Effect of different temperatures on the expression of the newly characterized heat shock protein 90 (Hsp90) in L3 of Anisakis spp. isolated from Scomber australasicus

Abstract

Anisakid nematodes are distributed worldwide in a wide variety of marine fishes and they are known to cause the zoonotic disease, anisakiasis. The temperature control is commonly applied for prevention and control of anisakiasis. To analyze the cellular response to temperature stress in Anisakis, the heat shock protein 90 (Hsp90) was chosen in the present study, as it plays a key role in many cellular processes and responds to stress conditions such as heat or cold shock. Anisakids were sampled from spotted mackerel Scomber australasicus caught from the coastal waters of Yilan, in northeastern Taiwan (25°N, 121°E). Anisakid nematodes were pre-identified morphologically and later molecularly by PCR-RFLP. In total, we obtained six species of the genus Anisakis, A. typica, A. pegreffii, 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. The Hsp90 genes of five species (except the recombinant genotype) were cloned by rapid amplification of cDNA ends (RACE) and their deduced amino acid sequences were further characterized. Quantitative real-time PCR and Western blot analysis were used to examine the expression levels of the Hsp90 in A. pegreffii under different temperature conditions. Quantitative RT-PCR showed that Hsp90 transcript levels increased slightly under heat shock (50°C) treatment, and increased gradually during the first 3h, 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.