Functional insights into the infective larval stage of Anisakis simplex s.s., Anisakis pegreffii and their hybrids based on gene expression patterns

List of species and specimen used in the phylogenetic tree of Additional file 1. Code of the voucher specimen and accession number for mitochondrial gene COII (*: sequences obtained from GenBank). Labeled are the specimens selected for RNA sequencing (first number, population; second number specimen). A. simplex s.s. – A. pegreffii refers to hybrids haplotype according Abollo et al. [23]. (DOCX 47 kb

Supplementary Material for: Ani s 11-Like Protein Is a Pepsin- and Heat-Resistant Major Allergen of Anisakis spp. and a Valuable Tool for Anisakis Allergy Component-Resolved Diagnosis

<br><em><strong>Background:</strong></em><i>Anisakis simplex</i> is a fish parasite responsible for gastrointestinal and allergic symptoms in humans. The Ani s 11-like protein has been proposed as an <i>Anisakis</i> allergen because its primary structure is similar to that of Ani s 11. The aims of this work were to analyse the frequency of detection of the Ani s 11-like protein and assess its diagnostic value. <b><i>Methods:</i></b> rAni s 11-like protein, rAni s 5 and rAni s 4 were expressed in <i>Escherichia coli</i> and rAni s 1 was produced in <i>Pichia pastoris</i>. Recombinant allergen detection patterns in 37 <i>Anisakis</i>-sensitised patients were determined. The stability to pepsin digestion and heat treatment of rAni s 11-like protein was also analysed by IgE immunoblotting. <b><i>Results:</i></b> Ani s 11-like protein is a major allergen detected by 78% of <i>Anisakis</i>-allergic patients, and 13.5% of patients detect only the rAni s 11-like allergen. This allergen is heat stable because it retains its capability of binding IgE after boiling for 30 min and it is resistant to pepsin digestion for 120 min. <b><i>Conclusions:</i></b> These data indicate that the Ani s 11-like protein is a pepsin- and heat-resistant major allergen (Ani s 11.0201) of <i>Anisakis</i> spp. and a valuable tool for <i>Anisakis</i> allergy component-resolved diagnosis

Additional file 3: of Functional insights into the infective larval stage of Anisakis simplex s.s., Anisakis pegreffii and their hybrids based on gene expression patterns

Differential expression analyses at the whole transcriptome level. Results obtained from the analyses of differential expression; 1) Hybrids vs A. pegreffi; 2) Hybrids vs A. simplex s.s.; 3) A. pegreffii vs A. simplex s.s. Rows summarizing significant differentially expressed transcripts supported by FDR < 0.05) are highlighted in light blue. (XLSX 12115 kb

Additional file 6: of Functional insights into the infective larval stage of Anisakis simplex s.s., Anisakis pegreffii and their hybrids based on gene expression patterns

Anisakis allergome. 1) Count file and results of differential expression for the 74 allergen families detected in A. simplex s.s., A. pegreffi and hybrids. 2) Blast results obtained in the search performed using 509 known allergens as queries against the reference Anisakis transcriptome. Nine hundred and thirty seven consensus sequences of the reference transcriptome were detected to significantly match to the queries and were classified into 74 allergen families of which we include additional information. This summarization is also available in AnisakisDB as a dynamic table from where sequences and annotations can be easily navigated and retrieved. (XLS 351 kb

Additional file 5: of Functional insights into the infective larval stage of Anisakis simplex s.s., Anisakis pegreffii and their hybrids based on gene expression patterns

Differential expression of ES transcripts. 1) Count file used as input in the differential expression test performed at the ES gene level; 2) Summarization of the 356 ES genes accompanied by logFC values and their FDR support obtained from the 3 performed differential expression analyses and additional information relative to consensus sequences grouped to each gene description, as well as associated GOs terms and identification of SignalP domain. This summarization is also available in AnisakisDB as a dynamic table from where sequences and annotations can be navigated and retrieved. (XLSX 91 kb

Additional file 1: of Functional insights into the infective larval stage of Anisakis simplex s.s., Anisakis pegreffii and their hybrids based on gene expression patterns

Phylogenetic relationships of the considered populations as an individual’s selection basis for RNA sequencing of A. simplex s.s., A. pegreffii and their hybrid haplotype. Tree was obtained based on maximum likelihood (ML) and Bayesian inference analysis using mitochondrial COII gene and the GTR + I + G evolutionary model. Numbers at nodes correspond to ML bootstrap proportions (BP) (above number) and Bayesian posterior probabilities (BPP) (under number). (PNG 621 kb

Additional file 4: of Functional insights into the infective larval stage of Anisakis simplex s.s., Anisakis pegreffii and their hybrids based on gene expression patterns

Differentially enriched metabolic pathways and GOs. Annotation of metabolic pathways and and GOs integrating results of differential enrichment using GOseq. (XLSX 232 kb