Nucleic acid synthesis during insect development—II. Control of DNA synthesis in the Cecropia silkworm and other Saturniid moths☆

The present report examines factors that control the rate of DNA synthesis during larval life, metamorphosis, pupal diapause, and adult life of Cecropia and other Saturniid moths. Autoradiographic determinations were made of the rate of incorporation of tritiated thymidine in various tissues during the life history. DNA synthesis was also studied in insects subjected to diverse endocrinological and surgical manoeuvres and treated with juvenile hormone. Using labelling techniques, the careers of epidermal cells were followed from one instar to the next. In addition, various tissues were labelled with tritiated thymidine, implanted into unlabelled hosts, and their developmental fate determined. DNA synthesis in adult moths was compared with DNA synthesis in other adult insects representing six orders. The effects of a potent inhibitor of DNA synthesis on development and moulting were also analysed. During each larval moult cycle and during metamorphosis of Saturniid moths there is a definite temporal pattern of DNA synthesis in each tissue and in regions within many tissues. During larval moults in some tissues such as chitogenous epithelia, Malpighian tubules, and nervous system, DNA synthesis occurred soon after the secretion of ecdysone. In other tissues such as imaginal wing disks and muscles, DNA synthesis during larval life was not correlated with ecdysone secretion. Almost every epidermal cell appeared to synthesize DNA during every normal moult cycle, but some tissues such as Malpighian tubules and prothoracic glands did not synthesize DNA during the pupal-adult moult although they did so during earlier moults. Apparently after experiencing a moult in the absence of a high concentration of juvenile hormone these tissues differentiate and ordinarily do not synthesize DNA again. However, two experimentally induced pupal-pupal moults in the presence of juvenile hormone and ecdysone restored the capacity of pupal Malpighian tubules to synthesize DNA. In insects with a pupal diapause DNA synthesis ceased after pupation in all tissues except haemocytes and to a small extent the gonads and fat body. This is not the result of low metabolism because the metabolic processes of a pupa could be increased up to fifteenfold by integumentary injury without stimulating DNA synthesis. The absence of DNA synthesis in most tissues appears to be a fundamental characteristic of diapause and results from the absence of ecdysone. DNA synthesis was also absent in adult moths and in surgical preparations deprived of a source of ecdysone such as isolated larval abdomens, ‘dauer’ larvae, and non-diapausing pupae which had their brains extirpated. The addition of ecdysone stimulated DNA synthesis in many tissues of both diapausing pupae and adults. In contrast, experimental administration of brain hormone or juvenile hormone alone and in the absence of ecdysone failed to stimulate DNA synthesis in any tissues examined. Injury to the integument of a diapausing pupa provoked DNA synthesis only in epidermal cells adjacent to the wound but did not stimulate general DNA synthesis. Injury to diapausing pupae also released an ‘injury factor’ which stimulated DNA synthesis in haemocytes but not in other tissues. Non-diapausing insects appeared to telescope into the larval-pupal transformation a number of the changes normally associated with adult development in diapausing insects such as DNA synthesis in certain muscles and parts of the epidermis. Artificial diapause could be imposed in a non-diapausing pupa either by brain removal or by injection of mitomycin C—an inhibitor of DNA synthesis—before or after some DNA synthesis had begun in the epidermis. However, after epidermal DNA synthesis was well along, an artificial diapause could no longer be imposed. Thus injecting mitomycin C into pupae that had already initiated adult development prevented further DNA synthesis and the cell divisions necessary for scale formation, but did not block the synthesis of an adult cuticle and moulting. No adult insect among six orders examined showed DNA synthesis in epidermis, tracheae, or fat body more than 3 days after ecdysis. This absence of DNA synthesis is correlated with the absence of ecdysone, and renewed DNA synthesis can be induced with ecdysone. Adult insects which live only a short time showed no DNA synthesis in any tissues except haemocytes. However, long-lived insects such as cockroaches, mealworms, dragon flies, and the Monarch butterfly showed DNA synthesis in several tissues during adult life. The developmental fate of various cells was determined by labelling experiments. It was shown that larval epidermal cells and their immediate descendants persisted in significant numbers from one larval instar to the next. However, most pupal epidermal cells appeared to be the remote descendants of larval cells. Similarly, the developmental fate of haemocytes and fat body was determined by implanting labelled pupal cells into unlabelled pupae which were allowed to transform into adults. The relation between DNA synthesis and subsequent synthetic activities of cells differed in various kinds of insects at different developmental stages. In Saturniid moths DNA synthesis seems to be an early obligatory step in the differentiation of most epidermal cells. In other insects (e.g. Rhodnius, Oncopeltus) some larval epidermal cells form adult structures without prior DNA synthesis. In the general case, the metamorphosis of epidermal cells may or may not require DNA synthesis and may be viewed in some insects as a progressive differentiation which is punctuated incidentally by moults and DNA synthesis. Although DNA synthesis may not be necessary for the metamorphosis of certain cells it appears to be necessary for the reversal of metamorphosis. Apparently DNA replication erases certain biases and enables cells to be reprogrammed. The mechanism of action of ecdysone was examined. Experimentally induced moulting in adult silkmoths is not preceded by DNA synthesis in most epidermal cells, hence stimulation of DNA synthesis cannot be the primary effect of ecdysone even though it is an early effect in many tissues at several developmental stages. From an analysis of available data it is concluded that ecdysone should be viewed primarily as a moulting hormone that initiates biosynthetic activities which lead to moulting. Its primary effect is to stimulate the synthesis or translation of certain messenger RNA's which were previously non-existent or non-functional and which are necessary for moulting. The nature of the proteins coded by these new messenger RNA's was considered.