Requirements for molting of the crochet epidermis of the tobacco hornworm larva in vivo and in vitro

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Abstract
In the tobacco hornworm,Manduca sexta, the epidermis which underlies the larval crochets is the first tissue to become independent of the prothoracic glands (PG) in a larval molt. In each successive larval molt, crochet forming cells increase in size, form hooks at their distal ends and, finally, secrete cuticle. This paper examines the endocrine requirements for competence to molt and describes parallel cultures in vivo and in vitro to define the hormonal control of crochet molting. When implanted into a fourth instar host larva prior to initiation of the last larval molt, competent crochet epidermis molted, forming crochets synchronously with its host. In the fourth instar, competence to form crochets is attained slowly during the first two days following ecdysis from the third instar. During the feeding phase of the fifth (last) instar, the crochet epidermis remains competent to molt (to form an extra “sixth instar” set of crochets) until the larva attains a weight of about 4.5 gm. Then, concurrent with the decline in the titer of juvenile hormone (JH) in the hemolymph, competence to form crochets declines. A similar loss of competence did not occur when fourth instar crochet epidermis was exposed to a declining JH titer by culture in either fourth instar isolated abdomens for 72 h or in fifth instar host larvae between 4 and 7 gm. Responses of crochet epidermis cultured in vitro also were examined. Competent fourth instar crochet epidermis formed crochets following 3–6 h exposure to ecdysone in vitro. Six ×10−7M β-ecdysone was required for 50% response, whereas a 10–50-fold higher concentration of α-ecdysone was necessary. Although formation of morphologically complete crochets in vitro proceeded with similar time course to that in situ, no molt-induced growth occurred in vitro. When crochet epidermis was exposed to ecdysone in vitro immediately after explantation, exogenous JH was not required for molting. But when tissue was first cultured for 72 h without hormones, subsequent molting in vitro could not be elicited, although molting still could occur when the tissue subsequently was implanted into a fourth instar host. Exposure to corpora allata or to JH during the 72 h of culture in vivo partially prevented the loss in capacity to respond to ecdysone in vitro, suggesting that JH may be one factor involved directly or indirectly in maintenance of tissue responsiveness.