Insect Biochemistry and Molecular Biology Insect Biochemistry and Molecular Biology 35 (2005) 11–22 Two structurally different defensin genes, one of them encoding a novel defensin isoform, are expressed in honeybee Apis mellifera Jaroslav Klaudiny a,Ã ,S ˇ tefan Albert b , Katarı´na Bachanova´ a , Ja´n Kopernicky´ c , Jozef S ˇ imu´ th a a Laboratory of Genetic Engineering, Institute of Chemistry, Slovak Academy of Sciences, Du´bravska´ cesta 9, 84538 Bratislava, Slovakia b Institute of Medical Radiation and Cell Research, Versbacherstr. 5, D-97078 Wu¨rzburg, Germany c Institute of Apiculture, Research Institute of Animal Production, Gasˇperı´kova´ 599, 03308 Liptovsky´ Hra´dok, Slovakia Received 30 July 2004; received in revised form 17 September 2004; accepted 29 September 2004 Abstract Two defensins showing high mutual similarity have previously been characterized in honeybee Apis mellifera: royalisin, a peptide isolated from the royal jelly, and defensin, found in the hemolymph of bacterially infected bees. Here we show that both these peptides are encoded by the same polymorphic gene, which we termed defensin1. Besides this gene, we identified an additional defensin gene coding for a novel honeybee defensin designated defensin2. The pre-pro-peptide sequence of defensin 2 was inferred from its cDNA. Mature defensin 2 peptide shows 55.8% identity with defensin 1. Sequences of genomic loci of the two defensin genes revealed their different structure. Defensin1 possesses an exon–intron structure unique among arthropoda defensin genes. Its second intron splits exactly the common structural module of defensins from a short amidated C-terminal extension found only in hymenopteran defensins. Transcription of defensin genes in some nurse honeybees tissues was studied by RT–PCR. Both defensins are expressed in heads and thoraces. Defensin1 but not defensin2 mRNA was detected in hyphopharyngeal, mandibular and thoracic salivary glands. Immune response elements were identified by computer analysis of the promoter regions of defensin genes. Their different representation in these genes reflects presumably observed tissue-specific expression of defensins. r 2004 Elsevier Ltd. All rights reserved. Keywords: Antimicrobial peptides; cDNA; Gene; Apis mellifera; Royal jelly; Insect immunity; Honeybee diseases; Mass spectrometry 1. Introduction Antimicrobial peptides are key elements of insect innate immunity (Boman, 1995; Bulet et al., 1999; Hoffmann et al., 1999). Most of them are produced upon infection or injury in the fat body or hemocytes and secreted subsequently into the hemolymph. In some insects, local expression of the peptides has also been reported [e.g. cuticle (Brey et al., 1993), midgut and salivary glands of blood-sucking insects (Lehane et al., 1997; Dimopoulos et al., 1998; Lowenberger et al., 1999a)]. Defensins comprise a widespread family of cystein-rich cationic antimicrobial peptides that act against a variety of microorganisms and constitute the primary defense system of most organisms (Raj and Dentino, 2002). Insect defensins are 36–51-amino-acid-long peptides possessing sequence similarity which is the basis of their common structure comprising an amino-terminal loop, an a-helix and two antiparallel b-strands stabilized by three disulfide bridges (Hanzawa et al., 1990; Bonmatin et al., 1992; Cornet et al., 1995). They are active against a broad spectrum of Gram-positive bacteria, although activity against Gram-negative bacteria and fungi has also been reported (Hetru et al., 1998; Yamauchi, 2001). ARTICLE IN PRESS www.elsevier.com/locate/ibmb 0965-1748/$-see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibmb.2004.09.007 Ã Corresponding author. Tel.: +421259410203; fax: +421259410222. E-mail address: chemjakl@savba.sk (J. Klaudiny).