JOURNAL OF NATURAL HISTORY, 2016 http://dx.doi.org/10.1080/00222933.2016.1193643

Morphology of tube-like threads related to aquatica (L., 1758) (Acariformes: Hydrachnidia: ) in the laboratory A.B. Shatrova, E.V. Soldatenkob and O.V. Gavrilovac aZoological Institute, Russian Academy of Sciences, St. Petersburg, Russia; bDepartment of Biology, Smolensk State University, Smolensk, Russia; cDepartment of Microbiology, St. Petersburg State University, St. Petersburg, Russia

ABSTRACT ARTICLE HISTORY Water (L., 1758) during maintenance in Received 9 April 2015 the laboratory for a long period of time in constant conditions Accepted 20 May 2016 fl periodically produced certain whitish occulent material consist- KEYWORDS ing of long rigid unbranched tube-like threads 1.3 ± 0.3 µm in Water mites; tube-like diameter crossing freely. These threads were studied using light- threads; TEM; SEM; optical as well as transmission electron microscopical and scan- Limnochares aquatica ning electron microscopical methods. Microbiological staining was also applied to the threads to exclude their bacterial or fungal origin. The thread wall is built of fine fibrils arranged at different angles to the long axis of threads that is reflected in a certain stratification of the wall. Threads are mostly hollow or may contain electron-dense homogeneous material. No cell components are present in the thread composition. Numerous dermal glands with their small slit-like orifice scattered throughout the body surface are thought to produce these threads. Most probably the thread formation is a reaction of mites to stress under laboratory conditions, and this is expected to be a type of defensive reaction.

Introduction Production of protein threads known as silk is widely distributed among , in particular spiders (Stubbs et al. 1992;Foelix1996; Vollrath et al. 1996;Craig1997, 2003; Downloaded by [University of Cambridge] at 00:01 09 August 2016 Vollrath 2000;DeBakkeretal.2006;Sponneretal.2007;Krafft and Cookson 2012; Blackledge 2013), (Hunt 1970;Schaller1971;Kovoor1987;Annamalai and Jayaprakash 2012), mites (Wallace and Mahon 1972; Alberti and Ehrnsberger 1977; Bolland 1983;Gerson1985; Manson and Gerson 1996; Alberti and Coons 1999) and (Schaller 1971; Kovoor and Zylberberg 1980; 1982; Kristensen 2003;Kebedeetal.2014; etc.). Organs involved in silk formation are thought to have developed several times and independently in every group (Craig 2003) and are localised either de novo on the abdominal region, as in spiders and some pseudoscorpions (Schaller 1971), or are trans- formed from prosomal glands, as in some pseudoscorpions and mites (Gerson 1985).

CONTACT A.B. Shatrov [email protected] Zoological Institute, Russian Academy of Sciences, Universitetskaya emb. 1, 199034, St. Petersburg, Russia © 2016 Informa UK Limited, trading as Taylor & Francis Group 2 A. B. SHATROV ET AL.

Among acariform mites, spinning ability is characterised for representatives of several terrestrial families, such as Tetranychidae, Eriophyidae, Camerobiidae, Cunaxidae and Bdellidae (Alberti and Coons 1999), providing a great diversity of important functions (Witte 1991; Alberti and Coons 1999; Clotuche et al. 2011; Kanazawa et al. 2011;LeGoff et al. 2011; Fernandez et al. 2012; Yano 2012; etc.). It is accepted that in mites, especially in Tetranychidae (Gerson 1985; Alberti and Coons 1999), as well as in preudoscorpions (Kovoor 1987; Annamalai and Jayaprakash 2012), in contrast to other , produc- tion of silk is realised by the reorganised salivary glands (Gerson 1985). By contrast to this supposition, it was shown that in representatives of the Serianus, males spin their signalling threads ‘with the opistosomal spinning glands which open at the anus’ (Schaller 1971, p. 426). In eriophyoid mites, spinning organs were not identified (Manson and Gerson 1996) although webbing around mites’ colonies of some species is found to be enormous. In the highly diverse water mites (Hydrachnidia), males of several families with indirect sperm transfer produce so-called ‘guiding/signalling threads’, supporting sper- matophores, by special glands of the genital tract (Witte 1984, 1991; Proctor 1992; Alberti and Coons 1999; Witte and Döring 1999). A similar strategy is also found in representatives of some terrestrial families of Parasitengona (Proctor et al. 2015). No other data on silk organs, or on silk formation, were obtained for the whole history of water mite observations. In previous work (Shatrov 2014;Shatrovetal.2014), we have found that several water mite species appear to produce various amounts of silk during maintaining in the laboratory. This silk consisted of long, rigid, unbranched, hollow, tube-like threads varying from 700 nm to 2.5 µm in diameter. Transmission electron micro- scope (TEM) examination of silk threads of Limnesia undulata (O.F. Müller) revealed that their walls are composed of fine fibrous material of different density and orientation. The threads’ lumen did not contain cell membrane or any cell compo- nents, although variously organised non-cellular core may be present (Shatrov 2014; Shatrov et al. 2014). Specific staining revealed neither DNA nor microbial walls in threads’ composition, so the microbial origin of threads was excluded. The observed silk formation did not correspond to the mite reproduction behaviour because it lasted from late summer until mid autumn when the mites had already completed detachment of both eggs and spermatophores. It was supposed that dermal glands,

Downloaded by [University of Cambridge] at 00:01 09 August 2016 an evolutionary acquisition of water mites, may be involved in the silk secretion (Shatrov 2013). We started keeping Limnochares aquatica (L.) in the laboratory, hoping to acquire additional data on the phenomenon of silk production by water mites. Generally, L. aquatica is a Holarctically distributed species with a well-known biology (Böttger 1972). Moreover, secretion supposedly of their dermal glands was biochemically tested, and behavioural experiments were performed with regard to the relationship of these mites with their potential natural enemies – predatory fishes (Kirstein and Martin 2009, 2010). It was shown that the secreted protein has a molecular weight of 30 kDa with additional components of a low weight. It was also demonstrated that fishes spat out both swallowed mites and food saturated with their secretion (Kirstein and Martin 2009, 2010), a behaviour which was also revealed earlier for some other species (Kerfoot 1982; Proctor and Garga 2004). On the other hand, in some Russian northern rivers, JOURNAL OF NATURAL HISTORY 3

the stomachs of fishes were found filled with red water mites of uncertain species (Sokolov 1940). The main purpose of the present study is a detailed morphological examination of threads secreted by water mites L. aquatica during their maintenance in the laboratory.

Materials and methods Mites, collecting site and laboratory maintenance Several adult mites of L. aquatica were captured 19 September 2014 in the artificial lake Dubrovenskoye (54°47ʹN, 31°56ʹE) – the widest part of the same river, the right tributary of the river Dnepr in the north vicinity of Smolensk city (around 9 km from the city centre). Three specimens of these mites – two males and one female (Tuzovskiy 1979) – were taken for the experiment. The remaining mites were also kept in the laboratory for control observations, but were out of the strict experiment, subject to only periodic examination. Three experimental specimens were placed separately in the same plastic 100-mL container with a pure bottled artesian water (certification of conformity N POCC RU. AE05.H02957, www.smolvoda.ru) distributed in Smolensk city. The containers were sterilised using hot steam, and afterwards during usage they were kept loosely covered with a small Petri dish to allow the intake of air. Approximately once a week, we changed the water in the containers for fresh water, and the containers were newly sterilised. The experiment continued until March 2015. For the whole time of the experiment, the mites did not feed and were active. The males died 15 May and 30 October 2015 after they moulted in January–February 2015. The female was still living up to January 2016, did not moult and periodically produced some amount of silk. During the experiment, the mites were active during the day and inactive at night. The water was kept absolutely free of plants, plant leaves and any other additional materials and substances. The containers were constantly maintained at a temperature of 20–21°С, and with natural lightning. The containers were examined daily or once every 2 days. To take photographs, the mites were replaced for a short time into a Petri dish, allowing a good visualisation. Photographs were taken with a Canon G11 digital camera with a resolution of 10 Mpx. Mites used for scanning electron microscope (SEM) study (five specimens) were Downloaded by [University of Cambridge] at 00:01 09 August 2016 captured 12 July 2009 in Lake Maloye Strechnoye (55°51ʹN, 31°77ʹE, Smolensk Province, Demidovskiy District, National Park ‘Smolenskoye Poozerye’, around 4 km from Przhevalskoye town) and were nearly immediately fixed.

Light-microscope observation For the light-microscope observation, the entire wisp or boll of threads or their smaller portions were removed from containers using a microscopic needle. Both temporary water and constant dry preparations were then made from these threads by mounting and stretching them as a thin one-row film on microscope slides. The specimens were then covered with a cover-glass and examined. Water preparations were examined immediately; dried preparations were observed later using oil immersion. These 4 A. B. SHATROV ET AL.

preparations were examined and photographed with a Leica DM LS-2 light-optical microscope combined with a Leica EC-3 digital camera. The techniques of bright field (BF) and differential-interferential contrast (DIC) were performed with a Leica 2500 microscope equipped with a Leica DFC 500 camera. Thread measurements were taken by a morphometrical program used in a CLSM Leica TCS SP 5.

Microbiological tests Different methods of specific staining were applied for threads to exclude their microbial origin: (1) staining of heat-fixed slides in aqueous gentian violet, the basic staining that interacts with bacterial (and fungal) cell wall and DNA; (2) DNA-specific staining of vital and fixed material with 4ʹ, 6-diamidino-2-phenylindole (DAPI). The results were exam- ined in the St. Petersburg State university using a CLSM Leica TCS SP 5 (Department of Microbiology, St. Petersburg State University, St. Petersburg, Russia).

Transmission electron microscope (TEM) examination For TEM observations, a standard double fixation in glutaraldehyde and osmium tetroxide was applied for portions of the threads’ wisps. The bunches of threads were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2–7.4) for several days, washed in 0.2 M cacodylate buffer for 1 hour, postfixed in 2% osmium tetroxide in 0.1 M cacodylate buffer for 2 h, dehydrated in ethanol and acetone series, and finally embedded in an araldite mixture. Ultra-thin sections mostly in a transverse plane to thread bunches were made on a Leica UC-6 ultramicrotome, mounted on copper grids with an oval hole provided with a formvar support and, after staining with uranile-acetate and lead citrate, were examined with a TEM Morgagni 268-D (FEI Company) at 80 kV (digital visualisation).

Scanning electron microscope (SEM) examination For SEM study, mites were treated as for TEM up to the acetone step, and then dried in air. After these procedures, the specimens were coated with a platinum layer in an Eiko IB-5 ion coater, and then examined with SEM Hitachi S-570, Hitachi TM-1000 and Quanta

Downloaded by [University of Cambridge] at 00:01 09 August 2016 250 (FEI Company) at 10–20 kV. Threads were mounted on cover glasses as a thin film, dried in air, covered with a platinum layer and examined with SEM Quanta 250. All instrumental procedures were performed on the basis of the Centre of Collective Use ‘TAXON’, ZIN RAS, St. Petersburg, Russia, and the Centre of Collective Use ‘CHROMAS’, St. Petersburg State University, St. Petersburg, Russia.

Results Laboratory observations Generally, two types of the phenomenon of thread formation may be recognised in relation to maintenance of L. aquatica in the laboratory – formation of flocks and formation of bolls. JOURNAL OF NATURAL HISTORY 5

The appearance of whitish flocks begun 25 September (i.e. the sixth day after capture of the mites), and lasted until 10 October 2014. This phenomenon occurred approximately in equal proportions in each of the three containers and was found to be directly related to mites. Initially, small, nearly transparent wisps or denser white ‘spots’ appeared at the mite dorsal surface in different body regions (Figure 1(a, b)), which later detached from the cuticle and associated into the much larger clots, flocks (Figure 1(c))orwisps,probablyby some unspecific actions of the mites. Two types of mite activity may be generally revealed in relation to the larger flocks. The first one is that the mite drags the flocks behind itself for some time by the fourth leg pair, to which the flock appears to be applied. The second type is that the mite puts the legs round the flock, thus keeping the large flock, compared with the mite body size, at its venter (Figure 1(c). The latter type of ‘activity’ is mostly seen in the dark time of the day, when the mites were mostly immobile. They ‘slept’ on the back or on the side, retaining the flock at the abdomen. When the clot was artificially removed from the mite, the latter was actually ‘disappointed’ by this loss and tried to put its legs round the air bubble on the bottom, comparable to the clot in size. The second type of the phenomenon of thread formation is the appearance of smaller, uniform whitish balls in the containers (Figure 1(d)), which began appearing 4 November 2015 and, after changing of both the container and the water for new ones, continued appearing for 10–12 days and then periodically until 11 February 2015. No special actions of the mites in relation to these balls were observed. For the whole time of observation until March 2015, apart from the periods of flock and ball formation, no conspicuous associations of threads were obviously recognised in the containers, and, on the other hand, no bacterial or fungal contamination of either the water or the mites was apparently revealed. The mites continued to be alive and active, and two of these (males) moulted 22 February 2015 (this process started on 28 December 2014 and 20 January 2015, respectively), confirming the fact that mites of this species may moult in the adult phase (Sokolov 1940; Böttger 1972).

Light microscope observation Both flocks and balls are composed of irregularly interlaced, colourless, rigid, uniform tube-like threads 1.3 ± 0.3 µm in diameter (Figure 2(a)). The threads do not branch and are either mostly devoid of any internal organic substance/core, remaining absolutely

Downloaded by [University of Cambridge] at 00:01 09 August 2016 empty (Figure 2(a)), or may contain (the tubes of balls) non-cellular organic core (Figure 2(c)). The length of the given thread cannot be estimated with certainty. Being freely interlaced, the threads may bend at different angles (Figure 2(b)) or may be occasionally broken into smaller portions. The rigidity of the threads cannot be obviously ascertained, but in comparison with those of L. undulata, the rupture strength appears to be much less expressed than in the latter species.

Microbiological tests Specific staining of threads with gentian violet does not reveal bacteria or other DNA material in the thread composition, although various accompanying bacteria may be occasionally present in the samples (Figure 2(d)). Staining with a fluorescent DNA- specified stain (DAPI) shows that no characteristic fluorescence is seen related to 6 A. B. SHATROV ET AL. Downloaded by [University of Cambridge] at 00:01 09 August 2016

Figure 1. Water mites Limnochares aquatica (L.) and threads’ associations during maintenance in the laboratory. (a) A mite in a drop of water with white spots on the dorsal surface (arrows); (b) Another, larger, mite in a drop of water revealing a small white flock on the posterior portion of the idiosoma (arrow); (c) A mite engaging with an extremely large flock association at its venter (arrowhead). Note the hardly distinguished possible detachment of the colourless threads from the dorsal body surface (arrow) forming figures of balls; (d) The balls of threads containing also inclusions of additional organic substances. Scale bars = 1 mm. JOURNAL OF NATURAL HISTORY 7 Downloaded by [University of Cambridge] at 00:01 09 August 2016

Figure 2. Threads of the water mites Limnochares aquatica (L.) in a light microscope. (a) Freely interlaced threads in oil immersion in a common microscope. Note that practically colourless threads are unbranched and totally devoid of any internal substances, being sometimes bent at different angle. Scale bar = 20 µm; (b) Differential-interferential contrast (DIC) showing threads’ replicas. Scale bar = 5 µm; (c) DIC of gentian-violet stained preparation showing both empty threads and thread with a core (arrow). Scale bar = 20 µm; (d) Bright field (BF) of gentian-violet stained preparation obviously indicating colourless threads and contaminating bacteria and bacterial chain (arrows) freely dispersed in the sample. Scale bar = 5 µm. 8 A. B. SHATROV ET AL.

threads. These tests clearly indicate that threads associated with L. aquatica activity in the laboratory have no nucleic acids or cell walls and so their bacterial origin should be excluded.

Transmission electron microscope (TEM) examination TEM investigations of ultra-thin sections made mostly in transverse plan to threads show round profiles of the tubes, with walls varying in thickness from 20 to 250 nm but mostly around 200 nm (Figures 3(a–e), 4(a)). These tubes and their walls do not include any cellular components. The walls are composed of fine fibrils orientated mostly along or at different angles to the axis of a thread that is well recognised in oblique or longitudinal sections of threads (Figure 3(d, f)). Contrarily, on transverse sections, the walls may show fine granulation or even certain stratification as a probable result of the different orientation of fibrils within the tube walls (Figure 3(c–e)). The external and, rarely, internal surface of the thread wall may show a certain ‘halo’ of loosely organised fibrils (Figure 3(d, f)). Occasionally, the internal volume of threads may demonstrate irregular lamellar profiles of unknown origin (Figure 3(f)). In threads with thinner walls, crumpled or incomplete profiles of threads may be frequently seen (Figure 3(a, c)) that may indirectly indicate that threads of this species, in comparison with L. undulata (Shatrov 2014; Shatrov et al. 2014), are not very rigid. The threads from flocks are mostly empty (Figure 3(a, b, e)), whereas the threads from balls may demonstrate a dense internal core of secreted organic material showing concentric layers (Figure 4(a)). This substance is spaced some distance from the wall which may be either thick (Figure 4(a)) or, contrarily, very thin.

Scanning electron microscope (SEM) examination SEM study of the thread ball reveals tubes mostly containing core (Figure 4(b)). By contrast, the threads from the cloth are mostly hollow and collapsed (Figure 5(a, b)). These threads are sometimes curved, bent or torn out. They retain their natural round shape quite rarely, only if they contain a core. It is characteristic that during collapse, the threads retain the thickened periphery where the tube walls fold in two (Figure 5(b)). Downloaded by [University of Cambridge] at 00:01 09 August 2016

Figure 3. Transmission electron microscopy (TEM) of threads of the water mites Limnochares aquatica (L.) from the thread cloth. (a) A group of transverse sectioned totally empty threads with variously organised walls. Note a somehow crumpled tube profile (at the bottom of the figure) as well as incomplete profiles (arrows). Scale bar = 2 µm; (b) transverse sectioned tube-like threads with variously organised walls. Scale bar = 1 µm; (c) transverse profiles of two tubes with walls of different thickness. Note that the thinner wall is incomplete (arrows). Also note an additional probably plant organism (arrowhead). Scale bar = 0.5 µm; (d) two threads, one of which, obliquely sectioned, reveals fibrils arranged along or at different angles to the thread’s wall (arrow). Note a certain ‘halo’ of loosely organised fibrils at the external wall surface (arrowhead). Scale bar = 1 µm; (e) a thread with the complex wall composed of hard-to-recognise variously packed fibrils. Scale bar = 0.5 µm; (f) longitudinal section of a thread with thick walls composed of fibrils arranged variously but mostly along the thread’s axis. Note a ‘halo’ on the walls’ surface (arrows) and a certain lamellar structure inside the thread (arrowhead). Scale bar = 0.5 µm. JOURNAL OF NATURAL HISTORY 9 Downloaded by [University of Cambridge] at 00:01 09 August 2016 10 A. B. SHATROV ET AL.

Figure 4. (a) Transmission (TEM) and (b) scanning (SEM) electron microscopy of threads of the water mites Limnochares aquatica (L.) from the thread balls. (a) Transverse section of the thread showing a dense core (arrowhead) at some distance from a thick wall (arrow). Scale bar = 1 µm; (b) bunch of the threads possessing a core (arrowheads) enclosed within the thread wall (arrows). Scale bar = 10 µm.

The mites (Figure 5(c)) demonstrate numerous small closed slit-like orifices of dermal glands (glandularia) irregularly distributed on both the ventral and, especially, the dorsal body surface (Figure 5(d)). It is quite characteristic that the smaller part of these orifices is provided with a sensory hair (Figure 5(f)), whereas the larger part possesses a short spur, or spine (Figure 5(e)), both situated at one pole of the orifices. No perceptible extrusions of any materials from the glands’ orifices were recognised, supposedly due to the fact that the examined mites were captured in mid-summer, when production of silk may be restricted (see Discussion).

Discussion

Downloaded by [University of Cambridge] at 00:01 09 August 2016 The present study shows that adult freshwater mites L. aquatica produce certain tube- like threads during their maintenance in the laboratory for a long period of time in constant room conditions. It is evident that these threads are not of bacterial, fungal or other-organism origin, and do not contain any cellular material. This assumption is strongly confirmed by the negative microbiological tests, by the total absence of any cellular elements in the threads’ composition and by the finding that no contamination of both mites and water occurred beyond the time of the threads’ production. The secreted material containing threads appears first at the mite cuticle, and this process is found to begin not long after the capturing of mites. The sources of this type of secretion are expected to be most likely dermal glands, and this secretion may be a probable reaction of mites to some new environmental conditions against the natural ones. These new conditions are evidently as follows – composition of the artesian water (biochemistry, gas saturation; the absence of additional plants and , etc.), JOURNAL OF NATURAL HISTORY 11 Downloaded by [University of Cambridge] at 00:01 09 August 2016

Figure 5. Scanning electron microscopy (SEM) of threads from the thread cloth and some external characteristics of the water mites Limnochares aquatica (L.). (a) Bunch of the hollow collapsed threads sometimes bending at different angles (arrow). Scale bar = 10 µm; (b) threads crossing freely and sometimes torn (arrow). Note a thread partly retaining its natural shape (arrowhead). Scale bar = 5 µm; (c) front view of a mite. Scale bar = 500 µm; (d) portion of the dorsal surface showing proposed dermal gland orifices (glandularia) provided either with a sensitive hair (arrows) or with a short spine (arrowheads). Scale bar = 100 µm; (e) slit-like orifice (arrow) provided with a sharp spine at the pole of the structure. Scale bar = 20 µm; (f) identical slit-like orifice (arrow) possessing a sensitive hair situated on the pedestal at the pole of the orifice. Scale bar = 20 µm. 12 A. B. SHATROV ET AL.

constant room temperature and the character of illumination in the laboratory. The response of an organism to each of these factors or to their various combinations could be quite different, and its analysis seems to be rather difficult at present. Because little is known about detailed behavioural characters of L. aquatica in the natural environment (see also Böttger 1972; Proctor et al. 2015), any suppositions concerning the meaning of thread production in the laboratory could be only hypothetical. By contrast, the role and functions of silk in terrestrial silk-producing mites are well known (Clotuche et al. 2011; Kanazawa et al. 2011;LeGoff et al. 2011; Fernandez et al. 2012; Yano 2012; etc.). In any case, the revealed phenomenon of the thread production is unlikely to be related to reproductive activity of mites, for several reasons. First, the mites were captured in autumn, when reproduction is already completed (Böttger 1972) and no evidence was revealed that the mites would restart egg or spermatophore deposition after their replacement into relatively warm water at the laboratory condition. Second, although in Limnochares a complete dissociation is found as a type of the sperm transfer mode (Proctor 1992) – that is, the most ancient/primitive mode of reproduction when no female needs to be present during sperm deposition by a male – no signalling threads have been found in Limnochares (Witte 1991). On the other hand, L. aquatica form zigzag tracks consisting of particular secretory signalling stripes (probably secreted by genital organs) leading to spermatophores if they are deposited (Witte 1991). Third, the production of these threads occurred in approximately equal proportions in all three examined mites – two males and one female. And, last, a fourth reason is that the formation of flocks was initiated from the dorsal body surface, where the dermal gland openings are most numerous, whereas the secretion of signalling threads occurs at the genital organs (Witte 1991). As concerns signalling/guiding threads in the Parasitengona, it has been found that the usage of these threads is reduced during the course of adaptation to hygric habitats (Witte 1991; Proctor 1992) because threads produced by non-hygric ancestors of some groups ‘were probably unstable upon contact with water’ (Witte 1991, p. 152). On the other hand, it was shown that in some water mite species, such as Thyas barbigera (Viets), the signalling threads immediately harden after detach- ment from the genital opening (Witte 1991). The same situation probably occurs in the case of L. aquatica, when the secretion material apparently hardens and transforms into long, rigid tubes highly resistant during the long preservation in water and other fixatives such as ethyl-alcohol, which is also shown for pseudoscorpion silk (Annamalai

Downloaded by [University of Cambridge] at 00:01 09 August 2016 and Jayaprakash 2012). Unfortunately, no information is yet available concerning the ultrastructural organisation of the water mite guiding threads, and so no serious comparison is really possible at present. Nearly for the same abovementioned reasons, the found secretion cannot be used for formation of the egg nest (Böttger 1972) that is observed in spring. In any case, the releasing of threads into water should differ from this process in air, as in spiders and insects. In this respect, silk threads of the water spider Argyroneta aquatica (Clerck) are to a great extent comparable with these of L. aquatica by their SEM appearance (De Bakker et al. 2006). Several types of threads in this spider are even generally thinner than threads of L. aquatica. It is assumed that spinning organs in Arthropoda have evolved independently and often de novo in different groups (Vollrath et al. 1996;Craig2003). Nevertheless, in tetranychid mites, the modified prosomal glands opening on the palp tips are known as specialised spinning organs JOURNAL OF NATURAL HISTORY 13

(Alberti and Crooker 1985; Alberti and Coons 1999). In other acariform groups, organs producing silk are still not known with certainty. Pseudoscorpions seem to have even two types of spinning organs – the opisthosomal spinning glands produce signalling threads (Schaller 1971), whereas the cheliceral glands are used in the formation of silken cocoons (Hunt 1970; Annamalai and Jayaprakash 2012). These findings obviously contradict the opinion of Gerson (1985) that in mites and pseudoscorpions webbing is provided by the modified salivary glands, in contrast with spiders where only abdominal glands are involved in silk production (Foelix 1996;Craig1997, 2003). In contrast to other related groups, water mites possess dermal glands – an evolutionary acquisition of this group, its synapomorphic character arising de novo after the mites came back into water. It is quite reasonable to suppose that dermal glands are involved in thread production in water mites (Shatrov 2013, 2014;Shatrov et al. 2014). A comparison of the organisation of spider silk (Stubbs et al. 1992; Vollrath et al. 1996; Vollrath 2000; Sponner et al. 2007; Blackledge 2013) and the ultrastructural characteristics of the water mite threads shows that the latter possess a simpler structure. Threads of water mites are mostly devoid of the core substance that is the inherent characteristic of the spider silk (Vollrath et al. 1996; Sponner et al. 2007; Blackledge 2013) and the regular fibrillar arrangement in the thread wall in spiders (Vollrath et al. 1996; Knight and Vollrath 2002)ismuchlesspronouncedinwater mites. In practice, these water mite threads, in comparison with spider silk, demon- strate an imperceptible elasticity and, on the other hand, a strong rigidity, because it is quite difficult to tear the threads during artificial manipulation, especially in L. undulata (Shatrov et al. 2014). The intimate process of secretion as such and forma- tion of threads remains mostly uncertain both in mites and in spiders (see Vollrath 2000; Knight and Vollrath 2002). Among the possible functions of silk in spiders (Foelix 1996;Krafft and Cookson 2012) and terrestrial mites (Hazan et al. 1974;Clotucheetal.2011; Kanazawa et al. 2011;LeGoff et al. 2011; Fernandez et al. 2012; Yano 2012), none can be positively applied to water mite threads. Because the intensive thread secretion did not con- tinue during the winter period, it may be supposed that threads are not used for the survivalofmitesinthisseasonunderartificial laboratory conditions (in constant temperature but in natural day lighting). Most likely, this type of secretion may be

Downloaded by [University of Cambridge] at 00:01 09 August 2016 a reaction of the mite organism to stress, and, in the most common sense, such a reaction may be referred to a defensive mechanism close in its meaning to a defence reaction from natural enemies – fishes (Kirstein and Martin 2009, 2010). These authors have shown that proteins with a relatively low molecular weight are the main components of the dermal gland secretion in L. aquatica with some traces of hydracarbonates. On the other hand, the capability of the mite threads to give a reaction with a Calcofluor White M2R fluorochrome (Shatrov 2014; Shatrov et al. 2014), the specific dye highly appropriate for revealing of silk (Johnson et al. 2006; Clotuche et al. 2009), may indicate the presence of some amount of polysaccharides in the thread composition. It cannot be excluded that chitin may be present in threads since their walls reveal certain similarities with a cuticle. The function of the thread balls still remains unclear. 14 A. B. SHATROV ET AL.

In general, the revealed phenomenon of production of the fine, hollow, rigid threads by water mites is of great importance. The capillary-like nano-tubes resistant in water and other solutions may be valuable and applied in different fields of modern physics, technology and engineering.

Acknowledgements

The work is realised on the basis of the Centre of Collective Use ‘TAXON’, ZIN RAS, St. Petersburg, Russia, and the Centre of Collective Use ‘CHROMAS’, St. Petersburg State University, St. Petersburg, Russia. We also thank A.E. Tenison and A.A. Mirolubov, engineers of the Department of Electron Microscopy, Zoological Institute RAS, for their qualified assistance with TEM and SEM.

Disclosure statement

No potential conflict of interest was reported by the authors.

Funding

This work was supported by the Russian Foundation for Basic Research [grant number N 15-04- 01203-a]; and the State Federal Scientific Project [grant number N 01201351187].

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