BACKGROUND: Helminth extracellular vesicles (EVs) are known to have a three-way communication function among parasitic helminths, their host and the host-associated microbiota. They are considered biological containers that may carry virulence factors, being therefore appealing as therapeutic and prophylactic target candidates. This study aims to describe and characterise EVs secreted by Sparicotyle chrysophrii (Polyopisthocotyla: Microcotylidae), a blood-feeding gill parasite of gilthead seabream (Sparus aurata), causing significant economic losses in Mediterranean aquaculture. METHODS: To identify proteins involved in extracellular vesicle biogenesis, genomic datasets from S. chrysophrii were mined in silico using known protein sequences from Clonorchis spp., Echinococcus spp., Fasciola spp., Fasciolopsis spp., Opisthorchis spp., Paragonimus spp. and Schistosoma spp. The location and ultrastructure of EVs were visualised by transmission electron microscopy after fixing adult S. chrysophrii specimens by high-pressure freezing and freeze substitution. EVs were isolated and purified from adult S. chrysophrii (n = 200) using a newly developed ultracentrifugation-size-exclusion chromatography protocol for Polyopisthocotyla, and EVs were characterised via nanoparticle tracking analysis and tandem mass spectrometry. RESULTS: Fifty-nine proteins involved in EV biogenesis were identified in S. chrysophrii, and EVs compatible with ectosomes were observed in the syncytial layer of the haptoral region lining the clamps. The isolated and purified nanoparticles had a mean size of 251.8 nm and yielded 1.71 × 108 particles · mL-1. The protein composition analysis identified proteins related to peptide hydrolases, GTPases, EF-hand domain proteins, aerobic energy metabolism, anticoagulant/lipid-binding, haem detoxification, iron transport, EV biogenesis-related, vesicle-trafficking and other cytoskeletal-related proteins. Several identified proteins, such as leucyl and alanyl aminopeptidases, calpain, ferritin, dynein light chain, 14-3-3, heat shock protein 70, annexin, tubulin, glutathione S-transferase, superoxide dismutase, enolase and fructose-bisphosphate aldolase, have already been proposed as target candidates for therapeutic or prophylactic purposes. CONCLUSIONS: We have unambiguously demonstrated for the first time to our knowledge the secretion of EVs by an ectoparasitic flatworm, inferring their biogenesis machinery at a genomic and transcriptomic level, and by identifying their location and protein composition. The identification of multiple therapeutic targets among EVs' protein repertoire provides opportunities for target-based drug discovery and vaccine development for the first time in Polyopisthocotyla (sensu Monogenea), and in a fish-ectoparasite model.

Faculty of Science University of South Bohemia Branišovská 1160 31 370 05 České Budějovice Czech Republic

Fish Pathology Group Institute of Aquaculture Torre de La Sal Consejo Superior de Investigaciones Científicas Ribera de Cabanes 12595 Castellón Spain

Laboratory of Functional Helminthology Institute of Parasitology Biology Centre Czech Academy of Sciences České Budějovice Czech Republic

Laboratory of Helminthology Institute of Parasitology Biology Centre Czech Academy of Sciences České Budějovice Czech Republic

Parasitology Reference and Research Laboratory National Centre for Microbiology Instituto de Salud Carlos 3 Majadahonda Madrid Spain

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Brabec J, Salomaki ED, Kolísko M, Scholz T, Kuchta R. The evolution of endoparasitism and complex life cycles in parasitic platyhelminths. Curr Biol. 2023;33:4269–4275. doi: 10.1016/j.cub.2023.08.064. PubMed DOI

Caña-Bozada V, Robinson MW, Hernández-Mena DI, Morales-Serna FN. Exploring evolutionary relationships within Neodermata using putative orthologous groups of proteins, with emphasis on peptidases. Trop Med Infect Dis. 2023;8:59. doi: 10.3390/tropicalmed8010059. PubMed DOI PMC

Ogawa K. Diseases of cultured marine fishes caused by Platyhelminthes (Monogenea, Digenea, Cestoda) Parasitology. 2015;142:178–195. doi: 10.1017/S0031182014000808. PubMed DOI

Muniesa A, Basurco B, Aguilera C, Furones D, Reverté C, Sanjuan-Vilaplana A, et al. Mapping the knowledge of the main diseases affecting sea bass and sea bream in Mediterranean. Transbound Emerg Dis. 2020;67:1089–1100. doi: 10.1111/tbed.13482. PubMed DOI

Mladineo I, Volpatti D, Beraldo P, Rigos G, Katharios P, Padrós F. Monogenean Sparicotyle chrysophrii: the major pathogen of the Mediterranean gilthead seabream aquaculture. Rev Aquac. 2024;16:287–308. doi: 10.1111/raq.12839. DOI

Stella E, Pastres R, Pasetto D, Kolega M, Mejdandžić D, Čolak S, et al. A stratified compartmental model for the transmission of Sparicotyle chrysophrii (Platyhelminthes: Monogenea) in gilthead seabream (Sparus aurata) fish farms. R Soc Open Sci. 2023;10:221377. doi: 10.1098/rsos.221377. PubMed DOI PMC

Sitjà-Bobadilla A, de Felipe MC, Álvarez-Pellitero P. In vivo and in vitro treatments against Sparicotyle chrysophrii (Monogenea: Microcotylidae) parasitizing the gills of gilthead sea bream (Sparus aurata L.) Aquaculture. 2006;261:856–864. doi: 10.1016/j.aquaculture.2006.09.012. DOI

Merella P, Montero FE, Burreddu C, Garippa G. In-feed trials of fenbendazole and other chemical/natural compounds against Sparicotyle chrysophrii (Monogenea) infections in Sparus aurata (Osteichthyes) Aquac Res. 2021;52:5908–5911. doi: 10.1111/are.15420. DOI

Jedličková L, Dvořáková H, Kašný M, Ilgová J, Potěšil D, Zdráhal Z, et al. Major acid endopeptidases of the blood-feeding monogenean Eudiplozoon nipponicum (Heteronchoinea: Diplozoidae) Parasitology. 2016;143:494–506. doi: 10.1017/S0031182015001808. PubMed DOI

Ilgová J, Jedličková L, Dvořáková H, Benovics M, Mikeš L, Janda L, et al. A novel type I cystatin of parasite origin with atypical legumain-binding domain. Sci Rep. 2017;7:1–12. doi: 10.1038/s41598-017-17598-2. PubMed DOI PMC

Jedličková L, Dvořáková H, Dvořák J, Kašný M, Ulrychová L, Vorel J, et al. Cysteine peptidases of Eudiplozoon nipponicum: a broad repertoire of structurally assorted cathepsins L in contrast to the scarcity of cathepsins B in an invasive species of haematophagous monogenean of common carp. Parasit Vectors. 2018;11:1–17. doi: 10.1186/s13071-018-2666-2. PubMed DOI PMC

Roudnický P, Vorel J, Ilgová J, Benovics M, Norek A, Jedličková L, et al. Identification and partial characterization of a novel serpin from Eudiplozoon nipponicum (Monogenea, Polyopisthocotylea) Parasite. 2018;25:61. doi: 10.1051/parasite/2018062. PubMed DOI PMC

Jedličková L, Dvořák J, Hrachovinová I, Ulrychová L, Kašný M, Mikeš L. A novel Kunitz protein with proposed dual function from Eudiplozoon nipponicum (Monogenea) impairs haemostasis and action of complement in vitro. Int J Parasitol. 2019;49:337–346. doi: 10.1016/j.ijpara.2018.11.010. PubMed DOI

Ilgová J, Kavanová L, Matiašková K, Salát J, Kašný M. Effect of cysteine peptidase inhibitor of Eudiplozoon nipponicum (Monogenea) on cytokine expression of macrophages in vitro. Mol Biochem Parasitol. 2020;235:111248. doi: 10.1016/j.molbiopara.2019.111248. PubMed DOI

Caña-Bozada V, Chapa-López M, Díaz-Martín RD, García-Gasca A, Huerta-Ocampo JÁ, de Anda-Jáuregui G, et al. In silico identification of excretory/secretory proteins and drug targets in monogenean parasites. Infect Genet Evol. 2021;93:104931. doi: 10.1016/j.meegid.2021.104931. PubMed DOI

Vorel J, Cwiklinski K, Roudnický P, Ilgová J, Jedličková L, Dalton JP, et al. Eudiplozoon nipponicum (Monogenea, Diplozoidae) and its adaptation to haematophagy as revealed by transcriptome and secretome profiling. BMC Genomics. 2021;22:1–17. doi: 10.1186/s12864-021-07589-z. PubMed DOI PMC

Mirabent-Casals M, Caña-Bozada VH, Morales-Serna FN, García-Gasca A. Predicted secretome of the monogenean parasite Rhabdosynochus viridisi: hypothetical molecular mechanisms for host-parasite interactions. Parasitologia. 2023;3:33–45. doi: 10.3390/parasitologia3010004. DOI

Drurey C, Coakley G, Maizels RM. Extracellular vesicles: new targets for vaccines against helminth parasites. Int J Parasitol. 2020;50:623–633. doi: 10.1016/j.ijpara.2020.04.011. PubMed DOI PMC

Rooney J, Northcote HM, Williams TL, Cortés A, Cantacessi C, Morphew RM. Parasitic helminths and the host microbiome – a missing ‘extracellular vesicle-sized’ link? Trends Parasitol. 2022;38:737–747. doi: 10.1016/j.pt.2022.06.003. PubMed DOI

Welsh JA, Goberdhan DCI, O’Driscoll L, Buzas EI, Blenkiron C, Bussolati B, et al. Minimal information for studies of extracellular vesicles (MISEV2023): from basic to advanced approaches. J Extracell Vesicles. 2024;13:e12404. doi: 10.1002/jev2.12404. PubMed DOI PMC

Kalra H, Drummen GPC, Mathivanan S. Focus on extracellular vesicles: introducing the next small big thing. Int J Mol Sci. 2016;17:170. doi: 10.3390/ijms17020170. PubMed DOI PMC

Henne WM, Buchkovich NJ, Emr SD. The ESCRT Pathway. Dev Cell. 2011;21:77–91. doi: 10.1016/j.devcel.2011.05.015. PubMed DOI

Hessvik NP, Llorente A. Current knowledge on exosome biogenesis and release. Cell Mol Life Sci. 2018;75:193–208. doi: 10.1007/s00018-017-2595-9. PubMed DOI PMC

Van Niel G, D’Angelo G, Raposo G. Shedding light on the cell biology of extracellular vesicles. Nat Rev Mol Cell Biol. 2018;19:213–228. doi: 10.1038/nrm.2017.125. PubMed DOI

Anand S, Samuel M, Kumar S, Mathivanan S. Ticket to a bubble ride: cargo sorting into exosomes and extracellular vesicles. Biochim Biophys Acta Proteins Proteom. 2019;1867:140203. doi: 10.1016/j.bbapap.2019.02.005. PubMed DOI

Bennett APS, de la Torre-Escudero E, Robinson MW. Helminth genome analysis reveals conservation of extracellular vesicle biogenesis pathways but divergence of RNA loading machinery between phyla. Int J Parasitol. 2020;50:655–661. doi: 10.1016/j.ijpara.2020.04.004. PubMed DOI

Palomba M, Rughetti A, Mignogna G, Castrignanò T, Rahimi H, Masuelli L, et al. Proteomic characterization of extracellular vesicles released by third stage larvae of the zoonotic parasite Anisakis pegreffii (Nematoda: Anisakidae) Front Cell Infect Microbiol. 2023;13:1–15. doi: 10.3389/fcimb.2023.1079991. PubMed DOI PMC

Consortium TU UniProt: the universal protein knowledgebase in 2023. Nucleic Acids Res. 2023;51:D523–D531. doi: 10.1093/nar/gkac1052. PubMed DOI PMC

Gertz EM, Yu YK, Agarwala R, Schäffer AA, Altschul SF. Composition-based statistics and translated nucleotide searches: improving the TBLASTN module of BLAST. BMC Biol. 2006;4:41. doi: 10.1186/1741-7007-4-41. PubMed DOI PMC

McGuffin LJ, Bryson K, Jones DT. The PSIPRED protein structure prediction server. Bioinformatics. 2000;16:404–405. doi: 10.1093/bioinformatics/16.4.404. PubMed DOI

Riera-Ferrer E, Del Pozo R, Piazzon MC, Sitjà-Bobadilla A, Estensoro I, Palenzuela O. Sparicotyle chrysophrii experimental infection of gilthead seabream (Sparus aurata): establishment of an in vivo model reproducing the pathological outcomes of sparicotylosis. Aquaculture. 2023;573:739588. doi: 10.1016/j.aquaculture.2023.739588. DOI

Mladineo I, Charouli A, Jelić F, Chakroborty A, Hrabar J. In vitro culture of the zoonotic nematode Anisakis pegreffii (Nematoda, Anisakidae) Parasit Vectors. 2023;16:51. doi: 10.1186/s13071-022-05629-5. PubMed DOI PMC

Webber J, Clayton A. How pure are your vesicles? J Extracell Vesicles. 2013;2:19861. doi: 10.3402/jev.v2i0.19861. PubMed DOI PMC

Rappsilber J, Mann M, Ishihama Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc. 2007;2:1896–1906. doi: 10.1038/nprot.2007.261. PubMed DOI

Cox J, Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol. 2008;26:1367–1372. doi: 10.1038/nbt.1511. PubMed DOI

Tyanova S, Temu T, Cox J. The MaxQuant computational platform for mass spectrometry-based shotgun proteomics. Nat Protoc. 2016;11:2301–2319. doi: 10.1038/nprot.2016.136. PubMed DOI

Tyanova S, Temu T, Sinitcyn P, Carlson A, Hein MY, Geiger T, et al. The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat Methods. 2016;13:731–740. doi: 10.1038/nmeth.3901. PubMed DOI

Paysan-Lafosse T, Blum M, Chuguransky S, Grego T, Pinto BL, Salazar GA, et al. InterPro in 2022. Nucleic Acids Res. 2023;51:D418–D427. doi: 10.1093/nar/gkac993. PubMed DOI PMC

Punta M, Coggill PC, Eberhardt RY, Mistry J, Tate J, Boursnell C, et al. The Pfam protein families database. Nucleic Acids Res. 2012;40:290–301. doi: 10.1093/nar/gkr1065. PubMed DOI PMC

Conesa A, Götz S, García-Gómez JM, Terol J, Talón M, Robles M. Blast2GO: A universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics. 2005;21:3674–3676. doi: 10.1093/bioinformatics/bti610. PubMed DOI

Supek F, Bošnjak M, Škunca N, Šmuc T. Revigo summarizes and visualizes long lists of gene ontology terms. PLoS ONE. 2011;6:e21800. doi: 10.1371/journal.pone.0021800. PubMed DOI PMC

Dalkiran A, Rifaioglu AS, Martin MJ, Cetin-Atalay R, Atalay V, Doğan T. ECPred: a tool for the prediction of the enzymatic functions of protein sequences based on the EC nomenclature. BMC Bioinformatics. 2018;19:334. doi: 10.1186/s12859-018-2368-y. PubMed DOI PMC

Rawlings ND, Barrett AJ, Thomas PD, Huang X, Bateman A, Finn RD. The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database. Nucleic Acids Res. 2018;46:D624–D632. doi: 10.1093/nar/gkx1134. PubMed DOI PMC

Cwiklinski K, De La Torre-Escudero E, Trelis M, Bernal D, Dufresne PJ, Brennan GP, et al. The extracellular vesicles of the helminth pathogen, Fasciola hepatica: biogenesis pathways and cargo molecules involved in parasite pathogenesis. Mol Cell Proteomics. 2015;14:3258–3273. doi: 10.1074/mcp.M115.053934. PubMed DOI PMC

Okumura M, Katsuyama AM, Shibata H, Maki M. VPS37 isoforms differentially modulate the ternary complex formation of ALIX, ALG-2, and ESCRT-I. Biosci Biotechnol Biochem. 2013;77:1715–1721. doi: 10.1271/bbb.130280. PubMed DOI

Qadeer A, Giri BR, Ullah H, Cheng G. Transcriptional profiles of genes potentially involved in extracellular vesicle biogenesis in Schistosoma japonicum. Acta Trop. 2021;217:105851. doi: 10.1016/j.actatropica.2021.105851. PubMed DOI

Krylova SV, Feng D. The machinery of exosomes: biogenesis, release, and uptake. Int J Mol Sci. 2023;24:1337. doi: 10.3390/ijms24021337. PubMed DOI PMC

Larios J, Mercier V, Roux A, Gruenberg J. ALIX- and ESCRT-III-dependent sorting of tetraspanins to exosomes. J Cell Biol. 2020;219:e201904113. doi: 10.1083/jcb.201904113. PubMed DOI PMC

Baietti MF, Zhang Z, Mortier E, Melchior A, Degeest G, Geeraerts A, et al. Syndecan-syntenin-ALIX regulates the biogenesis of exosomes. Nat Cell Biol. 2012;14:677–685. doi: 10.1038/ncb2502. PubMed DOI

Barrett J. Nutrition and Biosynthesis. Biochemistry of Parasitic Helminths. 1. London and Basingstoke: Macmillan Publishers Ltd; 1981. pp. 149–244.

Haraszti RA, Didiot MC, Sapp E, Leszyk J, Shaffer SA, Rockwell HE, et al. High-resolution proteomic and lipidomic analysis of exosomes and microvesicles from different cell sources. J Extracell Vesicles. 2016;5:32570. doi: 10.3402/jev.v5.32570. PubMed DOI PMC

Sotillo J, Pearson M, Potriquet J, Becker L, Pickering D, Mulvenna J, et al. Extracellular vesicles secreted by Schistosoma mansoni contain protein vaccine candidates. Int J Parasitol. 2016;46:1–5. doi: 10.1016/j.ijpara.2015.09.002. PubMed DOI

Kifle DW, Pearson MS, Becker L, Pickering D, Loukas A, Sotillo J. Proteomic analysis of two populations of Schistosoma mansoni -derived extracellular vesicles: 15k pellet and 120k pellet vesicles. Mol Biochem Parasitol. 2020;236:111264. doi: 10.1016/j.molbiopara.2020.111264. PubMed DOI

Sheng ZA, Wu CL, Wang DY, Zhong SH, Yang X, Rao GS, et al. Proteomic analysis of exosome-like vesicles from Fasciola gigantica adult worm provides support for new vaccine targets against fascioliasis. Parasit Vectors. 2023;16:62. doi: 10.1186/s13071-023-05659-7. PubMed DOI PMC

Chaiyadet S, Sotillo J, Smout M, Cooper M, Doolan DL, Waardenberg A, et al. Small extracellular vesicles but not microvesicles from Opisthorchis viverrini promote cell proliferation in human cholangiocytes. bioRxiv [Preprint] 2023;1–30. 10.1101/2023.05.22.540805.

Poddubnaya LG, Hemmingsen W, Gibson DI. Clamp ultrastructure of the basal monogenean Chimaericola leptogaster (Leuckart, 1830) (Polyopisthocotylea: Chimaericolidae) Parasitol Res. 2014;113:4023–4032. doi: 10.1007/s00436-014-4070-y. PubMed DOI

Konstanzová V, Koubková B, Kašný M, Ilgová J, Dzika E, Gelnar M. An ultrastructural study of the surface and attachment structures of Paradiplozoon homoion (Bychowsky & Nagibina, 1959) (Monogenea: Diplozoidae) Parasit Vectors. 2017;10:261. doi: 10.1186/s13071-017-2203-8. PubMed DOI PMC

Ramasamy P, Bhuvaneswari R. The ultrastructure of the tegument and clamp attachment organ of Gotocotyla bivaginalis (Monogenea, Polyopisthocotylea) Int J Parasitol. 1993;23:213–220. doi: 10.1016/0020-7519(93)90143-M. PubMed DOI

Mergo JCJr. Studies on the life history, development, occurrence, and microhabitat of Microcotyle spinicirrus, (Monogenea: Microcotylidae) Maccallum 1819. 1983.

Colorni A, Padrós F. Diseases and health management. In: Pavlidis MA, Mylonas CC, editors. Sparidae: Biology and Aquaculture of Gilthead Sea Bream and other Species. 1. Blackwell Publishing Ltd; 2011. pp. 321–357.

Wirtz-Peitz F, Knoblich JA. Lethal giant larvae take on a life of their own. Trends Cell Biol. 2006;16:234–241. doi: 10.1016/j.tcb.2006.03.006. PubMed DOI

Sojka D, Franta Z, Horn M, Caffrey CR, Mareš M, Kopáček P. New insights into the machinery of blood digestion by ticks. Trends Parasitol. 2013;29:276–285. doi: 10.1016/j.pt.2013.04.002. PubMed DOI

Delcroix M, Sajid M, Caffrey CR, Lim KC, Dvořák J, Hsieh I, et al. A multienzyme network functions in intestinal protein digestion by a platyhelminth parasite. J Biol Chem. 2006;281:39316–39329. doi: 10.1074/jbc.M607128200. PubMed DOI

Caffrey CR, Goupil L, Rebello KM, Dalton JP, Smith D. Cysteine proteases as digestive enzymes in parasitic helminths. PLoS Negl Trop Dis. 2018;12:e0005840. doi: 10.1371/journal.pntd.0005840. PubMed DOI PMC

Dalton JP, Neill SO, Stack C, Collins P, Walshe A, Sekiya M, et al. Fasciola hepatica cathepsin L-like proteases: biology, function, and potential in the development of first generation liver fluke vaccines. Int J Parasitol. 2003;33:1173–1181. doi: 10.1016/S0020-7519(03)00171-1. PubMed DOI

Peterkova K, Vorel J, Ilgova J, Ostasov P, Fajtova P, Konecny L, et al. Proteases and their inhibitors involved in Schistosoma mansoni egg-host interaction revealed by comparative transcriptomics with Fasciola hepatica eggs. Int J Parasitol. 2023;53:253–263. doi: 10.1016/j.ijpara.2022.12.007. PubMed DOI

Robinson MW, Dalton JP, Donnelly S. Helminth pathogen cathepsin proteases: it’s a family affair. Trends Biochem Sci. 2008;33:601–608. doi: 10.1016/j.tibs.2008.09.001. PubMed DOI

Riera-Ferrer E, Piazzon MC, Del Pozo R, Palenzuela O, Estensoro I, Sitjà-Bobadilla A. A bloody interaction: plasma proteomics reveals gilthead sea bream (Sparus aurata) impairment caused by Sparicotyle chrysophrii. Parasit Vectors. 2022;15:322. doi: 10.1186/s13071-022-05441-1. PubMed DOI PMC

Sitjà-Bobadilla A, Álvarez-Pellitero P. Experimental transmission of Sparicotyle chrysophrii (Monogenea: Polyopisthocotylea) to gilthead seabream (Sparus aurata) and histopathology of the infection. Folia Parasitol (Praha) 2009;56:143–151. doi: 10.14411/fp.2009.018. PubMed DOI

Chaimon S, Limpanont Y, Reamtong O, Ampawong S, Phuphisut O, Chusongsang P, et al. Molecular characterization and functional analysis of the Schistosoma mekongi Ca2+ -dependent cysteine protease (calpain) Parasit Vectors. 2019;12:383. doi: 10.1186/s13071-019-3639-9. PubMed DOI PMC

de la Torre-Escudero E, Bennett APS, Clarke A, Brennan GP, Robinson MW. Extracellular vesicle biogenesis in helminths: more than one route to the surface? Trends Parasitol. 2016;32:921–929. doi: 10.1016/j.pt.2016.09.001. PubMed DOI

Mathew R, Wunderlich J, Thivierge K, Cwiklinski K, Dumont C, Tilley L, et al. Biochemical and cellular characterisation of the Plasmodium falciparum M1 alanyl aminopeptidase (PfM1AAP) and M17 leucyl aminopeptidase (PfM17LAP) Sci Rep. 2021;11:2854. doi: 10.1038/s41598-021-82499-4. PubMed DOI PMC

Skinner-Adams TS, Stack CM, Trenholme KR, Brown CL, Grembecka J, Lowther J, et al. Plasmodium falciparum neutral aminopeptidases: new targets for anti-malarials. Trends Biochem Sci. 2010;35:53–61. doi: 10.1016/j.tibs.2009.08.004. PubMed DOI

Esser J, Gehrmann U, D’Alexandri FL, Hidalgo-Estévez AM, Wheelock CE, Scheynius A, et al. Exosomes from human macrophages and dendritic cells contain enzymes for leukotriene biosynthesis and promote granulocyte migration. J Allergy Clin. 2010;126:1032–1040. doi: 10.1016/j.jaci.2010.06.039. PubMed DOI

Haeggström JZ, Kull F, Rudberg PC, Tholander F, Thunnissen MMGM. Leukotriene A 4 hydrolase. Prostaglandins Other Lipid Mediat. 2002;68–69:495–510. doi: 10.1016/S0090-6980(02)00051-5. PubMed DOI

Toh SQ, Glanfield A, Gobert GN, Jones MK. Heme and blood-feeding parasites: friends or foes? Parasit Vectors. 2010;3:108. doi: 10.1186/1756-3305-3-108. PubMed DOI PMC

Nebert DW, Vasiliou V. Analysis of the glutathione S-transferase (GST) gene family. Hum Genomics. 2004;1:460–464. doi: 10.1186/1479-7364-1-6-460. PubMed DOI PMC

Atamna H, Ginsburg H. Heme degradation in the presence of glutathione. A proposed mechanism to account for the high levels of non-heme iron found in the membranes of hemoglobinopathic red blood cells. J Biol Chem. 1995;270:24876–24883. doi: 10.1074/jbc.270.42.24876. PubMed DOI

Sotillo J, Pearson MS, Becker L, Mekonnen GG, Amoahid AS, van Dam G, et al. In-depth proteomic characterization of Schistosoma haematobium: towards the development of new tools for elimination. PLoS Negl Trop Dis. 2019;13:e0007362. doi: 10.1371/journal.pntd.0007362. PubMed DOI PMC

Zhu L, Liu J, Dao J, Lu K, Li H, Gu H, et al. Molecular characterization of S. japonicum exosome-like vesicles reveals their regulatory roles in parasite-host interactions. Sci Rep. 2016;6:25885. doi: 10.1038/srep25885. PubMed DOI PMC

Chaiyadet S, Sotillo J, Smout M, Cantacessi C, Jones MK, Johnson MS, et al. Carcinogenic liver fluke secretes extracellular vesicles that promote cholangiocytes to adopt a tumorigenic phenotype. J Infect Dis. 2015;212:1636–1645. doi: 10.1093/infdis/jiv291. PubMed DOI PMC

Furuyama K, Kaneko K, Vargas PD. Heme as a magnificient molecule with multiple missions: heme determines its own fate and governs cellular homeostasis. Tohoku J Exp Med. 2007;213:1–16. doi: 10.1620/tjem.213.1. PubMed DOI

Jomova K, Valko M. Importance of iron chelation in free radical-induced oxidative stress and human disease. Curr Pharm Des. 2011;17:3460–3473. doi: 10.2174/138161211798072463. PubMed DOI

Glanfield A, McManus DP, Anderson GJ, Jones MK. Pumping iron: a potential target for novel therapeutics against schistosomes. Trends Parasitol. 2007;23:583–588. doi: 10.1016/j.pt.2007.08.018. PubMed DOI PMC

Carmona F, Palacios Ò, Gálvez N, Cuesta R, Atrian S, Capdevila M, et al. Ferritin iron uptake and release in the presence of metals and metalloproteins: chemical implications in the brain. Coord Chem Rev. 2013;257:2752–2764. doi: 10.1016/j.ccr.2013.03.034. DOI

Pakharukova MY, Savina E, Ponomarev DV, Gubanova NV, Zaparina O, Zakirova EG, et al. Proteomic characterization of Opisthorchis felineus exosome-like vesicles and their uptake by human cholangiocytes. J Proteomics. 2023;283–284:104927. doi: 10.1016/j.jprot.2023.104927. PubMed DOI

Prescott SM, Zimmerman G, Stafforini DM, McIntyre TM. Platelet-activating factor and related lipid mediators. Annu Rev Biochem. 2000;69:419–445. doi: 10.1146/annurev.biochem.69.1.419. PubMed DOI

Venable ME, Zimmerman GA, McIntyre TM, Prescott SM. Platelet-activating factor: a phospholipid autacoid with diverse actions. J Lipid Res. 1993;34:691–702. doi: 10.1016/S0022-2275(20)39691-7. PubMed DOI

Arai H, Koizumi H, Aoki J, Inoue K. Platelet-activating factor acetylhydrolase (PAF-AH) J Biochem. 2002;640:635–640. doi: 10.1093/oxfordjournals.jbchem.a003145. PubMed DOI

Sepulcre M, Pelegrín P, Mulero V, Meseguer J. Characterisation of gilthead seabream acidophilic granulocytes by a monoclonal antibody unequivocally points to their involvement in fish phagocytic response. Cell Tissue Res. 2002;308:97–102. doi: 10.1007/s00441-002-0531-1. PubMed DOI

Zhou Y, Zheng H, Chen Y, Zhang L, Wang K, Guo J, et al. The Schistosoma japonicum genome reveals features of host-parasite interplay. Nature. 2009;460:345–351. doi: 10.1038/nature08140. PubMed DOI PMC

Noverr MC, Erb-Downward JR, Huffnagle GB. Production of eicosanoids and other oxylipins by pathogenic eukaryotic microbes. Clin Microbiol Rev. 2003;16:517–533. doi: 10.1128/CMR.16.3.517-533.2003. PubMed DOI PMC

Noto H, Hara M, Karasawa K, Iso-O N, Satoh H, Togo M, et al. Human plasma platelet-activating factor acetylhydrolase binds to all the murine lipoproteins, conferring protection against oxidative stress. Arterioscler Thromb Vasc Biol. 2003;23:829–835. doi: 10.1161/01.ATV.0000067701.09398.18. PubMed DOI

Grigg ME, Gounaris K, Selkirk ME. Characterization of a platelet-activating factor acetylhydrolase secreted by the nematode parasite Nippostrongylus brasiliensis. Biochem J. 1996;317:541–547. doi: 10.1042/bj3170541. PubMed DOI PMC

Pawlowic MC, Zhang K. Leishmania parasites possess a platelet-activating factor acetylhydrolase important for virulence. Mol Biochem Parasitol. 2012;186:11–20. doi: 10.1016/j.molbiopara.2012.08.005. PubMed DOI PMC

Arme C, Fox MG. Oxygen uptake by Diclidophora merlangi (Monogenea) Parasitology. 1974;69:201–205. doi: 10.1017/S0031182000048046. PubMed DOI

Mazanec H, Koník P, Gardian Z, Kuchta R. Extracellular vesicles secreted by model tapeworm Hymenolepis diminuta: biogenesis, ultrastructure and protein composition. Int J Parasitol. 2021;51:327–332. doi: 10.1016/j.ijpara.2020.09.010. PubMed DOI

Sotillo J, Robinson MW, Kimber MJ, Cucher M, Ancarola ME, Nejsum P, et al. The protein and microRNA cargo of extracellular vesicles from parasitic helminths – current status and research priorities. Int J Parasitol. 2020;50:635–645. doi: 10.1016/j.ijpara.2020.04.010. PubMed DOI

Liu J, Zhu L, Wang J, Qiu L, Chen Y, Davis RE, et al. Schistosoma japonicum extracellular vesicle miRNA cargo regulates host macrophage functions facilitating parasitism. PLoS Pathog. 2019;15:e1007817. doi: 10.1371/journal.ppat.1007817. PubMed DOI PMC

Hartl FU, Bracher A, Hayer-Hartl M. Molecular chaperones in protein folding and proteostasis. Nature. 2011;475:324–332. doi: 10.1038/nature10317. PubMed DOI

Spiess C, Meyer AS, Reissmann S, Frydman J. Mechanism of the eukaryotic chaperonin: protein folding in the chamber of secrets. Trends Cell Biol. 2004;14:598–604. doi: 10.1016/j.tcb.2004.09.015. PubMed DOI PMC

Kubota H, Hynes G, Willison K. The chaperonin containing t-complex polypeptide 1 (TCP-1): multisubunit machinery assisting in protein folding and assembly in the eukaryotic cytosol. Eur J Biochem. 1995;230:3–16. doi: 10.1111/j.1432-1033.1995.tb20527.x. PubMed DOI

European Chemicals Agency - ECHA. Formaldehyde and formaldehyde releasers - Strategy for future work. 2018.

European Chemicals Agency - ECHA. Substance Evaluation Conclusion as require by REACH Article 48 and Evaluation Report for Formaldehyde. 2019.

Acosta D, Cancela M, Piacenza L, Roche L, Carmona C, Tort JF. Fasciola hepatica leucine aminopeptidase, a promising candidate for vaccination against ruminant fasciolosis. Mol Biochem Parasitol. 2008;158:52–64. doi: 10.1016/j.molbiopara.2007.11.011. PubMed DOI

Maggioli G, Acosta D, Silveira F, Rossi S, Giacaman S, Basika T, et al. The recombinant gut-associated M17 leucine aminopeptidase in combination with different adjuvants confers a high level of protection against Fasciola hepatica infection in sheep. Vaccine. 2011;29:9057–9063. doi: 10.1016/j.vaccine.2011.09.020. PubMed DOI

Mekonnen GG, Pearson M, Loukas A, Sotillo J. Extracellular vesicles from parasitic helminths and their potential utility as vaccines. Expert Rev Vaccines. 2018;17:197–205. doi: 10.1080/14760584.2018.1431125. PubMed DOI

Edgar RCS, Siddiqui G, Hjerrild K, Malcolm TR, Vinh NB, Webb CT, et al. Genetic and chemical validation of Plasmodium falciparum aminopeptidase PfA-M17 as a drug target in the hemoglobin digestion pathway. Elife. 2022;11:e80813. doi: 10.7554/eLife.80813. PubMed DOI PMC

Drinkwater N, Lee J, Yang W, Malcolm TR, McGowan S. M1 aminopeptidases as drug targets: broad applications or therapeutic niche? FEBS J. 2017;284:1473–1488. doi: 10.1111/febs.14009. PubMed DOI PMC

Fennell BJ, Naughton JA, Barlow J, Brennan G, Fairweather I, Hoey E, et al. Microtubules as antiparasitic drug targets. Expert Opin Drug Discov. 2008;3:501–518. doi: 10.1517/17460441.3.5.501. PubMed DOI

Cwiklinski K, Dalton JP. Omics tools enabling vaccine discovery against fasciolosis. Trends Parasitol. 2022;38:1068–1079. doi: 10.1016/j.pt.2022.09.009. PubMed DOI