1 3 Physiology of digestion and the molecular characterization of the major 4 digestive enzymes from Periplaneta americana 5 6 7 Fábio K. Tamaki a,1 Q1 , André C. Pimentel a,1 , Alcides B. Dias a , Christiane Cardoso a , Alberto F. Ribeiro b , 8 Clélia Ferreira a , Walter R. Terra a,⇑ 9 a Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, C.P. 26077, 05513-970 São Paulo, Brazil 10 b Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, C.P. 11461, 05513-970 São Paulo, Brazil 11 12 13 15 article info 16 Article history: 17 Received 26 June 2014 18 Received in revised form 22 August 2014 19 Accepted 23 August 2014 20 Available online xxxx 21 Keywords: 22 Proteases 23 Carbohydrases 24 Digestive enzymes sequences 25 Secretory mechanism 26 Enzyme immunocytolocalization 27 28 abstract 29 Cockroaches are among the first insects to appear in the fossil record. This work is part of ongoing 30 research on insects at critical points in the evolutionary tree to disclose evolutionary trends in the diges- 31 tive characteristics of insects. A transcriptome (454 Roche platform) of the midgut of Periplaneta 32 americana was searched for sequences of digestive enzymes. The selected sequences were manually 33 curated. The complete or nearly complete sequences showing all characteristic motifs and highly 34 expressed (reads counting) had their predicted sequences checked by cloning and Sanger sequencing. 35 There are two chitinases (lacking mucin and chitin-binding domains), one amylase, two a- and three 36 b-glucosidases, one b-galactosidase, two aminopeptidases (none of the N-group), one chymotrypsin, 37 5 trypsins, and none b-glucanase. Electrophoretic and enzymological data agreed with transcriptome 38 data in showing that there is a single b-galactosidase, two a-glucosidases, one preferring as substrate 39 maltase and the other aryl a-glucoside, and two b-glucosidases. Chromatographic and enzymological 40 data identified 4 trypsins, one chymotrypsin (also found in the transcriptome), and one non-identified 41 proteinase. The major digestive trypsin is identifiable to a major P. americana allergen (Per a 10). The lack 42 of b-glucanase expression in midguts was confirmed, thus lending support to claims that those enzymes 43 are salivary. A salivary amylase was molecularly cloned and shown to be different from the one from the 44 midgut. Enzyme distribution showed that most digestion occurs under the action of salivary and midgut 45 enzymes in the foregut and anterior midgut, except the posterior terminal digestion of proteins. A coun- 46 ter-flux of fluid may be functional in the midgut of the cockroach to explain the low excretory rate of 47 digestive enzymes. Ultrastructural and immunocytochemical localization data showed that amylase 48 and trypsin are released by both merocrine and apocrine secretion mainly from gastric caeca. Finally, a 49 discussion on Polyneoptera digestive physiology is provided. 50 Ó 2014 Published by Elsevier Ltd. 51 52 53 54 1. Introduction 55 Cockroaches, which are among the first neopteran insects to 56 appear in the fossil record, are extremely generalized in most 57 morphological features. They are usually omnivorous and are 58 included in the sub-order Blattodea that together with Mantodea 59 (mantids) form the Order Dictyoptera. After extensive molecular 60 phylogenetic analyses, Inward et al. (2007) showed that termites 61 are social cockroaches, no longer deserving classification as a 62 separate order (Isoptera) from cockroaches. Actually, termites 63 pertain to a sister family (Termitidae) of that of the woodroach 64 Cryptocercus (Cryptocercidae) (Lo et al., 2000). The branch 65 Cryptocercidae-Termitidae is a sister of Blattidae, forming 66 Blattoidea that is a sister of Blaberoidea (Blattelidae plus 67 Blaberidae), which in addition to Polyphagoidea form the 68 Blattodea. 69 The organization of the digestive process in the different insect 70 orders that corresponds to the basic plans of the ancestral forms 71 was reviewed several times (Terra, 1988, 1990; Terra and 72 Ferreira, 1994, 2012). Dictyoptera is supposed to be derived from 73 the Polyneoptera ancestors. Hence, its basic digestive organization 74 should be alike that of the Polyneoptera ancestor. One of the aims 75 of this paper is to provide support in this direction. 76 Data on compartmentalization of midgut pH and digestion in 77 Dictyoptera are fragmentary and contradictory (see Elpidina 78 et al., 2001 and references therein). In part this is a consequence http://dx.doi.org/10.1016/j.jinsphys.2014.08.007 0022-1910/Ó 2014 Published by Elsevier Ltd. ⇑ Corresponding author. Fax: +55 11 3838 2186. E-mail address: warterra@iq.usp.br (W.R. Terra). 1 These authors contribute equally to this paper. Journal of Insect Physiology xxx (2014) xxx–xxx Contents lists available at ScienceDirect Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys IP 3272 No. of Pages 14, Model 5G 3 September 2014 Please cite this article in press as: Tamaki, F.K., et al. Physiology of digestion and the molecular characterization of the major digestive enzymes from Periplaneta americana. Journal of Insect Physiology (2014), http://dx.doi.org/10.1016/j.jinsphys.2014.08.007