Uncoupling of sequential heteromorphic developmental programs

The regulatory basis for differences among species in the developmental rate at which successive life stages are reached ("heterochrony") is a subject of much controversy among vertebrate and invertebrate developmental biologists. The heterochrony in development of different insect species is characterized in part by the intercalcation between the embryo and adult of a (varied) number of heteromorphic larval instars. These heteromorphic larval instars exhibit changes of body form from one larval instar to the next, prior to the final metamorphic molt to the pupal form. The intractability of larval heteromorphosis to experimental dissection is due in part to the lack of suitable experimental probes that can test the nature of the coupling of each heterochronically expressed instar-specific program. The epistatic basis of expression of heteromorphic developmental programs was assessed by two-dimensional electrophoretic analysis of hemolymph proteins during the normal and experimentally manipulated feeding stages of the 3rd, 4th, and 5th (final) instar larvae, and during the prepupal stage, of Trichoplusia ni. Of the several hundred protein spots tracked, some were identified that were uniquely detected during a single stage, while others were observed during combinations of certain stages. The nature of coupling of sequential heterochronic expression of these proteins during successive instars or stages was tested by use of a parasite (Chelonus sp.) that injects regulatory material into the host embryo that later causes the subsequent precocious expression of the final instar larval program. Following the expression of a normal 3rd instar pattern, such larvae were observed to omit expression of the 4th instar program, including omission of the proteins heteromorphically specific to that instar, and instead then express an essentially normal final instar pattern. Thus, normal expression of the final instar feeding stage pattern was not invariantly coupled to prior expression of the penultimate instar-specific proteins or pattern. Also, expression of the full program of the final instar feeding stage was epistatic to teh penultimate instar program, i.e., the protein pattern unique to the penultimate larval instar was not co-expressed with the precociously expressed final instar pattern. Larvae developmentally redirected in this manner failed to fully express the final instar prepupal stage pattern of protein expression, due at least in part to failed expression of prepupal ecdysteroids, but this was shown not to arise from omission of any of the first 4 larval instars per se. The nature of the redirections in host development caused by this parasite finally provides means of probing the coupling of successive expression on heteromorphic programs during larval development.