The objective of this research was to identify a reliable biochemical indicator for diapause (dormancy) in the boll weevil, Anthonomus grandis. Hemolymph polypeptides from reproductive and diapausing weevils were compared using denaturing sodium dodecyl sulpfate (SDS)-polyacrylamide gel electrophoresis (PAGE). A 77-kDa protein, which proved to be a hexamerin (AgSP-1), strongly correlated with morphological diapause characters in both male and female adult weevils. N-terminal sequence analysis identified the first 25 amino acids of the mature protein and was used to develop an antibody to AgSP-1. Anti-AgSP-1 reacted only with hemolymph from diapausing weevils of both sexes but not with hemolymph from reproductive weevils. Also, the yolk protein, vitellogenin (VG), inversely correlated with AgSP-1. When hemolymph VG was high, AgSP-1 was absent or barely perceptible.Juvenile hormone regulates VG synthesis in most insect species. Juvenile hormone is reported to stimulate reproductive maturation in the boll weevil (Physiological Entomology 22 (1997) 261) and to be absent during diapause (Physiological Entomology 22 (1997a) 269). Therefore, the juvenile hormone (JH) mimic, methoprene, was used to assess the role of JH activity in the boll weevil for terminating diapause, stimulating reproductive maturation and possibly influencing AgSP-1 titers. Application of methoprene was not effective in activating reproductive development. Hemolymph from methoprene-treated, females contained VG and AgSP-1 titers that were similar to acetone-treated and untreated control weevils.Using a genomic DNA library and 3' RACE, two clones were isolated that yielded the complete sequence of AgSP-1 as well as a portion of the 5' untranslated region. Northern blot analysis confirmed the presence of a 2.5 kB transcript for AgSP-1 in the fat body of diapausing weevils. AgSP-1 was also present in the fat body of reproductive weevils, but to a lesser extent. No sex-related differences in gene expression were observed; diapausing weevils of both sexes showed similar levels of AgSP-1 expression. An inverse correlation was observed between the VG transcript and AgSP-1 mRNA. VG was highly expressed in the fat body of reproductive females and only slightly expressed in tissue from diapausing females. Our data suggests that AgSP-1 is a diapause-specific protein in adult weevils and that JH, alone, is not effective in terminating diapause.