Evaluation of bromocriptine and plumbagin against the monogenean Rhabdosynochus viridisi: Computational drug repositioning and in vitro approaches.

Monogeneans are parasitic platyhelminths that can harm the health of farmed fish. Few treatments are available against monogeneans, and the incentive to develop new antiparasitic agents is similar or even lower than the incentive for neglected parasitic diseases in humans. Considering that searching for and developing new antimonogenean compounds may require enormous investments of time, money, and animal sacrifice, the use of a computer-guided drug repositioning approach is a reasonable alternative. Under this context, this study aimed to evaluate the effectiveness of plumbagin and bromocriptine against adults and eggs of the monogenean Rhabdosynochus viridisi (Diplectanidae). Plumbagin is a phytochemical compound that has recently emerged as a potent antimonogenean; however, further investigation is required to determine its effects on different monogenean species. Bromocriptine was selected through a computational approach that included molecular docking analyses of 77 receptors of monogeneans (putative drug targets) and 77 ligands (putative inhibitors). In vitro experiments showed that bromocriptine does not exhibit mortality at concentrations of 0.1, 1, and 10 mg/L whereas plumbagin at 2 and 10 mg/L caused 100% monogenean mortality after 3 h and 30 min, respectively. The most effective concentration of plumbagin (10 mg/L) did not completely inhibit egg hatching. These findings underscore plumbagin as a highly effective agent against adult monogeneans and highlight the need for research to evaluate its effect(s) on fish. Although computational drug repositioning is useful for selecting candidates for experimental testing, it does not guarantee success due to the complexity of biological interactions, as observed here with bromocriptine. Therefore, it is crucial to examine the various compounds proposed by this method.

Description of Echthrogaleus spinulus n. sp. (Copepoda: Pandaridae) parasitic on a torpedo ray from the central Pacific Ocean utilising a morphological and molecular approach.

A new species of parasitic copepod, Echthrogaleus spinulus n. sp. (Pandaridae), is described from the torpedo ray Tetronarce tokionis (Tanaka) (Torpedinidae) captured in pelagic Hawaiian waters. The new species has pediger 4 bearing large dorsal plates with denticles on posterior margin, genital complex with posterolateral lobes widely curved medially and overlapping, leg 4 exopod incompletely 3-segmented, and the largest body size (maximum length 16 mm from anterior rim of frontal plates to tip of caudal rami, excluding setae). This morphology does not match any of the seven valid species of Echthrogaleus Steenstrup & Lütken, 1861. Analysis of 28S rDNA sequences separated the new material from the Central Pacific from samples of E. coleoptratus in the Atlantic and Eastern Pacific Oceans. However, due to the lack of DNA sequences in the databases, the new 28S rDNA sequence cannot used to confirm the species identity. The unique morphological characteristics of the Central Pacific female copepods combined with 28S rDNA sequencing was used as a basis to validate the new species.

De novo transcriptome assembly and identification of G-Protein-Coupled-Receptors (GPCRs) in two species of monogenean parasites of fish.

Genomic resources for Platyhelminthes of the class Monogenea are scarce, despite the diversity of these parasites, some species of which are highly pathogenic to their fish hosts. This work aimed to generate de novo-assembled transcriptomes of two monogenean species, Scutogyrus longicornis (Dactylogyridae) and Rhabdosynochus viridisi (Diplectanidae), providing a protocol for cDNA library preparation with low input samples used in single cell transcriptomics. This allowed us to work with sub-microgram amounts of total RNA with success. These transcriptomes consist of 25,696 and 47,187 putative proteins, respectively, which were further annotated according to the Swiss-Prot, Pfam, GO, KEGG, and COG databases. The completeness values of these transcriptomes evaluated with BUSCO against Metazoa databases were 54.1% and 73%, respectively, which is in the range of other monogenean species. Among the annotations, a large number of terms related to G-protein-coupled receptors (GPCRs) were found. We identified 109 GPCR-like sequences in R. viridisi, and 102 in S. longicornis, including family members specific for Platyhelminthes. Rhodopsin was the largest family according to GRAFS classification. Two putative melatonin receptors found in S. longicornis represent the first record of this group of proteins in parasitic Platyhelminthes. Forty GPCRs of R. viridisi and 32 of S. longicornis that were absent in Vertebrata might be potential drug targets. The present study provides the first publicly available transcriptomes for monogeneans of the subclass Monopisthocotylea, which can serve as useful genomic datasets for functional genomic research of this important group of parasites.

Title: Assemblage de novo du transcriptome et identification des récepteurs couplés aux protéines G (RCPG) chez deux espèces de Monogènes parasites de poissons. Abstract: Les ressources génomiques pour les Plathelminthes de la classe Monogenea sont rares, malgré la diversité de ces parasites dont certaines espèces sont hautement pathogènes pour leurs hôtes poissons. Ce travail visait à générer des transcriptomes assemblés de novo pour deux espèces de monogènes, Scutogyrus longicornis (Dactylogyridae) et Rhabdosynochus viridisi (Diplectanidae), fournissant un protocole pour la préparation de la bibliothèque d'ADNc avec des échantillons à faible apport utilisés en transcriptomique unicellulaire, ce qui a permis de travailler avec des quantités inférieures au microgramme d'ARN total avec succès. Ces transcriptomes se composent de 25 696 et 47 187 protéines putatives, respectivement, qui ont ensuite été annotées selon les bases de données Swiss-Prot, Pfam, GO, KEGG et COG. L'exhaustivité de ces transcriptomes évaluée avec BUSCO par rapport aux bases de données des Métazoaires était respectivement de 54,1 % et 73 %, ce qui est dans la gamme des autres espèces de monogènes. Parmi les annotations, un grand nombre de termes liés aux récepteurs couplés aux protéines G (RCPG) ont été trouvés. Nous avons identifié 109 séquences de type RCPG chez R. viridisi et 102 chez S. longicornis, y compris des membres de la famille spécifiques de Platyhelminthes. La rhodopsine était la plus grande famille selon la classification GRAFS. Deux récepteurs putatifs de la mélatonine trouvés chez S. longicornis représentent le premier signalement de ce groupe de protéines chez les Plathelminthes parasites. Quarante RCPG de R. viridisi et 32 de S. longicornis, qui sont absents chez les Vertébrés, pourraient être des cibles médicamenteuses potentielles. La présente sont fournit les premiers transcriptomes accessibles au public pour les monogènes de la sous-classe Monopisthocotylea, qui peuvent servir d'ensembles de données génomiques utiles pour la recherche génomique fonctionnelle de cet important groupe de parasites.

In silico identification of excretory/secretory proteins and drug targets in monogenean parasites.

The Excretory/Secretory (ES) proteins of parasites are involved in invasion and colonization of their hosts. In addition, since ES proteins circulate in the extracellular space, they can be more accessible to drugs than other proteins, which makes ES proteins optimal targets for the development of new and better pharmacological strategies. Monogeneans are a group of parasitic Platyhelminthes that includes some pathogenic species problematic for finfish aquaculture. In the present study, 8297 putative ES proteins from four monogenean species which genomic resources are publicly available were identified and functionally annotated by bioinformatic tools. Additionally, for comparative purposes, ES proteins in other parasitic and free-living platyhelminths were identified. Based on data from the monogenean Gyrodactylus salaris, 15 ES proteins are considered potential drug targets. One of them showed homology to 10 cathepsins with known 3D structure. A docking molecular analysis uncovered that the anthelmintic emodepside shows good affinity to these cathepsins suggesting that emodepside can be experimentally tested as a monogenean's cathepsin inhibitor.

Genome-wide identification of ABC transporters in monogeneans.

ATP-Binding Cassette (ABC) transporters are proteins that actively mediate the transport of a wide variety of molecules, including drugs. Thus, in parasitology, ABC transporters have gained attention as potential targets for therapeutic drugs. Among the parasitic Platyhelminthes, ABC transporters have been identified and classified in a few species of Trematoda and Cestoda but not in Monogenea. Monogeneans are mainly ectoparasites of marine and freshwater fish, although they can also be found on other aquatic organisms. Severe epizootics caused by monogeneans have been reported around the world, mainly in confined and/or overcrowded fish. The purpose of this study was to identify the ABC transporters in four species of monogeneans (Gyrodactylus salaris, Protopolystoma xenopodis, Eudiplozoon nipponicum and Neobenedenia melleni) for which genomic resources are publicly available. For comparative purposes, ABC transporters were also identified in endoparasitic (Schistosoma mansoni and Echinococcus granulosus) and free-living (Macrostomun lignano and Schmidtea mediterranea) platyhelminths. Thirty-two putative ABC transporters were identified in the genome of G. salaris, 40 in the genome of P. xenopodis, 46 in the transcriptome of E. nipponicum and 9 in a rather limited ESTs set available for N. melleni. Of the eight ABC subfamilies (A-H) known in metazoans, subfamily H was the only one not found in any monogenean species. In contrast, ABCC was the best represented subfamily. Phylogenetic analyses showed a few cases of one-to-one orthologous relationships, which agree with results from other metazoan species. We found some monogenean ABC members related to subfamilies B, C and G involved in drug resistance in humans. This information may be useful for future functional studies on ABC transporters in monogeneans.

Parasitic copepods (Crustacea, Hexanauplia) on fishes from the lagoon flats of Palmyra Atoll, Central Pacific.

We surveyed copepods parasitic on the fishes at Palmyra, a remote atoll in the Central Indo-Pacific faunal region. In total, we collected 849 individual fish, representing 44 species, from the intertidal lagoon flats at Palmyra and recovered 17 parasitic copepod species. The parasitic copepods were: Orbitacolaxwilliamsi on Mulloidichthysflavolineatus; Anuretesserratus on Acanthurusxanthopterus; Caligusconfusus on Carangoidesferdau, Carangoidesorthogrammus, Caranxignobilis, Caranxmelampygus, and Caranxpapuensis; Caliguskapuhili on Chaetodonauriga and Chaetodonlunula; Caliguslaticaudus on Rhinecanthusaculeatus, Pseudobalistesflavimarginatus, M.flavolineatus, Upeneustaeniopterus, Chrysipteraglauca, and Epinephalusmerra; Caligusmutabilis on Lutjanusfulvus and Lutjanusmonostigma; Caligusrandalli on C.ignobilis; Caligus sp. on L.fulvus; Caritusserratus on Chanoschanos; Lepeophtheiruslewisi on A.xanthopterus; Lepeophtheirusuluus on C.ignobilis; Dissonussimilis on Arothronhispidus; Nemesis sp. on Carcharhinusmelanopterus; Hatschekialongiabdominalis on A.hispidus; Hatschekiabicaudata on Chaetodonauriga and Chaetodonlunula; Kroyerialongicauda on C.melanopterus and Lernanthropus sp. on Kyphosuscinerascens. All copepod species reported here have been previously reported from the Indo-Pacific but represent new geographical records for Palmyra, demonstrating large-scale parasite dispersion strategies.