Glucose Absorption in the Cockroach

The physiology of absorption from the gut in insects has been little studied. In particular there is a paucity of information on the absorption of nutrient substances from the gut. Much of what is known on this subject is qualitative in nature, and has usually been indirectly derived from specialized investigations on digestion or storage of nutrient materials. The majority of these studies have been adequately summarized in the review by Waterhouse & Day (1953).

In the present investigation the absorption of ¹⁴C-labelled glucose from the gut of the adult cockroach (Periplaneta americana) has been studied, using a dye, Amaranth, as a marker. The dye is not absorbed from the lumen of the cockroach gut and thus the percentage absorption of glucose can be calculated from the glucose/dye ratio in the various parts of the gut. This technique has been recently used by Reynell & Spray (1956a) using phenol red as a marker to study intestinal absorption in the rat. Phenol red is, however, unsuitable for the study of poorly absorbed substances, since these authors were only able to get a 90 % recovery of the dye from the intestine. Amaranth, at least in the cockroach, is not limited in this way, for it was possible to recover this dye quantitatively.

In order to obtain individuals with the gut empty, adult cockroaches were isolated in glass jars and deprived of food for 1 week. They were given access to water except for the 24 hr. immediately preceding the experiment. Each cockroach was then allowed to drink 0·10 ml. of an experimental solution which was offered to it on a small planchette. After an appropriate period the insect was killed by immersion in boiling water, a procedure which appeared to produce no violent movements of the gut. The whole gut was then excised, quickly washed in saline and after ligaturing cut into appropriate portions, each of which was dropped into a known volume of a solution buffered to pH 10-0. These portions were homogenized and centrifuged at 4000 r.p.m. until a perfectly clear supernatant was obtained for analysis.

The experimental solutions contained 0·008M/1. of the dye Amaranth (Azo-Rubrin S) to which were added varying amounts of the test substances used. The concentration of this dye was determined in solution at pH 10.0 using a Unicam absorntiometer at an absorption maximum of 510 mμ.

The ¹⁴C-labelled glucose was assayed using a thin-windowed Geiger-Muller tube (G.E.C. CV 2139). 1·0 ml. of the radioactive solution was pipetted into a small glass container consisting of a short length of 3·7 cm. diameter glass tubing closed at one end by a sheet of 0·00025 in. terylene sheet cemented on with ‘Araldite’. The terylene sheet thus formed the floor of a cylindrical cell which was placed over the window of the G.M. tube. Because of its extreme thinness the stopping power of the terylene sheet was relatively low even to the low energy particles emitted by ¹⁴C.

The insects used in this investigation were reared and maintained during the experimental period at a temperature of 28·0 ± 1·0° C.

The dye Amaranth, which was used as a marker in these experiments, was selected from a number of substances which were tested. In a series of tests it was found that the recovery of Amaranth from the intestine, 6 hr. after feeding measured amounts of the dye, was 99·07 ± 1·80%. It was concluded, therefore, that this dye was not absorbed from the lumen of the intestine, neither was it absorbed in significant amounts on to the gut wall. In the present study the dye has been used to measure transit in the gut and, by comparing the ratio of dye to glucose in various parts of the gut, to determine the net percentage glucose absorption.

In order to establish the site of glucose absorption in the cockroach gut starved adult insects were fed 010 ml. of a solution of 0·008M/1. Amaranth and 0·05 M/1. ¹⁴C-labelled glucose. The percentage absorption in the various parts of the gut has been calculated from the glucose/dye ratio. Fig. 1 shows the amounts of glucose absorbed as the solution reached different regions of the gut after 0·25, 2·0 and 4·0 hr. It is clear that most of the glucose had disappeared from the lumen of the gut by the time the solution had reached the mid-gut caeca. There was no uptake from the crop and there appeared to be no significant absorption as the remaining glucose passed along the rest of the gut.

As the glucose absorption appears to be largely confined to the mid-gut caeca it is clearly of importance to know something of the rate at which the glucose solution reaches these absorptive surfaces. Some light has been thrown on this process by studying the rate of crop emptying. In these experiments starved insects were fed 0·10 ml. amounts of various glucose solutions containing 0·008M/1. Amaranth. These individuals were killed at intervals after feeding and from the amount of dye remaining in the crop the volume of the crop contents was determined. The concentration of the dye in the crop did not change during the experimental period, indicating that there was no appreciable dilution of the crop contents by liquid from the saliva or the mid-gut. Fig. 2 a shows the volume of the crop contents at various times after feeding the insect 0·01 ml. of a 0·11 M/1. glucose solution. If these volumes are plotted on a logarithmic scale with respect to time (Fig. 2b) then a straight line is obtained. Crop emptying is thus an exponential function of time. In this experiment the half-time for crop emptying was 2·73 hr. To study the effect of glucose concentration on crop emptying cockroaches were fed o-io ml. amounts of solutions at varying glucose concentrations. In Fig. 3 a semilogarithmic plot illustrates the rate of crop emptying at glucose concentrations of o·11, 0·55 and 2·77M/1. The rate of crop emptying is thus slower at the higher concentrations of glucose. The half-time for crop emptying was calculated according to the relation ⁠, where v is the initial volume and v, that remaining in the crop after time t. It will be seen that varies linearly with glucose concentration (Fig. 4).

The effect of osmotic pressure on crop emptying was studied by feeding cockroaches with solutions of different substances at similar osmotic pressures. The substances which were compared with glucose in this respect were sorbose, glycerol and sodium chloride. The results, which are summarized in Table 1, show that there was no significant difference in the half-time for crop emptying between any solutions of the substances used. Experiments were also performed to demonstrate the effect of one of these substances on glucose absorption. To do this one batch of starved individuals were fed 0·10 ml. of 0·05 M/1. glucose solution, while a second batch were given a similar solution containing in addition 0·20M/1. sodium chloride. After 1·0 hr. the whole gut was removed and the amount of glucose absorbed was calculated from the amount remaining in the gut. The results are summarized in Table 2.

The above experiments suggested that the rate of crop emptying might have a profound effect on the total glucose absorption by the insect. This effect was investigated by feeding starved insects 0·10 ml. volumes of the dye solution containing ¹⁴C-labelled glucose. After suitable experimental periods the amounts of dye and glucose in the gut were measured. The degree of crop emptying was determined from the amount of dye present in the crop, and the total amount of glucose absorbed was calculated from the glucose recovered from the crop and the remainder of the gut. The results of these experiments, which are illustrated in Fig. 5, show the relation of the percentage crop emptying to the percentage glucose absorbed from solutions at three different concentrations. These results show that a linear relationship exists between glucose absorption and the rate at which the solution leaves the crop.

It is clear that in this insect glucose absorption is confined to the mid-gut caeca, for no appreciable uptake could be demonstrated in any other part of the gut. There has in the past been some controversy as to the part played by the crop in the absorption of sugars. This will be dealt with in a subsequent paragraph.

The rate of crop emptying was shown to be linearly related to the concentration of the ingested glucose solution, so that the amount of fluid leaving the crop decreased with increasing concentration. The total amount of glucose absorbed also showed a linear relation with crop emptying. Thus the limiting process in the absorption of glucose is not the transfer of the substance across the intestinal wall, but the rate at which the liquid is allowed to leave the crop. Such a system would operate to prevent the saturation of any glucose-absorbing mechanism in the gut. This state of affairs can be compared with that found in mammals, where it has recently been shown in the rat that the rate of gastric emptying decreased with increased glucose concentration (Reynell & Spray, 1956b). The percentage glucose absorption was also found to be related to gastric emptying (Reynell & Spray, 1956a).

The effect of concentration of the ingested solution on the crop emptying would seem to be an osmotic effect, for it was shown that the rate of emptying remained constant when different substances were tested at the same osmotic pressure. The cockroach differs in this respect from the rat where the rate of gastric emptying could not be directly related to osmotic pressure (Reynell & Spray, 19566).

An effect resulting from this control of crop emptying in the cockroach is that a substance in solution can affect the absorption of a second substance, by reducing the volume of fluid available for absorption in the mid-gut. It was shown that the addition of 1·17% sodium chloride to a 0·05 M/1. glucose solution resulted in a 60·2% decrease in the amount of glucose absorbed.

It will now be appreciated why many of the early workers (e.g. Sanford, 1918) considered that the crop was responsible for sugar absorption. With their qualitative tests they could not detect appreciable amounts of sugar in the mid-gut, and concluded that it had not reached this part of the gut from the crop. Yet the sugar in the crop clearly decreased and therefore, they argued, it followed that it was being absorbed by this organ. The present investigation has shown that crop emptying is the limiting process in glucose absorption, so that the glucose in the mid-gut is always at a very low level, while there is a continuous decrease in the total amount held in the crop as it passes to the mid-gut. The possibility that this might be the explanation was appreciated by Cuènot (1896, p. 307). It has also been shown by Abbott (1926) that the isolated crop wall was relatively impermeable to glucose.