Analysis of contact-rehydration in terrestrial gastropods: absorption of ¹⁴C-inulin through the epithelium of the foot

Terrestrial slugs can recover from dehydration by absorbing water through the integument of the foot while in contact with a moist surface (Dainton, 1954; Prior, 1982, 1984). This process of contact-rehydration has been shown to involve a characteristic behavioural sequence known as drinking (Prior, 1982). Dehydrated slugs move onto a moist surface and assume a flattened posture which they maintain while water is absorbed through the foot. They remain in this posture until they have absorbed enough water to reach their rehydration set-point (see Table 1, Prior, 1984).

The rate of water uptake during the initial minutes of contact-rehydration has been shown to be as high as 20 µl cm⁻² min⁻¹ (see Fig. 3, Prior, 1984; based on the 2-3 min period with a 1·0 g slug). This rate is greater than that predicted from the increase in haemolymph osmotic pressure that occurs during dehydration. This suggested that the permeability of the foot may increase during contact-rehydration. The present experiments were designed to determine if water absorption during contact rehydration is due to water flow through an epithelial paracellular pathway. Support for this view is provided by our demonstration that during contact-rehydration there is absorption of the extracellular marker, ¹⁴C-inulin, into the haemolymph. A preliminary report of these results has appeared in abstract form (Prior, 1982).

Specimens of Limax maximus Linnaeus, 1758 and Lehmannia valentiana (Férussac, 1823) were maintained in the laboratory and fasted as previously described (Prior, Hume, Varga & Hess, 1983). Fully hydrated (i.e. 100% of initial body weight or IBW) slugs or dehydrated (i.e. 60–70%IBW) slugs weighing 1·0–l·5g were placed on wet filter paper (Whatman No. 1 circles, 5’5 cm diameter). The filter paper was saturated with distilled water containing 0·05 mmol l⁻¹ inulin-carboxyl, [carboxyl-¹⁴C] (i.e. ¹⁴C-inulin; 10 µCi ml⁻¹; 5000-5500 Da; New England Nuclear). The test period was started once the slug’s foot made contact with the wet filter paper (usually less than 10 s). After varying periods of time (10-240 s at 18-22 °C) on the wet filter paper, the slugs were rinsed in three changes of water and extracted in 10 ml of 70 % ethanol for 48 h. The slugs were cut into three or four pieces to facilitate extraction of the isotope. Preliminary experiments had shown that the absorbed isotope was readily extractable by this procedure. The slugs were removed from the ethanol, dried for 48 h (95 °C) and weighed (±0·1 mg). Radioactivity in the ethanol extracts was determined using liquid scintillation spectrometry. Appropriate corrections for quench were made. Absorption rates were calculated from the corrected isotope contents of the ethanol extracts and are expressed as µg ^I4C-inulin g⁻¹ ethanol-extracted dry weight per unit time (where indicated).

Large slugs (2–3 g) were used when samples of haemolymph were needed for analysis. The slugs were placed on filter paper containing 0·24 mmol l⁻¹C-inulin (50/tCiml⁻¹) and after 12 min were rinsed in three changes of water. A superior tentacle of each slug was cut and samples of haemolymph (5–30 mg) were collected, weighed (±0·1 mg) and immediately analysed for ¹⁴C content as described above. These slugs were then extracted in 15 ml of 70% ethanol as described above to determine total ¹⁴C-inulin absorbed. Results are expressed as the mean ± S.E. of three to five replicates. Differences were analysed using a Student’s t test and a P value of less than 0·05 was considered significant.

It was initially necessary to determine if during contact-rehydration the slug foot was permeable to an extracellular marker molecule. Slugs that were fully hydrated, or dehydrated to 60–70 % IBW, were placed on filter paper pads saturated with water that contained ¹⁴C-inulin. The dehydrated slugs assumed the characteristic drinking posture and partially rehydrated themselves during the 12 min test period. Haemolymph samples from these slugs contained 6·1 ± 1·3 µg of ¹⁴C-inulin 100 mg⁻¹ of haemolymph (Table 1). The remaining ¹⁴C-inulin that was absorbed during rehydration was readily extracted with 70 % ethanol. There was negligible additional isotope recovered following complete solubilization of the tissues. In contrast, there was no detectable radioactivity in the haemolymph samples from fully hydrated slugs after they had been on the filter paper for 12 min. These results indicate that during contact-rehydration ¹⁴C-inulin can move through the epithelium of the foot into the haemocoel via a paracellular pathway.

The absorption of ¹⁴C-inulin during contact-rehydration was compared to that of the uptake of water over a 4-min period (Fig. 1). The absorption rates of ¹⁴C-inulin by hydrated and dehydrated slugs at 40s were not significantly different. By 60s, however, dehydrated slugs had absorbed significantly more ¹⁴C-inulin than the hydrated control slugs. The progressive absorption of ¹⁴C-inulin by dehydrated slugs closely paralleled the absorption of water over the same period (compare Fig. 1A with IB and 1C). In contrast, the absorption of ¹⁴C-inulin by hydrated slugs did not increase over the 40–240 s period.

The foot epithelium of slugs is composed of a single layer of pyramidal cells which are joined by septate junctions near their microvillar borders (Fig. 2A–D). There are numerous mitochondria near these contacts and on the peripheral aspects of the nuclei. The extracellular spaces between the epithelial cells are continuous with the haemocoel (Fig. 2B, C). While the extracellular spaces are quite apparent in hydrated slugs (Fig. 2B), in dehydrated slugs they are greatly reduced (Fig. 2A). Even though the spaces in dehydrated slugs are difficult to see, after only 4 min of contact-rehydration they are pollen and appear to be continuous with the spaces between the cells in the region of the septate junctions (Fig. 2C, D).

It is well established that lateral or basolateral intercellular spaces represent major pathways for transepithelial ionic diffusion (Schultz & Fromm, 1982). In some epithelia these pathways may also represent a route for water flow in response to osmotic gradients (Schultz, 1977). In rabbit gallbladder, the distension of these spaces is correlated with experimentally induced fluid transport (Tormey & Diamond, 1967; Diamond & Bossert, 1967).

The present results support the hypothesis that in terrestrial slugs water absorption during contact-rehydration occurs through an epithelial paracellular pathway in the foot. It was shown in the preceding paper that contact-rehydration is mediated by integumental absorption of water (Prior, 1984). Because ¹⁴C-inulin and water rapidly accumulate in the haemolymph during contact-rehydration, we are led to conclude that there exists a paracellular pathway through which both water and relatively large molecules can pass.

The correlation between the time courses of absorption of ¹⁴C-inulin and water over the 4-min period indicated that there was movement of the substrate into the slugs by solvent drag (Fig. 1). There was a 50—60s delay before the onset of rapid absorption. This delay may be the time necessary for proper posturing of the slugs or could represent the time needed to initiate paracellular movement of substrate. The data in Fig. 1 do however indicate that ¹⁴C-inulin and water were absorbed together.

Ryder & Bowen (1977) provided ultrastructural evidence for the existence of a paracellular pathway in the foot of the slug, Agriolimax reticulatus. Sections were made of foot tissue from slugs that had been in contact with a substrate containing the extracellular markers, peroxidase and ionic lanthanum. Both markers passed between the epithelial cells and appeared in the zonulae adhaerens, septate desmosomes and in the intercellular spaces central to the septate desmosomes. This extensive system of intercellular spaces was a major feature in Newell’s (1977) description of the ultrastructure of the slug foot epithelium. Our observations have revealed a correlation between the hydration state of the slug and the width of the intercellular spaces (Fig. 2). Most notable was the considerable expansion of the spaces in dehydrated slugs during contact-rehydration. Without the use of histological markers we cannot, however, determine whether the spaces were expanded by redistribution of haemolymph due to the posturing that occurs during drinking behaviour or influx of water from the substrate.

In conclusion, contact-rehydration in slugs appears to be mediated by bulk flow of water through an epithelial paracellular pathway in the foot. Furthermore, the efficacy of the paracellular movement of solutes with water seems to be modulated by the state of hydration of the slug, the pathway being open only ii dehydrated slugs. The characteristics of this paracellular pathway and the mechanisms underlying its control are presently being investigated.

This research was supported by grants from NSF (PCM 78-16088) to GLU, the Whitehall Foundation (DJP) and an NIH-RCDA to DJP. We are pleased to acknowledge the facilities made available to us by Alan Stein and his associates at 803 South. We thank Dr Kook J. Lee for his technical assistance with the electron microscopy.

This is contribution no. 202 from the Tallahassee, Sopchoppy and Gulf Coast Marine Biological Association.