Amino acid alignment of Anisakis haemoglobin with haemoglobins of Ascaris, Pseudoterranova and Nippostrongylus brasilensis, and phylogenetic analysis.

A) The cDNA sequence of Anisakis haemoglobin was obtained by degenerate PCR and RACE-PCR (accession number: JX860676) and the amino acid sequence was deduced. Alignment of sequences was carried out using MUSCLE (http://www.ebi.ac.uk/Tools/muscle/index.html). The hydrophobic leader portion (sequence not obtained in Anisakis) and the C-terminal tail (important for assembly of octamers in Ascaris) are boxed in black, while hydrophobic haem-binding regions are boxed in red. The haemoglobin protein consists of two homologous domains, illustrated by dashed and dotted lines. The B10 tyrosine, E7 distal glutamine and F8 proximal histidine, important in binding of oxygen, are conserved between the three nematodes and are marked with boxes. B) A range of haemoglobin sequences across different species was obtained by BLAST search. The evolutionary history was inferred using the Minimum Evolution method [21]. The bootstrap consensus tree inferred from 2000 replicates is taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. The ME tree was searched using the Close-Neighbor-Interchange (CNI) algorithm at a search level of 0. The Neighbor-joining algorithm was used to generate the initial tree. The analysis involved 26 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 64 positions in the final dataset. Evolutionary analyses were conducted in MEGA5.