Abstract
The total proteomes of Anisakis simplex s.s., A. pegreffii and their hybrid genotype have been compared by quantitative proteomics (iTRAQ approach), which considers the level of expressed proteins. Comparison was made by means of two independent experiments considering four biological replicates of A. simplex and two each for A. pegreffii and hybrid between both species. A total of 1811 and 1976 proteins have been respectively identified in the experiments using public databases. One hundred ninety-six proteins were found significantly differentially expressed, and their relationships with the nematodes’ biological replicates were estimated by a multidimensional statistical approach. Results of pairwise Log2 ratio comparisons among them were statistically treated and supported in order to convert them into discrete character states. Principal component analysis (PCA) confirms the validity of the method. This comparison selected thirty seven proteins as discriminant taxonomic biomarkers among A. simplex, A. pegreffii and their hybrid genotype; 19 of these biomarkers, encoded by ten loci, are specific allergens of Anisakis (Ani s7, Ani s8, Ani s12, and Ani s14) and other (Ancylostoma secreted) is a common nematodes venom allergen. The rest of the markers comprise four unknown or non-characterized proteins; five different proteins (leucine) related to innate immunity, four proteolytic proteins (metalloendopeptidases), a lipase, a mitochondrial translocase protein, a neurotransmitter, a thyroxine transporter, and a structural collagen protein. The proposed methodology (proteomics and statistical) solidly characterize a set of proteins that are susceptible to take advantage of the new targeted proteomics.
Highlights
The genus Anisakis is formed by 10 known and at least two undescribed species (Anisakis sp. 1 and Anisakis sp. 2) based on L3 genotypes [1,2]
In total 1811 and 1976 proteins were respectively identified by both iTRAQ experiments (Supplementary File 1) both experiments share 1423 common proteins (Figure 1A)
Criterion for identification of differential regulation among them, were at least two peptides showing a 95% level of signification (p < 0.05) of differentially regulated proteins for 1% false discovery rate (FDR) at peptide quantitation level, measured by Log2 ratios of the relative protein/peptide abundances of all independent biological pools compared with their respective references (A. simplex-1 and A.simplex-2; A. simplex-3 and A. simplex-4)
Summary
The genus Anisakis is formed by 10 known and at least two undescribed species (Anisakis sp. 1 and Anisakis sp. 2) based on L3 genotypes [1,2]. Genes 2020, 11, 913 on differences found in the excretory system, esophageal intestinal region of larvae L3 [6,7], and the morphology of adult males [8,9], the use of the nuclear ribosomal internal transcribed spacer (ITS) has provided a consistent molecular diagnostic tool for larvae L3 for routine diagnosis [10,11] and for the detection of recombinant genotypes (hybrids) between A. simplex s.s. and A. pegreffii [12,13,14] Both species are the main responsible agents of anisakiasis [15,16,17,18,19] and can co-infect the same fish host [20]. These hybrids express allergenic and immunoreactive proteins with potential clinical or pathogenic implications [22]
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