Japan. J. Med. Sci. Biol., 19, 97-104, 1966 ON THE DELAYED PUPATION OF THE FLESHFLY, SARCOPHAGA PEREGRINA ROBINEAU-DESVOIDY TETSUYA OHTAKI Department of Medical Entomology, National Institute of Health, Tokyo (Received : February 19th, 1966) The larvae of Sarcophaga peregrine do not pupate in a wet condition. If they are placed in a glass vessel containing a certain amount of water, their pupation delays for 100 hr or more. The cause of this delayed pupation is neither a direct action of water to their integument nor disturbance of respiration, because the pupation takes place even in a wet vessel, if they have been previously exposed to a dry situation for a certain period. The mature larvae transferred into a dry vessel from a wet one always pupate 18 to 24 hr later. Ligation experiments show that the hormone inducing the pupation is released 6 hr after transference to a dry condition. When the ligation is made in the middle of the animal, behind the brain and the ring gland, the hind part of it readily pupates by an injection of ecdysone. The results of these experiments suggest that retardation of ecdysone release is the final reason for the delayed pupation. It seems likely that their removal from water contact induces the secretion of ecdysone from the ring gland. INTRODUCTION The larvae of a fleshfly, Sarcophaga peregrina Robineau-Desvoidy, which is one of the most common flies of medical importance in Japan, usually breed in carrion or in nightsoil pots. When they attain the full size, they cease feeding, leave the breeding site, and then creep into soil where the pupation takes place about one day later. It has been observed, however, that the pupation is delayed for several days when they can not find a favorable site to pupate. This delay of the pupation occurs both with wild flies in the field and with flies bred under experimental condition. If the larvae, reared with hog liver in a glass jar, are not transferred to a drier situation, the pupation never takes place concurrently throughout the same batch. Some of them may remain mature larvae for ten days or more without pupation. It seems probable that the delayed pupation brought by some environmental condition is owing to a possible prevention of hormone release from ring gland, for it has been well established by several investigators that the puparium formation is induced by a hormone which is produced by ring gland. Hence, the following questions may emerge : what is the real factor responsible for this delayed pupation ; is it due to a temporary absence of the hormone secretion ; and how is neuro-endocrine mechanism included in this prevention. Although it has been reported in Lucilia (Cousin, 1932, Evans, 1935, Mellanby, 1938) and in Drosophila (Takaoka, 1960) that delayed pupation may be caused by environmental conditions unfavorable for pupation, a convincing endocrinological 大 滝哲 也(国 立 予 防衛 生研 究所 衛 生昆 虫 部) 97 98 OHTAKI Vol. 19 explanation is still lacking to elucidate these phenomena. The present paper deals with a series of experiments which demonstrate retardation of pupation by simple contact with water on sarcophagid species. Studies involving some other species of the fly are included and a possible mode of action of neuro- endocrine systems on the delayed pupation is discussed. MATERIALS AND METHODS Materials used were mature larvae of the fleshfly, Sarcophaga peregrine Robineau-Desvoidy, which have been routinely maintained in our laboratory. About five hundred larvae, laid on cotton pads, were transferred to a glass jar (12cm in diameter and 15cm in height), and reared with 400g of hog liver. After three days, the larvae had grown to their maximal size, and ceased feeding. The crop of the larvae gradually became empty and filled with air. In order to get sufficient larvae of equal maturity for these experiments, they were kept in the same jar for the next two days, and a certain amount of water was added occasionally to avoid the drying of their residual food. Under this condition, individuals slow in maturing continued to develop to full maturation, but none of them pupated. After washing the larval body with fresh water and draining it on filter paper, the mature larvae were used for the following experiments: Experiment 1 : To learn the conditions which caused delayed pupation , a number of the larvae were transferred to small glass vessels (9cm in diameter and 6cm in height), which had a filter paper or sand on the bottom. Then, a required amount of water was added to the vessels. After the vessels were tightly covered, some of them were kept under continuous light and the others in complete darkness. Experiment 2 : The mature larvae were kept in glass vessels containing 5 ml of water for additional 48 hr in order to discard exceptional individuals which early pupated. Then, to know whether the dry condition may induce their pupation, the larvae were transferred to the vessels laid with filter paper alone, for varying period (1-12 hr). Experiment 3 and 4: Another group of the mature larvae was kept in a wet condition, the same as in Experiment 2, before using. To half of this group ligatures were applied, and injections of ecdysone were given to the remainder. The ligature was made with cotton thread after the method used by Fraenkel (1935). In the ecdysone test, a tight ligature was applied to the mature larvae at the level behind the brain complex. Immediately back of it, a suitable second ligature was made, and the larvae were divided into two pieces. The posterior half was mounted on a glass needle passing through the second ligature, and 0.002ml of water solution of ecdysone was injected in various concentrations. The assay for this test was the same as that of the Calliphora-test, using a quantitative assay of ecdysone which was described by Karlson (1956). The rearing of the materials and all experiments were carried out at 25•Ž. RESULTS Experiment 1. Delayed Pupation Caused by Simple Contact with Water The mature larvae were kept in the glass vessels containing various amounts of 1966 DELAYED PUPATION OF FLESHFLY 99 water, 2, 5 and 20ml, and in a vessel to which no water was added for control. After they were transferred to the vessels, the number of individuals beginning pupation was counted every hour for the next 120 hr. These experiments were repeated three times. The results obtained are shown in Fig. 1. Fig. 1. The pupation time of the mature larvae in the vessels containing 2ml and 5 ml of water, and filter paper alone. In the dry control*, all larvae pupated during a rather short range of time ; the earliest one started to pupate 18 hr after the transfer. The range of pupation time was a little more extended in continuous light than in darkness. When 2ml of water was poured into the vessel, only filter paper laid on the bottom was wet, and water did not show on the surface. Thus, the body surface of the larvae was not wet except for that portion which was in contact with the filter paper or the glass wall. Even under this condition, the pupation was remarkably delayed, and 50% pupation was not attained within 100 hr. When 5ml of water was poured into the vessel, it rose above the filter paper, and the larval body was entirely wet, but the posterior stigmata were not blocked by the water. Of 30 animals, only two pupated at 110 hr. The larvae in 20 ml of water were completely submerged in it and could not escape from the water. In this case, none pupated during the period of observation. In similar experiments to know whether dry or wet sand prevents the pupation or not, 50g of dried sand was previously placed into each vessel, and 5 and 20ml, respectively, of water were added. For dry control, vessels to which no water was added were provided. The larvae placed on sand with 5 ml of water burned themselves into the moistened sand, and pupated just above the bottom of vessels. In this case, the major portion of the body surface was not in direct contact with water, and their pupation * The humidity of all vessels was considered the same (saturated) as condensation was appeared on the top of all vessels. 100 OHTAKI Vol. 19 took place almost simultaneously, as shown in Fig. 2. Fig. 2. The pupation time of the mature larvae in the vessels containing sand and water. In the case of 50 g sand+20 ml of water, no one pupated during the period of observation. By adding 20ml of water to the sand, a thin layer of water appeared over the sand level, and the larvae could not creep into the sand. The pupation did not occur within 120 hr, showing that the situation was much like that in the vessels with only 5 ml of water. The larvae could not creep into the dry sand either, but most of them pupated in a short period of time. Experiment 2. Induction of Pupation by Drying When larvae with prolonged pupation, induced by contact with water, were transferred to a dry condition, their pupation took place 18 to 24 hr after transfer. If this retardation of the pupation is only due to a simple cause, such as a direct inhibitory action of water contact on the larval integument, the pupation would be prevented so long as the larvae are in contact with water, irrespective of previous drying. Therefore, a series of experiments was attempted to make sure that larvae, which had been subjected to dry condition for a certain period, could pupate under a wet condition.