Exoskeleton formation in Apis mellifera: Cuticular hydrocarbons profiles and expression of desaturase and elongase genes during pupal and adult development Q3 Tiago Falcón a , Maria Juliana Ferreira-Caliman b , Francis Morais Franco Nunes c , Érica Donato Tanaka a , Fábio Santos do Nascimento b , Márcia Maria Gentile Bitondi b, * a Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes 3900,14049-900 Ribeirão Preto, SP, Brazil b Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes 3900,14040-901 Ribeirão Preto, SP, Brazil c Centro de Ciências Biológicas e da Saúde, Universidade Federal de São Carlos, Rod. Washington Luís, km 235,13565-905 São Carlos, SP, Brazil article info Article history: Received 11 March 2014 Received in revised form 23 April 2014 Accepted 25 April 2014 Keywords: Cuticular hydrocarbons Apis mellifera Desaturase Elongase Cuticular envelope Insect exoskeleton abstract Cuticular hydrocarbons (CHCs) are abundant in the superficial cuticular layer (envelope) of insects where they play roles as structural, anti-desiccation and semiochemical compounds. Many studies have investigated the CHC composition in the adult insects. However, studies on the profiles of these com- pounds during cuticle formation and differentiation are scarce and restrict to specific stages of a few insect species. We characterized the CHCs developmental profiles in the honeybee workers during an entire molting cycle (from pupal-to-adult ecdyses) and in mature adults (forager bees). Gas chroma- tography/mass spectrometry (GC/MS) analysis revealed remarkable differences in the relative quantities of CHCs, thus discriminating pupae, developing and newly-ecdysed adults, and foragers from each other. In parallel, the honeybee genome database was searched for predicted gene models using known amino acid sequences of insect enzymes catalyzing lipid desaturation (desaturases) or elongation (elongases) as queries in BLASTP analysis. The expression levels of six desaturase genes and ten elongase genes potentially involved in CHC biosynthesis were determined by reverse transcription and real time poly- merase chain reaction (RT-qPCR) in the developing integument (cuticle and subjacent epidermis). Aiming to predict roles for these genes in CHC biosynthesis, the developmental profiles of CHCs and desaturase/elongase transcript levels were evaluated using Spearman correlation coefficient. This anal- ysis pointed to differential roles for these gene products in the biosynthesis of certain CHC classes. Based on the assumption that homologous proteins may share a similar function, phylogenetic trees were reconstructed as an additional strategy to predict functions and evolutionary relationships of the hon- eybee desaturases and elongases. Together, these approaches highlighted the molecular complexity underlying the formation of the lesser known layer of the cuticular exoskeleton, the envelope. Ó 2014 Published by Elsevier Ltd. 1. Introduction The cuticular exoskeleton of insects is mainly formed by proteins, the polysaccharide chitin, and lipids arranged as a complex multi- layered structure: the inner procuticle comprising the endocuticle and exocuticle, the epicuticle and an outermost envelope. These functional layers are sequentially produced and are secreted by the epidermis at each molt cycle, and differ from each other in biochemical composition and physiological properties. The envelope mainly consists of lipids that form a barrier against water loss and invading pathogens, and also serve as important cues for chemical communication, besides acting as sex pheromones (Wigglesworth, 1970; Blomquist and Dillwith, 1985; Gibbs, 2002; Châline et al., 2005). This lipid layer is largely enriched with hydrocarbons (Hepburn, 1985), which are synthesized in specialized cells called oenocytes (Piek, 1964; Diehl, 1973; Schal et al., 1998; Fan et al., 2003; Billeter et al., 2009). In honeybee workers the oenocytes are localized in close association with the epidermis and the parietal fat body that internally coat the exoskeleton, and are more frequently found closer to the sternites than the tergites (Ruvolo and Landim, 1993). Intermediates and end-products of metabolic pathways, such as fatty acids, in addition to specific enzyme classes, are involved in * Corresponding author. Tel.: þ55 1636023805. E-mail address: mmgbit@usp.br (M.M. Gentile Bitondi). Contents lists available at ScienceDirect Insect Biochemistry and Molecular Biology journal homepage: www.elsevier.com/locate/ibmb http://dx.doi.org/10.1016/j.ibmb.2014.04.006 0965-1748/Ó 2014 Published by Elsevier Ltd. Insect Biochemistry and Molecular Biology xxx (2014) 1e14 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 IB2576_proof ■ 10 May 2014 ■ 1/14 Please cite this article in press as: Falcón, T., et al., Exoskeleton formation in Apis mellifera: Cuticular hydrocarbons profiles and expression of desaturase and elongase genes during pupal and adult development, Insect Biochemistry and Molecular Biology (2014), http://dx.doi.org/ 10.1016/j.ibmb.2014.04.006