Comparative genomic analysis of the Tribolium immune system

Additional data file 1:

Immunity-related genes in T. castaneum

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Additional data file 2:

The sequences of three Tribolium (Tc), three Drosophila (Dm), two Apis (Am), six Anopheles (Ag) and two Bombyx βGRPs/GNBPs are aligned with Bacillus circulans (Bc) β-1,3-glucanase A1 as an outgroup. There was a family expansion in the lineage of A. gambiae. Pink arrowheads indicate nodes with bootstrap values greater than 800 from 1,000 trials, and the dashed line marks the outgroup.

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Additional data file 3:

The sequences of sixteen Tribolium (Tc), ten Drosophila (Dm), eight Anopheles (Ag) and eight Apis (Am) sequences are aligned. TcCTL3 (that is, Tc 3) contains two carbohydrate recognition domains and the first one is used for comparison. Different CTL subfamilies (GA, galactose; MA, mannose) are indicated, with the predicted orthologous groups marked by blue dots (for 1:1, 1:1:1 and 1:1:1:1 relationships). Pink arrowheads indicate nodes with significant bootstrap values (>800 of 1,000 trials). Note that many Dm- and Ag-CTLs, not included in this analysis, are results of major lineage-specific expansions [29].

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Additional data file 4:

The amino acid sequences from three Tribolium (Tc), seven Drosophila (Dm), seven Anopheles (Ag), two Apis (Am) and one Phlebotomus (Pp) galectins are examined. The phylogenetic tree, derived from the aligned sequences, shows family expansions in Anopheles (pink) and Drosophila (blue). Pink arrowheads at nodes denote bootstrap values greater than 800 from 1,000 trials. Green lines connect the putative orthologous pairs or trio.

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Additional data file 5:

The sequences of seven Tribolium (Tc), fourteen Drosophila (Dm), nine Anopheles (Ag), nine Aedes (Aa) and one Apis (Am) FREPs are aligned for constructing this unrooted tree. For simplicity, other family members from Drosophila, Anopheles and Aedes are excluded from the analysis. Lineage-specific expansions (shaded yellow for Tribolium, blue for Drosophila and pink for Anopheles) are confirmed in the complete tree that includes all FREPs from these four species (data not shown). Nodes with pink arrowheads have bootstrap values exceeding 800 in 1,000 trials. Green bars connect the putative orthologs with 1:1 or 1:1:1 relationship. The chromosomal locations (lower corner) of Tribolium FREP-1 through -4 are shown.

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Additional data file 6:

The sequences of four Tribolium (Tc), six Drosophila (Dm), fifteen Anopheles (Ag) and three Apis (Am) TEPs are aligned. Lineage-specific family expansions are indicated with color shades (blue for Drosophila and pink for Anopheles). Pink arrowheads at nodes denote bootstrap values greater than 800 for 1,000 trials, and green bars link the predicted 1:1 and 1:1:1:1 orthologs.

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Additional data file 7:

The amino acid sequences of seven Tribolium (Tc), six Drosophila (Dm), five Anopheles (Ag), two Apis (Am) and one Bombyx (Bm) spätzles are aligned for building the unrooted tree. Pink arrowheads indicate nodes with significant bootstrap values (>800 of 1,000 trials), and green bars connect the putative orthologous pairs or trios.

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Additional data file 8:

The entire sequences of Tribolium (Tc), Tenebrio (Tm), Holotrichia (Hd), and Drosophila (Dm), Anopheles (Ag), Apis (Am), Bombyx (Bm) and Manduca (Ms) proPOs are compared. Tribolium proPO3, >99% identical in amino acid sequence to Tc-proPO2, is not included in the analysis. The phylogenetic tree, derived from the multiple sequence alignment, shows the extensive family expansion (shaded pink) in the malaria mosquito. Pink arrowheads point to nodes with high bootstrap values (>800 from 1,000 trials), and green lines link the predicted 1:1 or 1:1:1 orthologs.

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Additional data file 9:

(a) GTX, (b) SOD and (c) TPX. The Tribolium (Tc), Drosophila (Dm), Anopheles (Ag) and Apis sequences are studied. As shown in the trees, duplication and divergence have given rise to gene clusters (shaded yellow for Tribolium and blue for Drosophila). Pink arrowheads denote nodes with high bootstrap values (>800 in 1,000 trials), whereas green lines connect the putative orthologs with 1:1, 1:1:1 or 1:1:1:1 relationships.

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Additional data file 10:

The sequences of four Tribolium (Tc), twelve Drosophila (Dm), five Anopheles (Ag), one Bombyx (Bm), one Manduca (Ms), and two Apis (Am) lysozymes are aligned and used to derive the tree (upper panel). Lineage-specific expansion (shaded in different colors) occurs quite extensively in this family of enzymes. For instance, four Tribolium lysozyme genes are found as a gene cluster (lower panel) at the same genomic location. Pink arrowheads at nodes indicate bootstrap values greater than 800 from 1,000 trials. A green bar links the putative orthologous pair.

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Additional data file 11:

Functions, families, and counts of putative defense proteins from D. melanogaster, A. gambiae, A. mellifera and T. castaneum

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Additional data file 12:

Oligonucleotide primers used in expression analysis by real-time PCR

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