Evidence for an immune function of lepidopteran silk proteins Ahmed M. Korayem a,b , Thomas Hauling a , Christine Lesch a , Marco Fabbri c,1 , Malin Lindgren a,d , Olga Loseva a , Otto Schmidt c , Mitchell S. Dushay d,e , Ulrich Theopold a, * a Department of Molecular Biology and Functional Genomics, Stockholm University, SE-106 91 Stockholm, Sweden b Department of Zoology, Faculty of Science, Assiut University, 71516 Assiut, Egypt c School of Agriculture, Food and Wine, University of Adelaide, Glen Osmond 5064, SA, Australia d Department of Natural Sciences, So ¨ derto ¨rns ho ¨ gskola, SE-141 89 Huddinge, Sweden e Department of Comparative Physiology, Uppsala University, Norbyva ¨ gen 18A, SE-752 36 Uppsala, Sweden Received 23 October 2006 Available online 14 November 2006 Abstract Hemolymph coagulation stops bleeding and protects against infection. Clotting factors include both proteins that are conserved dur- ing evolution as well as more divergent proteins in different species. Here we show that several silk proteins also appear in the clot of the greater wax moth Galleria mellonella. RT-PCR analysis reveals that silk proteins are expressed in immune tissues and induced upon wounding in both Galleria and Ephestia kuehniella, a second pyralid moth. Our results support the idea that silk proteins were co-opted for immunity and coagulation during evolution. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Innate immunity; Insect immunity; Coagulation; Silk; Molecular evolution; Galleria; Ephestia; Drosophila; Bleeding Arthropod silks have a long tradition as materials with medical applications. In medieval times, spider webs were applied as wound dressing and insect silk was used for ban- dages and sutures with low allergenic potential [1,2]. With the possibility to produce recombinant silk and to recapit- ulate the spinning process in vitro [3], silks have attracted increasing interest as biodegradable polymers with remark- able tensile strength and elasticity. Several silk components have been expressed in non-arthropod species to facilitate large-scale production and industrial usage [2]. Molecular characterization of lepidopteran silks showed that they are composed of several proteins including heavy chain fibroin as a major structural component as well as light chain fibroin and P25 and seroin [4–6]. These proteins form the fibrous network that is responsible for the remarkable strength of insect silk. A serine-rich protein called sericin serves as a glue that links the structural components together [7]. Insect silk is produced by silk glands, which are labial glands with several sections that are specialized to produce different silk proteins and facilitate the spinning process [3]. The clot is a matrix that forms immediately after wounding to stop bleeding and prevent bacterial infections [9]. We have previously shown that Drosophila gp150, one of the major proteins produced in the labial glands of Dro- sophila larvae, is also a major component of the hemo- lymph clot [8]. Gp150 had previously been described as a glue-like secretory protein (I71–7 [10]), and we found it to be expressed in hemocytes and to participate in coagula- tion. In the clot gp150 appears to form dot-like aggregates within the fibrous network [8]. This, and the lack of conser- vation of many clotting factors between Drosophila and other insect species [18] led to the idea that coagulation— the formation of a clot matrix—evolved in insects by the co-option of proteins that already participated in the for- 0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.11.022 * Corresponding author. Fax: +46 8 166488. E-mail address: uli@molbio.su.se (U. Theopold). 1 Present address: Instituto Clinico Humanitas, Via Manzoni, 52, 20089 Rozzano, Italy. www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 352 (2007) 317–322 BBRC