1. Ecdysone is in a highly dynamic state after its injection or its secretion by the ring-gland of Sarcophaga peregrina. Hormonal activity is rapidly destroyed by an inactivating mechanism which is present in the tissues but not in the blood. 2. Inactivation is blocked by low temperatures or anaerobic conditions-a finding that implicates chemical and, more particularly, oxidative reactions. The mechanism in question could be demonstrated in larval fragments but not in crude or fractionated homogenates. 3. When injected into mature larvae, 1 µg of α-ecdysone loses 50% of its activity in 1 hour and 98% in 8 hours. Lower doses show even briefer "half-lives." 4. The rapid inactivation of ecdysone can account for its low titer in both the blood and tissues. Thus at the "critical period" for puparium formation, the entire larva contains only 2.5 nanograms, corresponding to only 7% of a Sarcophaga unit. 5. The evidence points to the accumulation, not of the hormone itself, but the covert biochemical and biophysical effects of the hormone. The covert effects undergo spatial and temporal summation within the target organs and finally discharge the overt developmental response. 6. The role of the blood is to serve, not as a reservoir, but as a pipeline through which ecdysone flows from the ring-gland to its sites of action and swift inactivation.

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.

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A continuous cell line was established from embryonic tissues of the fleshfly, Sarcophaga peregrina, and was designated as NIH-SaPe-4. The primary culture was initiated in October, 1977. and the cell line was passed 68 times during the following year. The cells were heterogeneous in morphology. Most cells were diploid and their chromosomes consisted of 4 metacentric, 6 submetacentric and 2 micro chromosomes. The population doubling time of the cell line was about 30 hr. The cells grew faster in Mitsuhashi-Maramorosch's medium than in Schneider's medium. The cells were either stored in the usual medium at 5°C for about 3 months, or in a medium containing 10% glycerol at -880°C for a longer period. Cell growth was suppressed by 20-hydroxyecdysone when at a greater concentration than 0.01 ,ug/ml, whereas insulin showed no effects on cell growth at a strength of 0.4 and 0.04 IU/ml.

The temperate race (D) of S. peregrina undergoes pupal diapause in response to certain environmental conditions of photoperiod (13L:11D-11L:13D) and temperature (under 20 degrees C). The tropical race (ND) does not do so under the same circumstances. The tendency toward diapause was suppressed 30-40% of the hybrids of crossings D female x ND male and ND female x Dmale even under such short day and low temperature conditions as 11L:13D-9L:15D, 20 DEGREES C. For entering diapause, the hybrids rrequire a shorter day length (13L:11D) than that of (D) parents (15L:9D).

Using the immunoblot technique, we analyzed the quality and quantity of IgG, IgG4, and IgE specific to mosquito salivary gland (hereafter abbreviate as SG) components of Aedes albopictus in the sera of volunteers with common reactions and of 3 patients with severe reactions. In the volunteers with delayed reactions only or with both delayed and immediate reactions, IgG against SG components of A. albopictus formed several faint or moderately stained bands. Those with immediate reactions showed several intense bands and many other weak bands. In volunteers, who had been bitten by Aedes sp. frequently but had no skin reaction, and in severe cases, many intense IgG bands were observed. IgG4 bound to SG components were found in the sera of the common reaction group at the levels of 24 and 48 kD, but, in one severe case, no bands were observed, although the total IgG was very high. IgE levels specific to SG components were much higher in severe cases than in the volunteers. These results indicate that high titers of specific IgG and IgE and lack of IgG4 for particular components of SG may lead to severe allergic reactions in severe cases. Immunoblotting analysis of the antibodies also verified the possibility of developing in vitro tests to identify causative species of the mosquito for severe cases.

An in vitro study in which isolated prothoracic glands of the Bombyx silkworm were cultured has provided definite evidence that the prothoracic gland is the site where molting hormone is synthesized. The hormone behaved very similarly to free ecdysone on thin-layer chromatography. Analysis by liquid chromatography and mass fragmentography revealed that the hormone is identical with alpha-ecdysone.