A diuretic hormone of unusual structure was isolated from extracts of whole heads of the mealworm Tenebrio molitor. The hormone is a 37-aa peptide of 4371 Da, with the sequence SPTISITAPIDVLRKTWEQERARKQM-VKNREFLNSLN. This peptide increases cAMP production in Malpighian tubules of T. molitor. The amino acid sequence reveals that this peptide is a member of the family of sauvagine/corticotropin-releasing factor/urotensin I-related insect diuretic hormones. The C-terminal sequence of this peptide is quite different from other members of this family, which have a hydrophobic C terminus (isoleucinamide or valinamide). When aligned comparably, T. molitor diuretic hormone has a more hydrophilic C terminus, leucylasparagine (free acid). In contrast to all other known diuretic hormones of this family, this peptide has exceptionally low stimulatory activity on cAMP production in Malpighian tubules of Manduca sexta. However, at nanomolar concentrations it stimulates cAMP production in Malpighian tubules of T. molitor. Diuretic hormones of this family have been isolated previously from Lepidoptera, Orthoptera, Dictyoptera, and Diptera. This appears to be the first diuretic hormone isolated from a coleopteran insect.The regulation of water balance in insects has been heavily studied (1). With a variety of approaches, biologically active factors which promote urine production have been described from a large number of insect species (2). Recently, a number of insect diuretic hormones (DHs) have been isolated and characterized (3-9) which are homologous to a family of peptides including sauvagine, corticotropin-releasing factor (CRF), and urotensin. For simplicity, these DHs are referred to as the CRF-related DHs; all appear to act on the Malpighian tubules (Mt) via cAMP as second messenger (10). The leucokinins (11) and achetakinins (12), which were originally isolated as myotropic peptides, have been shown to stimulate fluid secretion from Mt (13), although this effect is not mediated via cAMP (14). Serotonin may also function as a DH; it stimulates fluid secretion by Mt of many insect species (14-18) and causes elevation of cAMP levels in Mt of some, but not all, of the stimulated species (14,16,(18)(19)(20).CRF-related DHs have now been isolated from four orders of insects, Lepidoptera, Orthoptera, Dictyoptera, and Diptera. coleopteran DH and preliminary results on its unusual biological activity. § MATERIALS AND METHODS Experimental Insects. T. molitor were kept under crowded conditions at 27°C on a 14 hr/10 hr light/dark cycle and were fed on bran and raw potatoes. Pupae were decapitated 24-48 hr after pupation. The heads were collected in liquid nitrogen and stored at -80°C until extraction. For the cAMP bioassay, Mt were taken from newly emerged adults of both sexes 3-12 hr after adult eclosion.Bioassay. DH activity in fractions from all purification steps was detected by the increase of cAMP in isolated Mt dissected from newly emerged adult T. molitor. Single Mt of T. molitor were incubated as described (5, 21...