RESEARCH ARTICLE Infective larvae of Anisakis simplex (Nematoda) accumulate trehalose and glycogen in response to starvation and temperature stress Elz ̇ bieta Lopien ́ ska-Biernat 1, *, Robert Stryin ́ ski 1 , Malgorzata Dmitryjuk 1 and Barbara Wasilewska 2 ABSTRACT Anisakis simplex L3 larvae infect fish and other seafood species such as squid or octopi; therefore, humans consuming raw or undercooked fish may become accidental hosts for this parasite. These larvae are induced to enter hypometabolism by cold temperatures. It is assumed that sugars (in particular trehalose and glycogen) are instrumental for survival under environmental stress conditions. To elucidate the mechanisms of environmental stress response in A. simplex, we observed the effects of starvation and temperature on trehalose and glycogen content, the activity of enzymes metabolizing those sugars, and the relative expression of genes of trehalose and glycogen metabolic pathways. The L3 of A. simplex synthesize trehalose both in low (0°C) and high temperatures (45°C). The highest content of glycogen was observed at 45°C at 36 h of incubation. On the second day of incubation, tissue content of trehalose depended on the activity of the enzymes: TPS was more active at 45°C, and TPP was more active at 0°C. The changes in TPP activity were consistent with the transcript level changes of the TPP gene, and the trehalose level, while glycogen synthesis correlates with the expression of glycogen synthase gene at 45°C; this suggests that the synthesis of trehalose is more essential. These results show that trehalose plays a key role in providing energy during the thermotolerance and starvation processes through the molecular and biochemical regulation of trehalose and glycogen metabolism. KEY WORDS: Glycogen, Nematoda, Trehalose, Stress protection, Anisakis simplex INTRODUCTION Anisakis simplex is a parasitic aquatic nematode that colonizes crustaceans, cephalopods, fish and marine mammals. The occurrence of Anisakis nematodes has been reported in all major oceans and seas (Sakanari and McKerrow, 1989). The accidental intake of these nematodes, generally after the consumption of raw or undercooked parasitized fish, (herring, hake, horse mackerel and cod) can cause digestive disorders and/or allergies in humans (Valls et al., 2005; Audicana and Kennedy, 2008). Anisakis larvae produce proteolytic enzymes, penetrate the host’s gastric and intestinal mucosa and cause mucosal infections which are referred as anisakiasis (Sakanari and McKerrow, 1990). According to the sanitary authorities of the USA and the EU, fish products should be cooked at 60°C for 10 min or longer to prevent infection. Fish products that are not intended for cooking or processing at temperatures higher than 60°C should be deep frozen at -20°C for 24 h or at -35°C for >15 h or at -23°C for a minimum of 7 days. Decapitation and evisceration of freshly caught fish and storage at low temperature until consumption are recommended (Audicana et al., 2002; Garcia et al., 2012; Nieuwenhuizen and Lopata, 2014). The regulations issued by the USA and EU sanitary authorities can be found online at http://www.fda.gov/ and https://europa.eu/ european-union/index_en. Trehalose and glycogen play important roles in free-living, entomopathogenic and parasitic nematodes exposed to thermal, oxidative and desiccation stress (Wharton et al., 2000; Grewal and Jagdale, 2002; Jagdale et al., 2005). For these reasons, carbohydrate metabolism in L3 larvae of A. simplex continues to attract the interest of researchers with the aim of determining new anthelmintics. The results of our previous study of L3 larvae of A. simplex (Lopień ska- Biernat et al., 2006), which do not feed and rely solely on external sources of energy, support analyses of the hypometabolic state of this larvae. The mechanisms which are implicated in the regulation of carbohydrate metabolism under stress and which ensure high survival have to be studied in greater detail. Trehalose [α, D-glucopyranosyl-(1→1)-α, D-glucopyranoside] is a ubiquitous nonreducing disaccharide (Eastmond et al., 2002) in all kingdoms, excluding mammals (El-Bashiti et al., 2005). Living organisms rely on trehalose as a source of energy which stabilizes cell membranes, is responsible for the liquid-crystal phase of phospholipid bilayers, participates in protein folding and stabilizes native proteins (Singer and Lindquist, 1998; Silva et al., 2005; Zhu et al., 2010). Nematodes synthesize trehalose in two steps. In the first step, trehalose-6-phosphate synthase (TPS, EC 3.1.3.12) catalyzes the synthesis of trehalose-6-phosphate (T6P) from glucose-6-phosphate (G6P) and uridine diphosphate glucose (UDPG). In the second step, the dephosphorylation of T6P to trehalose is catalyzed by trehalose-6-phosphate phosphatase (TPP, EC 2.4.1.15) (Pellerone et al., 2003; El-Bashiti et al., 2005). Lopieńska-Biernat et al. (2014, 2015) demonstrated TPP and TPS activity and the expression of tps and tpp genes in A. simplex. The expression and activity of TPS and TPP under exposure to stress have never been studied in A. simplex. Trehalose undergoes hydrolytic and phosphorolytic degradation. During hydrolysis, trehalose is broken down by the enzyme trehalase (EC 3.2.1.28), whereas phosphorolytic decomposition is catalyzed by trehalose Received 6 November 2018; Accepted 20 February 2019 1 Department of Biochemistry, Faculty of Biology and Biotechnology, University Warmia and Mazury in Olsztyn, Oczapowskiego 1A, 10-719 Olsztyn, Poland. 2 Department of Animal Anatomy and Physiology, Faculty of Biology and Biotechnology, University Warmia and Mazury in Olsztyn, Plac Ló dzki 3, 10-727 Olsztyn, Poland. *Author for correspondence (ela.lopienska@uwm.edu.pl) E.L.-B., 0000-0003-3265-5207; R.S., 0000-0001-5518-8854; M.D., 0000-0002- 9986-3847 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. 1 © 2019. 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