Anthelmintic resistance in parasitic nematodes, particularly in Haemonchus contortus, poses a significant threat to livestock health and productivity. Since resistance mechanisms have not yet been fully elucidated, the present study employed metabolomic and proteomic analyses of H. contortus adults (females and males separately) from drug-susceptible (ISE) and benzimidazole-resistant (IRE) strains, with a focus on resistance-specific differences. Using nuclear magnetic resonance (NMR)-based metabolomics and mass spectrometry-based proteomics, significant alterations in metabolic and protein expression profiles associated with resistance were identified. Resistant adults exhibited reduced levels of multiple amino acids and tricarboxylic acid cycle intermediates, alongside elevated levels of trehalose, myo-inositol, NAD+, and glycerophosphocholine, suggesting enhanced stress resistance and altered energy metabolism. Proteomic analysis revealed over 3000 proteins, with substantial sex-related differences. Moderate resistance-related differences in protein expression were observed, indicating adaptive biochemical pathways supporting resistance. While some resistance-associated changes in the metabolome and proteome were observed in both sexes, several were distinctly sex-specific. This finding provides the first evidence of such sex-dependent strategies in H. contortus, reinforcing the notion that anthelmintic resistance is a multifaceted and highly complex phenomenon, with many aspects yet to be fully understood. In addition, certain nematode-specific enzymes upregulated in the IRE strain - such as cysteine synthases and transthyretin-like proteins - might be considered as potential targets for future anthelmintic development.

Biomedical Research Centre University Hospital Hradec Králové Hradec Králové Czech Republic

Core Facility for Mass Spectrometry and Proteomics Center for Molecular Biology of Heidelberg University Heidelberg Germany

Department of Biochemical Sciences Faculty of Pharmacy Charles University Heyrovského 1203 Hradec Králové Czech Republic

Laboratory of Molecular Structure Characterization Institute of Microbiology Czech Academy of Sciences Vídeňská 1083 Prague Czech Republic

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Merlin A, Ravinet N, Briot L, Chauvin A, Hebert L, Valle-Casuso JC, Delerue M (2024) Prevalence and seasonal dynamic of gastrointestinal parasites in equids in France during two years. Prev Vet Med 223:106100 PubMed DOI

Brinzer RA, Winter AD, Page AP (2024) The relationship between intraflagellar transport and upstream protein trafficking pathways and macrocyclic lactone resistance in PubMed DOI PMC

Kotze AC, Prichard RK (2016) Anthelmintic resistance in PubMed DOI

Rychlá N, Navrátilová M, Kohoutová E, Raisová Stuchlíková L, Štěrbová K, Krátký J, Matoušková P, Szotáková B, Skálová L (2024) Flubendazole carbonyl reduction in drug-susceptible and drug-resistant strains of the parasitic nematode PubMed DOI PMC

Ruano ZM, Carolino N, Mateus TL (2017) Gastrointestinal parasites as a threat to grazing sheep. Large Anim Rev 23(6):231–238

Emery DL, Hunt PW, Le Jambre LF (2016) PubMed DOI

Ehsan M, Hu RS, Liang QL, Hou JL, Song XK, Yan RF, Zhu XQ, Li XR (2020) Advances in the development of anti- PubMed DOI PMC

Teixeira M, Matos A, Albuquerque F, Bassetto CC, Smith WD, Monteiro JP (2019) Strategic vaccination of hair sheep against PubMed DOI

Mravcakova D, Varadyova Z, Kopcakova A, Cobanova K, Gresakova L, Kisidayova S, Babjak M, Dolinska MU, Dvoroznakova E, Konigova A, Vadlejch J, Cieslak A, Slusarczyk S, Varady M (2019) Natural chemotherapeutic alternatives for controlling of haemonchosis in sheep. BMC Vet Res 15(1):302 PubMed DOI PMC

Xue WX, Snoeck S, Njiru C, Inak E, Dermauw W, Van Leeuwen T (2020) Geographical distribution and molecular insights into abamectin and milbemectin cross-resistance in European field populations of PubMed DOI

Zajickova M, Nguyen LT, Skalova L, Stuchlikova LR, Matouskova P (2020) Anthelmintics in the future: current trends in the discovery and development of new drugs against gastrointestinal nematodes. Drug Discov Today 25(2):430–437 PubMed DOI

Babjak M, Konigova A, Varady M (2021) Multiple anthelmintic resistance at a goat farm in Slovakia. Helminthologia 58(2):173–178 PubMed DOI PMC

Rodrigues JFV, Monteiro JP, Almeida TM, Molento MB (2025) A systematic review of the molecular mechanisms related to anthelmintic resistance in PubMed DOI

Munguia B, Saldana J, Nieves M, Melian ME, Ferrer M, Teixeira R, Porcal W, Manta E, Dominguez L (2022) Sensitivity of PubMed DOI PMC

Sales N, Love S (2016) Resistance of PubMed DOI

Harder A (2016) The biochemistry of PubMed DOI

Chaudhry U, Redman EM, Raman M, Gilleard JS (2015) Genetic evidence for the spread of a benzimidazole resistance mutation across southern India from a single origin in the parasitic nematode PubMed DOI

Sangster NC, Cowling A, Woodgate RG (2018) Ten events that defined anthelmintic resistance research. Trends Parasitol 34(7):553–563 PubMed DOI

Barrere V, Alvarez L, Suarez G, Ceballos L, Moreno L, Lanusse C, Prichard RK (2012) Relationship between increased albendazole systemic exposure and changes in single nucleotide polymorphisms on the beta-tubulin isotype 1 encoding gene in PubMed DOI

Kotze AC, Cowling K, Bagnall NH, Hines BM, Ruffell AP, Hunt PW, Coleman GT (2012) Relative level of thiabendazole resistance associated with the E198A and F200Y SNPs in larvae of a multi-drug resistant isolate of PubMed DOI PMC

Avramenko RW, Redman EM, Melville L, Bartley Y, Wit J, Queiroz C, Bartley DJ, Gilleard JS (2019) Deep amplicon sequencing as a powerful new tool to screen for sequence polymorphisms associated with anthelmintic resistance in parasitic nematode populations. Int J Parasitol 49(1):13–26 PubMed DOI

Dilks CM, Hahnel SR, Sheng Q, Long L, McGrath PT, Andersen EC (2020) Quantitative benzimidazole resistance and fitness effects of parasitic nematode beta-tubulin alleles. Int J Parasitol Drugs Drug Resist 14:28–36 PubMed DOI PMC

Doyle SR, Laing R, Bartley D, Morrison A, Holroyd N, Maitland K, Antonopoulos A, Chaudhry U, Flis I, Howell S, McIntyre J, Gilleard JS, Tait A, Mable B, Kaplan R, Sargison N, Britton C, Berriman M, Devaney E, Cotton JA (2022) Genomic landscape of drug response reveals mediators of anthelmintic resistance. Cell Rep 41(3):111522 PubMed DOI PMC

Vosála O, Krátky J, Matousková P, Rychlá N, Sterbová K, Stuchlíková LR, Vokrál I, Skálová L (2025) Biotransformation of anthelmintics in nematodes in relation to drug resistance. Int J Parasitol Drugs Drug Resist 27:100579 PubMed DOI PMC

Laing R, Doyle SR, McIntyre J, Maitland K, Morrison A, Bartley DJ, Kaplan R, Chaudhry U, Sargison N, Tait A, Cotton JA, Britton C, Devaney E (2022) Transcriptomic analyses implicate neuronal plasticity and chloride homeostasis in ivermectin resistance and response to treatment in a parasitic nematode. PLoS Pathog 18(6):e1010545 PubMed DOI PMC

Tuersong W, Liu X, Wang YF, Wu SM, Qin PX, Zhu SN, Liu F, Wang CQ, Hu M (2023) Comparative metabolome analyses of ivermectin-resistant and -susceptible strains of PubMed DOI PMC

Liu Y, Wang XM, Luo XP, Wang R, Zhai BT, Wang PL, Li JY, Yang XY (2023) Transcriptomics and proteomics of PubMed DOI PMC

Roos MH, Otsen M, Hoekstra R, Veenstra JG, Lenstra JA (2004) Genetic analysis of inbreeding of two strains of the parasitic nematode PubMed DOI

Yilmaz E, Ramunke S, Demeler J, Krucken J (2017) Comparison of constitutive and thiabendazole-induced expression of five cytochrome P450 genes in fourth-stage larvae of PubMed DOI PMC

Kellerová P, Navrátilová M, Nguyen LT, Dimunová D, Raisová Stuchlíková L, Štěrbová K, Skálová L, Matoušková P (2020) UDP-glycosyltransferases and albendazole metabolism in the juvenile stages of PubMed DOI PMC

Jacob D, Deborde C, Lefebvre M, Maucourt M, Moing A (2017) NMRProcFlow: a graphical and interactive tool dedicated to 1D spectra processing for NMR-based metabolomics. Metabolomics 13(4):36 PubMed DOI PMC

Pang ZQ, Lu Y, Zhou GY, Hui FA, Xu L, Viau C, Spigelman AF, Macdonald PE, Wishart DS, Li SZ, Xia JG (2024) MetaboAnalyst 6.0: towards a unified platform for metabolomics data processing, analysis and interpretation. Nucleic Acids Res 52(W1):W398–W406 PubMed DOI PMC

Wessel D, Flugge UI (1984) A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem 138(1):141–143 PubMed DOI

Kulak NA, Geyer PE, Mann M (2017) Loss-less nano-fractionator for high sensitivity, high coverage proteomics. Mol Cell Proteomics 16(4):694–705 PubMed DOI PMC

Zurawska M, Basik M, Aguilar-Mahecha A, Dadlez M, Domanski D (2023) A micro-flow, high-pH, reversed-phase peptide fractionation and collection system for targeted and in-depth proteomics of low-abundance proteins in limiting samples. MethodsX 11:102306 PubMed DOI PMC

Batth TS, Francavilla C, Olsen JV (2014) Off-line high-pH reversed-phase fractionation for in-depth phosphoproteomics. J Proteome Res 13(12):6176–6186 PubMed DOI

Eng JK, McCormack AL, Yates JR (1994) An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J Am Soc Mass Spectrom 5(11):976–989 PubMed DOI

Kall L, Canterbury JD, Weston J, Noble WS, MacCoss MJ (2007) Semi-supervised learning for peptide identification from shotgun proteomics datasets. Nat Methods 4(11):923–925 PubMed DOI

Plubell DL, Wilmarth PA, Zhao Y, Fenton AM, Minnier J, Reddy AP, Klimek J, Yang X, David LL, Pamir N (2017) Extended multiplexing of tandem mass tags (TMT) labeling reveals age and high fat diet specific proteome changes in mouse epididymal adipose tissue. Mol Cell Proteomics 16(5):873–890 PubMed DOI PMC

Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, Smyth GK (2015) Limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res 43(7):e47 PubMed DOI PMC

Lespine A, Blancfuney C, Prichard R, Alberich M (2024) P-glycoproteins in anthelmintic safety, efficacy, and resistance. Trends Parasitol 40(10):896–913 PubMed DOI

Mukherjee A, Kar I, Patra AK (2023) Understanding anthelmintic resistance in livestock using “omics” approaches. Environ Sci Pollut Res 30:125439–125463 PubMed DOI

Doyle SR, Cotton JA (2019) Genome-wide approaches to investigate anthelmintic resistance. Trends Parasitol 35(4):289–301 PubMed DOI

Shaver AO, Andersen EC (2024) Integrating metabolomics into the diagnosis and investigation of anthelmintic resistance. Trends Parasitol 40(12):1097–1106 PubMed DOI

Mukherjee S, Mukherjee N, Gayen P, Roy P, Babu SPS (2016) Metabolic inhibitors as antiparasitic drugs: pharmacological, biochemical and molecular perspectives. Curr Drug Metab 17(10):937–970 PubMed DOI

Harder A (2016) The Biochemistry of PubMed

Liu JY, Zheng RQ, Wang Y, Liu YH, Jiang S, Wang XZ, He K, Pan X, Zhou T, Li T, Xia Q, Zhang WN (2022) The endogenous metabolite glycerophosphocholine promotes longevity and fitness in PubMed DOI PMC

Li XT, Qian XJ, Chen H, Wang XD, Wu X (2024) Anti-aging effect of glycerophosphocholine in PubMed DOI PMC

Sun XJ, Chen WD, Wang YD (2017) DAF-16/FOXO transcription factor in aging and longevity. Front Pharmacol 8:548 PubMed DOI PMC

Kim SH, Kim BK, Park S, Park SK (2019) Phosphatidylcholine extends lifespan via DAF-16 and reduces amyloid-beta-induced toxicity in PubMed PMC

Yang NC, Chin CY, Zheng YX, Lee I (2023) The attenuation of insulin/IGF-1 signaling pathway plays a crucial role in the myo-inositol-alleviated aging in PubMed DOI PMC

Mouchiroud L, Houtkooper RH, Moullan N, Katsyuba E, Ryu D, Cantó C, Mottis A, Jo YS, Viswanathan M, Schoonjans K, Guarente L, Auwerx J (2013) The NAD+/sirtuin pathway modulates longevity through activation of mitochondrial UPR and FOXO signaling. Cell 154(2):430–441 PubMed DOI PMC

Honda Y, Tanaka M, Honda S (2010) Trehalose extends longevity in the nematode PubMed DOI

Chen YR, Jin BS, Yu J, Wu LW, Wang YY, Tang B, Chen HL (2024) The nematode PubMed DOI PMC

Hibshman JD, Doan AE, Moore BT, Kaplan REW, Hung A, Webster AK, Bhatt DP, Chitrakar R, Hirschey MD, Baugh LR (2017) PubMed DOI PMC

Zheng YT, Young ND, Wang T, Chang BCH, Song JN, Gasser RB (2025) Systems biology of PubMed DOI

Tan TK, Lim YAL, Chua KH, Chai HC, Low VL, Bathmanaban P, Affendi S, Wang D, Panchadcharam C (2020) Characterization of benzimidazole resistance in PubMed DOI

Mesa-Torres N, Calvo AC, Oppici E, Titelbaum N, Montioli R, Miranda-Vizuete A, Cellini B, Salido E, Pey AL (2016) PubMed DOI

Holt SJ, Riddle DL (2003) SAGE surveys PubMed DOI

Lüersen K, Müller S, Hussein A, Liebau E, Walter RD (2000) The γ-glutamylcysteine synthetase of PubMed DOI

Muller S, Gilberger TW, Fairlamb AH, Walter RD (1997) Molecular characterization and expression of PubMed DOI PMC

Vozdek R, Hnízda A, Krijt J, Será L, Kozich V (2013) Biochemical properties of nematode PubMed DOI

Saldanha JN, Parashar A, Pandey S, Powell-Coffman JA (2013) Multiparameter behavioral analyses provide insights to mechanisms of cyanide resistance in PubMed DOI PMC

Saxena VK, Vedamurthy GV, Swarnkar CP, Kadam V, Onteru SK, Ahmad H, Singh R (2021) De novo pathway is an active metabolic pathway of cysteine synthesis in PubMed DOI

Shi H, Huang X, Chen X, Yang Y, Wu F, Yao C, Du Guangxu, A (2021) PubMed DOI PMC

Wen T, Zhang Y, Wu X, Ye J, Qiu Y, Rui L (2022) Studies on the requirement of transthyretin protein (BxTTR-52) for the suppression of host innate immunity in PubMed DOI PMC

Maruszewska-Cheruiyot M, Szewczak L, Krawczak-Wójcik K, Kierasińska M, Stear M, Donskow-Łysoniewska K (2023) The impact of intestinal inflammation on nematode’s excretory-secretory proteome. Int J Mol Sci 24:14127 PubMed DOI PMC

ASEP repository (https://hdl.handle.net/11104/0370184).

ProteomeXchange Consortium. (https://proteomecentral.proteomexchange.org).

Perez-Riverol Y, Bandla C, Kundu DJ, Kamatchinathan S, Bai J, Hewapathirana S, John NS, Prakash A, Walzer M, Wang S, Vizcaino JA (2025) The PRIDE database at 20 years: 2025 update. Nucleic Acids Res 53(D1):D543–D553 PubMed DOI PMC

Claeys T, Van Den Bossche T, Perez-Riverol Y, Gevaert K, Vizcaino JA, Martens L (2023) Lessdrf is more: maximizing the value of proteomics data through streamlined metadata annotation. Nat Commun 14(1):6743 PubMed DOI PMC