The brain and subesophageal ganglion (BR-SG) of the commercial silk worm, Bombyx mori, were stained immunohistochemically at the larval stage for circadian clock neurons with antibodies against Doubletime (DBT) of B. mori and Period (PER) of Periplaneta americana. The BR-SGs were also stained with antisera against [Arg7]-corazonin, which has been known to be present in B. mori and co-localized with PER in Manduca sexta, and against [His7]-corazonin, a homolog identified in other species. From co-localization of [Arg7]-corazonin and PER-like reactivities in the pars lateralis, [Arg7]-corazonin is suspected to be a downstream regulator of the circadian clock in M. sexta. DBT- and corazonin-like immunohistochemical reactivities were found in both the neurosecretory cells of the pars intercerebralis (PIC) and pars lateralis (PL) in B. mori. Small numbers of neurons shared both reactivities against anti-DBT and anti-corazonin. The majority of the immunopositive cells were common to both corazonins, but some cells were unique in expressing either reactivity against [His7]-corazonin or [Arg7]-corazonin only. The results suggest that there is a diversity in the clock output pathway among lepidopterans and that [His7]-corazonin may be present in B. mori, as well as [Arg7]-corazonin, although the former has not been chemically identified in this species. Corazonin may be a downstream regulator of circadian clocks in B. mori because of the co-localization of [His7]-corazonin at PIC and [Arg7]-corazonin at PL with anti-DBT.
Heartbeat reversal patterns have been monitored in the body of diapausing pupae of M. sexta 2 h before and 3 h after the injections of [Arg7]-corazonin, using noninvasive thermographic and optocardiographic methods. Large dosages (10-6 M final concentrations of corazonin in the body) caused almost immediate, adrenaline-like enhancement of the anterograde heartbeat. During the relatively short, acute phase of the tachycardia induced by corazonin, the systolic anterograde contractions of the heart increased in average from 10.5 to 24 pulses per min, culminating at 2.5 min after the injections. Duration of the acute period of tachycardia was only 7 to 20 min, which was followed by a period of slightly elevated, residual anterograde heartbeat which persisted occasionally for 1 to 3 h. Smaller dosages of corazonin (10-7M concentrations in the body) occasionally also produced a less intensive cardiotropic effect, while the more diluted samples were completely inactive. In pupae of the beetle T. molitor, injections of corazonin (10-6 M in the body) had no effect on the rate of in vivo heartbeat at all. Pharmacological analysis of the effects of corazonin in M. sexta indicated that the cardiostimulating effects of corazonin did not conform with the expected action of a peptidic neurohormone. A possibility that these effects might be artifacts produced by the low molecular breakdown products of corazonin has been discussed.