Catechols involved in sclerotization of cuticle and egg pods of the grasshopper, Melanoplus sanguinipes, and their interactions with cuticular proteins

N-Acetyldopamine (NADA) is the major catechol in the hemolymph of nymphal a nd adult grasshoppers, Melanoplus sanguinipes (F.), and mainly occurs as an acid-labile conjugate indicated to be a sulfate ester. Its concentration i ncreases in last instar nymphs and peaks during adult cuticle sclerotizatio n. Dopamine (DA), the precursor of NADA and melanic pigments, is about 10 t imes lower in concentration than NADA, but shows a similar pattern of accum ulation. NADA also predominates in cuticle, but its concentration is lowest during the active period of sclerotization, reflecting its role as a precu rsor for quinonoid tanning agents. Two other catechols, 3,4-dihydroxybenzoi c acid (DOBA) and 3,4-dihydroxyphenylethanol (DOPET), also occur in hemolym ph and cuticle, and their profiles suggest a role in cuticle stabilization. Solid-state NMR analysis of sclerotized grasshopper cuticle (fifth instar exuviae) estimated the relative abundances of organic components to be 59% protein, 33% chitin, 6% catechols, and 2% lipid. About 99% of the catechols are covalently bound in the cuticle, and therefore are involved in sclerot ization of the protein-chitin matrix. To determine the types of catechol co valent interactions in the exocuticle, samples of powdered exuviae were hea ted in HCL under different hydrolytic conditions to release adducts and cro ss-linked products. 3,4-Dihydroxyphenylketoethanol (DOPKET) and 3,4-dihydro xyphenylketoethylamine (arterenone) are the major hydrolysis products in we ak and strong acid, respectively, and primarily represent NADA oligomers th at apparently serve as cross-links and filler material in sclerotized cutic le. Intermediate amounts of norepinephrine (NE) are released, which represe nt N-acetylnorepinephrine (NANE), a hydrolysis product of NADA bonded by th e b-carbon to cuticular proteins and possibly chitin. Small quantities of h istidyl-DA and histidyl-DOPET ring and side-chain C-N adducts are released by strong acid hydrolysis. Therefore, grasshopper cuticle appears to be scl erotized by both o-quinones and p-quinone methides of NADA and dehydro-NADA , which results in a variety of C-O and C-N covalent bonds linked primarily through the side-chain carbons of the catechol moiety to amino acid residu es in cuticular proteins. The primary catechol extracted from both the fema le accessory glands/calyx and the proteinaceous frothy material of the egg pod is DOBA, which also commonly occurs in cockroach accessory glands and o othecae, presumably as a tanning agent precursor. 3,4-Dihydroxyphenylalanin e (DOPA) was also detected in extracts of the accessory glands/calyx of gra sshoppers, and may serve as a precursor for DOBA synthesis. (C) 1999 Wiley- Liss, Inc.