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. 2026 Mar 26;43:e00334. doi: 10.1016/j.fawpar.2026.e00334

Local host fish-Anisakis spp. parasite lipid interplay: A lipidomic characterization of Anisakis larvae and parasitized tissue of its host fish, the European hake (Merluccius merluccius)

João P Monteiro a,b,c,⁎,1, Tiago Sousa a,b,1, Fernando Atroch d,e, Luís Filipe Rangel d, Camilo Ayra Pardo d, Paula Ramos e,f, Maria João Santos d,e, Felisa Rey a,b, Ricardo Calado g,⁎, M Rosário Domingues a,b,⁎
PMCID: PMC13084367  PMID: 42003893

Abstract

Anisakid nematodes are widespread marine parasites with zoonotic potential and possible impact on fish nutritional quality. In this study, we investigated the lipid composition of L3 Anisakis larvae, the host fish tissue adjacent to the presence of parasites (belly flaps) and the tail (used as a control due to the absence of Anisakis larvae) of parasitized European hake (Merluccius merluccius), combining fatty acid profiling and advanced lipidomics. Fatty acid analysis revealed distinct profiles between the Anisakis spp. parasites and hake tissues, with Anisakis larvae showing higher levels of stearic acid (18:0), vaccenic acid (18:1n-7), and linoleic acid (18:2n-6), while hake tissues, namely the tail, contained more palmitic acid (16:0), palmitoleic acid (16:1n-7), docosahexaenoic acid (22:6n-3), and omega-3-rich polyunsaturated fatty acids in general. Principal component analysis positioned the parasitized tissue of the hake belly flaps as an intermediate group, suggesting possible local metabolic interference. Lipidomics identified a total of 477 different lipid species and highlighted the existence of the following differences: saturated triacylglycerols and ether-linked phosphatidylcholines, phosphatidylethanolamines, and sphingomyelins species were more prevalent in Anisakis larvae, while some highly unsaturated phosphatidylcholines and monounsaturated triglycerydes were more abundant in hake tissues; specific sphingomyelins and ether lipids were exclusive of the fish tissue adjacent to the parasites (belly flaps) and may represent potential biomarkers of Anisakis spp. infection. Together, these results suggest that Anisakis larvae may influence/modulate local lipid metabolism, with parasite influence being more pronounced in fish tissue closer to the infection site with regard to distant unparatized tissue. Ultimately, this work supports the use of lipid profiling for better understanding host-parasite interactions in marine fish and unveils applications for parasite infection detection.

Keywords: Fish parasitism, Food safety, Anisakis spp. L3 larvae, Lipidomics, Nutritional quality, Zoonosis

Graphical abstract

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Highlights

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    Distinct lipid profiles found between Anisakis larvae and hake host tissues.

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    Larvae richer in saturated TAGs and ether-linked phospholipids.

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    Hake tissues richer in omega-3 and highly unsaturated phospholipids.

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    Local metabolic interference suggested in parasitized belly tissues.

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    Lipid profiling reveals biomarkers of Anisakis infection in European hake.

1. Introduction

The European hake (Merluccius merluccius) is a marine fish species for which particularly high levels of L3 Anisakis larvae infection in terms of intensity, abundance and prevalence have consistently been reported (Fuentes et al., 2022; Santos et al., 2022; Ramilo et al., 2023; Cipriani et al., 2025). This species is widely distributed in the Northeast Atlantic and the Mediterranean Sea (Casey and Pereiro, 1995), and holds great commercial and nutritional importance, particularly in Europe, where it is highly valued (Santos et al., 2022; Ospina-Alvarez et al., 2024; Cipriani et al., 2025). Therefore, the presence of parasites, such as Anisakis spp., not only poses public health concerns due to their zoonotic potential, but they may also compromise fish quality, ultimately affecting the economic value of hake capture and trade.

The epidemiological and pathological aspects of anisakid infections have already been thoroughly addressed in multiple studies (Aibinu et al., 2019; Ozuni et al., 2021; Kumas et al., 2024). Nonetheless, our understanding of host-parasite biological interactions is still limited and the nutritional exchanges that take place during parasitic infections remain poorly known. Previous studies have suggested a possible interplay in lipid content between anisakid larvae and their fish hosts, despite the L3 stage of Anisakis larvae being generally considered to be non-developing and relatively nutritionally independent (Mika et al., 2010) and that an imposed decline in the fish health condition may ultimately shape its nutritional quality (Buchmann and Mehrdana, 2016; Jouini et al., 2023).

In a previous study, clear differences were reported between the fatty acid profiles of Anisakis spp. L3 larvae and adjacent host tissue tissues (belly flaps), therefore suggesting distinct metabolic pathways and a controlled and restrained lipid acquisition by the parasite (Monteiro et al., 2025b). Moreover, it was reported that Anisakis simplex L3 larvae tend to accumulate in lipid-rich regions of its host, with larval distribution correlating positively with the lipid content of host fish tissue (Strømnes and Andersen, 1998; Strømnes, 2014). These findings suggest a putative importance of lipids of the fish host in the context of parasitic infection. Moreover, studying lipids is particularly relevant when assessing the potential impact of parasitism on fish nutritional quality, as key quality parameters (especially in species of high dietary value for human consumption) are usually related to their fatty acid composition, namely the levels of omega-3 polyunsaturated fatty acids, such as eicosapentaenoic acid and docosahexaenoic acid (Patted et al., 2024; Schuchardt et al., 2024; Glencross et al., 2025).

In this study, we build upon previous observations by applying a comprehensive advanced lipidomics approach to elucidate the local interplay and potential lipid exchanges between Anisakis spp. L3 larvae and their fish host, the European hake (M. merluccius). By investigating these processes at molecular level, we aim to uncover mechanisms through which anisakid parasites may influence their host fish tissue composition and potentially shape its nutritional value. We also seek to assess the extent to which the lipid profile of these parasites depends on that of its host fish. Altogether, this study represents an important step forward in our need to decipher the complex biochemical networks underpinning host-parasite interactions and their broader implications for fish physiology, nutritional quality, and overall food safety for human consumption.

2. Material and methods

2.1. Chemicals

High-grade liquid chromatography (HPLC)-grade dichloromethane (CH2Cl2), 96% absolute ethanol (CH3CH2OH), and methanol (CH3OH) were obtained from Fisher Scientific Ltd. (Loughborough, UK). Milli-Q purified water was obtained using the Synergy® system from Millipore Corporation (Billerica, MA, USA). Whatman No. 1 filter paper was acquired from Sigma-Aldrich, St. Louis, MO, USA. The 37 Component FAME Mix was purchased from Supelco (Sigma-Aldrich, St. Louis, MO, USA) along with the internal standard methyl nonadecanoate (≥99% purity) from Sigma-Aldrich (St. Louis, MO, USA). Lipid internal standards for lipidomic analyses were acquired from Avanti Polar Lipids, Inc. (Alabaster, AL, USA), including 1,2-dimyristoyl-sn-glycero-3-phosphate (dMPA), 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (dMPG), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (dMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylinositol (dPPI), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (dMPE), 1,2-dimyristoyl-sn-glycero-3-phosphatidylserine (dMPS), 1-nonadecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC), N-heptadecanoyl-D-erythro-sphingosine (Cer), 1′,3′-bis[1,2-di-tetradecanoyl-sn-glycero-3-phospho]-sn-glycerol (CL), and N-heptadecanoyl-D-erythro-sphingosylphosphorylcholine (SM). All other reagents were purchased from leading commercial suppliers guaranteeing product quality.

2.2. Samples

Five European hake (M. merluccius) specimens captured in the Bay of Biscay region (in the summer of 2023) were supplied fresh by a local fish wholesaler. The fish were eviscerated and samples were collected from the belly flaps (the muscle tissue adjacent to the visceral cavity where anisakid parasites preferentially accumulate), as well as along from the tail muscle, here used as a negative control after confirming the absence of Anisakis spp. larvae (Fig. 1), were dissected and stored at −20 °C until samples were freeze-dried for subsequent analysis. Anisakis spp. parasites either on the surface or slightly encysted on the belly flaps were collected and weighed for further processing (Monteiro et al., 2025b). The worms were morphologically identified, until genus level, using the works of Berland, 1961, Berland, 1989 and Moravec (1994). Overall, from each one of the five M. merluccius specimens (with 100% prevalence and a number of worms in one belly flap of 287 ± 31.9, Table 1), samples from their belly flaps (peritoneum and parasitized fish tissue, after complete removal of any visible signs or traces of encysted parasites), tail (here considered as non-parasitized fish tissue after a thorough confirming of the absence of parasites) and parasites present at the surface of the samples were collected for analysis (Table 1).

Fig. 1.

Fig. 1

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Schematic representation of host fish tissue locations sampled from European hake specimens. Silhouette images of European hake adapted from Creazilla.com.

Table 1.

Description of European hake (Merluccius merluccius) specimens sampled in the present study and the data regarding Anisakis spp. parasitism density.

Hake specimen Size (total length, mm) Eviscerated Weight (g) Number of anisakid parasites
(n of worms/wet weight in g)		 Number of Anisakis spp.
in one belly flap
1 670 1525 6.44 322
2 680 1489 7.33 308
3 685 1511 7.10 298
4 610 1180 7.32 249
5 655 1504 6.14 258
Mean 660 ± 27.0 1442 ± 131.4 6.9 ± 0.5 287 ± 28.5
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2.3. Lipid extraction

Total lipids from belly flap muscle, tail tissue, and Anisakis spp. larvae were extracted using a modified Bligh and Dyer protocol (Bligh and Dyer, 1959), using fully optimized protocols for lipid extraction from tissue as previously described in detail (Monteiro et al., 2021). Briefly, approximately 10 mg of freeze-dried tissue or parasite material was homogenized and subjected to sequential extraction with methanol and dichloromethane, followed by phase separation and recovery of the organic phase. Lipid extracts were dried under a nitrogen stream and stored at −20 °C until further analysis.

2.4. Phospholipid quantification in total lipid extracts

Total phospholipid content in lipid extracts was quantified using a modified Bartlett and Lewis phosphorus assay (Bartlett and Lewis, 1970), following an optimized procedure previously described in detail (Monteiro et al., 2021). Briefly, lipid extracts were digested with perchloric acid, and inorganic phosphorus was determined colorimetrically after reaction with ammonium molybdate and ascorbic acid. Absorbance was measured at 797 nm, and phospholipid content was calculated using a calibration curve prepared with known phosphate standards.

2.5. Gas chromatography–mass spectrometry (GC–MS)

Fatty acid composition of lipid extracts from belly flaps, tail tissues, and Anisakis spp. larvae was determined by GC–MS following alkaline transmethylation, as previously described (Monteiro et al., 2025b). Briefly, lipid extracts were transmethylated using methanolic KOH in the presence of methyl nonadecanoate (C19:0) as an internal standard. Fatty acid methyl esters (FAMEs) were recovered in n-hexane, purified to remove cholesterol, and analyzed on an Agilent 8860 GC system coupled to a 5977B mass selective detector using a DB-FFAP column. Fatty acids were identified by comparison with commercial FAME standards and mass spectral libraries. Relative fatty acid composition was calculated by area normalization, and quantification was performed using the internal standard. Nutritional and physiological indices, including average chain length (ACL) and peroxidizability index (PInd), were calculated as previously described (Monteiro et al., 2025b).

2.6. Reverse-phase liquid chromatography–mass spectrometry (C18–LC–MS)

Total lipid extracts were analyzed by reversed-phase liquid chromatography coupled to high-resolution mass spectrometry (C18 RP-LC–MS) using a Dionex Ultimate 3000 UHPLC system coupled to a Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific), as previously described (Monteiro et al., 2025a; Monteiro et al., 2025c). Lipid separation was performed on a C18 column (Ascentis® Express 90 Å) using a binary gradient of aqueous and organic mobile phases containing ammonium formate and formic acid. Samples were analyzed in both positive and negative electrospray ionization modes under high-resolution full-scan and data-dependent MS/MS acquisition. Molecular lipid species were identified using Lipostar software (v2.1.5; Molecular Discovery Ltd.) based on accurate mass, retention time, and MS/MS fragmentation patterns, against a database generated from the LIPID MAPS structure repository. Lipid assignments were manually validated based on class-specific fragments and fatty acyl diagnostic ions. Semi-quantification of the detected lipid species was performed by normalizing peak areas to class-specific internal standards, and relative lipid abundances were calculated as the proportion of each species within its respective lipid class.

2.7. Statistical analysis

Figures were produced using GraphPad Prism version 7.00 for Windows (GraphPad Software, La Jolla, CA, USA), Python version 3.11 (Python Software Foundation, Python Language Reference, available at http://www.python.org). Principal component analyses (PCA) and hierarchical clustering heat map analyses (including the 50 most significant polar lipid discriminating species between groups as identified through ANOVA statistical analysis, or all the SM species semi-quantified in the study) were performed using MetaboAnalyst version 6.0 (available at https://www.metaboanalyst.ca). All experimental data are presented as means ± standard deviations (SDs) for five samples of host fish belly flaps and tails, as well as Anisakis spp. parasites (N = 5). Differences were considered statistically significant at p < 0.05 (* p ≤ 0.05; ** p ≤ 0.01).

3. Results

3.1. Phospholipid contents

The percentage of phospholipids (PL) in the total lipid content was not significantly different between European hake belly flaps (15.87 ± 2.43 μg of PL / mg of tissue dry weight, DW) and tail muscles (14.05 ± 2.86 μg of PL / mg DW). However, significant differences were observed between the parasite and both muscle and tail considering the phospholipid content, with Anisakis spp. parasites showing a significantly higher phospholipid content with regard to both fish tissues (23.04 ± 5.35 μg of PL / mg of tissue dry weight, DW; p < 0.05).

3.2. Fatty acid profiles

The fatty acid profiles of Anisakis spp. parasites, the adjacent belly flap muscle and tail samples were characterized by the presence of 30 different fatty acids. In fish tissues (belly flaps and tail), the predominant fatty acids recorded were docosahexaenoic acid, palmitic acid and oleic acid (Fig. 2).

Fig. 2.

Fig. 2

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Fatty acid (FA) content in European hake tail and belly flap muscle tissues and the Anisakis spp. parasites, as expressed in μg of FA per mg of dry weight (DW) of biomass. Data are presented as mean ± standard deviation (SD) from five replicate samples (N = 5). Differences were considered statistically significant at p < 0.05 (* p ≤ 0.05; ** p ≤ 0.01). Blue *: statistically significant differences between parasites and tail tissue; red *: statistically significant differences between parasites and belly flaps (no statistically significant differences were observed between the tail and belly flap tissue groups). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

For the anasikid parasites, docosahexaenoic acid (C22:6n-3) was the most abundant fatty acid, but was present in remarkably lower amounts when compared to host fish tissues, what contributes to an observable a more even distribution between all the different fatty acids present in the Anisakis spp. parasites. Other major FAs recorded in Anisakis spp. parasites worth referring include linoleic acid and stearic acid (Fig. 2).

The differences between tail muscle and Anisakis spp. parasites were more pronounced than those between belly flaps and parasites, with the fatty acid profile of host fish belly flaps consistently presenting fatty acid quantities between the ones observed for the other two groups; this feature is particularly noticeable in the case of docosahexaenoic acid, which is scarcer in Anisakis spp. parasites (although it is the most abundant fatty acid in the parasites) (Fig. 2). The only exceptions to this trend were oleic acid (C18:1n-9) and vaccenic acid (C18:1n-7), for which belly flap samples presented a lower abundance in both cases.The lipid indices calculated from the fatty acid composition - ACL (average chain length), MUFA (monounsaturated fatty acids), PUFA (polyunsaturated fatty acids), SFA (saturated fatty acids), emphasized further the differences between European hake tissues and those of Anisakis spp. parasites (Table 2), with the exception of the total content in saturated fatty acids (SFA), in which case parasites presented an intermediate value.

Table 2.

Indexes/factors derived from the fatty acid profiles of European hake tail and belly flaps, as well as anisakid parasites. Data are presented as mean ± standard deviation (with five replicate samples per group). Different letters in the same row indicate statistically significant differences.

Index/Factor Tail Belly Flap Parasites
n-3 40.68 ± 7.91%ab 43.78 ± 2.36%a 29.87 ± 2.99%b
n-6 3.43 ± 0.54%a 5.95 ± 2.85%ab 17.07 ± 1.03%b
n-6/n-3 0.09 ± 0.01%a 0.14 ± 0.07%ab 0.58 ± 0.07%b
SFA 24.37 ± 2.36% 28.73 ± 2.59% 28.53 ± 2.45%
MUFA 30.37 ± 9.89% 20.45 ± 6.03% 22.00 ± 0.57%
PUFA 45.25 ± 8.17% 50.82 ± 3.60% 49.46 ± 2.94%
ACL 19.44 ± 0.15%a 19.39 ± 0.12%a 18.96 ± 0,12%b
PI 318.23 ± 64.58 ab 349.24 ± 20.52 a 251.91 ± 19.60b
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ACL: average chain length; MUFA: monounsaturated fatty acids; PI: peroxidizability index; PUFA: polyunsaturated fatty acids; SFA: saturated fatty acids.

Anisakis spp. parasites exhibited a lower omega-3 content (29.87 ± 2.99%) compared to fish tissues (40.68 ± 7.91% in the tail and 43.78 ± 2.36% in the belly flaps), primarily due to the higher docosahexaenoic acid levels in fish. In contrast, the parasites had a significantly higher omega-6 content (17.07 ± 1.03%) compared to the tail (3.43 ± 0.54%) and belly flaps (5.95 ± 2.85%) tissues. As a result, the parasite displayed a significantly higher n-6/n-3 ratio than that of host fish tissues.

3.3. Tail, belly flaps and parasite lipidomes

The lipidome of tail muscle, belly flaps, and Anisakis spp. parasites was characterized using HPLC-MS, leading to the identification and semi-quantification of a total of 477 distinct lipid species.

The tail samples presented a total of 460 different lipid species, the belly flaps a total of 477 and Anisakis spp. parasites 474. A total of 457 lipid species were found to be common across all three sample groups (Fig. 3). These included 9 acyl carnitines, 83 phosphatidylcholines (PC), 19 lysophosphatidylcholines (LPC), 60 phosphatidylethanolamines (PE), 12 lysophosphatidylethanolamines (LPE), 18 phosphatidylinositols (PI), 15 phosphatidylserines (PS), and 5 phosphatidylglycerols (PG). Additionally, 17 ceramides (Cer), 5 hexosylceramides (HexCer), 17 sphingomyelins (SM), and 2 sphingoid bases (SPB) were detected (Supplementary Table 1). Among the neutral lipids, it was possible to identify 149 triacylglycerols (TG), 41 diacylglycerols (DG), and 3 monoacylglycerols (MG), along with 3 cholesteryl esters (CE) and a single coenzyme (Q10) (Supplementary Table 1).

Fig. 3.

Fig. 3

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Venn diagram representing the number of unique and shared lipid species identified in the lipid profiles of European hake tail and belly flap muscle tissues and the Anisakis spp. parasites. The diagram includes the names of the 3 lipid species shared between tail tissue and belly flaps, as well as the 17 lipid species common to both belly flaps and parasites.

Despite the general overlap in the lipid species present in the three groups, rather relevant differences were observed between the tail and the parasites and host fish tissue with parasites (belly flaps). Specifically, 17 species were found to be common to the parasites and belly flaps, while being absent from the host fish tail muscle (Fig. 3). These lipid species included PE 33:0, SM 39:0, PE 35:0, PE O-36:0, HexCer 41:0; O4, SM 32:0, PE O-35:1/P-35:0, SM 41:0, TG 53:0a, SM 33:0, PI O-38:5, PE 34:2, Cer 41:0; O4, SM 30:0, PS O-36:1/P-36:0, LPE 24:0, and PG 40:6. In turn, three species (PC 36:8, PG 38:4, and TG 65:7), were shared between the tail and belly flaps, but were absent from the parasites (Fig. 3). Notably, no lipid species were found to be shared only between the tail of the host fish and the Anisakis spp. parasites (Fig. 3).

Among glycerophospholipids, PC was the most diverse class (84 different lipids in the class). The most abundant PC species in the tail and belly flap tissues was PC 38:6, while in contrast, the predominant PC species in the parasites was PC 34:1 (Fig. 4; Supplementary Table 1). Triglycerides represented the most diverse lipid class overall (150 different lipids in the class), with TG 52:2 being particularly abundant across all groups (Fig. 4; Supplementary Table 1). In terms of specific TG profiles, the total fatty acyl carbon chains ranged from 37 to 66 carbons, with TG 62:15 being the most unsaturated species identified (Fig. 4). The parasites exhibited a higher proportion of saturated TG species compared to the fish tissues (Fig. 4). In contrast, the fish tissues, particularly the tail, displayed greater levels of unsaturation, with notably higher proportions of TG species containing seven or eight double bonds (Fig. 4).Regarding the PE and PC profiles, parasites continued to show a trend toward lower degrees of unsaturation, predominantly presenting higher contents in species with only one or two double bonds. Conversely, fish tissues exhibited a higher prevalence of more unsaturated lipid species, particularly those with five to seven double bonds (Fig. 4).

Fig. 4.

Fig. 4

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Heatmaps showing lipid unsaturation profiles in the triacylglycerols (TG; panels a–c), phosphatidylcholines (PC; panels d–f), and phosphatidylethanolamines (PE; panels g–i) classes present in European hake tail and belly flap muscle tissues and the Anisakis spp. parasites. Each heatmap plots the number of carbon atoms (y-axis) versus the number of double bonds (x-axis) for the corresponding lipid species. Colour intensity represents the relative abundance within each lipid class, with darker blue shades indicating higher levels. Panels a, d, and g correspond to tail tissue; panels b, e, and h to belly flap tissue; and panels c, f, and i to the associated parasites. Data is expressed as mean values of five individual replicates (N = 5). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

The PCA analysis of the results of the semi-quantification of lipids in the different groups, confirmed the differences between the tail and Anisakis spp. parasite groups, with each occupying opposing regions of the plot, while belly flaps tissue samples appear again in an intermediate position between the other two experimental groups (Fig. 5A). A hierarchical clustering heat map analysis, including the 50 most significant polar lipid discriminating species between groups as identified through ANOVA statistical analysis, (Fig. 5B).The lower leaflets of the heatmap clearly highlight a distinct separation between the parasite and the European hake tissues, primarily driven by upregulated levels of PC species (PC 34:4, PC 42:11, PC 38:7, PC 39:7, PC 38:6, PC 36:6, PC 36:5) and TG species (TG 47:1, TG 54:1, TG 51:2, TG 49:2, TG 49:1, TG 51:1, TG 50:3, TG 56:6) in the tails and belly flaps, as seen in the bottom right leaflet (Fig. 5B). In contrast, the upper left leaflet reveals significant dissimilarities between the tail and the Anisakis spp. parasites, primarily driven by higher levels of SM species (SM 44:2, SM 34:1, SM 32:1, SM 40:2, SM 41:2, SM 36:0, SM 33:2, SM 38:2), along with increased relative abundance in certain PE species (PE O-36:1, PE O-35:3/PE P-35:2, PE 36:2, PE O-36:2, PE O-38:6/PE P-38:5b, PE 36:1; Fig. 5B). Lastly, the separation between the belly flaps and either the parasite or the host fish tail appears to be less distinct in the upper leaflet, with the results displaying overlapping features from both groups (Fig. 5B). Overall, the most distinct separation between groups is clearly observed between the tails and the parasites groups, with belly flaps showing an intermediate transitional pattern. Moreover, the lipid species contributing the most for the observed differences between groups generally consisted of particular lipid species more unsaturated in the host fish tissues, and others more saturated in the Anisakis spp. parasites.

Fig. 5.

Fig. 5

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A) Principal Component Analysis (PCA) score plot depicting differences in the abundance of lipid species detected in European hake tail and belly flap muscle tissues and the Anisakis spp. parasites. B) Hierarchical clustering heatmap of the 50 most significantly different lipid species, identified through ANOVA statistical analysis between groups: tail tissue (dark blue), belly flap tissue (red), and parasites (green). Only lipid species shared across all three groups were included to enable direct comparison. Lipid abundance values were normalized using log base 10. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Among the lipid classes detected and analyzed in this work, sphingomyelins (SM) emerged as a key discriminating class. In fact, SMs were the only class effectively spatially discriminating between the two European hake tissues, parasitized (belly flaps) and non-parasitized (tail) when performing PCA analysis (Supplementary Fig. 1).

4. Discussion

Understanding the impact of anisakid parasitism in fish hosts physiology and nutritional quality is especially relevant in a context where anisakid infections (Anisakis spp. in particular) are increasingly reported across a wide range of marine fish species (Fiorenza et al., 2020; Caldeira et al., 2021; Ganucci Cancellieri et al., 2023) and fishery products (Ramos, 2020), with a high prevalence being specifically recorded in European hake (M. merluccius) (Pascual et al., 2018; Santos et al., 2022; Koutsoumanis et al., 2024). Recent surveys have reported infection rates of more than 95% in specimens caught off the Portuguese coast (Santos et al., 2022), raising concerns not only about zoonotic risks, but also regarding potential shifts on fish tissue composition and quality. This high prevalence of Anisakis spp. infection presented has an inherent limitation to this work in particular. Although more than 240 European hake specimens were screened, all individuals sampled for this work were infected with Anisakis spp. larvae in the belly flap region, precluding the inclusion of a truly unparasitized control group. To partially address this constraint, muscle tissue from the tail region of the same individuals, consistently free of visible parasites, was used as an internal reference. This within-host comparison allowed parasitized and non-parasitized tissues to be contrasted while minimizing inter-individual variability, thereby mitigating the absence of a completely unparasitized control group. It is worth highlighting that although the presence of Anisakis pegreffii in hake has already been reported locally in the Bay of Biscay, Anisakis simplex is by far the most prevalent species in this fish host (according to previous studies) (Diez et al., 2022). In a previous study (Monteiro et al., 2025b), the fatty acid profiles of Anisakis spp. L3 stage larvae were compared with those of adjacent belly flap tissue of European hake to explore local lipid dynamics and a possible nutritional interplay between the fish host and its anisakid parasites. The results reported are in line with those of the present work, as they showed a clear separation between the two profiles: the parasite featuring higher relative levels of stearic acid (18:0), vaccenic acid (18:1n-7), and linoleic acid (18:2n-6), while the host fish tissue (belly flaps) was richer in palmitic acid (16:0) and notably in docosahexaenoic acid (22:6n-3) (Monteiro et al., 2025b). These differences suggested that, despite the coexistence and physical contact between parasite and host fish tissues, the fatty acid composition of the parasite larvae does not mirror that of its host fish, indicating either the presence of selective regulatory mechanisms, as expected from a very distant evolutionary relationship between nematodes and teleosts, or a limited degree of nutrient exchange at this stage. Furthermore, physiologically relevant indexes derived from the fatty acid composition, such as the peroxidizability index (PInd) and the average chain length (ACL), were significantly higher in the host, pointing toward a greater structural complexity and susceptibility to oxidative damage in the tissue lipids of the host fish (Monteiro et al., 2025b). Overall, the findings in this work validate the results of that previous work, with the L3 stage Anisakis spp. parasites presenting higher amounts of stearic acid (18:0), vaccenic acid (18:1n-7), and linoleic acid (18:2n-6) when compared to both European hake tissues (belly flaps and tail). On the other hand, fish tissues presented higher amounts of palmitic acid (16:0), palmitoleic acid (16:1n-7), erucic acid (22:1n-9), and the omega-3 fatty acid docosahexaenoic acid (22:6n-3). Once again we highlight the presence of significant amounts of omega-3 fatty acids (eicosapentaenoic acid in particular), apparently absent in helminths parasitizing land-based animals (Smith et al., 1996; Mondal et al., 2009; Ghosh et al., 2010; Becker et al., 2017) as an evidence of the dependence of anisakid parasites on hosts for obtaining lipid components (although this may also occur at a previous developmental stage). In fact stearic acid (18:0) was shown to consistently be one of the major fatty acids in terrestrial helminths (Wangchuk et al., 2023) while for the Anisakis spp. parasites eicosapentaenoic acid (22:6n-3) is the major fatty acid present, hinting at a dependence and lipid exchange with hosts. Additionally, the parasitized belly flap tissue samples exhibited a significantly higher proportion of saturated fatty acids (SFA) when compared to the tail, suggesting a possible dysregulation of fatty acid biosynthesis or impaired desaturase activity in the affected tissue.

Notably, no significant differences were observed between the two European hake muscle regions. However, for all fatty acids that differed significantly between parasite and host, the values in the non-parasitized tissue (tail) tended to be more divergent from those observed in the parasite, whereas the heavily parasitized belly flaps often displayed intermediate levels. These findings may reflect a localized interference of the parasite with host lipid metabolism and its regulation. Furthermore, the greater dispersion observed among belly flap samples, relative to the more tightly clustered tail samples, may indicate a disruption or loosening of the normal regulatory mechanisms controlling lipid and fatty acid composition in the parasitized tissue.

A previous study suggested that parasite intensity could have a meaningful impact on the nutritional composition of fish, particularly on their fatty acid profile (Jouini et al., 2023). Our findings appear to support this hypothesis to some extent, based on the differences observed between parasitized and non-parasitized European hake tissues. However, it remains unclear whether these differences result directly from the presence or activity of the parasites, or whether they reflect intrinsic anatomical and compositional variation between the belly flaps and the tail muscle regions of the fish.

There are a significant number of studies using Omics tools to study helminthic parasites (Ma et al., 2020; McVeigh, 2020; Yeshi et al., 2020; Wang et al., 2021; Wangchuk et al., 2023). Some of these studies have even identified many genes and proteins associated with the biosynthesis and transport of lipids (IHGC, 2019; Wang et al., 2021; Wangchuk et al., 2023). However, specific lipidomic approaches to study and characterize this group of parasites are scarce, especially in the marine realm.

Our study allowed the disclosure of a group of 17 lipid species that were only present in both parasite and adjacent European hake muscle (the belly flaps) while absent in the tissue without parasites (tail), comprising a variety of different classes, especially PEs (5) and SMs (4), and notably tendentially saturated lipid species. Therefore, these lipid species exclusively present in the parasites and in the adjacent parasitized tissue and not detected in the unparasitized tails of European hake specimens surveyed may represent putative or prospective biomarkers of Anisakis spp. infection in fish tissues. The analysis of the lipidomic profiles revealed that Anisakis spp. L3 larvae exhibited a higher abundance of saturated triacylglycerol (TG) species compared to both European hake tissues. Notably, the total saturated fatty acid (SFA) content was similar between the parasites and the parasitized tissue (belly flaps), suggesting that in the parasite, saturated fatty acids are preferentially stored in the form of TGs. This accumulation of saturated triacylglycerol species in the parasite may reflect a physiological adaptation. Saturated TGs are more resistant to oxidative degradation than their unsaturated counterparts (Musakhanian et al., 2022), which could be advantageous for the parasites to prevent molecular damage in challenging low-turnover environments. Additionally, saturated TGs offer a dense and stable form of energy storage (Ayling, 2014; Evans and Hauton, 2016), potentially supporting long-term viability during this extended larval stage, if in fact the turnover and dependence upon the host is limited at this life stage (Wang et al., 2018). Regarding the other major lipid classes, phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs), European hake tissues showed higher levels of molecular species with five to seven double bonds, particularly those with six unsaturations. This higher prevalence of more highly unsaturated PC and PE species in the hake tissues should reflect a higher abundance in docosahexaenoic acid with regard to Anisakis spp. parasites, as observed when determining the fatty acid profiles.

The PCA of lipid composition revealed some degree of variability within groups. In the case of the parasites, this variability may reflect differences in their acquisition history, as larvae were likely obtained from different previous hosts. For fish specimens, intra-group variability may be driven by differences in diet, environmental conditions, or even behavioral factors (Diez et al., 2022). As observed for the fatty acid profiles, PCA of the semi-quantified lipid species also positioned the parasitized tissue (belly flaps) as an intermediate group between the Anisakis spp. larvae and the non-parasitized tissue of the European hake (their tail muscle). Similarly, hierarchical clustering heat map analysis revealed a clear separation between parasite and tail samples, with belly flaps once again forming a transitional cluster. Among the most statistically different lipid species, several phosphatidylcholines (PCs), including highly unsaturated species, and numerous mono- and disaturated triacylglycerols (TGs) were more abundant in European hake tissues. In contrast, ether-linked PCs and PEs, as well as a notable set of eight sphingomyelins (SMs), were enriched in the parasites. It is interesting that some ether-lipids are enriched in the parasites, as the plasmalogen form of these ether lipids, characterized by the presence of a vinyl-ether bond at the sn-1 position of a fatty alcohol, is known to be ubiquitous in animal membranes (Vítová et al., 2021). Other studies have already reported the importance of ether lipids in terrestrial helminthic parasites (Simbari et al., 2016; Wang et al., 2018; Yeshi et al., 2020). These molecules have been proposed to act as endogenous antioxidants and to assist in membrane bilayer organization and stability (Hossain et al., 2020). Therefore, similar to the increased saturation observed in the TG species, the enrichment in ether lipids in Anisakis spp. parasites may represent a complementary resilience strategy, enhancing membrane protection and limiting oxidative damage under metabolically constrained or stress-prone conditions.

Among the major lipid classes analyzed, sphingomyelins (SMs) revealed particularly intriguing patterns. When assessing group-level differences within each lipid class using PCA, SMs were the only class that allowed clear discrimination between parasitized (belly flaps) and non-parasitized (tail) European hake tissues. This observation may have physiological significance, considering that SMs are not only structural components of membranes, but also key lipids involved in cell signaling (Chakraborty and Jiang, 2013; Jamil and Cowart, 2023; Wang et al., 2024), membrane domain organization (components of lipid rafts) (Ando et al., 2015; Bieberich, 2018), and immune response and disease progression (Avota et al., 2019; Lee et al., 2023; Thomas et al., 2023). An altered SM profile in the parasitized region may reflect a local tissue response to the presence of parasites, potentially associated with inflammation, membrane remodeling, or even immune modulation induced by the parasite. Moreover, the fact that the PCA analysis of SM contents showed that the samples in the belly flaps group present a remarkably more scattered pattern with regard to the other two experimental groups, may suggest a local deregulation of these lipids as a result of the presence and action of the parasites. Therefore, these findings suggest that SM metabolism may be particularly sensitive to parasitic influence and warrant further investigation as possible markers of local physiological disruption. Some particular saturated SM species, namely SM 30:0, SM 32:0, SM 33:0, SM 39:0 and SM 41:0, present in the parasite and parasitized European hake tissue, and absent in the unparasitized tail tissue, may represent specific prospective biomarkers of Anisakis spp. parasitism, at least for European hake.

Ultimately, the disclosed differences between anisakid larvae and host tissue, as well as the differences observed between parasitized and unparasitized fish host tissue open the possibility of using lipid profiling as a tool to detect parasitism in fish. This approach can be particularly useful after the capture of the fish (in post-mortem conditions) when larvae are known to encyst into the belly flaps and the dorsal muscle previously to the refrigeration treatment and storage provided by current legislation (Cipriani et al., 2015; Cipriani et al., 2024). Improving analytical methods for detecting anisakid parasites in fish and fishery products is a priority encompassed in goals of the EU Regulation 2017/625 on official controls and therefore lipid profiling may contribute in a meaningful way to pursue this goal.

5. Conclusions

The present study provides new insights at lipid species-level of interactions between Anisakis larvae and one of their most common host marine fish the European hake. Anisakis spp. L3 larvae exhibited a distinct lipid signature, including higher levels of saturated triacylglycerols and specific ether lipids, which may reflect physiological adaptations to oxidative stress and long-term energy storage during their L3 life stage. Importantly, sphingomyelins emerged as a lipid class showing a clear separation between parasitized and non-parasitized host fish tissues, thus suggesting potential roles of these biomolecules in host-parasite signaling or immune modulation.

Both fatty acids and lipidome analyses revealed marked differences between parasites and host fish tissues, as well as between parasitized and non-parasitized regions of the host fish (belly flaps vs. tail). These findings support, to some extent, previous hypotheses that parasite intensity may influence the lipid composition of fish and ultimately its nutritional value.

Although lipidomics is still in its infancy in parasitological research, this work illustrates its promise as a powerful tool for exploring host–parasite biochemical interplay. As multi-omic approaches continue to evolve, a deeper understanding of the structure and function of specific lipid species and classes in socioeconomically important zoonotic fish parasites may pave the way for novel strategies in the diagnosis, treatment, and control of parasite infections.

Clinical trial number

Not applicable.

CRediT authorship contribution statement

João P. Monteiro: Writing – original draft, Methodology, Investigation, Formal analysis, Conceptualization. Tiago Sousa: Writing – original draft, Methodology, Investigation, Data curation. Fernando Atroch: Writing – review & editing, Investigation. Luís Filipe Rangel: Writing – review & editing, Investigation. Camilo Ayra Pardo: Writing – review & editing, Investigation. Paula Ramos: Writing – review & editing, Investigation. Maria João Santos: Writing – review & editing, Investigation. Felisa Rey: Writing – review & editing, Investigation. Ricardo Calado: Writing – review & editing, Project administration, Funding acquisition, Conceptualization. M. Rosário Domingues: Writing – review & editing, Resources, Project administration, Conceptualization.

Ethical approval

Not applicable (animals were supplied by a retailer and were meant for commercialization).

Funding

This study was performed within the scope of project “BLUE BIOECONOMY PACT” (Project N°. C644915664–00000026), co-funded by Next Generation EU European Fund, under the incentive line “Agendas for Business Innovation” within Component 5 - Capitalization and Business Innovation of the Portuguese Recovery and Resilience Plan (RRP) specifically, in the STOParasitas project framed in the Vertical FISH (WP7).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work is funded by national funds through FCT – Fundação para a Ciência e a Tecnologia I.P., under the project/grant UID/50006 + LA/P/0094/2020 (doi.org/10.54499/LA/P/0094/2020) (Centro de Estudos do Ambiente e Mar (CESAM)) and UID/50006 - Laboratório Associado para a Química Verde - Tecnologias e Processos Limpos. The authors are thankful to the project “BLUE BIOECONOMY PACT” (Project No. C644915664–00000026), co-funded by Next Generation EU European Fund, under the incentive line “Agendas for Business Innovation” within Component - Capitalization and Business Innovation of the Portuguese Recovery and Resilience Plan (RRP), specifically in the STOParasitas project framed in Vertical FISH (WP7). The authors also acknowledge to the University of Aveiro and to the Portuguese Mass Spectrometry Network – RNEM (LISBOA-01-0145-FEDER-402-022125). Thanks to Molecular Discovery Ltd. for the Lipostar version 2.1.5 license.

The authors also acknowledge the FCT/MCTES for individual funding in the scope of the Individual Call to Scientific Employment Stimulus CEECIND/03501/2017/CP1420/CT0010 (https://doi.org/10.54499/CEECIND/03501/2017/CP1420/CT0010) and (CEECIND/00580/2017) to Felisa Rey (https://doi.org/10.54499/CEECIND/00580/2017/CP1459/CT0005).

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.fawpar.2026.e00334.

Contributor Information

João P. Monteiro, Email: jpspmonteiro@yahoo.com.

Ricardo Calado, Email: rjcalado@ua.pt.

M. Rosário Domingues, Email: mrd@ua.pt.

Appendix A. Supplementary data

Supplementary Table 1

Molecular species identified by mass accuracy HPLC−MS and characterized by MS/MS analyses

mmc1.docx (323KB, docx)

Data availability

Not applicable.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Table 1

Molecular species identified by mass accuracy HPLC−MS and characterized by MS/MS analyses

mmc1.docx (323KB, docx)

Data Availability Statement

Not applicable.

Articles from Food and Waterborne Parasitology are provided here courtesy of Elsevier

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