Developmental expression, synthesis, and secretion of insecticyanin by the epidermis of the tobacco hornworm, Manduca sexta

Abstract

Insecticyanin was found to be synthesized in several isoelectric forms and stored in the pigment granules in the epidermis. Both major epidermal forms (INS-a, pl 5.5; INS-b, pl 5.7) were found in the cuticle, but only the most basic form, INS-b, was present in the hemolymph. In vitro the epidermis synthesized and secreted both forms into both the cuticle and the medium. Isolation of two cDNA clones for insecticyanin followed by hybridization to epidermal mRNA showed the presence of only one 1.1 kb mRNA, but transcription of the longer cDNA yielded a RNA which produced INS-a but no INS-b. Insecticyanin mRNA was present during the intermolt feeding stages of the 4th and 5th instars and absent during the larval molt and after the onset of metamorphosis. Exposure of either day 2 4th-instar or day 1 5th-instar larval epidermis to 20-hydroxyecdysone (20HE) in vitro caused a dose-dependent decline in this mRNA that was not prevented by simultaneous exposure to JH. When synthesis resumes just before ecdysis, INS-b appears before INS-a; then on the final day of feeding, synthesis of INS-a ceases before that of INS-b. Culture experiments showed that exposure of day 15th epidermis to a small pulse of 20HE followed by an ecdysteroid-free period was best to mimic the in vivo situation, indicating that the small rise of ecdysteroid in the absence of JH on day-2 was responsible for this differential cessation.

The bilin‐binding protein of Pieris brassicae

“…This pattern of BBP expression corresponds well to the synthesis of insecticyanin in the larvae of M. sextu (Riddiford et al, 1990). Since expression of insecticyanin was shown to be suppressed by a rise in the ecdysteroid titer during each molting phase, this may well be the case for BBP, too.…”

“…In Munducu the biliverdin-binding proteins insecticyanin a and b are synthesized and secreted by the epidermis (Riddiford et al, 1990), although it is not known whether synthesis also occurs in other tissues. In analogy, the epidermis was assumed to be the source of BBP in Pieris, as well (Huber et al, 1987a).…”

“…The cDNA and the genomic DNA sequences for two isoforms of insecticyanin were recently reported (Li and Riddiford, 1992). The developmental pattern of expression in epidermal cells was investigated and it was shown that insecticyanin is synthesized in the epiderm i s from which it is secreted both into the cuticle and into the hemolymph (Riddiford et al, 1990).…”

Grasshopper Lazarillo, a GPI-anchored Lipocalin, increases Drosophila longevity and stress resistance, and functionally replaces its secreted homolog NLaz

“…Lepidopteran Lipocalins like Bilin binding protein from Pieris brasssicae and Samia cynthia or Insecticyanin from Manduca sexta are expressed in epidermis, and some of them have been demonstrated to play roles in cuticle coloration (Riddiford et al, 1990;Saito, 1998;Sehringer and Kayser, 2006) and prevention of oxidative damage (Schmidt and Skerra, 1994). Brain Lipocalins have been identified both in Diptera and Lepidoptera.…”

A molt timer is involved in the metamorphic molt inManduca sextalarvae

“…They begin to void their gut contents, which are no longer formed into discrete pellets, and their dorsal body coloration changes as insecticyanin is removed from the dorsal epidermis (17) and ommochrome pigments are deposited into the epidermis beside the heart (18,19). These changes are linked together as a common response to a small surge of ecdysteroid in the absence of JH that irreversibly commits the larva to a metamorphic pathway (20,21).…”