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.

The phylogenetic relationships within Neodermata were examined based on putative orthologous groups of proteins (OGPs) from 11 species of Monogenea, Trematoda, and Cestoda. The dataset included OGPs from BUSCO and OMA. Additionally, peptidases were identified and evaluated as phylogenetic markers. Phylogenies were inferred using the maximum likelihood method. A network analysis and a hierarchical grouping analysis of the principal components (HCPC) of orthologous groups of peptidases were performed. The phylogenetic analyses showed the monopisthocotylean monogeneans as the sister-group of cestodes, and the polyopisthocotylean monogeneans as the sister-group of trematodes. However, the sister-group relationship between Monopisthocotylea and Cestoda was not statistically well supported. The network analysis and HCPC also showed a cluster formed by polyopisthocotyleans and trematodes. The present study supports the non-monophyly of Monogenea. An analysis of mutation rates indicated that secreted peptidases and inhibitors, and those with multiple copies, are under positive selection pressure, which could explain the expansion of some families such as C01, C19, I02, and S01. Whilst not definitive, our study presents another point of view in the discussion of the evolution of Neodermata, and we hope that our data drive further discussion and debate on this intriguing topic.