ABSTRACT
For meal-worms, which are able to survive under extremely dry living conditions, the normal situation is that the K+ concentration of the haemolymph regulates the process of water conservation via the rectal complex (Grimstone, Mullinger & Ramsay, 1968; Koefoed, 1975). The aim of this paper has been to study the handling of sodium and potassium by the midgut epithelium of the larva of Tenebrio molitor and to discover if the transport of these ions is reflected in the structure of the epithelium.
For meal-worms, which are able to survive under extremely dry living conditions, the normal situation is that the K+ concentration of the haemolymph regulates the process of water conservation via the rectal complex (Grimstone, Mullinger & Ramsay, 1968; Koefoed, 1975). The aim of this paper has been to study the handling of sodium and potassium by the midgut epithelium of the larva of Tenebrio molitor and to discover if the transport of these ions is reflected in the structure of the epithelium.
Meal-worms measuring from 1·8 to 1·9 cm were obtained from stock cultures maintained on dry bran. Such meal-worms were considered normal. Their average weight was o-1 g. Freshly moulted larvae were not used.
A piece of the midgut was emptied and within 2 min mounted in the apparatus described in Fig. 1.
The apparatus was mounted on a stereomicroscope and shielded by a grounded ‘Faraday cage’. Five mm Amaranth (Merck. 1248) was added to the solution washing the gut lumen to detect possible leakages.
The Ringer solution (including the Ringer’s with Amaranth) contained : Na, 66 m-equiv/l, and K, 36 m-equiv/1 (both as chlorides), which corresponds to the concentration of these ions in the haemolymph of ‘normal’ meal-worms (Ramsay, 1964). The sucrose content of the Ringer was 166 mm. Samples were taken at 5–10 min intervals: from the lumen side 50μl and from the haemocoel side 150μl. Each time the 150 μl taken from the haemocoel side were replaced. The specimen was weighed on a Cahn Electrobalance. The total potassium and sodium content of the specimen was measured on a flame photometer (Unicam SP 90 B). The extracellular space was measured by addition of [14C]inulin. All the experiments were done at room temperature.
The midgut is circular in cross-section. The epithelium consists of very tall and narrow, uniform columnar cells plus some regenerating cells. The conspicuous basal lamina consists of many electron-dense particles with some space of lesser electron density between. The basal plasma membranes are highly folded, forming long and narrow channels with extremely narrow openings toward the basal lamina. Many of the channels run through most of the cell, having connexions with each other and perhaps with the apical membrane between the microvilli. The cells are joined by desmosomes, septate desmosomes and gap junctions. Mitochondria are associated with the previously mentioned channels, and the cells contain rough endoplasmic reticulum, free ribosomes, Golgi complexes and many lysosomes. This fine structure corresponds to the previously described typical form of midgut epithelium fine structure in more primitive insects with high content of sodium in the haemolymph relative to potassium and with active transport of sodium from lumen to haemocoel side (O’Riordan, 1969).
No significant potential difference across the midgut epithelium was recorded. Measurements of p.d. from ten meal-worms showed an average of 0·4 mV ± 0·2, lumen negative.
No sure signs of survival were found, therefore the total concentration of potassium was measured in pieces of the midgut from the same meal-worm immediately after dissection and after the midgut piece had been set up in normal bathing solution for 3 h. The figures are listed in Table 1. If the midgut was dead the total concentration of potassium in the empty gut could be expected to approach the concentration of the ions in the bathing solution with time. Table 1 shows that this was only found in one case. This gut (no. 3) must be presumed dead.
The extracellular space, expressed as inulin space in microlitres, was much larger on the haemocoel side (mean value, 0·49 μl/mm2) than on the lumen side (mean value 0·095 μl/mm2). The very conspicuous basal lamina (Fig. 2) may explain this difference.
The Na+ was labelled with 22Na on the haemocoel side and 24 Na on the lumen side. The K+ was labelled with 42K on either the haemocoel side or the lumen side of the gut. Table 2 demonstrates a net flux of Na+ from lumen to haemocoel side; since the bathing solutions on the two sides were identical, the transport must be considered to be active. There is no significant net transport of potassium over the meal-worm midgut. Twelve measurements of the flux in each direction gave mean values of 0·24 ± 0·04 μequiv/h/mm2 for K+ flux from lumen to haemocoel side and 0·31 ±0·04 μ-equiv/h/mm2 for K+ flux in the opposite direction. Regarding potassium it was necessary to compare different specimens.
Table 3 shows that the in vitro exchange of cellular sodium must be solely from the haemocoel side, and that of cellular potassium was about twice as fast with the haemo-coel side as with the lumen side. It is a general trait for the insect midgut that the epithelium shows asymmetry with respect to the cellular exchange of both ions and uncharged molecules like amino acids. There is a very rapid cellular exchange with the haemocoel side solution and a slow or non-existing exchange with the lumen solution regardless of the direction of a transport of the ions or molecules. In the midgut of the larvae of Hyalophora cecropia, for example, this is found for K+, which is actively transported over the epithelium from haemocoel side to lumen side of the at (Harvey & Zerahn, 1969), as well as for some amino acids, which are actively transported in the opposite direction (Nedergaard, 1977).
The experiments were done without Ca2+ and Mg2+ in the bathing solutions, but the authors did not find that the p.d. was affected by addition of 5 mmol/1 of Ca2+ and 5 mmol/1 of Mg2+, both as chlorides to the bathing solutions. Altering the Na+ concentration on either side of the epithelium between 0 and 87 μequiv/ml, and the K+ concentration between o and 40 μequiv/ml, similarly had no effect on the p.d.
The only cations in the bathing solutions were sodium and potassium, so that the finding that there was neither significant net flux of potassium nor significant potential difference over the epithelium suggests that chloride may be involved in the transport of sodium.
ACKNOWLEDGEMENT
We thank Professor S. O. Andersen, Professor J. Ringsted and Professor H. H. Ussing for helpful criticism in preparing the manuscript, Dr Leon Pape for correcting the language, and Mrs Britta Kondrup for excellent technical assistance.