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 and adult grasshoppers, Melanoplus sanguinipes (F.), and mainly occurs as an acid-labile conjugate indicated to be a sulfate ester. Its concentration increases in last instar nymphs and peaks during adult cuticle sclerotization. Dopamine (DA), the precursor of NADA and melanic pigments, is about 10 times lower in concentration than NADA, but shows a similar pattern of accumulation. NADA also predominates in cuticle, but its concentration is lowest during the active period of sclerotization, reflecting its role as a precursor for quinonoid tanning agents. Two other catechols, 3,4-dihydroxybenzoic acid (DOBA) and 3,4-dihydroxyphenylethanol (DOPET), also occur in hemolymph 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 sclerotization of the protein-chitin matrix. To determine the types of catechol covalent interactions in the exocuticle, samples of powdered exuviae were heated in HCl under different hydrolytic conditions to release adducts and cross-linked products. 3,4-Dihydroxyphenylketoethanol (DOPKET) and 3,4-dihydroxyphenylketoethylamine (arterenone) are the major hydrolysis products in weak and strong acid, respectively, and primarily represent NADA oligomers that apparently serve as cross-links and filler material in sclerotized cuticle. Intermediate amounts of norepinephrine (NE) are released, which represent N-acetylnorepinephrine (NANE), a hydrolysis product of NADA bonded by the b-carbon to cuticular proteins and possibly chitin. Small quantities of histidyl-DA and histidyl-DOPET ring and side-chain C-N adducts are released by strong acid hydrolysis. Therefore, grasshopper cuticle appears to be sclerotized 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 residues in cuticular proteins. The primary catechol extracted from both the female accessory glands/calyx and the proteinaceous frothy material of the egg pod is DOBA, which also commonly occurs in cockroach accessory glands and oothecae, presumably as a tanning agent precursor. 3,4-Dihydroxyphenylalanine (DOPA) was also detected in extracts of the accessory glands/calyx of grasshoppers, and may serve as a precursor for DOBA synthesis.