Nematodes belonging to the species A. simplex (s. s.) and A. pegreffii are considered to be highly significant pathogenic organisms. Accidental consumption of the third larval stage of these parasites (L3), may cause acute clinical symptoms, which can be relieved by removing the larvae (Mladineo et al., 2018). Infective L3 larvae of Anisakis spp. usually do not molt into human. However, few cases of the presence of L4 stage larvae were confirmed. in situA. simplex (s. s.). The L4 stage larvae of A. simplex (type I) was described in human patients in Japan (Weerasooriya et al., 1986). In a case of a patient with multiple infections of A. simplex larvae, the presence of L4 stage was also confirmed (Kagei and Isogaki, 1992). The recent case study from Korea showed that among 32 Anisakis (type I) larvae detected, 7 were in the fourth stage (L4), which had molted in the stomach of the patients (Sohn et al., 2015).

According to a ranking of foodborne parasites based on a decision analysis approach, A. simplex (s. s.) was ranked among the top ten most important parasites in Europe and anisakiasis is the fifth most common foodborne parasitic infection in southwestern Europe (Bouwknegt et al., 2017). Severe cases of anisakiasis have been reported in countries with high fish consumption, with one Spanish patient having over 200 larvae removed (Jurado-Palomo et al., 2010) and a Portuguese patient having over 140 larvae removed (Baptista-Fernandes et al., 2017). The actual number of infections is estimated from 7,700 to 8,320 cases occurring annually in Spain (Bao et al., 2019) and around 7,000 cases of the disease are currently reported annually in Japan (Suzuki et al., 2021; Yorimitsu et al., 2013).

The disease is often misdiagnosed due to its non-specific symptoms, which can mimic other gastrointestinal conditions, such as acute appendicitis or ulcer, whereas in mild cases patients are reluctant to seek medical care and the illness remains unreported. The reasons for these differences are not yet fully understood, but factors such as the environment, genetics, and awareness of the population may play a role. The increasing migration and popularity of consuming raw fish dishes also contribute to the prevalence of anisakiasis (Aibinu et al., 2019). Unfortunately, there is currently no drug available that has been proven to be effective against the parasite in humans. In cases of chronic intestinal anisakiasis, treatment with albendazole and mebendazole has been commonly used, but with conflicting results (Gómez-Mateos et al., 2021). Some sources suggest that high doses of these drugs over a long period of time may be effective (Łopieńska-Biernat et al., 2018; Pacios et al., 2005), while others explore alternatives such as natural essential oils (Navarro et al., 2008). However, a definitive cure for anisakiasis remains elusive, and further study is needed to understand the mechanisms of action and survival of the parasites. The focus on phenotypes suggests that there may be common effects among anti-infective drugs, and integrating biological information from different drug targets could be beneficial (Moffat et al., 2017). Given the complexity of the problem and the presence of drug resistance in parasites, it is crucial to investigate the effectiveness of commonly used drugs and to justify the need for further research in this area.

Programs to control parasitic nematodes in humans and animals rely heavily on the widespread administration of three classes of anthelmintics: macrocyclic lactones, imidazothiazoles and benzimidazoles. Each of these drug groups has a different mechanism of action that allows to fight the parasite (James and Davey, 2009). Macrocyclic lactones (e.g., ivermectin) affect glutamate and γ-aminobutyric acid (GABA) gated ion channels in nerve and muscle cells, increase the permeability of cell membranes for chloride ions, resulting in hyperpolarization and parasite paralysis. Imidazothiazoles (e.g., pyrantel) affect the neuromuscular transmission, as agonists of acetylcholine receptors increase the flow in ion channels, causing depolarization of muscle cells, resulting in spastic paralysis of the parasite. Benzimidazoles (e.g., albendazole, thiabendazole) interfere with the formation of microtubules in cells, and inhibit fumarate reductase, which is involved in the citrate reduction cycle, causing parasite death (Holden-Dye et al., 2013; Martin et al., 2012; Whittaker et al., 2017). Despite the specific mechanisms of action of each group of drugs, in many parasitic nematodes the effectiveness of anthelmintics is equivocal and varies between species. In this work, we focused on the first two classes, with pyrantel and ivermectin as representatives.

The majority of genes involved in xenobiotic metabolic processes or drug resistance mechanisms are those that participate in oxidation-reduction processes (such as cytochromes P450, CYPs), ABC transporters, multidrug resistance protein genes (pgp), and members of the nuclear hormone receptor family (nhr) (Ardelli, 2013; Prichard et al., 2012). Transcriptomic data from helminth parasites will undoubtedly contribute to our understanding of helminth genomes and support post-genomic progress in many other medically and economically important species. The complete, multigenic nature of parasite response to drugs and, in particular, knowledge of their functionality could provide an important clue to the genetic basis of drug resistance. Differences in the background genetics of the parasite and the nature of the selection pressures to which worms are subjected under different environmental conditions may mean that resistance to individual drug classes is based on species-specific genetic changes and mechanisms (Chaudhry et al., 2015).

Therefore, our study is the first of its kind to analyze the expression patterns of specific genes related to drug transformation and resistance in two developmental stages of A. simplex (s. s.). The main hypothesis is that A. simplex (s. s.) is resistant to usually used anthelmintics (PYR and IVM) or the mechanism of drug action differs from that known from the literature. In this work, we focused on the two classes of anthelmintics: macrocyclic lactones, and imidazothiazoles, with ivermectin (IVM) and pyrantel (PYR) as representatives, respectively. Consequently, our study aimed to evaluate the gene expression of ABC transporters, antioxidant enzymes, GABA, and nicotinic acetylcholine receptors, and also examined the oxidative status of A. simplex (s. s.).

Similar analyzes on the species Pseudoterranova decipiens (s. s.) and Contracaecum osculatum (s. s.) have been published by our team (Polak et al., 2022), and this work is the culmination of research on representatives of the family Anisakidae.