Evidence for Humoral Regulation of Digestive Secretion in the Beetle, Tenebrio Molitor

As a broad generalization it may be said that feeding results in an increase in the midgut enzyme activity of insects (the evidence for this is reviewed by Waterhouse, 1957). When due allowance was made for a spontaneous enzyme increase which occurs after the larval moult or on emergence from the pupa, this was shown to be true of Tenebrio (Dadd, 1956).

Information on the mechanisms whereby feeding might regulate secretion is meagre. Day & Powning (1949) adduced evidence to support the view that humoral regulation might occur in some insects. They cited Tenebrio in their arguments; for, having shown that in cockroaches feeding caused an increase in both enzyme activity and the rate of mitoses in the midgut epithelium, and inferring from this that increased mitosis might be taken as an index of secretory activity, they demonstrated that the injection of blood from fed into starved Tenebrio accelerated the rate of mitosis. Unfortunately similar cross-injection experiments with cockroaches were unsuccessful, and as no attempt was made to study directly the enzyme activity of Tenebrio midguts, the hypothesis that a hormone-like midgut regenerative factor might stimulate enzyme secretion remained tentative.

In choosing to study digestive secretion in Tenebrio one aim was to discover whether a factor in the blood, produced in response to feeding, could affect secretion as well as epithelial mitosis. Supposing that regulation of secretion always involved such a factor, its presence in the blood could also be expected during the period when endogeneous events bring about the spontaneous rise in digestive activity from zero at emergence to a high level some days later. As this spontaneous enzyme activity arises during the final differentiation of the adult (as judged by its gradual hardening and darkening from the soft, white, immediate post-emergent condition) it seemed possible that the initiation of secretory activity might be under the same general hormonal control as adult differentiation.

These considerations suggested that the following experimental approaches might yield an insight into the nature of the mechanisms controlling digestive secretion. First, the experimental situation used by Day & Powning to detect the influence of a blood factor on mitosis could be followed to discover whether enzyme activity was similarly affected. Measurements of enzyme activity were therefore compared in starved adults which had been injected with blood from either fed or starved donors. As an extension of this, it was hoped that the effect of injecting starved insects with blood taken from recently emerged adults at the height of their initial spontaneous enzyme secretion would indicate whether a similar blood factor (should such exist) was present at that time.

A different line of approach arose from the speculation that the initial spontaneous secretion might be controlled by the hormones concerned in the events of metamorphosis and adult differentiation. To illuminate this possibility, the course of enzyme development was followed in insects whose heads had been ligatured off at appropriate times in the late pupa or shortly after emergence, it being expected that such treatment would interfere with the release of cephalically derived hormones concerned in emergence and adult differentiation. By ligaturing off the heads of pupae one day before emergence it proved possible to produce malformed adults in which the normal spontaneous development of digestive enzyme failed to occur. A few experiments were made in which the effect of injecting such recipients with blood from normal post-emergent adult donors was examined. It was hoped thereby to find whether such blood contained a factor which could rehabilitate the secretory activity suppressed by ligation.

The work discussed here formed part of a study, the bulk of which has already been published. A detailed account of the general methods used in defining the developmental and experimental history of individual insects, the estimation of midgut protease, and the assessment of the data obtained will be found in the earlier paper (Dadd, 1956).

In blood injection experiments, adults which had been unfed for either 13 or 16 days from emergence were used as recipients. Blood drawn from starved, recently fed, or newly emerged beetles was injected into the recipients and their total midgut protease was estimated 24 hr. later when they were 14 or 17 days post-emergent. Starved donors were at least 9 days unfed from emergence. Both flour- and cellulosefed donors were used, and were mostly known to have fed (following 14 days starvation) within the 4 hr. preceding the withdrawal of blood. Newly emerged donors were 3 or 4 days post-emergent, this being the time of greatest enzyme increase during the initial spontaneous secretory period.

The following injection technique was adopted. Without etherization, the forelegs of the donor were cut off at the coxae, when drops of clear blood welled out (under ether no such welling occurred). A further copious drop was usually obtained by snicking the membranous cuticle around the genitalia. These drops were sucked into a syringe made by inserting a glass pipette with a fine capillary termination into a convenient length of pressure tubing, one end of which could be held in the mouth and gentle suction, or blowing, applied. The volume of blood was not measured; all that could be obtained from one donor was used for each recipient. The recipient, which had meanwhile been lightly etherized, was deprived of an elytron and wing; under the binocular microscope the syringe was inserted beneath one of the posterior abdominal tergites and the blood was gently blown forward. The wound left by the removal of the wing allowed excess pressure to displace some of the recipient’s own blood. Insertion of the syringe was facilitated if the tip of the capillary was so. fractured as to leave one side projecting as a sharp point. The beetles were considered to have survived this operation satisfactorily if, on the following day when due for enzyme estimation, they appeared normally active. If moribund, or if on dissection the gut appeared damaged, they were discarded.

Insects used in the experiments on cephalic interference were at first merely ligatured with cotton thread at the neck. Later, and in the majority of cases, they were first ligatured loosely, then decapitated, and the ligature finally tightened, as it was found that rupturing of the cuticle behind the ligature was thereby reduced, particularly when dealing with pupae. If such rupture occurred, post-operative survival was short, presumably through loss of blood and desiccation. Both methods of interference gave similar results and are treated together. Successfully decapitated and ligatured beetles survived actively for about 14 days, and pupae operated on the day before emergence moulted (imperfectly, because of the ligature) and survived up to 10 days as active, although variously malformed, adults.

Decapitated pupae never darkened completely after moulting; if they survived longer than a day or two they usually became a light to medium reddish brown colour. Decapitated adults eventually developed various shades of darker brown, or the normal nearly black colour, depending on the time of decapitation.

When decapitated pupae were subsequently injected with blood after moulting, this was done the day following the moult. The double operation resulted in a high proportion of failures (rather more than 50%). Such doubly operated beetles were only used if they appeared active on the fifth post-emergent day when maximal enzyme activity would normally be expected. Doubly operated beetles injected with blood from pupae in mid-instar were used as controls for these experiments, it being supposed that, should a secretion-inducing factor exist, its presence would be less likely in the pupa than at any other stage. This proved to be a doubtful assumption.

Values for total midgut protease of starved adult Tenebrio injected 24 hr. previously with blood from starved, fed, or recently emerged donors are listed in Table 1. The data given for non-injected starved and fed beetles are taken from previously described experiments which were run concurrently.

For starved recipients of both ages (14 and 17 days post-emergent) the mean enzyme values of those injected with blood from donors which had recently fed were about 50 % greater than the means for those injected with blood from starved donors. Increases of the same order were recorded for recipients of blood from recently emerged donors. The magnitude of the difference between starved recipients of ‘starved’ or ‘fed’ blood is thus about one-third that between normal (non-injected) starved and recently fed adults of comparable age.

These results accord well with the hypothesis that regulation of secretion might be mediated by a blood factor occurring spontaneously in adults just after emergence, or later in response to feeding. The comparatively small increases associated with the injection of active blood when compared with those occurring normally in response to feeding may be understood in terms of the probable low concentration of injected factor. A normal concentration could hardly be expected short of a complete replace-ment of recipient’s blood by that of the donor, and it is unlikely that anything approaching this was effected by the technique used here.

It is interesting to note that, although too meagre to be more than tentative, the data for recipients injected with blood from cellulose-fed donors indicate that the occurrence of feeding, rather than the nature of the food, is of importance in activating the blood. This is consistent with the ability of inert foods (cellulose powder or water) to cause an increase in midgut protease activity of the same order as that caused by flour (Dadd, 1956).

Total midgut protease values were determined for adults of various post-emergence ages which had been ligatured at the neck and, in most cases, decapitated at times shortly before or after emergence. The results are presented graphically in Fig. 1.

Decapitation of pupae a day before they moulted quite clearly suppressed the normal spontaneous secretory activity of the emergent adults almost entirely. Of twentyeight individuals thus dealt with, twenty failed to develop measurable midgut protease. The comparatively high mean value shown for the sixth day was due to one insect having an activity of 12 units, perhaps indicative of faulty ligaturing or incomplete decapitation; the remaining seven having detectable enzyme gave values in the range 1− 3 units.

Decapitation after emergence clearly delayed the development of enzyme and reduced the highest level attained to a degree dependent on the time of decapitation. When this occurred within 1 day after emergence the peak enzyme activity was about half normal and took twice as long to develop. Decapitation during the second and third days after emergence caused progressively less interference with secretory activity.

These results support the idea that a factor originating in the head might initiate and subsequently maintain secretion. Further insight into this possibility was sought by injecting newly emerged adults obtained from decapitated pupae with blood from normal post-emergent adults. These experiments were on the whole unsatisfactory, for the double operation killed over half the material, and this implies that those which survived were probably in a grossly abnormal condition. Nevertheless, the results are of considerable interest, although of tentative significance, and are set out in Table 2.

Of thirteen non-injected individuals, nine had no midgut protease, and the remaining four only traces. The mean value for the group was 0·3 unit. The midguts of all except one of eleven individuals injected with blood from normal newly emerged adults developed protease activity. Two had moderately high values (11 and 20 units) and the mean value for the group was 3·8 units. Seven out of nine recipients of pupal blood developed low levels of protease, the mean value for the group being 1·2 units. To the extent that these meagre data have significance, they indicate that the cephalic factor concerned in the initiation and regulation of the secretory activity of the midgut is carried in the blood.

In support of their hypothesis that a blood factor might regulate digestive secretion, Day & Powning (1949) had to rely on a circumstantial argument. This shortcoming is alleviated by the evidence from adult Tenebrio that injection of the blood of fed, But not starved, donors caused an increase in the enzyme activity of the midguts of starved recipients. Moreover, a similar effect followed the injection of blood from recently emerged adults, suggesting that a factor similar to that occurring in response to feeding is spontaneously present during the final stages of adult differentiation.

This last observation has a particular bearing on whether one may envisage either a special hormone concerned solely in the regulation of digestive secretion, or a humoral factor related to, dependent on, or actually one of the well-known hormones which primarily function in the regulation of growth, metamorphosis and adult maturation. The latter possibility seems more likely in view of the spontaneous development of midgut enzyme activity, while adult characters reach their full expression during the few days following emergence. The relationships between the time of decapitation and the extent of both enzyme development and postemergence hardening and darkening support this interpretation.

Because of the well-substantiated influence that the known endocrine organs have on differentiation, and consequently on the rate of mitosis in many tissues, Day & Powning (1949) speculated that the midgut regenerative stimulating factor in the blood might originate from one of them. Their attempts to detect, histologically, heightened secretory activity in the corpora allata and cardiaca of fed cockroaches were negative, but in this connexion the early observation that digestive efficiency was reduced in decapitated Rhodnius is noteworthy (Wigglesworth, 1948). More recently, inhibition of midgut protease activity by removal of brain neurosecretory cells has been demonstrated in adult female Calliphora (Thomsen & Moller, 1959). Morphological evidence (Thomsen, 1954) suggested that the neurohormone might in this case reach the midgut via the nervi oesophagei rather than the blood, and the inhibition of enzyme activity was interpreted primarily as part of a general influence which it appears to have on protein synthesis, for removal of brain neurosecretory cells also inhibits the growth of ovaries, accessory glands, corpora aflata and oenocytes (Thomsen, 1952, 1956). Evidently the effect on digestion is here only part of a complex of physiological events regulated by this hormone.

The idea that digestive secretion might be controlled by the same humoral factors whose balanced interplay governs developmental physiology commends itself for its simplicity, for the postulation of additional, specific hormones merely on the basis of the demonstration of a humoral effect is to be avoided until the possibilities of the well-defined hormones are exhausted, more particularly in view of the widening spheres of influence which extended research attributes to them. Moreover, it helps to account for the relationships which are found between feeding and digestive secretion on the one hand, and feeding, growth, metamorphosis and sexual maturation on the other. It may be envisaged that were feeding to so alter the endocrine balance as to regulate these functions in a nicely integrated way, food would most conveniently be made metabolically available for utilization at the appropriate stages of growth, development and reproduction.

This does not exclude other more specific means of digestive regulation. An arrangement of the foregoing sort is feasibly applicable to Tenebrio and those Orthoptera whose secretory responses are comparatively gradual (Waterhouse, 1957), but could clearly not account for the immediate secretory response to feeding found in the carnivorous beetle Dytiscus (Duspiva, 1939; Dadd, 1956). It is to be expected that in carnivores which take food intermittently, dependent on the vagaries of finding prey, a more precise regulation of secretion must obtain, perhaps under direct nervous control or brought about by a direct response to food or secretogogues therefrom. A mechanism of the latter type seems most likely in the mosquito Aedes, for whereas no evidence of a postprandial, blood-borne, humoral stimulus to secretion was found, a differential secretory response to various food substrates occurred (Fisk, 1950; Fisk & Shambaugh, 1952, 1954; Shambaugh, 1954). In these cases where food would not normally be always available, or where quite different foods are taken for different physiological purposes, it is not to be expected that digestive secretion should be closely integrated into the developmental physiology of the insect.