Oxygen Consumption of Castrated Females of the Blow-Fly, Calliphora Erythrocephala Meig

In a previous investigation on the influence of the corpus allatum on the oxygen consumption of adult Calliphora erythrocephala (Thomsen, 1949), it was found that removal of the corpus allatum from the female fly had the effect that the oxygen uptake was lowered by 24 %.

As allatectomy, when performed on a young Calliphora, also results in an inhibition of the growth of the ovaries (Thomsen, 1940, 1942, 1952), the author realized that the decrease of the oxygen consumption of allatectomized flies might be due to the inhibited growth of the ovaries. If this were the case the corpus allatum could not be said to have a direct effect on the oxygen uptake, but only an indirect one. This possibility might be tested by measuring the oxygen uptake of castrated females. The reason why such experiments were not undertaken at that time was that it was feared that the severe operation would interfere too much with the metabolism. Since then the operational technique has been improved so that by now it is only necessary to make a small incision in order to castrate the flies, and as will be seen in what follows, this incision does not influence the oxygen uptake.

As in the former investigation, the oxygen consumption was measured by means of the Warburg apparatus, and the experiments were performed in the same way and at the same temperature, 25° C. (cf. Thomsen, 1949). It may be useful to stress the fact that, as in the previous experiments, each flask contained a single fly only. The flies used for the experiments were of the same age, i.e. 7 days (except in one case in which they were 8 days old), all the flies used in an experiment having emerged from the puparia within 2–3 hr. They were fed on meat, sugar and water and kept at 25°C.

The oxygen consumption of a total of 102 flies was measured. Of these thirtyseven flies were normal, unoperated females, twenty-nine were castrated females, and thirty-six were operated controls to the castrated females. Of the 102 experiments six were excluded because the flies were exceptionally active. One of these flies was a castrated one, two were operated controls, and three were normal, unoperated females.

In order to remove the ovaries two small incisions were made in the hind part of the third abdominal segment. The wound was sealed with paraffin wax, and care was taken that the neighbouring spiracle was not closed. Similar incisions were made in the operated controls, but the ovaries were not removed. In the case of the normal unoperated female a little paraffin wax was placed on the abdomen. (This was done because the oxygen consumption was expressed per unit of live weight.) The castration was undertaken when the flies were from 5 to 9 hr. old. In all, forty-one flies were castrated and twelve of these (29%) died before the respiration experiment. Of the forty-seven operated controls, eleven (23 %) died. The mortality of the normal unoperated females was 33 %, but this was due to a very high mortality rate in one series only, caused by addition of too much water to the food. Since the flasks containing the flies were submerged in the water-bath, it was not possible to observe the flies continuously during the experiment. As a difference in the activity of the experimental animals and their controls might obscure the result, an attempt was made to measure the basal metabolism, as in the former investigation.

As it was most likely that the resting periods corresponded to the lowest readings, in each experiment the mean of the three lowest rates of oxygen consumption measured over 10 min. periods was accepted as a measure of the basal metabolism. In order to be reasonably sure of obtaining the lowest possible values, all experiments were excluded in which the difference between the three lowest readings of the manometer was greater than 2 mm. Thus since the readings of the manometers usually changed by about 10 mm. for every 10 min. period of ‘basal metabolism’, the three lowest readings were only accepted and averaged if they deviated by less than 20 %. In the previous paper 12 % of the experiments were discarded for this reason, ranging in a single experiment from 4 to 17%. In the present work, the percentage of experiments which had to be excluded for this reason was 27 % for the operated controls, and 18 % for the normal, unoperated females ; for the castrated flies the percentage was only 10. For the operated controls in particular the percentage excluded was very high, and on this account an additional set of calculations was made in which all the readings of each experiment were used. In these calculations the experiments were evaluated in the following way : the cumulative oxygen consumption measured over a 2 hr. period of time with readings every 10 min. was plotted against time. The average rate of oxygen consumption was calculated from the slope of the straight line which to the eye fitted the points best.

After the experiments the flies were dissected, and the developmental stage of the ovaries recorded. The outline of the corpora aflata of the living flies was drawn, and the area calculated (cf. Thomsen, 1942, p. 334). Furthermore, the size of the fat-body was estimated.

The results of the experiments carried out under this heading are summarized in Table 1 and Figs. 1 and 2. It is quite obvious from the figures that there is no significant difference in oxygen consumption between the three categories. It is also to be noted that the results of the present experiments are in good agreement with the earlier work (Thomsen, 1949).

It may therefore be concluded that the oxygen consumption of female flies is not lowered by castration.

It was previously found that the corpus allatum of castrated females hypertrophies (Thomsen, 1940, 1942). This finding was corroborated in the present study. The figures for the castrated females and their operated controls are given in Fig. 3. The size of the corpora allata of the normal, unoperated females was of the same order as that of the operated controls. As removal of the corpus allatum resulted in a decrease in the oxygen uptake it might have been expected that females with enlarged corpora allata would have a high oxygen consumption ; however, as the oxygen consumption of the castrated females—tending to have large corpora allata—and of the operated controls proved to be the same, it is not surprising that no correlation could be found when the size of the corpora allata was plotted against the oxygen uptake. This fact suggests that the hypertrophy of the corpus allatum might be due to a storage of the corpus allatum hormone rather than an indication of a higher activity of the organ.

To our knowledge the only thorough investigation of the fat-body of a castrated insect has been made by Pfeiffer (1945), in her comprehensive paper on the effect of corpora allata on the metabolism of adult Melanoplus differentialis. Therefore, even if we have not as yet studied the effect of castration upon the fat-body of Calliphora females in detail, some rough estimates of the size of the fat-body of these females and their controls might be of some interest.

In Table 2 is given the estimated size and appearance of the fat-body of the castrated females and their controls. In this case the controls comprise both operated and unoperated controls. The group ‘controls T contains all the females with fully mature ovaries, whereas ‘controls II’ comprise the females in which for unknown reasons the development of the eggs was more or less delayed. From many dissections it has been found that even in females with mature ovaries the variation in the size of the fat-body is very great and this is also to be seen from the figures of Table 2 (controls I). The number of females with a hypertrophied fat-body (+ + +) is just as great as the number of females with a depleted fat-body (+), the majority being in between (++). In the castrated females, however, the number of flies with a hypertrophied fat-body (+++) is five times greater than the number of flies with a depleted fat-body (+). Furthermore, 21 % of the castrated females had a greatly hypertrophied fat-body (++++), whereas none of the fat-bodies of the controls with mature ovaries belonged to this group. Admittedly the number of castrated females is rather small, but it looks as if they tend to accumulate more material in their fat-body than normal females.

The group ‘controls II’ only comprises a small number of flies, and therefore one should be cautious in drawing any conclusions from the findings in this group. But it has been observed repeatedly that the fat-body very often hypertrophies when the development of the ovaries is delayed for unknown reasons. A similar tendency is seen in the flies of ‘controls II’.

In all cases in which a hypertrophy of the fat-body was found, it was shown to be due to a great abundance of fat and in some cases of glycogen. A more thorough investigation of the fat-body is planned.

In the previous paper (Thomsen, 1949) there was some discussion as to whether the decrease in the oxygen uptake found in allatectomized females was due to the inhibited growth of the ovaries or whether it was due to the removal of a more general stimulating action of the corpus allatum on the tissues of the female fly.^* However, normal unoperated females and males showed very nearly the same rate of oxygen consumption, and also allatectomy of the male resulted in a decrease in the oxygen consumption. It was therefore suggested that the influence of the corpus allatum on the metabolism of the female fly was most likely a general one.

This supposition has been corroborated in the present study, in which it was found that castration of females does not lower the oxygen uptake.

In accordance with this result no correlation was found between the magnitude of the oxygen consumption and the developmental stage of the ovaries.

How the corpus allatum exerts its effect on the respiratory metabolism cannot be explained for the time being, but must await further investigations. As to the influence of the corpus allatum on metabolism see Wigglesworth (1954, pp. 80–3).

The tendency of the castrated females of Calliphora to contain rather a large amount of stored fat is in accordance with the finding of Pfeiffer (1945, p. 202) in Melanoplus. In Calliphora (Thomsen, 1942,1952), as in some other insects, the fatbody of allatectomized females hypertrophies. Therefore the fact that castrated females may at the same time have an enlarged corpus allatum and a hypertrophied fat-body supports the view that the hypertrophy of the corpus allatum is due to a storage of hormone rather than to a higher activity (p. 694).

However, where the hypertrophy of the corpora allata is brought about by severing of the nervi corporis cardiaci, as in the experiments of Scharrer (1952) on Leuco-phaea maderae, the evidence is in favour of an increase in the activity of the corpora aflata rather than of a decrease.

The experiments of Thomsen (1949) were discussed by Pflugfelder (1952, p. 248) who made the same comments as the author herself as to whether the effect of the corpus allatum on the oxygen consumption was most likely to be a direct or an indirect one. (Unfortunately Pflugfelder did not realize that the measurements of the oxygen uptake were made on single flies). Pflugfelder’s data on Dixippus, mostly obtained on larvae, are somewhat difficult to compare with the data on adult Calliphora. The fact that in allatectomized Dixippus the oxygen uptake decreases just after the removal of the corpus allatum, but increases later on, is interesting because it indicates the possibility that some other organ might gradually take over the function of the corpus allatum. It is very natural to suspect that the pericardial glands or the ventral glands, or both, might be the responsible organs. In the adult Calliphora the side-lobes of the ring-gland (the peritracheal gland), homologous with the above-mentioned glands of Dixippus, degenerate during the first 3–4 days of the imaginal stage, so in the 7-day-old Calliphora, allatectomized when young (i.e. less than 8 hr. old), this organ cannot replace the function of the corpus allatum.

Quite recently L’Hélias (1954) has confirmed the results of Pflugfelder in so far that she found a decrease in the basal metabolism of the fifth stage of Dixippus morosus, allatectomized in the fourth stage, followed by an increase in the last stage.

The fact that the oxygen uptake of castrated females and of females with normally developed ovaries was found to be of the same order is in line with the view that the synthesis of the yolk material is performed somewhere outside the ovaries (presumably in the fat-body). This was originally suggested by Wigglesworth (1943), see also Pfeiffer (1945, p. 211). This idea is likewise favoured by Telfer & Williams (1952) and by Telfer (1954) who stated that a protein, antigen 7, of the ovaries of Cecropia, is probably synthesized by some tissue other than the ovaries.

In this connexion it is interesting that according to Clavert (1953) at least some of the yolk (protein and fat) of the bird’s egg is synthesised in the liver and conveyed to the oocyte via the blood.

We want to thank Dr E. Zeuthen very much for many valuable discussions. It is a pleasure to thank the Carlsberg Foundation, which has supported this work with a grant.