Study on heat shock proteins of Anisakis spp.
Date Issued
2015
Date
2015
Author(s)
Chen, Hui-Yu
Abstract
Anisakis nematodes were found in a variety of marine fishes around the world and they are known to cause anisakiasis in human hosts. Anisakis third-stage larvae (L3, n = 502) were isolated from 250 spotted mackerel Scomber australasicus caught from the coastal waters of Yilan, in northeastern Taiwan (25◦ N, 121◦ E). Anisakis nematodes were pre-identified morphologically and later molecularly by PCR-RFLP. A simple molecular taxonomic key, utilizing RFLP by two restriction enzymes HinfI and HhaI, enabled the differentiation of the genus Anisakis. We obtained six species of the genus Anisakis, A. pegreffii, A. typica, A. paggiae, A. brevispiculata, A. physeteris, and a recombinant genotype between A. pegreffii and A. simplex sensu stricto. Thereby we provide new host and locality records for A. paggiae, A. brevispiculata and A. physeteris. A. pegreffii was determined to be the dominant species (prevalence = 57.2%) and important agent of human anisakiasis. The phylogeny of the Maximum Likelihood and Neighbor Joining trees show two well supported clades: one includes the species of A. pegreffii and the other includes A. paggiae, A. physeteris and A. brevispiculata, while A. typica has basal position to all other Anisakis spp. analyzed. To analyze the cellular response to temperature stress in Anisakis, the heat shock protein 90 (Hsp90) was chosen in the study, as it plays a key role in many cellular processes and responds to stress conditions such as heat or cold shock. The Hsp90 genes of five species (except the recombinant genotype) were cloned by rapid amplification of cDNA ends (RACE). Quantitative RT-PCR showed that Hsp90 transcript levels increased slightly under heat shock (50˚C) treatment, and increased gradually during the first 3 h, and thereafter, returned to its baseline value at 37˚C. Under cold shock (4˚C) treatment, the mRNA expression of Hsp90 did not change significantly. In addition, we found a clear time-dependent Hsp90 protein expression pattern of A. pegreffii exposed to high temperature. Our results suggest that the mRNA and protein expression patterns of Hsp90 are related to the temperature, and are especially significantly increased under heat stress. On the other hand, to elucidate the possible roles of Hsp70 and Hsp90 in the life cycle of the parasitic nematode Anisakis, particularly third- and fourth-stage larvae, which are transferred from cold-blooded fish to warm-blooded marine mammals or accidentally to human hosts, and we examined the expression profiles of Hsp70 and Hsp90 in different developmental stages of A. pegreffii. On heatmap and phylogenetic analysis, ApHsp70 and ApHsp90 shared the highest amino acid sequence identity with other nematodes and formed a monophyletic clade. The three-dimensional (3D) structure prediction of the newly characterized ApHsp70 and known ApHsp90 gene showed highly conserved motifs between A. pegreffii and other species. Quantitative real-time PCR and western blot analysis revealed higher mRNA and protein expression for ApHsp70 and ApHsp90 in fourth- than third-stage larvae, with higher mRNA and protein expression for ApHsp70 than ApHsp90. Our results suggest that ApHsp70 and ApHsp90 may play important roles in Anisakis in response to the thermal stress , and might be important molecules in the development of A. pegreffii, which has implications for its control.
Subjects
Anisakis nematode
molecular taxonomic key
Hsp90
Hsp70
stress
development
SDGs
Type
thesis
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