Evolutionary analysis of species-specific duplications in flatworm genomes.

Platyhelminthes, also known as flatworms, is a phylum of bilaterian invertebrates infamous for their parasitic representatives. The classes Cestoda, Monogenea, and Trematoda comprise parasitic helminths inhabiting multiple hosts, including fishes, humans, and livestock, and are responsible for considerable economic damage and burden on human health. As in other animals, the genomes of flatworms have a wide variety of paralogs, genes related via duplication, whose origins could be mapped throughout the evolution of the phylum. Through in-silico analysis, we studied inparalogs, i.e., species-specific duplications, focusing on their biological functions, expression changes, and evolutionary rate. These genes are thought to be key players in the adaptation process of species to each particular niche. Our results showed that genes related with specific functional terms, such as response to stress, transferase activity, oxidoreductase activity, and peptidases, are overrepresented among inparalogs. This trend is conserved among species from different classes, including free-living species. Available expression data from Schistosoma mansoni, a parasite from the trematode class, demonstrated high conservation of expression patterns between inparalogs, but with notable exceptions, which also display evidence of rapid evolution. We discuss how natural selection may operate to maintain these genes and the particular duplication models that fit better to the observations. Our work supports the critical role of gene duplication in the evolution of flatworms, representing the first study of inparalogs evolution at the genome-wide level in this group.

Does the body size, sex, and reproductive modes of leaf litter anurans affect the diversity of parasites?

We describe the composition of endoparasites associated with leaf litter anurans from an Atlantic Forest area, in southeastern Brazil. We tested if body size, sex, and reproductive modes of anuran hosts influence endoparasite abundance and richness. We sampled 583 individuals from 11 anuran species and recorded 1,600 helminths from 14 taxa. The helminths that infected the greatest number of anuran host species were the nematodes Cosmocerca parva (8 spp.), Physaloptera sp. (8 spp.), and Cosmocerca brasiliense (7 spp.), and the most abundant helminth species were Physaloptera sp. (14.6%), Cosmocerca brasiliense (13.7%) and Cosmocerca parva (12.6%). Both helminth abundance and richness were positively affected by anuran body size and dependence on water for reproduction. Larger hosts can contain a higher abundance of parasites because they may provide more physical space than smaller ones, or it can simply be a function of age. Besides, parasite species richness can be highly correlated with the amount of time a host spends in association with aquatic habitats, a conservative aspect of both parasite and host natural history. Within host species, there was a positive and significant influence of body size on helminth abundance. Haddadus binotatus females had greater helminth abundance than males, probably due to sex-related differences in behavior and/or in physiology. Our data suggest that reproductive modes could also influence helminth infection parameters in other anuran communities and should be considered in detail in future analyses.

Exploring Evolutionary Relationships within Neodermata Using Putative Orthologous Groups of Proteins, with Emphasis on Peptidases.

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.

Trans-splicing in the cestode Hymenolepis microstoma is constitutive across the life cycle and depends on gene structure and composition.

Spliced leader (SL) trans-splicing is a key process during mRNA maturation of many eukaryotes, in which a short sequence (SL) is transferred from a precursor SL-RNA into the 5' region of an immature mRNA. This mechanism is present in flatworms, in which it is known to participate in the resolution of polycistronic transcripts. However, most trans-spliced transcripts are not part of operons, and it is not clear if this process may participate in additional regulatory mechanisms in this group. In this work, we present a comprehensive analysis of SL trans-splicing in the model cestode Hymenolepis microstoma. We identified four different SL-RNAs which are indiscriminately trans-spliced to 622 gene models. SL trans-splicing is enriched in constitutively expressed genes and does not appear to be regulated throughout the life cycle. Operons represented at least 20% of all detected trans-spliced gene models, showed conservation to those of the cestode Echinococcus multilocularis, and included complex loci such as an alternative operon (processed as either a single gene through cis-splicing or as two genes of a polycistron). Most insertion sites were identified in the 5' untranslated region (UTR) of monocistronic genes. These genes frequently contained introns in the 5' UTR, in which trans-splicing used the same acceptor sites as cis-splicing. These results suggest that, unlike other eukaryotes, trans-splicing is associated with internal intronic promoters in the 5' UTR, resulting in transcripts with strong splicing acceptor sites without competing cis-donor sites, pointing towards a simple mechanism driving the evolution of novel SL insertion sites.

New species of Polystomoides (Monogenoidea: Polystomatidae) parasitizing the urinary bladder of a freshwater turtle in Brazil / Nova espécie de Polystomoides (Monogenoidea: Polystomatidae) parasitando a bexiga urinária de uma tartaruga de água doce no Brasil

Trachemys dorbigni is the most abundant freshwater turtle species in Rio Grande do Sul, southern Brazil. Chelonians are known to host a wide variety of pathogens, including viruses, bacteria, hemoparasites and helminths. Among these, nine genera of polystomatid flatworms (Monogenoidea; Polystomatidae) infect freshwater turtles: Apaloneotrema, Aussietrema, Fornixtrema, Manotrema, Pleurodirotrema, Polystomoidella, Polystomoides, Uropolystomoides and Uteropolystomoides. However, little is known about the biology of these parasites in the Neotropical Realm. Through investigative cystoscopy, specimens of Polystomatidae were located inside the urinary bladder of the host T. dorbigni. Retrieved specimens were fixed and stained whole mounts prepared for taxonomic identification. In the present paper, a new species of Polystomoides (Monogenoidea: Polystomatidae) parasitizing the urinary bladder of a freshwater turtle of the species T. dorbigni in Brazil is described. Polystomoides santamariensis n. sp. differs from the congeneric species on the length of the genital spines, which are longer. Given the enormous diversity of freshwater turtles around the world, it is likely that a large number of chelonian polystomatids are still unknown.(AU)

Trachemys dorbigni é a espécie de tartaruga de água doce mais abundante do Rio Grande do Sul, no Sul do Brasil. Sabe-se que os quelônios são hospedeiros de uma grande variedade de patógenos, incluindo vírus, bactérias, hemoparasitos e helmintos. Dentre estes, oito gêneros de Polystomatidae (Platyhelminthes: Monogenoidea) infectam tartarugas de água doce: Apaloneotrema, Aussietrema, Fornixtrema, Manotrema, Pleurodirotrema, Polystomoidella, Polystomoides, Uropolystomoides e Uteropolystomoides. No entanto, pouco ainda se sabe sobre a biologia destes parasitos na Região Neotropical. Durante uma citoscopia investigativa, foram observados espécimes de Polystomatidae localizados no interior da bexiga urinária de T. dorbigni. Os espécimes coletados foram fixados e corados para identificação taxonômica. No presente artigo, uma nova espécie de Polystomoides (Monogenoidea: Polystomatidae) parasitando a bexiga urinária de tartaruga de água doce da espécie T. dorbigni no Brasil é descrita. Polystomoides santamariensis n. sp. difere das demais espécies no tamanho dos espinhos genitais, que são maiores. Devido a enorme diversidade de tartarugas de água doce ao redor do mundo, é provável que um grande número de polistomatídeos de quelônios ainda seja desconhecido.(AU)

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.

Fish-parasite interactions: A dataset of continental waters in Mexico involving fishes and their helminth fauna.

Most of the available knowledge in the literature on Mexican fishes and their parasites refers to information within political divisions and/or hydrological basins in the country. Indeed, only a few studies have analyzed the helminth fauna of these vertebrates as a biological group distributed nation-wide. This lack of available knowledge prevents the study of several basic and applied aspects involving fish-parasite interactions at different spatial and temporal scales. In this dataset, we compiled all the available geographic information on fish-helminth parasite interactions involving native and exotic fish species recorded in continental waters throughout the Mexican territory. After an exhaustive filtering and the curation of information, our data set contains 5999 records of 361 freshwater fish species (roughly 70% of known freshwater fish species occurring in Mexico) and 483 endo- and ectoparasitic helminths collected over an 85-year period (from 1936-2021) in 1070 localities distributed throughout Mexico. These records are mainly concentrated in only a few states located to the south and east of the country; although all states have been sampled and all major basins in Mexico are represented. The fish order with the highest number of records was Perciformes (n = 2325, 38.75%) while the fish family with the highest number of records was Cichlidae (n = 1741, 29.02%). Native species of fishes corresponded to 92.14% of the records (n = 5528) and fish-associated parasites were found in 41 habitat types in/on their host bodies. Regarding fish parasites, we found that most of the records are from the phylum Platyhelminthes (n = 4495, 74.92%). At the class level, we observed that Trematoda reached the highest number of records (n = 2965, 49.42%). Moreover, we found that Diplostomidae (n = 917, 15.25%) were the family of trematodes with the highest number of records. Most parasites were registered in their adult stage (n = 3730, 62.17%), followed by larval stages (n = 2267, 37.78%). We hope that the fish-parasite interactions data set will encourage researchers worldwide to explore different ecological and coevolutionary aspects of fishes and their helminth parasites, as well as provide useful information for the better implementation of conservation initiatives. There are no copyright restrictions; please cite this data paper when using its data in publications or teaching events.

Evolutionary Adaptations of Parasitic Flatworms to Different Oxygen Tensions.

During the evolution of the Earth, the increase in the atmospheric concentration of oxygen gave rise to the development of organisms with aerobic metabolism, which utilized this molecule as the ultimate electron acceptor, whereas other organisms maintained an anaerobic metabolism. Platyhelminthes exhibit both aerobic and anaerobic metabolism depending on the availability of oxygen in their environment and/or due to differential oxygen tensions during certain stages of their life cycle. As these organisms do not have a circulatory system, gas exchange occurs by the passive diffusion through their body wall. Consequently, the flatworms developed several adaptations related to the oxygen gradient that is established between the aerobic tegument and the cellular parenchyma that is mostly anaerobic. Because of the aerobic metabolism, hydrogen peroxide (H2O2) is produced in abundance. Catalase usually scavenges H2O2 in mammals; however, this enzyme is absent in parasitic platyhelminths. Thus, the architecture of the antioxidant systems is different, depending primarily on the superoxide dismutase, glutathione peroxidase, and peroxiredoxin enzymes represented mainly in the tegument. Here, we discuss the adaptations that parasitic flatworms have developed to be able to transit from the different metabolic conditions to those they are exposed to during their life cycle.

Unraveling post-translational modifications in Echinococcus granulosus sensu lato.

Echinococcus granulosus sensu lato (s.l.) is the helminth parasite responsible for cystic echinococcosis, a neglected tropical disease currently affecting millions of people worldwide. Incomplete knowledge on the parasite biochemistry contributes, at least partially, to the limited development of useful biotechnological advances for the infection control. In this sense, little information is available regarding post-translational modifications (PTMs) occurring in E. granulosus s.l. proteins, which ultimately may affect the performance of biotechnological products to be developed. Therefore, we report here a proteomic analysis of the parasite PTMs identified through FindMod software applied to a set of tegumental proteins previously characterized by mass spectrometry (MALDI-TOF/TOF) analysis of protein spots from a 2D electrophoresis gel. Manual searches for already annotated proteins exhibiting such PTMs were also performed within proteome databases of E. granulosus s.l. and other platyhelminthes. In addition, key enzymes involved in PTMs modifications were searched for within E. granulosus s.l. proteome. Finally, the presence of selected PTMs was further confirmed by a high-resolution proteomic approach (nanoLC-MS/MS). A set of 22 different PTMs most likely to be present in the parasite was suggested, 9 of them with high confidence as they were identified in the same m/z fragment by both proteomic techniques (acetylation, deamidation, deamidation followed by methylation, mono- and di-hydroxylation, mono- and di-methylation, S-nitrosylation and phosphorylation). Interestingly, 5 PTMs were herein identified for the first time in E. granulosus s.l. proteins. Our results expand the scarcely studied topic of PTMs in platyhelminthes.

Function of lipid binding proteins of parasitic helminths: still a long road.

Infections with parasitic helminths cause severe debilitating and sometimes lethal diseases in humans and domestic animals on a global scale. Unable to synthesize de novo their own fatty acids and sterols, helminth parasites (nematodes, trematodes, cestodes) rely on their hosts for their supply. These organisms produce and secrete a wide range of lipid binding proteins that are, in most cases, structurally different from the ones found in their hosts, placing them as possible novel therapeutic targets. In this sense, a lot of effort has been made towards the structure determination of these proteins, but their precise function is still unknown. In this review, we aim to present the current knowledge on the functions of LBPs present in parasitic helminths as well as novel members of this highly heterogeneous group of proteins.

Parasites with zoonotic potential found in commercially important fish in Tamaulipas, Northeastern Mexico.

Human population is exposed to numerous parasitic ichthyozoonoses. Although Tamaulipas state (northeastern Mexico) is well known for its fishing and aquaculture industry, there are few reports of this type of zoonosis. Therefore, it is imperative to investigate whether the parasites that affect these fish may represent a zoonotic risk for the inhabitants of the area. The objective of this study was to identify molecular and/or morphologically muscle parasites of fish from coastal locations in Tamaulipas, Mexico, and assess the risk of infection for humans. Between 2017 and 2018, 764 individual fish belonging to 28 species were examined for parasites. Collected worms were processed for their identification using morphological characteristics. In addition, partial sequences of the large subunit (28S) ribosomal RNA gene were obtained from some species to corroborate their identity. Prevalence and mean intensity of all registered infections were calculated. A total of seven species of parasites were found: cestodes (Poecilancistrium caryophyllum), trematodes (Clinostomum tataxumui, Clinostomum cichlidorum), nematodes (Eustrongylides sp., Contracaecum sp.) and pentastomids (Sebekia purdieae, Sebekia sp.). Parasites infected 10 species belonging to different fish families (Ariidae, Centrarchidae, Centropomidae, Cichlidae, Eleotridae, Ictaluridae, Mugilidae and Sciaenidae). Congeneric species of parasites or related to those registered in this study have been identified as zoonotic agents in other regions of the world. Despite the low levels of infection (2.6-16.6% prevalence and 1-5.5 parasites per infected host), there is a latent risk of transmission to humans, so it is recommended to avoid eating raw or undercooked fish meat.

Gleasonian structure in the helminth metacommunity of the opossum Didelphis albiventris in two extremes of the Atlantic Forest.

Didelphis albiventris is the opossum with the largest geographic distribution in Brazil and has a wide spectrum of helminth parasites. This study aimed to describe the species composition and analyse the structure of helminth communities of D. albiventris in two extremes of the Atlantic Forest in Brazil. The influence of host sex, body mass, age, helminth species richness and locality on the abundance and prevalence of the most prevalent helminth species was investigated. Ninety-five per cent of the animals were infected with at least one species. Nine helminth species were found: the nematodes Aspidodera raillieti, Cruzia tentaculata, Trichuris didelphis and Trichuris minuta in the large intestine; Turgida turgida in the stomach; Travassostrongylus orloffi and Viannaia hamata; and the trematodes Brachylaima advena and Rhopalias coronatus in the small intestine. Three helminth morphospecies were also recovered: the nematodes Hoineffia sp. and Viannaia sp. and a cestode in the small intestine. Cruzia tentaculata and V. hamata were the species with the highest mean abundances and intensities, while the most prevalent species were A. raillieti, C. tentaculata, T. turgida and V. hamata, forming the central nucleus of the helminth component community. The analysis of the helminth metacommunity structure indicated a Gleasonian pattern for the total set of infracommunities, corroborating the beta diversity indices, which indicated more species replacement than loss between localities and infracommunities, although at a low level. The results indicated a stronger influence of host attributes than geographical distance on the community structure.

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.

Presence of the invasive land flatworm emPlatydemus/em emmanokwari/em (Platyhelminthes, Geoplanidae) in Guadeloupe, Martinique and Saint Martin (French West Indies).

The land flatworm Platydemus manokwari (Platyhelminthes, Geoplanidae) is recorded from the islands of Guadeloupe, Martinique, and Saint Martin in the Caribbean arc. Photographs and records were obtained mainly from citizen science and ranged from the end of 2018 to February 2021; several specimens were deposited in the collections of the Muséum National d'Histoire Naturelle in Paris, France. Thirty records were from Guadeloupe, but only one from Martinique and from Saint Martin, respectively. The COI sequences of 3 specimens from Guadeloupe show that they belong to the World haplotype also found in many countries. We also report P. manokwari from Fort Myers, Florida, USA, with molecular characterization, which was also the World haplotype. This is the first published record of P. manokwari for Guadeloupe, Martinique and Saint Martin and the second for islands in the Caribbean, after Puerto Rico.

Mitochondrial genome of Rhabdosynochus viridisi (Monogenea: Diplectanidae), a parasite of Pacific white snook Centropomus viridis.

We report the nearly complete mitochondrial genome of Rhabdosynochus viridisi - the first for this genus - achieved by combining shotgun sequencing of genomic and cDNA libraries prepared using low-input protocols. This integration of genomic information leads us to correct the annotation of the gene features. The mitochondrial genome consists of 13,863 bp. Annotation resulted in the identification of 12 protein-encoding genes, 22 tRNA genes and two rRNA genes. Three non-coding regions, delimited by three tRNAs, were found between the genes nad5 and cox3. A phylogenetic analysis grouped R. viridisi with three other species of diplectanid monogeneans for which mitochondrial genomes are available.

A new schistosome (Digenea: Schistosomatidae) from the nasal tissue of South America black-necked swans, Cygnus melancoryphus (Anatidae) and the endemic pulmonate snail Chilina gibbosa.

To date, 9 species of Schistosomatidae have been found parasitizing the nasal tissues of mammal and bird hosts in the Eastern Hemisphere, 5 species in Rwanda (Africa), 2 in Australia (Oceania) and 2 in Eurasia. During a parasitological survey of black necked swans, Cygnus melancoryphus, an anatid endemic to South America, schistosome worms in the nasal tissue were found; the first in the Americas. Morphological results based on male worms and in isolated eggs. The worms have a spiny tegument, filiform body with rounded posterior end, two muscular suckers, a robust gynaecophoric channel with thickened cross bands, and around 130 testes. The eggs are elongate with an asymmetrical bulge, with a slender process at one end and a longer curved process at the other. Diagnostic morphological characteristics do not match with any schistosome genus. Part of the mitochondrial cox1 and nuclear DNA 28S partial genes were sequenced and compared to Schistosomatidae in GenBank. The genetic results confirm the distinctiveness of the specimens since they do not group with any described genus or undescribed lineage other than cercariae of "Chilina lineage 1" that emerge from the Patagonian Chilina gibbosa, a freshwater snail endemic to South America. Based on morphological and genetic characterization of these schistosomes, these specimens represent a new genus and species that parasitizes black necked swans as adults in the nasal tissue, and C. gibbosa is the first intermediate host, both hosts being endemic to South America.

Quantifying Cell Proliferation During Regeneration of Aquatic Worms.

Many species of aquatic worms, including members of the phyla Nemertea, Annelida, Platyhelminthes, and Xenacoelomorpha, can regenerate large parts of their body after amputation. In most species, cell proliferation plays key roles in the reconstruction of lost tissues. For example, in annelids and flatworms, inhibition of cell proliferation by irradiation or chemicals prevents regeneration. Cell proliferation also plays crucial roles in growth, body patterning (e.g., segmentation) and asexual reproduction in many groups of aquatic worms. Cell proliferation dynamics in these organisms can be studied using immunohistochemical detection of proteins expressed during proliferation-associated processes or by incorporation and labeling of thymidine analogues during DNA replication. In this chapter, we present protocols for labeling and quantifying cell proliferation by (a) antibody-based detection of either phosphorylated histone H3 during mitosis or proliferating cell nuclear antigen (PCNA) during S-phase, and (b) incorporation of two thymidine analogues, 5'-bromo-2'-deoxyuridine (BrdU) and 5'-ethynyl-2'-deoxyuridine (EdU), detected by immunohistochemistry or inorganic "click" chemistry, respectively. Although these protocols have been developed for whole mounts of small (<2 cm) marine and freshwater worms, they can also be adapted for use in larger specimens or tissue sections.

Three new species of Urocleidoides (Monogenoidea: Dactylogyridae) parasitizing characiforms (Actinopterygii: Characiformes) in Tocantins River, states of Tocantins and Maranhão, and new record for U. triangulus in Guandu River, state of Rio de Janeiro, Brazil

Three new species of Urocleidoides Mizelle & Price, 1964 are described from the gills of characiform fishes in the Tocantins River and its tributaries. Urocleidoides boulengerellae sp. nov. is described from Boulengerella cuvieri (Spix & Agassiz, 1829) and differs from all its congeners by the dorsal bar with a long posteromedial projection; male copulatory organ with 2-3 counterclockwise rings and a base with a flange; an accessory piece comprising a robust Y-shaped unit and a sheath-like unit; and a highly sclerotized vaginal canal. Urocleidoides paratriangulus sp. nov., described from Psectrogaster amazonica Eigenmann & Eigenmann, 1889, Cyphocharax gouldingi Vari, 1992, Caenotropus labyrinthicus (Kner, 1858) and Mylesinus paucisquamatus Jégu & Santos, 1988, is most similar to Urocleidoides triangulus (Suriano, 1981) Rossin & Timi, 2016 based on the shape of the anchors and bars but differs from U. triangulus in the morphology of the projection of the dorsal bar, the number of rings of male copulatory organ, and by the smaller size of members of hook pairs 1 and 5 compared with those of the remaining pairs. Urocleidoides tocantinensis sp. nov. is easily distinguished from all other species of the genus by the morphology of the vagina, which present a vaginal vestibule with a membranous cap. Urocleidoides triangulus is reported from its type host in the Guandu River, state of Rio de Janeiro. The present study increases the number of Urocleidoides species to 37 recognized species that fit all the generic characters.

Three new species of Urocleidoides (Monogenoidea: Dactylogyridae) parasitizing characiforms (Actinopterygii: Characiformes) in Tocantins River, states of Tocantins and Maranhão, and new record for U. triangulus in Guandu River, state of Rio de Janeiro, Brazil

ABSTRACT Three new species of Urocleidoides Mizelle & Price, 1964 are described from the gills of characiform fishes in the Tocantins River and its tributaries. Urocleidoides boulengerellae sp. nov. is described from Boulengerella cuvieri (Spix & Agassiz, 1829) and differs from all its congeners by the dorsal bar with a long posteromedial projection; male copulatory organ with 2-3 counterclockwise rings and a base with a flange; an accessory piece comprising a robust Y-shaped unit and a sheath-like unit; and a highly sclerotized vaginal canal. Urocleidoides paratriangulus sp. nov., described from Psectrogaster amazonica Eigenmann & Eigenmann, 1889, Cyphocharax gouldingi Vari, 1992, Caenotropus labyrinthicus (Kner, 1858) and Mylesinus paucisquamatus Jégu & Santos, 1988, is most similar to Urocleidoides triangulus (Suriano, 1981) Rossin & Timi, 2016 based on the shape of the anchors and bars but differs from U. triangulus in the morphology of the projection of the dorsal bar, the number of rings of male copulatory organ, and by the smaller size of members of hook pairs 1 and 5 compared with those of the remaining pairs. Urocleidoides tocantinensis sp. nov. is easily distinguished from all other species of the genus by the morphology of the vagina, which present a vaginal vestibule with a membranous cap. Urocleidoides triangulus is reported from its type host in the Guandu River, state of Rio de Janeiro. The present study increases the number of Urocleidoides species to 37 recognized species that fit all the generic characters.

Three new species of Urocleidoides (Monogenoidea: Dactylogyridae) parasitizing characiforms (Actinopterygii: Characiformes) in Tocantins River, states of Tocantins and Maranhão, and new record for U. triangulus in Guandu River, state of Rio de Janeiro, Brazil

Three new species of Urocleidoides Mizelle & Price, 1964 are described from the gills of characiform fishes in the Tocantins River and its tributaries. Urocleidoides boulengerellae sp. nov. is described from Boulengerella cuvieri (Spix & Agassiz, 1829) and differs from all its congeners by the dorsal bar with a long posteromedial projection; male copulatory organ with 2-3 counterclockwise rings and a base with a flange; an accessory piece comprising a robust Y-shaped unit and a sheath-like unit; and a highly sclerotized vaginal canal. Urocleidoides paratriangulus sp. nov., described from Psectrogaster amazonica Eigenmann & Eigenmann, 1889, Cyphocharax gouldingi Vari, 1992, Caenotropus labyrinthicus (Kner, 1858) and Mylesinus paucisquamatus Jégu & Santos, 1988, is most similar to Urocleidoides triangulus (Suriano, 1981) Rossin & Timi, 2016 based on the shape of the anchors and bars but differs from U. triangulus in the morphology of the projection of the dorsal bar, the number of rings of male copulatory organ, and by the smaller size of members of hook pairs 1 and 5 compared with those of the remaining pairs. Urocleidoides tocantinensis sp. nov. is easily distinguished from all other species of the genus by the morphology of the vagina, which present a vaginal vestibule with a membranous cap. Urocleidoides triangulus is reported from its type host in the Guandu River, state of Rio de Janeiro. The present study increases the number of Urocleidoides species to 37 recognized species that fit all the generic characters.(AU)