Characterization of the Arion vulgaris pedal gland system

Abstract The most common European gastropod species, Arion vulgaris, is one of the most troublesome pests for private garden owners and commercial agriculturists. The sticky and hard to remove secretion produced by these animals allows them to overcome most artificial and natural barriers. However, this highly adherent biopolymer has recently shown great potential for novel wound‐healing applications in medicine. Nevertheless, our knowledge of the underlying gland system is still limited and few studies on the ventral gland system are available. We studied the lateral and ventral pedal glands in Arion vulgaris to determine their secretory content histochemically and through lectin assays. Using these histological and histochemical methods we differentiate five gland types with different mucus composition in the lateral pedal region of the foot of Arion vulgaris. These contain sulphated and carboxylated mucosubstances (positive Alcian blue staining) but lack hexose‐containing mucosubstances (negative PAS staining). In the ventral pedal region, four gland types can be differentiated producing sulphated and carboxylated mucosubstances. Within the ventral mucus, a high affinity for the lectins PNA and WGA is observed. While the lateral glands are histochemically negative for PAS, a positive staining with the lectin JAC is observed. Arion vulgaris shows clear morphological differences from other arionid species. This raises the question whether the variation in the chemistry of the secretory material and mucus composition is the result of different functions and/or is related to the animals' different environmental conditions. A comparison of some glands of Arion vulgaris with those of the helicid species Helix pomatia and Cepaea hortensis indicates morphological similarities.

Although gastropod mucus appears to offer a range of industrial applications, surprisingly little is known about the mucus-producing glands in this group of molluscs. In an earlier study on the pedal gland system in the helicid species Helix pomatia and Cepaea hortensis, we reported differences concerning gland number, size, and content (Greistorfer et al., 2017;von Byern et al., 2018). While in Helix three different glands were observed in the dorsal epithelium (Greistorfer et al., 2017), in Cepaea four glands could be distinguished in the same location (von Byern et al., 2018). The glands also differ histochemically between the two species: Compared to Cepaea, Helix glands contain different sugar moieties and lack basic proteins. In both species, two ventrally located gland types were described that are positive for acidic glycoproteins only, but with different sugar moieties (Greistorfer et al., 2017;von Byern et al., 2018).
With this study, we extend our investigation of the gastropod pedal gland and aim to characterise Arionidae. These gastropod group lack the protective shell of helicid species and therefore secrete an extremely stiff mucus when threatened by predators (Mair & Port, 2002;Martin & Deyrup-Olsen, 1986). However, large differences in their mucus-producing glands are known to exist both between and within species: In Arion ater, two (Barr, 1927) to three (Wondrak, 1967) types of glands have been described in the ventral and lateral epithelia. In the related species Arion rufus, four ventral glands have been found (Chétail & Binot, 1967), but these authors did not provide any details on the lateral subepithelial glands in their Arion species. More recently, Wondrak (2012)  4. An anterior part of the ventral mantle, reactive to Alcian blue, while Safranin O stains only part of the granules. This gland reacts to none of the applied lectins. According to Wondrak (2012), the latter two gland regions might be involved in the production of the lateral mucus, although they are ventrally located.
Although previous examinations of the pedal gland system in the Arionidae provide a good overview of the number of ventral glands, the gland system of Arion vulgaris has only been marginally examined. The aim of the current study of the lateral and ventral pedal glands in Arion vulgaris is to determine their secretory content histochemically and by lectin affinity tests. A comparison with earlier studies on Helix pomatia and Cepaea hortensis (Greistorfer et al., 2017;von Byern et al., 2018) is expected to show structural and chemical differences in the gland systems and mucus of these three common European terrestrial gastropods.

| Morphology
For the ultrastructural characterization, two samples were immersed in 2.5% glutaraldehyde buffered with sodium cacodylate (0.1 mol l −1 , pH 7.4, plus 10% sucrose) for 5 hr at room temperature. Afterwards, the samples were washed in the same buffer, post-fixed for 1 hr in 1% osmium tetroxide (again in 0.1 mol l −1 sodium cacodylate buffer at pH 7.4), stepwise dehydrated, and finally embedded in Epon epoxy resin (AGAR 100, Co. Agar Scientific Ltd, United Kingdom). Polymerisation took place at 60 C for 3 days.
Semithin sections (1 μm thickness) of the resin samples were cut with a Leica UC7 ultramicrotome (Co. Leica Microsystems GmbH, Germany) and stained with Toluidine blue. Ultrathin sections (70 nm thick) were made with an ultra-diamond knife (Co. Diatome AG, Switzerland) on a Leica UC7 ultramicrotome, mounted on copper grids, stained with Richardson solution, and visualised with a Zeiss Libra 120 transmission electron microscope (TEM) (Co. Carl Zeiss AG, Germany) at 120 kV.
For scanning electron microscopy (SEM), two animals were frozen in liquid nitrogen, freeze-dried (Mod. LyovacGT2, Co. Leybold-Heraeus GmbH, Germany), coated with gold in a sputter coater (Mod. 108, Co. Agar Scientific Ltd, UK), and observed with a scanning electron microscope JEOL IT 300 at 15 kV. For histochemical characterization two samples were fixated and proceeded as explained in Greistorfer et al. (2017).
To visualise the glands in the dorsal and ventral epithelium a schematic drawing was made using Illustrator CS6 (Adobe Systems, San Jose). All measurements, including the gland dimension, were made with Photopshop CS6 (Adobe Systems, San Jose). The gland length was measured on semithin sections (1 μm), the granules within the glands and the dimensions/height of the epithelia were measured on ultrathin sections (70 nm thick). Every gland was measured from the opening of the duct to the bottom of the gland. Only glands with complete extension were taken. The granules were measured from top to bottom and from right to left. For every final data, six measurements were taken and then the mean value was calculated.
To give a good overview and compare the glands to each other these measurements of the glands/granules were made. A detailed statistic evaluation is missing, because the gland size in different body regions varied a lot and it was not possible to get sufficient measurements. To keep the measurement inaccuracy low, only glands with an open duct were measured.

| Mucus chemistry
For the chemical analyses of the pedal glands, samples from all regions of the foot (anterior, mid-region and posterior) of two animals were fixed in Carnoy's solution (Kiernan, 1999) for 3 hr at 25 C and then sectioned into 1 mm slices with a vibratome (Leica VT 1200S, Co. Leica Biosystems GmbH, Germany). Afterwards, the samples were cleared in methylbenzoate, transferred to benzene, and infiltrated overnight with paraffin. Sections (5-7 μm thick) were cut with a rotary microtome (Leica RM2265, Co. Leica Biosystems GmbH, Germany), mounted on glass slides with Ruyter fluid (Ruyter, 1931) and dried at room temperature before use. Samples of the ventral mucus were collected by having four individual species crawl over glass slides until it was fully covered. The lateral mucus samples were collected by attaching the glass slides to the animals' bodies, again until it was fully covered. All collected mucus samples were air-dried overnight before staining. Following the methodological description of Greistorfer et al. (2017), histochemical analyses included the detection of hexosecontaining mucosubstances by periodic acid Schiff (PAS) staining.

T A B L E 1
Summary of the different lectins tested on the trail mucus as well as the lateral and ventral glands of Arion vulgaris    Welsch, 2010), and at pH 6, 8, 9.5, and 10.5 for basic proteins using Biebrich scarlet staining (Kiernan, 1999;Spicer & Lillie, 1961). A combination of Alcian blue and PAS staining was used to verify the presence of acidic glycoproteins, while Safranin O staining (Böhm & Oppel, 1919) was applied to confirm polyanionic proteoglycans in the glands.

| Mucus element analysis
For the histochemical analyses, fresh lateral and ventral mucus from four individuals was collected on standard SEM stubs, consisting of aluminium and copper (Co. Gröpl, Austria) until the whole stub was covered. All samples were then air-dried and analysed using energy dispersive x-ray spectroscopy (EDX) with the x-ray microanalysis soft- F I G U R E 1 Arion vulgaris, schematic overview of the pedal gland system. The lateral epithelium is covered by a microvilli layer (mv), while ventrally a ciliary border (ci) is additionally present in the periphery and centre of the sole. Laterally, five gland types (A1l, A2l, A3l, A4l, and A5l) are present, which differ in their secretory content (see inserts). In the ventral region, the gland types A1v, A2v, and A3v are frequently present, while A4v occurs mainly in the cilia-free periphery of the sole. The gland type At only occurs in the peripheral groove (marked by a red arrow), the transition region between the lateral and ventral epithelium. Scale bar in main image = 250 μm, in the inserts A2l, A4l, A1v, A2v = 1 μm, and in the inserts A1l, A3l, A5l, A3v, A4v and At = 0.5 μm

| Gland terminology and imaging
According to the gastropod gland nomenclature of Smith (2006), the gland cells were named as follows: The first letter of the genus name Arion, a sequential number, and the acronym "l" for lateral or "v" for ventral.
As done earlier for Helix pomatia (Greistorfer et al., 2017) and This sole centre is the part of the arionid foot which clings to various types of slippery substrate.
All gland types of the foot of Arion vulgaris are unicellular, subepithelial, and embedded in connective tissue. Their nucleus is situated laterally or centrally in the basal area of the gland. Laterally, five different gland types (A1l, A2l, A3l, A4l, and A5l) can be differentiated by their appearance (Figure 1), size, and secretory content, while ventrally four different glands (named A1v, A2v, A3v, and A4v) are observed. In the transition region, one gland type (At) is present.
Throughout the lateral epithelium and the subepithelium, yellowbrownish pigments can be observed, but these are absent ventrally.

| Lateral gland morphology
A1l is a common gland type, frequently present in the lateral area of Similar to A1l, the A5l glands also appear large in size (approximate length ≈161 μm) and are commonly distributed, but not as frequently as A2l (Figure 2a). These glands are filled with fine and homogeneous granular material (Figure 3d) which does not appear as tightly packed as the granular material of A1l, A3l, and A4l.
The gland type At is only observed in the transition region between the lateral and ventral areas of the body surface (Figure 1).
With a length of ≈38 μm, it is the smallest gland type found in the Arion vulgaris pedal system and contains roundish granules of 1 μm in diameter, which aggregate near the apical pole (Figure 3f).

| EDX-analyses of the lateral mucus
The EDX measurements indicate the presence of carbon (51-57 at.%), nitrogen (11-17 at.%), and oxygen (19-26 at.%) in high amounts in the lateral mucus (Table 2). For the elements chlorine (1-7 at.%, locally crystals contain values above 40 at.%) and potassium (2-8 at.%, locally crystals contain values above 44 at.%), different range values could be measured as these elements are unevenly distributed in the sample (Fig-ure S1a-c). Sodium, magnesium, phosphorus, sulphur, and calcium are also present in the mucus, however, at very low concentrations.

| Ventral gland morphology
Gland type A1v (≈82 μm long) is columnar-shaped with a narrow duct towards the epithelium surface (Figure 2b). Its granules appear irregular in size (length up to 4 μm) and are stratified like "ice-floes"; the content is homogeneous and enclosed by an electron-dense membrane ( Figure 3g). confirms the presence of sugar in the connective tissue (black arrowheads) but not within the lateral glands (e.g., gland type A3l, marked by a black asterisk). The black arrows mark the yellow-brownish pigmentation. (b) With Safranin O staining, the larger gland type (gt, which refers to a non-identified gland type as a correlation with one of the five potential gland types failed) shows no reactivity to glycosylated proteins (mucus), as given by the bluish-green background staining. (c) With Alcian blue staining (here shown for the pH level 1.0), some of the larger gland type (gt) and its duct (black arrowhead) react for acidic proteins. The black arrows mark the pigments within the lateral region, and the black asterisks point out gland type A3l, which show no reactivity to acidic mucosubstance staining. (d) With Toluidine blue at pH 4.3, the contents of large (gt) and small gland cells (white arrows) are strongly stained. Furthermore, a gamma metachromasia (green staining, black #) is visible in some smaller subepithelial glands. Scale bars in images a to d = 100 μm Gland type A2v is of similar shape and length (≈87 μm) as A1v and neighbors it ( Figure 2b). However, the granular material of A2v clearly differs by having roundish, electron-translucent granules (≈2 μm) with densely scattered inclusions (Figure 3g).
Gland type A3v is tubular to goblet-shaped with a length of ≈75 μm (Figure 2b). Its granules are of the same diameter (≈2 μm) as those of A2v, but they are polygonal in shape and tightly packed within the gland. The content is moderately electron-dense but lacks darker inclusions (Figure 3g).
Gland type A4v can only be observed in the cilia-free peripheral region (Figure 2b). It is the longest gland (≈127 μm) in the ventral region and appears pear-shaped. Its secretory material is finely granulated, but not as homogeneous or dense as the content of A2l and A5l ( Figure 3h). Beside UEA II, also a small affinity to STL (specific for Nacetylglucosamine) (Table 1) could be confirmed in the gland region.

| Ventral gland chemistry
All other applied lectins showed no clear affinity with the gland or its secretion.

| Lateral subepithelial gland system
Published data varies concerning the number of epithelial mucus gland types, not only in arionid species but Arionoidea in general. Moreover, the lateral gland types are frequently disregarded as they do not contribute to its locomotion or adhesion, nor to shell building, as has been described for the dorsal glands of helicid species (Campion, 1961;Greistorfer et al., 2017). Summarising the data from the literature (Table 3), the current study confirms five gland types located laterally in Arion vulgaris, although in other arionid species only four (Limax ecarinatus, Cook & Shirbhate, 1983), three (Limax maximus, Herfs, 1921) or two gland types (Arion ater, Barr, 1927;Wondrak, 1967;Lehmania poirieri, Arcadi, 1967;Meghimatium fruhstorferi, Yamaguchi et al., 2000) have been observed.  (Chétail &Binot, 1967), A. vulgaris, andA. rufus (Wondrak, 2012) and the oval-shaped gland type of Limax ecarinatus (Cook & Shirbhate, 1983). No information about such a gland type or its chemical content has been presented for the other species nor its involvement in mucus formation been confirmed.

| Ventral subepithelial gland system
While in helicid species only two gland types are classified ventrally (Table 2), in some arionid species up to five different gland types have been documented (Table3). In contrast, Limax maximus and L. poirieri contain only one gland type in the sole epithelium (Arcadi, 1967;Herfs, 1921). As in the case of the lateral gland types, the ventral glands also differ with regard to their secretory content. In most spe- Some lectins (PNA, WGA) did not show an affinity to the lateral mucus but a reactivity in the glands. It could not be excluded that the lectin amount in the mucus was lower than in the gland cells.
In arionid species, it is proposed that the ventral (trail) mucus for locomotion is not only produced by the ventral gland types, as been supposed for the helicid species (Greistorfer et al., 2017;von Byern et al., 2018). Additionally, the suprapedal gland system, located within the ventral body cavity, contributes to the secretion of trail mucus (Barr, 1927;Wondrak, 2012). The system contains only one gland type, which stains positive for sulphated and carboxylated acidic mucosubstances and specific sugars moieties such as galactose (lectin GSL-1 B4 and lectin RCA) and N-acetyl-α-D-galactosaminyl (lectin Helix pomatia), but is negative for PAS staining (Wondrak, 2012). In our study, we did not re-examine the suprapedal gland, but focused on the weakly characterised subepithelial gland system.
Beside similarities to other arionid species, the Arion vulgaris specimens also share similarities to the gland morphologies of helicid species like Helix pomatia and Cepaea hortensis ( Table 2) Cepaea (as well as in Helix), the lectin GNA shows affinity to the ventral mucus. Dorsally, the helicid gland types likewise react to acidic mucosubstances (Helix and Cepaea) or basic proteins (Cepaea only; Greistorfer et al., 2017;von Byern et al., 2018), likely serving as hydrogel-like lubricants that reduce the friction force between the soft skin and the hard shell (Herfs, 1921;Werneke, Swann, Farquharson, Hamilton, & Smith, 2007). In Arion, only acidic mucosubstances could be detected laterally, suggesting a defence system against predators or bacteria, as discussed earlier (Barnhart, 1983;Pawlicki et al., 2004).
In a detailed report, Campion (1961) described how helicid snails produce and secrete calcium to build their shell and epiphragm. Distinct calcium depots can be observed among the dorsal glands in Helix and Cepaea (Greistorfer et al., 2017;von Byern et al., 2018). On the other hand, calcium and metals like iron or copper or other elements (e.g., potassium, sulphur, phosphorus) could also be detected in the mucus of terrestrial gastropods such as Arion subfuscus (Braun, Menges, Opoku, & Smith, 2013) and marine species such as Lottia limatula (Smith, Quick, & St.Peter, 1999). These elements are not only involved in shell formation, but also play a role in the cross-linkage and viscosity of the mucus (Pawlicki et al., 2004;Werneke et al., 2007), as described for the Mytilus byssal system (Waite, Holten-Andersen, Jewhurst, & Sun, 2005). More precise analyses with ICP-MS are necessary to study the presence of heavy metals (iron, copper, manganese) in the presently investigated Arion mucus, as measured for A.
subfuscus (Braun et al., 2013), since the performed EDX measurement may not be sufficiently sensitive.
Currently, the high levels of potassium and chlorine found locally

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