Analysis of Cystatin a family of cystine peptidases. (A) Phylogenetic tree based on maximum likelihood analysis of a Dermacentor variabilis midgut protein and published secreted and cytoplasmic cystatin sequences. The transcripts identified in this analysis are in bold (DvM). Phylogenetic analysis was conducted on protein alignments using Tree Puzzle version 5.2. Values at nodes represent calculated internal branch node support (1000 replications). Multiple sequence alignment (CLUSTALX) of protein sequences identified in a cDNA library of unfed/2 d fed or 6 d fed D. variabilis midguts (DvM) and published secreted (B) and cytoplasmic (C) cystatin sequences found on Genbank. Arrowheads indicate putative papain binding domains. S-S indicates cysteines involved in disulfide bridge formation. The underlined amino acids highlights the conserved QNVLG domain. Shading represents 100% identity (black) or similarity (grey) among the sequences. Alignments were conducted using CLUSTALX.D. variabilis (Dv), Ixodes scapularis (Is), I. ricinus (Ir), Ornithodoros moubata (Om), Haemaphysalis longicornis (Hl), Dictyostelium discoideum (Dd), and Lepidoglyphus destructor (Ld).