Studies on Insect Metamorphosis: IV.—Effect of Carbon Dioxide

The problem of how the apparently healthy organs of the larva of the holometabolic insects, at the height of their maximum growth, become disintegrated and the rudiments of the imaginal structures, dormant up to this time, begin proliferating and developing into functional organs, is a very interesting one and has attracted the attention of numerous physiologists and entomologists. Bataillon (1893), as a result of his elaborate researches, established that there was an accumulation of CO₂ in the blood and body cavity of meta-morphosing silkworms. Previously, he (1891) had observed the same state of things in the Anura. Bert (1885) had previously noticed respiratory abnormalities in silkworms ready to pupate. On the basis of these observations Bataillon advanced the theory that in the metamorphosis of insects the first event is the destruction of larval organs, i.e., histolysis, and that this is brought about by the suffocation effect of the accumulated CO₂ Terre (1900) endorsed this view, while Perez (1903) and Mercier (1907) were sceptical about it. In order to test the asphyxia theory of Bataillon I carried out the following experiments:–

1. 220 larvæ of the Blowfly (Calloplzora erythrosephata), which had almost ceased feeding and were ready to pupate, were divided into two lots of 110 individuals each. One series was put in a jar (6” high and 7.5” in circumference) into which 500 c.c. of CO₈ (at N.T.P.) were introduced, after which the mouth of the jar was at once closed; thus the larvæ were practically in an atmosphere of almost pure carbon dioxide. The other series was put in a jar of the same capacity as the above, but containing no CO₂ and with its mouth uncovered-this served as control.

The above experiment was started on 14/2/25 at 10 a.m. As will appear from Table 1., on the 16th, while there were 19 pupaæ in the control, there were only 5 in the CO₁ jar. On the 17th (10 a.m.) there were 95 pupæ in the former, and I 3 only in the latter. By the evening of the same date all the individuals of the control series completed pupation, while only 1 5 of the CO, lot had changed. During the next six days only 6 more individuals of this series metamorphosed. On the 24th the lid of the CO, jar was removed for twenty minutes ; many larvæ were found clinging to the lid near its margins. After this dilution of. COs, as compared with the previous days, many more individuals began to pupate—on the 25th the total number reached 52, on the 26th it was 70. On the 27th the number was 85, and on the 28th 97; thus from day to day, there was again decrease in the number of pupæ formed, so much so that on the 3rd March the total reached only 100; on this day the lid was removed permanently, and the next three days brought the number to 107, the remaining 3 did not pupate at all.

Thus, evidently, suffocation instead of accelerating meta-morphosis, which it should have done according to the asphyxia theory of Bataillon, retarded it to a great extent.

II. In another experiment, 50 full-grown larvæ were put in a jar with the same concentration of CO₂ as in the above experiment, but in this case the mouth of the vessel was made almost air-tight. In an interval of three days (Table II.), while 49 out of 50 individuals kept in the normal atmosphere (control) pupated, none of the CO₂ lot metamorphosed. Then the lid of the CO, jar was taken off for twenty-four hours; after this the larvæ began to change and 48 finished pupation within the next five days; the remaining 2 died.

III. In the third experiment, in three jars containing 35 larvæ each, 300 c.c., 200 c.c., 100 c.c. of CO₂ respectively were introduced. A fourth jar with the same number of individuals but without any CO₂ in it served as control. The experiment was started on 18/2/25 (Table III.). By the 20th (4 p.m.) all the larvæ in the control metamor-phosed, in the CO₂ jars there were 3, 1, 1 pupæ in 100 c.c., 200 c.c., 300 c.c. jars respectively. On the 22nd (1 p.m.) the totals in these jars reached 34, 29, 25 respectively. The last larvæ in the 100 c.c. jar and the last 6 in the 200 c.c. jar pupated during the night between the 22nd and 23rd; the larvæ in the 300 c.c. jar finished pupation on the 23rd at 4 p.m. ; one of this lot died.

These results indicate that the degree of retardation of metamorphosis depends upon the concentration of CO₂ m the surrounding atmosphere.

The results of the above experiments entirely contradict the asphyxia theory of Bataillon. It is not implied that his observations about the greater amount of CO₁ in the body of the pupating larvæ was not correct, for recently Bishop (1924) has also shown that there is greater tension of C0₁ in the spinning larvæ of the Bee (Apis sp.). However, if one closely follows the descriptions of these authors it appears that the maximum amount of CO₂ noticed is on the day the individual pupates (Bataillon, p. 34 and Curve IV.). But it is now generally admitted that metamorphosis in the real sense of the word, i.e., the beginning of changes in the internal organs, starts a few days before the individual actually pupates; in a previous communication (1925) it was shown that the insect larvæ, after attaining maximum growth, begin to lose weight, indicating the beginning of histolysis inside the body, and this they do a few days before they change into pupre. If the accumulated CO₂ is the cause of metamorphosis, this accumulation must be shown to occur just when, or a little after, the larvæ attain their maximum weight. An accumulation just before actual pupation, when histolysis is in full swing, cannot be considered the cause of this phenomenon, it seems to be rather its by-product.