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.

Molecular Effects of Silver Nanoparticles on Monogenean Parasites: Lessons from Caenorhabditis elegans.

The mechanisms of action of silver nanoparticles (AgNPs) in monogenean parasites of the genus Cichlidogyrus were investigated through a microarray hybridization approach using genomic information from the nematode Caenorhabditis elegans. The effects of two concentrations of AgNPs were explored, low (6 µg/L Ag) and high (36 µg/L Ag). Microarray analysis revealed that both concentrations of AgNPs activated similar biological processes, although by different mechanisms. Expression profiles included genes involved in detoxification, neurotoxicity, modulation of cell signaling, reproduction, embryonic development, and tegument organization as the main biological processes dysregulated by AgNPs. Two important processes (DNA damage and cell death) were mostly activated in parasites exposed to the lower concentration of AgNPs. To our knowledge, this is the first study providing information on the sub-cellular and molecular effects of exposure to AgNPs in metazoan parasites of fish.

The Efficacy of Geraniol and ß-Citronellol against Freshwater and Marine Monogeneans.

Monogeneans are parasitic flatworms that may be a threat for finfish aquaculture. In this study, the anthelmintic activity of two terpenes, geraniol and ß-citronellol, was tested in vitro against ancyrocephalin and diplectanid monogeneans. Experiments were performed in both water and a culture medium. We observed that monogeneans in culture medium may be more tolerant to treatments compared with bioassays performed only in water. Concentrations of 300 mg/L of both compounds were required to kill 100% of monogeneans at 1 h postexposure. The toxicity of ß-citronellol to fish was not evaluated. However, geraniol at 300 mg/L and 150 mg/L killed juvenile Nile Tilapia Oreochromis niloticus and White Snook Centropomus viridis, respectively, after a few minutes. Therefore, the present work suggests that other alternatives should be studied for use against monogeneans in aquaculture.

Effects of temperature on the life cycle of Neobenedenia sp. (Monogenea: Capsalidae) from Seriola rivoliana (Almaco jack) in Bahía de La Paz, BCS Mexico.

Seriola rivoliana cultivated in Mexico are infected by Neobenedenia sp. (Monogenea: Capsalidae), resulting in dermal ulceration and subsequent bacterial invasion that can cause fish death. This study assesses the effects of temperature over hatching success, oncomiracidia longevity, and infection success. The experimental design consisted of culturing the parasite at temperatures ranging between 16 and 32 °C. The oncomiracidia infection success, time to sexual maturity, and size at sexual maturity of Neobenedenia sp. were examined only at three temperatures (20 °C, 24 °C, and 30 °C). Experiments were conducted under controlled conditions in the laboratory. The oncomiracidia development was found to be faster at warmer temperatures (4-5 days between 24 and 30 °C) than in colder treatments (7-11 days between 18 and 20 °C). Hatching success and oncomiracidia longevity were higher at 24 °C and 26 °C. At 20 °C, 24 °C, and 30 °C, infection success was greater than 90%. Additionally, the laid eggs were observed at 9, 12, and 15 days at 30 °C, 24 °C, and 30 °C, respectively. The results of this study will allow for improving the temporal schedule of applications of treatments against Neobenedenia sp. by the function of temperatures. In conclusion, it is recommended to treat fish more frequently if the temperature in cultures is higher than 24 °C, because Neobenedenia sp. development is faster. As an alternative, the fish could be moved to deeper and cooler waters.

In vitro efficacy of two terpenes against ancyrocephalid monogeneans from Nile tilapia.

Terpenes are naturally produced compounds with a broad range of biological activities. Currently, there is limited information regarding the anthelminthic effect of terpenes against monogenean parasites of fish. The aim of this work was to evaluate the in vitro efficacy of two terpenes [α-terpinene and (+)-limonene oxide] against ancyrocephalid monogeneans found on farmed Nile tilapia (Oreochromis niloticus). (+)-Limonene oxide was more effective in killing these parasites than α-terpinene, with 86 and 90% mortality at concentrations of 36 and 55.4 mg/L, respectively, with a 5-h treatment. The estimated 5-h EC50 of (+)-limonene oxide was 4.8 mg/L. Even though this compound has the potential to be used as an anthelmintic compound in finfish aquaculture, before in vivo experiments are performed, additional studies are needed to find a more effective concentration, as well as to evaluate other terpenic compounds.

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.

Rapid infection and proliferation of dactylogyrid monogeneans on gills of spotted rose snapper (Lutjanus guttatus) after transfer to a sea-cage.

Finfish mariculture is typically threatened by parasite and disease outbreaks. Therefore, it is important to identify parasite species of potential risk for this activity. Snappers are valuable food fish worldwide. In the Eastern Pacific, spotted rose snapper (Lutjanus guttatus [Steindachner, 1869]) is a firm candidate for sea-cage aquaculture. In the current study, the parasitism of caged L. guttatus by dactylogyrids was evaluated for the first time during a complete farming period. Twenty five thousand juvenile fish produced at the Research Center for Food and Development (CIAD, Mazatlan Unit) were reared in a sentinel sea-cage from February to November 2012 in Mazatlan Bay, Mexico. A fish sample (n=15) was obtained every month. Dactylogyrids from the left second gill arch were identified and quantified. A total of 18,704 dactylogyrids distributed in three species, Euryhaliotrema perezponcei García-Vargas, Fajer-Ávila and Lamothe-Argumedo, 2008, E. mehen (Soler-Jiménez, García-Gasca and Fajer-Ávila, 2012), and Haliotrematoides guttati García-Vargas, Fajer-Ávila and Lamothe-Argumedo, 2008 (Monogenea: Dactylogyridae) was found, which were able to infect caged L. guttatus since the first month of the farming period. Prevalence of these parasite species was 100% all the time, except for initial low values for E. mehen and H. guttati. The mean intensity of infection of each dactylogyrid species varied significantly between sampling months. Euryhaliotrema perezponcei was the most abundant parasite, reaching the highest mean intensity in May, June and July (154.3, 296.9 and 176.6 parasites/host, respectively). No clear seasonality of infection was observed; however, the influence of the water temperature on the observed infection levels is discussed. There was no mortality, change on behavior or pathological signs. However, given the rapid infection and proliferation of dactylogyrids, particularly E. perezponcei on L. guttaus reared in a sentinel sea-cage, outbreak of these parasites could be expected when this fish species is cultured on a large scale.

New records of sea lice (Copepoda: Caligidae) from marine fishes in Jaramijó, an area with potential for sea-cage aquaculture in Ecuador.

Farming of finfish in sea cages is gaining popularity worldwide. These systems are a suitable environment for the emergence, establishment and transmission of parasites or pathogens, such as sea lice (Copepoda: Caligidae), known to cause serious diseases and economic losses in finfish aquaculture worldwide. In coastal waters of Jaramijó, Ecuador, there are plans to culture spotted rose snapper (Lutjanus guttatus) and longfin yellowtail (Seriola rivoliana); however, the information about the occurrence of sea lice on fish from this country is scarce. To address this problem, a parasitological survey of economically important fish caught by artisanal fishermen was conducted between June 2013 and May 2014. A total of 608 fish belonging to 66 species were examined. Sea lice were found on 23 fish species. The diversity of these parasites consisted of 22 species of Caligus and 5 species of Lepeophtheirus. Most sea lice species (66%) occurred in a single fish species only, with low infection levels. The most frequently encountered species were Caligus asperimanus Pearse, 1951, Caligus mutabilis Wilson, 1905 and Caligus rufimaculatus Wilson, 1905. Taxonomic remarks are presented for some of the species recorded during this survey. All but two sea lice records are new to Ecuador, considerably expanding the geographical range of some species.

Three new gonad-infecting species of Philometra (Nematoda: Philometridae) parasitic in Lutjanus spp. (Lutjanidae) in the northern Gulf of Mexico off Florida, USA.

Based on light and scanning electron microscopical studies, three new gonad-infecting species of Philometra Costa, 1845 (Nematoda: Philometridae) are described from marine fishes of the genus Lutjanus Bloch (Perciformes: Lutjanidae) in the northern Gulf of Mexico: P. longispicula sp. n. from the ovary of the northern red snapper L. campechanus (Poey) (type host) and silk snapper L. vivanus (Cuvier); P. latispicula sp. n. from the ovary and rarely testes of the grey snapper L. griseus (Linnaeus); and P. synagridis sp. n. (only males available) from the ovary of the lane snapper Lutjanus synagris (Linnaeus). These species are mainly characterised by the lengths of spicules (378-690 microm, 135-144 microm and 186-219 microm, respectively) and spicule shapes, structure of the distal portion of the gubernaculum and the structure of the male caudal end. These are the first valid, nominal species of gonad-infecting philometrids reported from fishes of the family Lutjanidae in the western Atlantic region.

Philometra floridensis (Nematoda: Philometridae) damages ovarian tissue without reducing host (Sciaenops ocellatus) fecundity.

The parasitic nematode Philometra floridensis infects the ovary of its only host, the economically important fish species Sciaenops ocellatus, but the factors influencing host susceptibility and potential pathogenic effects are unknown. Here we report new information on these topics from evaluations of infected and uninfected hosts collected from the northeastern Gulf of Mexico. Fish length and age were evaluated vis-à-vis nematode prevalence to check for ontogenetic differences in host susceptibility. To evaluate health and reproductive consequences of infection, we looked for effects in Fulton's condition factor (K) and batch fecundity estimates (BF), and we evaluated ovarian tissue histologically to check for oocyte atresia and other host responses. We observed localized pathological changes in fish ovarian tissue associated with female nematodes, including leucocytic exudates, granulomatous inflammation, and Langhans-type multinucleated giant cells; the hosts, however, appeared to maintain high fecundity and actually exhibited, on average, better health index scores and higher relative fecundity than did uninfected fish. These differences are likely explained by the parasite's tendency to disproportionately infect the largest, actively spawning fish and by the localization of pathogenic changes, which could have masked effects that otherwise would have been reflected in mass-based health indicators. Although we did not detect negative effects on measures of overall health or reproductive output, further research is needed to better elucidate the relationship between these parasites and other factors affecting host reproductive potential, such as egg quality.

A new species of Euryhaliotrematoides Plaisance & Kritsky, 2004 (Monogenea: Dactylogyridae) from the gills of the spotted rose snapper Lutjanus guttatus (Steindachner) (Perciformes: Lutjanidae).

Euryhaliotrematoides mehen n. sp. (Monogenea: Dactylogyridae) is described based on specimens collected from the gills of the spotted rose snapper Lutjanus guttatus (Steindachner) off Mazatlan, Sinaloa, on the northwestern coast of Mexico. The synapomorphy of this genus is the presence of a funnel-shaped base of the male copulatory organ. This new species differs from all other species of the genus by possessing a male copulatory organ with a base with a thickened margin and a membranous accessory piece resembling a scarf and covering about 60% of its distal region. PCR products of the 28S rRNA (831 bp) and 18S rRNA (662 bp) genes were sequenced and submitted to GenBank (accession numbers HQ615997 and JF938069, respectively). BLASTn searches revealed no 100% identical hits with the previously registered monogenean sequences.

The microecology of dactylogyrids (Monogenea: Dactylogyridae) on the gills of wild spotted rose snapper Lutjanus guttatus (Lutjanidae) from Mazatlan Bay, Mexico.

The spatial distribution and coexistence of monogenean dactylogyrids was assessed on the gills of 63 specimens of wild spotted rose snapper, Lutjanus guttatus (Steindachner), caught in the Mazatlan Bay, Sinaloa, Mexico. Five species are reported: Euryhaliotrema perezponcei Garcia-Vargas, Fajer-Avila et Lamothe-Argumedo, 2008, Euryhaliotrematoides sp., Haliotrematoides spinatus Kritsky et Mendoza-Franco, 2009, H. plectridium Kritsky et Mendoza-Franco, 2009, and H. guttati Garcia-Vargas, Fajer-Avila et Lamothe-Argumedo, 2008. All except E. perezponcei and H. guttati represent new geographical records for the Pacific coast. The most prevalent dactylogyrid species was E. perezponcei (100%), H. plectridium and H. spinatus had > 80% prevalence, and H. guttati and Euryhaliotrematoides sp. had the lowest prevalence. The mean abundance of H. plectridium and E. perezponcei was close to 60 parasites/fish, whereas Euryhaliotrematoides sp. and H. guttati had the lowest abundance. The dactylogyrid species exhibited a tendency for attachment to gill arch 2: 25% attachment occurring on gill arch 1, 30% on 2, 27% on 3 and 18% on 4, and showed a significant preference for the central sector of the gill (42%). Haliotrematoides plectridium had a preference for attachment to gill arches 2 and 3 and the central sector. Haliotrematoides spinatus tended to settle on the gill arches 2 and 3 and had a preference for the central sector. Euryhaliotrema perezponcei tended to settle on the gill arches 1 and 2 and the anterior gill sector. Euryhaliotrematoides sp. and H. guttati did not show a preference for any gill arch or sector. The intraspecific aggregation was stronger than the interspecific aggregation, indicating that all the dactylogyrid species on spotted rose snapper were aggregated, and there was no evidence of competition among the species.

Rhabdosynochus Spp. (Monogenoidea: Diplectanidae) infecting the gill lamellae of snooks, Centropomus spp. (Perciformes: Centropomidae), in Florida, and redescription of the type species, R. rhabdosynochus.

Examination of the gill lamellae of 3 species of Centropomus spp. (Centropomidae) from 9 localities around the southern coast of Florida revealed 3 species of Rhabdosynochus (Monogenoidea, Diplectanidae). The common snook, Centropomus undecimalis, was infected with Rhabdosynochus rhabdosynochus (30 of 37 infected; prevalence 81%), Rhabdosynochus hargisi (9 of 37; 24%), and Rhabdosynochus hudsoni (20 of 37; 46%); the fat snook, Centropomus parallelus, with R. rhabdosynochus (12 of 22; 55%) and R. hudsoni (8 of 22; 36%) (new host records for both); and the tarpon snook, Centropomus pectinatus, with R. hudsoni (3 of 4; 75%) (new host record). Snooks infected with R. rhabdosynochus were collected only from tidal waters of salinity ≤10.4 ppt, where the parasite dominated the monogenoidean community. Rhabdosynochus hargisi and R. hudsoni showed greater salinity tolerance; the former dominated where salinity was ≥28.8 ppt. Thus, salinity appears to be a determinate factor in partitioning the monogenoidean community, while water depth and temperature and host population (as defined by collection locality) did not appear to affect occurrence of Rhabdosynochus spp. on snooks in Florida. With the exception of Tampa Bay for R. hargisi , each locality represented a new geographic record for the respective diplectanid. The type species, R. rhabdosynochus, is redescribed based on specimens collected from its type host, C. undecimalis.