Partial characterization and isolation of earwig 'allatostatins': biological activities in earwigs and cockroaches

Allatostatins are a family of neuropeptides first isolated from the cockroa ch, Diploptera punctata, that inhibit juvenile hormone production in that s pecies (but do not do so in earwigs), and inhibit hindgut muscle contractio ns in some insects, including the earwig, Euborellia annulipes. We examined whether material from earwig brains is similar to cockroach allatostatins biochemically, immunologically and physiologically. Brain extracts from adu lt female earwigs were separated by high performance liquid chromatography (HPLC), followed by radioimmunoassay using antibodies to cockroach allatost atin (Dip-AST). Fractions that co-eluted with cockroach allatostatins were immunoreactive, and at least two peaks of immunoreactivity were detected. M aterial from each peak at 10 nM Dip-AST equivalents inhibited juvenile horm one biosynthesis in vitro by corpora allata of 2-day virgin D. punctata coc kroaches; 1 nM was less effective, and non-immunoreactive fractions failed to inhibit juvenile hormone biosynthesis. Both crude and Sep-Pak (Waters) p urified extracts of brains of earwigs containing 1 nM Dip-AST equivalents f ailed to suppress hindgut contractions in vitro of 2-day earwigs and of bro oding female earwigs. In contrast, 1 nM cockroach allostatin (Dip-AST 7) re versibly inhibited hindgut contractions in vitro. These results suggested t he: presence of another brain factor, such as proctolin, that counteracts t he inhibitory effects of Dip-AST. In support of this hypothesis, proctolin stimulated hindgut contractions in vitro at I nM, the effects of equal conc entrations of allatostatin and proctolin varied with the stage of the femal e. Furthermore, HPLC-separated fractions that co-eluted with cockroach alla tostatin and were immunoreactive with antibodies to Dip-AST suppressed hind gut contractions in vitro of 3-day female earwigs. Finally, crude brain ext racts of earwigs suppressed earwig juvenile hormone biosynthesis in vitro i n glands of low, but not in glands of high, activity. Thus, earwig blain ex tract after HPLC separation has Dip-AST-like material that inhibits cockroa ch corpora allata and suppresses earwig hindgut contractions. Sep-Pak-extra cted earwig brain material, however, does not inhibit earwig gut contractio n. Although synthetic Dip-AST 7 does not inhibit juvenile hormone synthesis by earwig corpora allata, there is heat-stable material in earwig brain ex tract that does have this action. (C) 1998 Elsevier Science Inc. All rights reserved.