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Abstract 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. Graphical Abstract

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. [ABSTRACT FROM AUTHOR]

The morphological characterisation of Diplozoidae spp. is highly reliant on the details of the sclerotised components of the hooks and clamps in the haptor. Only six species of Paradiplozoon (Diplozoinae) have been described from Africa, four of which have adequate morphological and even comparative ITS2 rDNA data available. However, the descriptions of Paradiplozoon ghanense (Thomas, 1957) and Paradiplozoon aegyptense (Fischthal & Kuntz, 1963) lack essential taxonomic information, specifically the details for their haptoral sclerites. As such, all available material from museum collections for these two species were studied using light microscopy to supplement the original morphometric descriptions. The holotype and paratypes of P. aegyptense were studied, but only voucher material for P. ghanense could be sourced. However, this voucher material for P. ghanense was deposited by the species authority and bore a striking resemblance to the illustrations and collection details from the original description. They were thus identified as the type series for the taxon, with a lectotype and paralectotype designated. Both P. ghanense and P. aegyptense could be readily distinguished from other taxa based on the supplementary data generated here, supporting their distinctness. The haptoral sclerites of P. aegyptense were most similar to those of Paradiplozoon krugerense Dos Santos & Avenant-Oldewage, 2016, also described from Labeo spp., while the sclerites of P. ghanense were most similar to Paradiplozoon bingolense Civáňová, Koyun & Koubková, 2013 and Paradiplozoon iraqense Al-Nasiri & Balbuena, 2016. Additionally, a voucher of P. aegyptense collected from the alestid type host of P. ghanense was reidentified as the latter species here. This greatly simplified the known host specificity for Paradiplozoon spp. in Africa, with P. aegyptense now exclusively reported from Cypriniformes (Cyprinidae and Danionidae), and P. ghanense restricted to Characiformes (Alestidae). The occurrence of all diplozoids from non-cyprinoid hosts was also investigated and several records of diplozoids occurring on non-cyprinoid hosts were collated and scrutinised. Excluding the two instances of diplozoids described and exclusively occurring on Characiformes fishes (P. ghanense and Paradiplozoon tetragonopterini (Sterba, 1957)), most other non-cyprinoid collections appear sporadic and unsubstantiated, but warrant further investigation supported by diligent taxonomic data. Even though the morphometric descriptions of both P. ghanense and P. aegyptense were fully reported on here, additional material will be needed to study their genetic profiles and phylogeny. [ABSTRACT FROM AUTHOR]

The morphological characterisation of Diplozoidae spp. is highly reliant on the details of the sclerotised components of the hooks and clamps in the haptor. Only six species of Paradiplozoon (Diplozoinae) have been described from Africa, four of which have adequate morphological and even comparative ITS2 rDNA data available. However, the descriptions of Paradiplozoon ghanense (Thomas, 1957) and Paradiplozoon aegyptense (Fischthal & Kuntz, 1963) lack essential taxonomic information, specifically the details for their haptoral sclerites. As such, all available material from museum collections for these two species were studied using light microscopy to supplement the original morphometric descriptions. The holotype and paratypes of P. aegyptense were studied, but only voucher material for P. ghanense could be sourced. However, this voucher material for P. ghanense was deposited by the species authority and bore a striking resemblance to the illustrations and collection details from the original description. They were thus identified as the type series for the taxon, with a lectotype and paralectotype designated. Both P. ghanense and P. aegyptense could be readily distinguished from other taxa based on the supplementary data generated here, supporting their distinctness. The haptoral sclerites of P. aegyptense were most similar to those of Paradiplozoon krugerense Dos Santos & Avenant-Oldewage, 2016, also described from Labeo spp., while the sclerites of P. ghanense were most similar to Paradiplozoon bingolense Civáňová, Koyun & Koubková, 2013 and Paradiplozoon iraqense Al-Nasiri & Balbuena, 2016. Additionally, a voucher of P. aegyptense collected from the alestid type host of P. ghanense was reidentified as the latter species here. This greatly simplified the known host specificity for Paradiplozoon spp. in Africa, with P. aegyptense now exclusively reported from Cypriniformes (Cyprinidae and Danionidae), and P. ghanense restricted to Characiformes (Alestidae). The occurrence of all diplozoids from non-cyprinoid hosts was also investigated and several records of diplozoids occurring on non-cyprinoid hosts were collated and scrutinised. Excluding the two instances of diplozoids described and exclusively occurring on Characiformes fishes (P. ghanense and Paradiplozoon tetragonopterini (Sterba, 1957)), most other non-cyprinoid collections appear sporadic and unsubstantiated, but warrant further investigation supported by diligent taxonomic data. Even though the morphometric descriptions of both P. ghanense and P. aegyptense were fully reported on here, additional material will be needed to study their genetic profiles and phylogeny.

The morphological characterisation of Diplozoidae spp. is highly reliant on the details of the sclerotised components of the hooks and clamps in the haptor. Only six species of Paradiplozoon (Diplozoinae) have been described from Africa, four of which have adequate morphological and even comparative ITS2 rDNA data available. However, the descriptions of Paradiplozoon ghanense (Thomas, 1957) and Paradiplozoon aegyptense (Fischthal & Kuntz, 1963) lack essential taxonomic information, specifically the details for their haptoral sclerites. As such, all available material from museum collections for these two species were studied using light microscopy to supplement the original morphometric descriptions. The holotype and paratypes of P. aegyptense were studied, but only voucher material for P. ghanense could be sourced. However, this voucher material for P. ghanense was deposited by the species authority and bore a striking resemblance to the illustrations and collection details from the original description. They were thus identified as the type series for the taxon, with a lectotype and paralectotype designated. Both P. ghanense and P. aegyptense could be readily distinguished from other taxa based on the supplementary data generated here, supporting their distinctness. The haptoral sclerites of P. aegyptense were most similar to those of Paradiplozoon krugerense Dos Santos & Avenant-Oldewage, 2016, also described from Labeo spp., while the sclerites of P. ghanense were most similar to Paradiplozoon bingolense Civáňová, Koyun & Koubková, 2013 and Paradiplozoon iraqense Al-Nasiri & Balbuena, 2016. Additionally, a voucher of P. aegyptense collected from the alestid type host of P. ghanense was reidentified as the latter species here. This greatly simplified the known host specificity for Paradiplozoon spp. in Africa, with P. aegyptense now exclusively reported from Cypriniformes (Cyprinidae and Danionidae), and P. ghanense restricted to Characiformes (Alestidae). The occurrence of all diplozoids from non-cyprinoid hosts was also investigated and several records of diplozoids occurring on non-cyprinoid hosts were collated and scrutinised. Excluding the two instances of diplozoids described and exclusively occurring on Characiformes fishes ( P. ghanense and Paradiplozoon tetragonopterini (Sterba, 1957)), most other non-cyprinoid collections appear sporadic and unsubstantiated, but warrant further investigation supported by diligent taxonomic data. Even though the morphometric descriptions of both P. ghanense and P. aegyptense were fully reported on here, additional material will be needed to study their genetic profiles and phylogeny., Competing Interests: Annemarie Avenant-Oldewage is an Academic Editor for PeerJ., (© 2024 Dos Santos and Avenant-Oldewage.)