Immune-gene candidates from other insects were used in several ways to query the honey bee genome, primarily using the BLAST family of search functions (www.ncbi.nlm.nih.gov). Most searches were initiated by BLASTP queries against the consensus protein list (GLEAN3, derived from HBGP assembly 2.0) using BLASTP and algorithms (BLOSUM and PAM variants) appropriate to gene size and structure. Honey bee orthologues were also identified by searching honey bee genome assemblies 2.0 and 3.0 directly using TBLASTN and either local databases or the BeeBase BLAST server (http://racerx00.tamu.edu/blast/blast.html). Searches for missing genes were also carried out a on smaller coverage set of honey bee contigs that were too short to be included in the assembly, as well as the unassembled reads from the project (http://www.hgsc.bcm.tmc.edu/projects/honeybee/). Given honey bee candidates, searches were repeated in the hope of identifying paralogues missed by interspecific comparisons. PSI-BLAST was used to identify honey bee genes on the basis of conserved domains, followed by RPS-BLAST to confirm the significance of these domain matches. Putative matches were aligned and, in the case of serine proteases, scavenger receptors and C-type lectins, screened for additional motifs using pfam categories (http://sanger.ac.uk/software/pfam). Tentative matches were aligned and checked for gene-prediction errors (in the case of genes from the official protein list) as part of the annotation of immune candidate genes for the Honey Bee Genome Project (Honey Bee Genome Sequencing Consortium, 2006). All protein matches were ported to Apis mellifera assembly 3.0 using the alignment program BLAT (Jim Kent, University California, Santa Cruz) to establish scaffold locations (Supplementary Material Table S1 ).