A total of 146 samples of European hake M. merluccius with similar size (18 ± 1.5 cm) were bought from fisherman at the port of Bizerte (Tunisian northern waters). The fish were immediately transported to the laboratory in polystyrene, ice-cooled boxes. Only immature specimens were included in this study in order to avoid any inference with the reproduction cycle. All specimens were immediately examined to look for the presence of L. lusci. Specimens infected with L. lusci were further dissected and only samples without other macro parasites (such as worms) were kept to avoid any bias. The fish were determined to be free from infection with L. lusci or other macro parasites were taken as control samples. All the fish were filleted. The fillet samples from the parasitized and unparasitized fish were weighed (5.0 ± 0.5 g), and then stored at −30 °C until analysis.

Total lipids were extracted from the fillet samples according to the method of Folch et al., (1957), using a mixture of the chloroform-methanol (2:1) solvent containing 0.01% butylated hydroxytoluene (BHT) as antioxidant. After extraction, the solvent was evaporated under a nitrogen stream and the lipids were dried by vacuum desiccation. Aliquots of total lipids were subjected to transesterification as proposed by Cecchi et al., (1985). The obtained fatty acid methyl esters (FAMEs) were analyzed in an HP 6890 GC (Agilent Technologies, Sacramento, CA, USA) equipped with an Innowax 250 capillary column (0.25 inside diameter ×30 m length, 0.25 μm film; Agilent Technologies). The carrier gas was nitrogen and the injector temperature was set at 250 °C. The oven was programmed to rise from 50 °C to 180 °C at a rate of 4 °C·min⁻¹, then to reach 220 °C with a ramp rate of 1.33 °C·min⁻¹, and finally to stabilize at 220 °C for 7 min. Chromatographic data were recorded using a Hewlett-Packard ChemStation software and FAMEs were identified by comparison of their retention times with those of known standards (Supelco 47085U PUFA No: 3 and Supelco 37 component FAME mix 47885-U). Methyl nonadecanoate (C19:0, sigma) was used as an internal standard and fatty acids were reported as mg·g⁻¹ wet weight.

Data from the fatty acid composition analyses were used to assess the nutritional profile of the lipid fraction. Three indices were used: (i) index of atherogenicity (IA), (ii) index of thrombogenicity (IT) and (iii) hypocholesterolemic/ hypercholesterolenic fatty acid ratio (HH).

The IA and IT indices, which are important tools to estimate the probability of developing coronary heart disease, were calculated according to the Ulbricht and Southgate (1991) equations:

IA = ([12: 0 + (4 × 14:0) + 16:0])/([ΣMUFA + ΣPUFA (ω – 6) + (ω – 3)])

IT = (14:0 + 16:0 + 18:0)/((0.5 × ΣMUFA) + (0.5 × Σn – 6PUFA) + (3 × Σω – 3PUFA) + (ω – 3)/(ω – 6)

Where ΣMUFA is the sum of monounsaturated fatty acids and ΣPUFA is the sum of polyunsaturated fatty acids.

The hypocholesterolemic/hypercholesterolemic index (related to cholesterol metabolism) was determined according to Santos-Silva et al., (2002) equation as follows:

H/H = (18:1ω – 9 + 18: 2ω – 6 + 20: 4ω – 6 + 18: 3ω – 3 + 20: 5ω – 3 + 22: 5ω – 3 + 22: 6ω – 3)/(14: 0 + 16: 0)

Further ratios related to fatty acid content were also calculated as PUFA/SFA (polyunsaturated FA/saturated FA) and ω-3 / ω-6.

Means of three replicates (separately prepared) were calculated by Microsoft Excel software (2016 version). Statistical analyses were carried out using R software version 3.3.3 (R Core Team, 2017). Data were analyzed for normality and variance homogeneity through Kolmogorov-Smirnov and Levene’s tests, respectively. One-way analysis of variance (ANOVA) and Tukey HSD’s test (at p < 0.05) were performed so as to detect significant statistical differences. Raw data were arcsine-transformed before analysis when necessary to meet requirements for normal distribution and homogeneity of variances.

Table 1 shows the fatty acid profiles in the fillet of the European hake M. merluccius unparasitized and parasitized by the copepod L. lusci with different intensities. In the unparasitezed group, the total fatty acid content (∑FA), expressed on a wet weight basis, comprised about 0.5% of the hake fillet (0.519 mg per g) and followed a relative pattern with PUFA > SFA > MUFA. Our findings are in agreement with those of Huynh and Kitts (2009) who reported that unlike other lean fish, the pacific hake exhibited a typical pattern with the highest amount of SFA than MUFA. It was found that Palmitic acid (C16:0) was the predominant fatty acid among the SFA fraction; whereas the most abundant MUFA was oleic acid (C18:1), followed by palmitoleic acid (C16:1) with 0.453 and 0.217 mg·g⁻¹ respectively. The PUFA content reached 2.670 mg·g⁻¹ (constituted about half of the total fatty acids) with higher prevalence of PUFA in the n-3 family (about 70% of total PUFA) and with a predominance of docosahexanoic acid (DHA, C22:6ω-3) and to lesser extent, eicosapentaenoic acid (EPA, C20:5ω-3). Our data are comparable to those previously reported for other lean and low-fat fish in which the amounts of DHA generally exceeded those of EPA (Mendez and Gonzalez, 1997; Huynh and Kitts, 2009). In this context, Huynh and Kitts (2009) have reported that the highest DHA amount seems to be an inherent characteristic of lean fish, probably due to their greater ability to retain this fatty acid.

Variations in contents and composition of fatty acids of fish due to parasite infections have been confirmed (Khan, 1988; Hajji et al., 2015). Herein, the decrease in the total FA content of M. merluccius fillet was quantitatively substantial and depended on the L. lusci load. In fact, the FA content dropped by 50, 70 and 83% when the fish was infected by one single (L1), two (L2) and three (L3) parasites, respectively, in comparison to the control. Our results are in line with the findings of Khan (1988), who reported that infection by the copepod L. branchialis resulted in a significant reduction (up to the half) in the fat content of cod.

Within the fatty acid composition, we noticed that there was a tendency for a significant decrease in the SFA, MUFA and PUFA concentrations in all parasitized groups and in a parasite load dependant manner (p < 0.05). Thereby, the L3 group was the most affected one, the SFA, MUFA and PUFA contents decrease by 77.84 and 85%, respectively, compared to the control group. The same observations were made for the majority of fatty acids when analyzed individually. The pronounced effects of L. lusci were observed in fish infected with two or more parasites. In fact, the intraspecific competition for host resources and the metabolic demands of the adult parasites resulted in the associated decreased values mainly in essential fatty acids. Herein, comparing with the control group (L0), the arachidonic acid (C20:4ω-6, ARA), EPA and DHA contents dropped by around 85% in L2 and by 90% in L3. Those fatty acids play key roles in growth, development processes and the modulation of the immune response in copepods (Fast et al., 2004; Leiknes et al., 2016). The hijack of these fatty acids from the host is an “easy way out” for parasites to acquire essential nutrients and the ability of selective diversion of these essential fatty acids from the host was signalled for other parasitic copepod (Hajji et al., 2015; Telahigue et al., 2017).

In the current study, the substantial changes observed in the fatty acid composition in relationship to the parasite load can negatively affect the nutritional quality of the fish for the human consumer. Health-related lipid indices such as atherogenic index (IA), thrombogenic index (IT), hypocholesterolemic:hypercholesterolemic ratio (H/H), polyunsaturated fatty acid: saturated fatty acids (PUFA/SFA), ω-3/ω-6 ratio, EPA and DHA levels are extensively used to assess the nutritional quality and the potential of fish oil to prevent diseases. The nutritional quality indices determined for different fish groups in this study are shown in Table 2.

The polyunsaturated and saturated fatty acid (PUFA/SFA) ratio is an index usually adopted to evaluate the nutritional quality of lipids in a diet. Diets with a PUFA/SFA ratio lower than 0.45 have been viewed as undesirable for the human diet due to their ability to increase cholesterol in the blood (Department of Health and Social Security, 1994). In our study, despite the fact that L. lusci infection has induced a significant reduction in this index (0.88, 0.87 and 0.93 in L1, L2 and L3 groups respectively) when compared to the control (1.46), all recorded values were above the recommended rate.

The ratio of ω-3/ω-6 is another good index widely used to assess the nutritional value of fish oil. As shown in Table 2, the obtained values for M. merluccius ranged from 4.30 and 5.35, and were insignificantly different among all the studied groups. In all cases, the reported values exceed the optimal nutritional ratio recommendations which vary from 1/1 to 1/4. Our findings are in agreement with Hossain (2011), who reported that in general, fish lipids and fish products have a much higher ω-3/ω-6 ratio than what is recommended. According to the same author, the elevated ω-3/ω-6 ratio is highly beneficial for the daily human diet. However, other authors stress that this index is of limited value without consideration of which fatty acids are present (Arts et al., 2001). Furthermore, according to Wijendran and Hayes (2004), the intake of PUFA (mainly EPA and DHA) expressed in terms of mass (% en or g/d) is a more optimal approach to a dietary ω-3 and ω-6 fatty acid balance than a simple ratio.

The beneficial effects of EPA and DHA in animal models and humans are very well documented. They are known to exert anti-inflammatory action (Calder 2010) and are considered as potent hypotriacyl glycerolemic and antiarrhythmic agents (Billman et al., 1999). According to the US Department of Agriculture and U.S. Department of Health and Human Services (2010), an average of 250 mg/day of EPA+DHA is required for individuals at both higher and average cardiovascular disease risks. However, the American Heart Association recommends about 1.0 g/day of EPA and DHA to reduce the risk of death from coronary heart disease (Kris-Etherton et al., 2003). On the other hand, a daily intake of 0.2 g of EPA and DHA is proposed by the British Nutrition Foundation (2009) for people with balanced and healthy diets. In our study, the results reported in Table 2 show that among the analyzed groups, the unparasitezed fish had the highest combined EPA and DHA level (160.48 mg/100 g wet wt). The recorded level is below those previously reported for other Gadidae species such as M. productus (240 mg/100 g) (Huynh and Kitts, 2009) and M. hubbsi (440 mg/100 g) (Mendez and Gonzalez, 1997). In fact, the amount of EPA+DHA in fish is highly variable and strongly dependent on several parameters such as species, dietary intake, lipid content and the size of the fish (Huynh and Kitts, 2009; Tocher et al., 2010; Strandberg et al., 2017). The current study revealed that the L. lusci infection considerably reduced the EPA+DHA contents of M. merluccius fillet (p < 0.05), which fell to 40.37, 20.25 and 10.43 mg/100 g wet wt in L1, L2 and L3 groups, respectively (Table 2). These amounts account for 16, 8 and 4%, respectively, of the minimum recommended daily requirements of 250 mg/day.

To further ascertain the global dietetic quality of lipids and their potential effect on the development of coronary disease, other indices based on the functional effects of different fatty acids are often used. Ulbricht and Southgate (1991) proposed two indices: IA (atherogenic index) and IT (thrombogenic index), which might better characterize the atherogenic and the thrombogenic potential of fatty acids (Hosseini et al., 2014). It is assumed that lipids with higher values of IA and TI (>1.0) are detrimental to human health (Stancheva et al., 2014).

In addition to these indices, the hypocholesterolemic and hypercholesterolemic fatty acid ratio (H/H), which considers the specific effects of fatty acids on cholesterol metabolism, is a confirmed indicator of the cholesterolemic value of the lipid source (Fernandes et al., 2014). From a nutritional standpoint, high H/H values are considered more beneficial for human health. Among the current studied groups, the unparasitezed one presented the greatest H/H (1.81) followed by the L1 group (0.96) while L2 and L3 groups showed the lowest values (Table 2).

Thus, unlike the H/H index, the lower the value of both indices, the better the nutritional quality of the fatty acids and vice versa. In the current study, the IA and IT indices of unparasitezed fish were 0.66 and 0.26, respectively (Table 2). The L. lusci infection had significantly increased the IA as well as the IT in all the parasitized groups (p < 0.05) when compared to the control group. However, a more pronounced negative effect was observed for IA since it exceeded the recommended value for a healthy diet. In the parasitized group, the higher values of IA and IT are related to the lower contents of PUFA and MUFA when compared to the control. In addition, the low levels of the H/H ratio of the parasitized hake are related to the low content of the hypocholesterolemic fatty acids (C18:2ω-6, C20:4ω-6, C18:3ω-3, C20:5ω-3 and C22:6ω-3). Hence, the consumption of parasitized hake could increase the intake of cholesterol, atherogenic and thrombogenic fats that have negative health implications particularly for patients on a special healthy diet. It is obvious that these substances are strongly connected with the increase in the incidence of pathogenic phenomena, such as atheroma and/or thrombus formation (Rafieian-Kopaei et al., 2014). Therefore, the present study emphasizes the need to take into consideration the prevalence of parasitic species such as L. Lusci, which can reach more than 30%, for recommended fish species in health and disease prevention.