odonates, water beetles and water bugs (Biesiadka and Cichocka, 1994;Zawal, 2002Zawal, , 2003Zawal et al., 2017).

Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. The study was supported only by both the statutory funds of the University of Szczecin and University of Łódź.

Table 3
Place of attaching of parasites on the host body in particular host species: total number of larvae/maximum number of the larvae on one host/average number of larvae on one host.  Table 4 Place of attaching of particular species of parasites on the host body: total number of larvae/maximum number of the larvae on one host/average number of larvae on one host.   Słowińska, et al. IJP: Parasites and Wildlife 12 (2020) 165-171 Availability of data and material The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations of competing interest
There is no conflict of interest.