198 Yu and Kanost Archives of Insect Biochemistry and Physiology 42:198–212 (1999) © 1999 Wiley-Liss, Inc. Developmental Expression of Manduca sexta Hemolin Xiao-Qiang Yu and Michael R. Kanost* Department of Biochemistry, Kansas State University, Manhattan, Kansas Hemolin is hemolymph protein that is a member of the immu- noglobulin superfamily. Its induced expression after bacterial infection suggests that it functions in the immune response. In this paper, we describe the expression of the Manduca sexta hemolin gene at certain developmental stages in the absence of microbial challenge. Hemolin was present at a very low level in hemolymph of naive larvae until the beginning of the wan- dering stage prior to pupation, when its concentration in hemolymph increased dramatically. At the same time, hemolin could be found in the fluid contained in the midgut lumen. The appearance of hemolin mRNA in fat body and midgut at the beginning of the wandering stage correlated with the pres- ence of hemolin in the hemolymph and midgut lumen. Hemolin was present in hemolymph through the pupal and adult stages. Hemolin was also present in newly deposited eggs, and per- sisted in eggs throughout embryonic development. A hemolin cDNA isolated from an adult fat body library had the same se- quence as those previously obtained from larval libraries. Hemolin purified from hemolymph of bacteria-injected larvae, from hemolymph of naive wandering stage larvae and adult moths, and from midgut fluid of wandering stage larvae had the same apparent mass, which was consistent with the mass predicted from the hemolin cDNA sequence. Hemolin from hemolymph of wandering stage larvae did not contain any de- tectable carbohydrate, but hemolin from the hemolymph of bac- teria-injected larvae and from naive adult moths was associated with carbohydrate, although of different amounts and compo- sition. These results suggest that a single hemolin gene is de- velopmentally regulated and is also induced when insects are exposed to microbial infection. M. sexta hemolin apparently lacks post-translational covalent glycosylation, but instead is associated under some conditions with non-covalently bound carbohydrates. Arch. Insect Biochem. Physiol. 42:198–212, 1999. © 1999 Wiley-Liss, Inc. Key words: insect immunity; immunoglobulin superfamily; hemolymph; to- bacco hornworm; postlarval protein; midgut; fat body Abbreviations used: Con A = concanavalin A; HIC = hydro- phobic interaction chromatography; HPLC = high performance liquid chromatography; Ig = immunoglobulin; MALDI-MS = matrix assisted laser desorption ionization mass spectroscopy; MOPS = 3-(N-morpholino)-propanesulfonic acid; PBS = phos- phate-buffered saline; PAGE = polyacrylamide gel electro- phoresis; PTU = 1-phenyl-2-thiourea; SDS = sodium dodecyl sulfate; TBS = Tris-buffered saline. Contract grant sponsor: National Institutes of Health; Con- tract grant number: AI31084. *Correspondence to: Michael R. Kanost, Department of Bio- chemistry, Kansas State University, Manhattan, KS 66506. E-mail: kanost@ksu.edu Received 6 May 1999; accepted 28 July 1999