Elsevier

Veterinary Parasitology

Volume 205, Issues 3–4, 15 October 2014, Pages 540-550
Veterinary Parasitology

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

https://doi.org/10.1016/j.vetpar.2014.09.013Get rights and content

Highlights

  • Molecular characterization of Hsp90 of different Anisakis species.

  • Experiment to test relevance of Hsp90 for temperature resistance of A. pegreffii.

  • Quantification of Hsp90 gene expression on RNA and protein level.

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 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.

Introduction

Marine nematodes of the genus Anisakis are food-borne parasites that can infect over 200 pelagic fish species and cephalopods such as squid. Humans may become accidental hosts if they ingest raw or undercooked infected seafood such as Japanese sushi, sashimi, Hawaiian lomi-lomi, Latin American ceviche and Dutch salted or smoked herring (Bouree et al., 1995, Alonso-Gómez et al., 2004). Even dead parasites in frozen or well-cooked seafood can induce allergic reactions in sensitive patients to this parasite (Rodríguez-Mahillo et al., 2010, Tejada et al., 2013). Three species of the family Anisakidae are known to cause infections in humans: A. simplex sensu stricto, A. pegreffii and Pseudoterranova decipiens (Quiazon et al., 2011, Arizono et al., 2012, Mattiucci et al., 2013). The digestive symptoms of anisakiasis include acute gastric, abdominal pain, nausea, vomiting, abscessing or eosinophilic granulomas and stomach ulcers. The excretory-secretory products of larvae and dead larvae may also induce severe allergic reactions such as rash, itching, anaphylaxis, acute urticaria and angioedema (Audicana et al., 2002, Baird et al., 2014, Sharp and Lopata, 2014). To date, twelve allergens of A. simplex are described and five of those (Ani s 1, 4, 5, 8 and 9) are highly resistant to heat (Audicana and Kennedy, 2008, Rodriguez-Perez et al., 2008, Quiazon et al., 2013).

Several papers have been published concerning the survival and resistance of anisakids under different treatment conditions (Brutti et al., 2010, Tejada et al., 2014). The survival capability of the third-stage Anisakis larvae was tested in various conditions, such as in microwaving, freezing, heating, salting, as well as application of anthelmintic drugs and condiments (Wang et al., 2010). The common method to kill the larvae in fish is the application of high or low temperature. Lanfranchi and Sardella (2010) found that Anisakis can survive up to 330 days in NaCl solution (0.85%) at 4–5.5 °C. Vidaček et al. (2010) reported that larvae were immobile and dead after heating at 60 °C for ≥3 min, 70 °C for ≥1 min and 80 °C for ≥30 s. According to the US Food and Drug Administration (FDA) recommended that fish should be cooked to an internal temperature of at least 63 °C and 74 °C for stuffed fish (FDA/CFSAN, 2001). Anisakis species have been found to be alive after freezing at −20 °C for short periods and therefore the FDA recommends freezing fish at −20 °C for at least 168 h or blast-freezing at −35 °C for at least 15 h.

Heat shock proteins (Hsps) are known to exist in almost all living organisms, ranging from bacteria to humans. These proteins are involved in development, growth, signal transduction, cellular metabolism and innate immune response. They play critical roles in responses to stress such as low or high temperature, heavy metal exposure or parasite infections (Asea et al., 2000, Devaney, 2006, Saunders and Verdin, 2009, Shiny et al., 2011, Yang et al., 2013, De Jong et al., 2014). According to their molecular weight, Hsps have been grouped into several families: Hsp100 (100 kDa), Hsp90 (83–90 kDa), Hsp70 (67–70 kDa), Hsp60 (60 kDa), Hsp40 (40 kDa) and small heat shock proteins (16–25 kDa) (Lindquist and Craig, 1988).

Hsp90 is an essential molecular chaperone in eukaryotic organisms and abundant in unstressed cells, where it accounts for 1–2% of the cytosolic proteins (Pratt, 1998). Hsp90 exists as a homodimer which consists of a highly conserved N-terminal ‘ATPase domain’, a charged linker region, that connects the N-terminus with the middle domain followed by ‘middle region’ and the C-terminal dimerization domain, which were involved client proteins and cochaperones binding (Johnson, 2012, Eckl and Richter, 2013). Its functions are related to cell growth and differentiation, apoptosis, signal transduction, oncogenic transformation, antigen presentation and cancer (Graefe et al., 2002, Tsan and Gao, 2004, Péroval et al., 2006, Kumar et al., 2007, Tsutsumi and Neckers, 2007, Lamoureux et al., 2013). Hsp90 also plays an important role in response to stress condition such as heat or cold shock and induces stress proteins to protect cells. On the other hand, Hsp90 is a useful marker for phylogenetic analysis in nematodes (Skantar and Carta, 2004, Him et al., 2009). To date, no Hsp90 gene of Anisakis spp. has been characterized, therefore, we cloned, characterized and compared the full-length cDNA of Hsp90 of five Anisakis spp. Additionally, we investigated the expression levels of Hsp90 after cold and heat shock. For that, we used the third-stage larvae of A. pegreffii as a model, which is commonly found in many marine fishes and causes human anisakiasis.

Section snippets

Sampling and experimental design

Two hundred and fifty spotted mackerel (Scomber australasicus) were obtained from professional fisherman in the coastal waters of Yilan, north-eastern Taiwan (25° N, 121° E) during the 2012–2013. Fresh specimens were stored on ice and immediately transported to the laboratory. Live Anisakis spp. third-stage larvae (L3) were recovered from the visceral cavity and intestine of fish. After washing in physiological saline, species and developmental stages of Anisakid nematodes were pre-identified

Morphological and PCR-RFLP analysis of Anisakis spp.

The third-stage of Anisakis larvae were obtained from S. australasicus, which were collected in Taiwanese waters. Through a combination of morphological and molecular analyses the species A. typica, A. pegreffii belonging to Type I and A. paggiae, A. brevispiculata, A. physeteris belonging to Type II were detected. Additionally, the molecular analysis revealed a recombinant genotype of A. simplex s.s. and A. pegreffii. S. australasicus is a new host record for A. paggiae, A. brevispiculata, A.

Discussion

In our previous study, we found three Anisakis nematodes in S. australasicus collected from Taiwanese waters: A. pegreffii, A. typica or a recombinant genotype of A. pegreffii and A. simplex sensu stricto (Chou et al., 2011). In addition, A. simplex s.s. and A. pegreffii had previously been recovered from this species of fish in Japanese waters (Quiazon et al., 2008). In the present study, A. paggiae, A. physeteris and A. brevispiculata are reported for the first time from this host, providing

Conclusion

In the present investigation, new host and locality records were provided for A. paggiae, A. physeteris and A. brevispiculata from S. australasicus in the coastal waters of northeastern Taiwan. We report for the first time the full-length cDNA sequence of Hsp90 and the molecular characteristics of this gene in five species of the genus Anisakis. In an experimental approach, we have examined the expression patterns of Hsp90 at different time-temperature combinations in A. pegreffii. Our findings

Acknowledgments

We wish to thank Yin-Chun Jian for providing the fish samples. We thank Prof. Lian-Chen Wang (University of Chang Gung) for the kind gift of the 5′/3′ RACE kit and technical assistance. We thank Li-Yuan Hung (Genomics Research Center, Academia Sinica) and Christoph Koch (University of Duisburg-Essen) for revising an early draft of the manuscript. We would like to sincerely thank Prof. Bernd Sures (University of Duisburg-Essen) for reading the manuscript and his helpful comments. The project was

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