An inside out journey: biogenesis, ultrastructure and proteomic characterisation of the ectoparasitic flatworm Sparicotyle chrysophrii extracellular vesicles.

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.

The invasive land flatworm Arthurdendyus triangulatus has repeated sequences in the mitogenome, extra-long cox2 gene and paralogous nuclear rRNA clusters.

Using a combination of short- and long-reads sequencing, we were able to sequence the complete mitochondrial genome of the invasive 'New Zealand flatworm' Arthurdendyus triangulatus (Geoplanidae, Rhynchodeminae, Caenoplanini) and its two complete paralogous nuclear rRNA gene clusters. The mitogenome has a total length of 20,309 bp and contains repetitions that includes two types of tandem-repeats that could not be solved by short-reads sequencing. We also sequenced for the first time the mitogenomes of four species of Caenoplana (Caenoplanini). A maximum likelihood phylogeny associated A. triangulatus with the other Caenoplanini but Parakontikia ventrolineata and Australopacifica atrata were rejected from the Caenoplanini and associated instead with the Rhynchodemini, with Platydemus manokwari. It was found that the mitogenomes of all species of the subfamily Rhynchodeminae share several unusual structural features, including a very long cox2 gene. This is the first time that the complete paralogous rRNA clusters, which differ in length, sequence and seemingly number of copies, were obtained for a Geoplanidae.

Insecticidal Effects of Transgenic Maize Bt-Cry1Ab, Bt-Vip3Aa, and Bt-Cry1Ab+Vip3Aa against the Oriental Armyworm, Mythimna separata (Walker) in Southwest China.

The oriental armyworm, Mythimna separata (Walker), an important migratory pest of maize and wheat, is posing a severe threat to maize production in Asian countries. As source areas of spring-summer emigratory populations, the control of M. separata in southwestern China is of great significance for East Asian maize production. To assess the toxicity of Bt maize against the pest, bioassays of Bt-(Cry1Ab+Vip3Aa) maize (event DBN3601T), Bt-Cry1Ab maize (event DBN9936), and Bt-Vip3Aa maize (event DBN9501) were conducted in Yunnan province of southwest China. There were significant differences in insecticidal activity between the three Bt maize events, and DBN3601T presented the highest insecticidal role. The results also indicated that the insecticidal effect of various Bt maize tissues took an order in leaf > kernel > silk, which is highly consistent with the expression amounts of Bt insecticidal protein in leaf (69.69 ± 1.18 µg/g), kernel (11.69 ± 0.75 µg/g), and silk (7.32 ± 0.31 µg/g). In field trials, all larval population densities, plant damage rates, and leaf damage levels of DBN3601T maize were significantly lower than the conventional maize. This research indicated that the DBN3601T event had a high control efficiency against M. separata and could be deployed in southwest China for the management of M. separata.

Genome-wide identification of histone lysine methyltransferases and their implications in the epigenetic regulation of eggshell formation-related genes in a trematode parasite Clonorchis sinensis.

Epigenetic writers including DNA and histone lysine methyltransferases (DNMT and HKMT, respectively) play an initiative role in the differentiation and development of eukaryotic organisms through the spatiotemporal regulation of functional gene expressions. However, the epigenetic mechanisms have long been suspected in helminth parasites lacking the major DNA methyltransferases DNMT1 and DNMT3a/3b. Very little information on the evolutionary status of the epigenetic tools and their role in regulating chromosomal genes is currently available in the parasitic trematodes. We previously suggested the probable role of a DNMT2-like protein (CsDNMT2) as a genuine epigenetic writer in a trematode parasite Clonorchis sinensis. Here, we analyzed the phylogeny of HKMT subfamily members in the liver fluke and other platyhelminth species. The platyhelminth genomes examined conserved genes for the most of SET domain-containing HKMT and Disruptor of Telomeric Silencing 1 subfamilies, while some genes were expanded specifically in certain platyhelminth genomes. Related to the high gene dosages for HKMT activities covering differential but somewhat overlapping substrate specificities, variously methylated histones were recognized throughout the tissues/organs of C. sinensis adults. The temporal expressions of genes involved in eggshell formation were gradually decreased to their lowest levels proportionally to aging, whereas those of some epigenetic tool genes were re-boosted in the later adult stages of the parasite. Furthermore, these expression levels were significantly affected by treatment with DNMT and HKMT inhibitors. Our data strongly suggest that methylated histones are potent epigenetic markers that modulate the spatiotemporal expressions of C. sinensis genes, especially those involved in sexual reproduction.

Biocontrol efficiency and characterization of insecticidal protein from sugarcane endophytic Serratia marcescens (SM) against oriental armyworm Mythimna separata (Walker).

The plant endophytic bacteria are a great source of nature insecticides. However, no such endophytic bacteria have been found in sugarcane, to address this gap, we isolated and identified a strain of Serratia marcescens with moderate insecticidal activity from sugarcane. Taken armyworm Mythimna separata as example, the mortality rates of oral infection and injection infection were 47.06 % and 91 %, respectively. The SM has significant negative affect on the growth, development, and reproduction of M. separata. After determining that these insecticidal substances, 33 potential virulence proteins were screened through the identification and prediction of bacterial proteins. Later we confirmed serralysin was a vital toxic protein from SM that caused M. separata death by prokaryotic expression. In addition, we also found that the intestinal tissue cells infected with SM or serralysin were severely diseased, which may be a major factor in M. separata demise. Finally, through gene expression level, protein molecular docking, we found the aminopeptidase-N would be one of the potential receptors of serralysin. Taken together, our findings indicate that sugarcane endophyte S. marcescens may be beneficial for pest control in sugarcane and explain its insecticidal mechanism. This study provides new ideas and materials for the biological control of pests.

Structure of putative epidermal sensory receptors in an acoel flatworm, Praesagittifera naikaiensis.

Acoel flatworms possess epidermal sensory-receptor cells on their body surfaces and exhibit behavioral repertoires such as geotaxis and phototaxis. Acoel epidermal sensory receptors should be mechanical and/or chemical receptors; however, the mechanisms of their sensory reception have not been elucidated. We examined the three-dimensional relationship between epidermal sensory receptors and their innervation in an acoel flatworm, Praesagittifera naikaiensis. The distribution of the sensory receptors was different between the ventral and dorsal sides of worms. The nervous system was mainly composed of a peripheral nerve net, an anterior brain, and three pairs of longitudinal nerve cords. The nerve net was located closer to the body surface than the brain and the nerve cords. The sensory receptors have neural connections with the nerve net in the entire body of worms. We identified five homologs of polycystic kidney disease (PKD): PKD1-1, PKD1-2, PKD1-3, PKD1-4, and, PKD2, from the P. naikaiensis genome. All of these PKD genes were implied to be expressed in the epidermal sensory receptors of P. naikaiensis. PKD1-1 and PKD2 were dispersed across the entire body of worms. PKD1-2, PKD1-3, and PKD1-4 were expressed in the anterior region of worms. PKD1-4 was also expressed around the mouth opening. Our results indicated that P. naikaiensis possessed several types of epidermal sensory receptors to convert various environmental stimuli into electrical signals via the PKD channels and transmit the signals to afferent nerve and/or effector cells.

Flatworm cocoons in the abyss: same plan under pressure.

While knowledge of early ontogeny in abyssal animals is highly limited in general, it was completely lacking for abyssal, free-living platyhelminths. We discovered flatworm egg capsules (or 'cocoons') on rocks collected at depths of 6176-6200 m on the abyssal slope of the Kuril-Kamchatka Trench, northwestern Pacific. The egg capsules were black and spherical, around 3 mm in diameter, and contained three to seven individuals (n = 4) at the same developmental stage, either the spherical (putative early embryo) or vermiform (putative late embryo) stages. A molecular phylogenetic analysis based on 18S and 28S rRNA sequences revealed that the flatworms belong in suborder Maricola in Tricladida and suggested that they may have colonized from shallow to deep waters. This study provides the deepest record for free-living flatworms and the first information on their early life stages in the abyssal zone, which were very similar to those in shallow-water forms. This similarity in development between the relatively benign shallow-water and the extreme abyssal environments suggests that triclads adapting to the latter faced primarily physiological and/or ecological adaptive challenges rather than developmental ones.

Geography, phylogeny and host switch drive the coevolution of parasitic Gyrodactylus flatworms and their hosts.

BACKGROUND: Gyrodactylus is a lineage of monogenean flatworm ectoparasites exhibiting many features that make them a suitable model to study the host-parasite coevolutionary dynamics. Previous coevolutionary studies of this lineage mainly relied on low-power datasets (a small number of samples and a single molecular marker) and (now) outdated algorithms. METHODS: To investigate the coevolutionary relationship of gyrodactylids and their fish hosts in high resolution, we used complete mitogenomes (including two newly sequenced Gyrodactylus species), a large number of species in the single-gene dataset, and four different coevolutionary algorithms. RESULTS: The overall coevolutionary fit between the parasites and hosts was consistently significant. Multiple indicators confirmed that gyrodactylids are generally highly host-specific parasites, but several species could parasitize either multiple (more than 5) or phylogenetically distant fish hosts. The molecular dating results indicated that gyrodactylids tend to evolve towards high host specificity. Speciation by host switch was identified as a more important speciation mode than co-speciation. Assuming that the ancestral host belonged to Cypriniformes, we inferred four major host switch events to non-Cypriniformes hosts (mostly Salmoniformes), all of which occurred deep in the evolutionary history. Despite their relative rarity, these events had strong macroevolutionary consequences for gyrodactylid diversity. For example, in our dataset, 57.28% of all studied gyrodactylids parasitized only non-Cypriniformes hosts, which implies that the evolutionary history of more than half of all included lineages could be traced back to these major host switch events. The geographical co-occurrence of fishes and gyrodactylids determined the host use by these gyrodactylids, and geography accounted for most of the phylogenetic signal in host use. CONCLUSIONS: Our findings suggest that the coevolution of Gyrodactylus flatworms and their hosts is largely driven by geography, phylogeny, and host switches.

The ribosomal transcription units of five echinostomes and their taxonomic implications for the suborder Echinostomata (Trematoda: Platyhelminthes).

Five newly obtained nuclear ribosomal transcription unit (rTU) sequences from Echinostomatidae and Echinochasmidae are presented. The inter- and intrafamilial relationships of these and other families in the suborder Echinostomata are also analyzed. The sequences obtained are the complete rTU of Artyfechinostomum malayanum (9,499 bp), the near-complete rTU of Hypoderaeum conoideum (8,076 bp), and the coding regions (from 5'-terminus of 18S to 3'-terminus of 28S rRNA gene) in Echinostoma revolutum (6,856 bp), Echinostoma miyagawai (6,854 bp), and Echinochasmus japonicus (7,150 bp). Except for the longer first internal transcribed spacer (ITS1) in Echinochasmus japonicus, all genes and spacers were almost identical in length. Comprehensive maximum-likelihood phylogenies were constructed using the PhyML software package. The datasets were either the concatenated 28S + 18S rDNA sequences (5.7-5.8 kb) from 60 complete rTUs of 19 families or complete 28S sequences only (about 3.8-3.9 kb) from 70 strains or species of 22 families. The phylogenetic trees confirmed Echinostomatoidea as monophyletic. Furthermore, a detailed phylogeny constructed from alignments of 169 28S D1-D3 rDNA sequences (1.1-1.3 kb) from 98 species of 50 genera of 10 families, including 154 echinostomatoid sequences (85 species/42 genera), clearly indicated known generic relationships within Echinostomatidae and Echinochasmidae and relationships of families within Echinostomata and several other suborders. Within Echinostomatidae, Echinostoma, Artyfechinostomum, and Hypoderaeum appeared as monophyletic, while Echinochasmus (Echinochasmidae) was polyphyletic. The Echinochasmidae are a sister group to the Psilostomidae. The datasets provided here will be useful for taxonomic reappraisal as well as studies of evolutionary and population genetics in the superfamily Echinostomatoidea, the sole superfamily in the suborder Echinostomata.

Contrasting the form and strength of pre- and postcopulatory sexual selection in a flatworm.

Sexual traits may be selected during multiple consecutive episodes of selection, occurring before, during, or after copulation. The overall strength and form of selection acting on traits may thus be determined by how selection (co-)varies along different episodes. However, it is challenging to measure pre- and postcopulatory phenotypic traits alongside variation in fitness components at each different episode. Here, we used a transgenic line of the transparent flatworm Macrostomum lignano expressing green fluorescent protein (GFP) in all cell types, including sperm cells, enabling in vivo sperm tracking. We assessed the mating success, sperm-transfer efficiency, and sperm fertilizing efficiency of GFP(+) focal worms in which we measured 13 morphological traits. We found linear selection on sperm production rate arising from pre- and postcopulatory components and on copulatory organ shape arising from sperm fertilizing efficiency. We further found nonlinear (mostly concave) selection on combinations of copulatory organ and sperm morphology traits arising mostly from sperm-transfer efficiency and sperm fertilizing efficiency. Our study provides a fine-scale quantification of sexual selection, showing that both the form and strength of selection can change across fitness components. Quantifying how sexual selection builds up along episodes of selection allows us to better understand the evolution of sexually selected traits.

piRNA generation is associated with the pioneer round of translation in stem cells.

Much insight has been gained on how stem cells maintain genomic integrity, but less attention has been paid to how they maintain their transcriptome. Here, we report that the PIWI protein SMEDWI-1 plays a role in the filtering of dysfunctional transcripts from the transcriptome of planarian stem cells. SMEDWI-1 accomplishes this through association with the ribosomes during the pioneer round of translation, and processing of poorly translated transcripts into piRNAs. This results in the removal of such transcripts from the cytoplasmic pool and at the same time creates a dynamic pool of small RNAs for post-transcriptional surveillance through the piRNA pathway. Loss of SMEDWI-1 results in elevated levels of several non-coding transcripts, including rRNAs, snRNAs and pseudogene mRNAs, while reducing levels of several coding transcripts. In the absence of SMEDWI-1, stem cell colonies are delayed in their expansion and a higher fraction of descendants exit the stem cell state, indicating that this transcriptomic sanitation mediated by SMEDWI-1 is essential to maintain stem cell health. This study presents a new model for the function of PIWI proteins in stem cell maintenance, that complements their role in transposon repression, and proposes a new biogenesis pathway for piRNAs in stem cells.

Dactylogyrids (Platyhelminthes, Monogenea) from the gill lamellae of doradids (Siluriformes) with description of five new species of Cosmetocleithrum and new geographical distribution for known species from the Neotropical Region, Brazil.

Five new species of Cosmetocleithrum were described parasitizing the gill filaments of neotropical doradid fishes. Cosmetocleithrum undulatum n. sp., Cosmetocleithrum brachylecis n. sp. and Cosmetocleithrum ludovicense n. sp. are described from Platydoras brachylecis from a market-place of São Luís, State of Maranhão, Brazil. Cosmetocleithrum sacciforme n. sp. and Cosmetocleithrum basicomplexum n. sp. are described from Oxydoras niger from Juruá River, State of Acre, Brazil. Cosmetocleithrum undulatum and Cosmetocleithrum brachylecis resemble Cosmetocleithrum falsunilatum Feronato, Razzolini, Morey & Boeger, 2022 mainly by the unique male copulatory organ (MCO) morphology but differ from these and all congeneric species mainly by the morphology of the MCO, accessory piece and hooks pairs. Cosmetocleithrum ludovicense is closer to Cosmetocleithrum confusus Kritsky, Thatcher & Boeger, 1986 and to Cosmetocleithrum akuanduba Soares, Santos Neto & Domingues, 2018 but differs from those mainly by the morphology of the accessory piece. Cosmetocleithrum sacciforme differs from all congeneric species mainly by the morphology of the accessory piece formed by a single plate of saccular appearance. Cosmetocleithrum basicomplexum also shares morphological characters with Cosmetocleithrum gigas Morey, Cachique & Babilonia, 2019 considering the size of the body and shape of the anchors, but differs mainly in the morphology of the bars and hooks. Besides the new species, new data are presented for Cosmetocleithrum leandroi Soares, Neto & Domingues, 2018, C. akuanduba and C. confusus regarding morphological characteristics and biogeography.

Title: Dactylogyridae (Platyhelminthes, Monogenea) des lamelles branchiales de Doradidae (Siluriformes) avec description de cinq nouvelles espèces de Cosmetocleithrum et nouvelle répartition géographique d'espèces connues de la région néotropicale au Brésil. Abstract: Cinq nouvelles espèces de Cosmetocleithrum sont décrites, parasitant les filaments branchiaux de poissons Doradidae néotropicaux. Cosmetocleithrum undulatum n. sp., Cosmetocleithrum brachylecis n. sp. et Cosmetocleithrum ludovicense n. sp. sont décrits de Platydoras brachylecis provenant d'un marché de São Luís, État du Maranhão, Brésil. Cosmetocleithrum sacciforme n. sp. et Cosmetocleithrum basicomplexum n. sp. sont décrits d'Oxydoras niger de la rivière Juruá, État d'Acre, Brésil. Cosmetocleithrum undulatum et Cosmetocleithrum brachylecis ressemblent à Cosmetocleithrum falsunilatum Feronato, Razzolini, Morey & Boeger, 2022 principalement par la morphologie unique de l'organe copulateur mâle (OCM), mais diffèrent de ces espèces et de toutes les espèces congénères principalement par la morphologie de l'OCM, de la pièce accessoire et des paires de crochets. Cosmetocleithrum ludovicense est proche de Cosmetocleithrum confusus Kritsky, Thatcher & Boeger, 1986 et de Cosmetocleithrum akuanduba Soares, Santos Neto & Domingues, 2018 mais en diffère principalement par la morphologie de la pièce accessoire. Cosmetocleithrum sacciforme se distingue de toutes les espèces congénères principalement par la morphologie de la pièce accessoire formée d'une seule plaque d'aspect sacculaire. Cosmetocleithrum basicomplexum partage également des caractères morphologiques avec Cosmetocleithrum gigas Morey, Cachique & Babilonia, 2019 compte tenu de la taille du corps et de la forme des anchors, mais en diffère principalement par la morphologie des barres et des crochets. Outre les nouvelles espèces, de nouvelles données sont présentées pour Cosmetocleithrum leandroi Soares, Neto & Domingues, 2018, C. akuanduba et C. confusus concernant les caractéristiques morphologiques et la biogéographie.

Species of Dactylogyridae (Platyhelminthes: Monogenoidea) infecting marine fishes of Moreton Bay, Queensland, Australia, with proposals of Pleuronectitrema n. gen. and Ecnomotrema n. gen. and descriptions of 13 new species.

Twenty-nine dactylogyrid species were reported from teleosts collected during a survey of the parasites of fishes of Moreton Bay, Queensland, Australia, in 2016. Two new genera, Ecnomotrema n. gen. and Pleuronectitrema n. gen., were proposed, and 13 new species were illustrated and described: Atherinicus difficilis n. sp., Diversohamulus similis n. sp., and Ecnomotrema cetiosum n. sp. from Atherinomorus vaigiensis; Haliotrema apiculum n. sp. from Pempheris schwenkii; Haliotrema tugulduriforme n. sp. from Scarus ghobban; Lethrinitrema australiense n. sp., and Lethrinitrema lituus n. sp. from Lethrinus nebulosus; Tetrancistrum siganioides n. sp. from Siganus fuscescens; Ligophorus bostrychus n. sp. from Planiliza subviridis; Neohaliotrema gemmula n. sp. from Abudefduf vaigiensis; Neohaliotrema moretonense n. sp. from Ab. vaigiensis and Ab. bengalensis; and Pleuronectitrema spirula n. sp. from Pseudorhombus arsius (all from Moreton Bay); Pleuronectitrema kuwaitense Kritsky & Sey n. sp. was described from specimens collected from P. arsius in Kuwait during 1996. Ten new host records were recorded: Chauhanellus duriensis Lim, 1994 and Hamatopeduncularia thalassini Bychowsky & Nagibina, 1969 from Pararius proximus; Diplectanotrema sp. 1 and sp. 2 from Sillago maculata and Goniistius vestitus, respectively; Diversohamulus tricuspidatus Bychowsky & Nagibina, 1969 from At. vaigiensis; Hal. cf. dempsteri (Mizelle & Price, 1964) Young, 1968 from Prionurus microlepidotus; Hal. spirale Yamaguti, 1968 from Upeneus tragula; Ligophorus kaohsianghsieni (Gussev, 1962) Gussev, 1985 from Planiliza subviridis; and Neohaliotrema malayense Lim & Gibson, 2010 from Ab. bengalensis and Ab. whitleyi. Twenty-five new faunal records for Moreton Bay were recorded, including the new species listed above and C. duriensis, Diplectanotrema sp. 1 and sp. 2, Diversohamulus tricuspidatus Bychowsky & Nagibina, 1969, Glyphidohaptor phractophallus Kritsky, Galli, & Yang, 2007, Hal. cf. dempsteri, Hal. johnstoni Bychowsky & Nagibina, 1970, Hal. spirale, Yamaguti, 1968 Hamatopeduncularia thalassini Bychowsky & Nagibina, 1969, Lethrinitrema nebulosum Sun, Li, & Yang, 2014, Ligophorus kaohsianghsieni (Gussev, 1962) Gussev, 1985, Ligophorus parvicopulatrix Soo & Lim, 2012, and Neohaliotrema malayense Lim & Gibson, 2010. Three new combinations were proposed: Hal. spariense Roubal, 1981 as Euryhaliotrema spariense (Roubal, 1981) n. comb.; and Hal. arsiosa Venkatanarasaiah, 1984 and Hal. youngi Venkatanarasaiah, 1984 as Pleuronectitrema arsiosa (Venkatanarasaiah, 1984) n. comb. and Pleuronectitrema youngi (Venkatanarasaiah, 1984) n. comb., respectively; Haliotrema sp. of Zhang is transferred to Pleuronectitrema as Pleuronectitrema sp. Hal. ctenochaeti Yamaguti, 1968 was replaced with Hal. asymphylum n. nom. to remove it from homonymy with Hal. ctenochaeti Young, 1968. Pseudohaliotrematoides zancli Yamaguti, 1968 was transferred to Haliotrema as Haliotrema hawaiiense n. nom. Hal. zancli Yamaguti, 1968 was considered a junior subjective synonym of Hal. dempsteri.

Title: Espèces de Dactylogyridae (Platyhelminthes, Monogenoidea) infectant les poissons marins de Moreton Bay, Queensland, Australie, avec la proposition de Pleuronectitrema n. gen. et Ecnomotrema n. gen. et la description de 13 nouvelles espèces. Abstract: Vingt-neuf espèces de Dactylogyridae sont signalées chez des téléostéens collectés lors d'une étude des parasites des poissons de Moreton Bay, Queensland, Australie, en 2016. Deux nouveaux genres, Ecnomotrema n. gen. et Pleuronectitrema n. gen., sont proposés, et 13 nouvelles espèces sont illustrées et décrites : Atherinicus difficilis n. sp., Diversohamulus similis n. sp., et Ecnomotrema cetiosum n. sp. d'Atherinomorus vaigiensis ; Haliotrema apiculum n. sp. de Pempheris schwenkii ; Haliotrema tugulduriforme n. sp. de Scarus ghobban ; Lethrinitrema australiense n. sp. et Lethrinitrema lituus n. sp. de Lethrinus nebulosus, Tetrancistrum siganioides n. sp. de Siganus fuscescens ; Ligophorus bostrychus n. sp. de Planiliza subviridis ; Neohaliotrema gemmula n. sp. d'Abudefduf vaigiensis ; Neohaliotrema moretonense n. sp. d'Ab. vaigiensis et Ab. bengalensis ; et Pleuronectitrema spirula n. sp. de Pseudorhombus arsius (tous de Moreton Bay). Pleuronectitrema hawaiiense Kritsky & Sey n. sp. est décrit à partir de spécimens collectés de P. arsius au Koweït en 1996. Dix nouvelles combinaisons parasites-hôtes sont répertoriées : Chauhanellus duriensis Lim, 1994 et Hamatopeduncularia thalassini Bychowsky & Nagibina, 1969 de Pararius proximus ; Diplectanotrema sp. 1 et sp. 2, respectivement de Sillago maculata et Goniistius vestitus ; Diversohamulus tricuspidatus Bychowsky & Nagibina, 1969 d'At. vaigiensis ; Hal. cf. dempsteri (Mizelle & Price, 1964) Young, 1968 de Prionurus microlepidotus ; Hal. spirale Yamaguti, 1968 d'Upeneus tragula ; Ligophorus kaohsianghsieni (Gussev, 1962) Gussev, 1985 de Planiliza subviridis ; et Neohaliotrema malayense Lim & Gibson, 2010 d'Ab. bengalensis et Ab. whitleyi. Vingt-cinq nouveaux signalements fauniques pour la baie Moreton sont rapportés, y compris les nouvelles espèces énumérées ci-dessus et C. duriensis, Diplectanotrema sp. 1 et sp. 2, Diversohamulus tricuspidatus Bychowsky & Nagibina, 1969, Glyphidohaptor phractophallus Kritsky, Galli et Yang, 2007, Hal. cf. dempsteri, Hal. johnstoni Bychowsky et Nagibina, 1970, Hal. spirale, Hamatopeduncularia thalassini Bychowsky & Nagibina, 1969, Lethrinitrema nebulosum Sun, Li, & Yang, 2014, Ligophorus kaohsianghsieni (Gussev, 1962) Gussev, 1985, Ligophorus parvicopulatrix Soo & Lim, 2012, et Neohaliotrema malayense Lim et Gibson, 2010. Trois nouvelles combinaisons sont proposées : Hal. spariense Roubal, 1981 comme Euryhaliotrema spariense (Roubal, 1981) n. comb. ; Hal. arsiosa Venkatanarasaiah, 1984 et Hal. youngi Venkatanarasaiah, 1984, respectivement comme Pleuronectitrema arsiosa (Venkatanarasaiah, 1984) n. comb. et Pleuronectitrema youngi (Venkatanarasaiah, 1984) n. comb. ; Haliotrema sp. de Zhang est transféré à Pleuronectitrema sous le nom de Pleuronectitrema sp. Hal. ctenochaeti Yamaguti, 1968 est remplacé par Hal. asymphylum n. nom. pour supprimer l'homonymie avec Hal. ctenochaeti Young, 1968. Pseudohaliotrematoides zancli Yamaguti, 1968 est transféré à Haliotrema sous le nom de Haliotrema hawaiiense n. nom. Hal. zancli Yamaguti, 1968 est considéré comme un synonyme subjectif junior de Hal. dempsteri.

Microcotylidae (Monogena) parasites of snappers (Perciformes: Lutjanidae) collected from the northeast coast of Brazil.

Monogeneans are a diverse group of flatworms, being ectoparasites of marine and freshwater fish, with great morphological and ecological variety. Analyzes of monogenetics in fish with great habitat diversity such as snappers are scarce in the literature, which already emphasizes the need for an update in this regard. The presente study found, morphologically characterized and mapped the geographic distribution and in known hosts the species Microcotyloides incisa and Microcotyloides impudicus, describing the first occurrence of these monogeneans for South America and in new hosts. The survey of hosts in the literature shows that M. incisa seems to have a greater Affinity with congener species of Lutjanidae and fish from other families, which may indicate a preference or specificity for fish of this family because they are congeners of their host type. The introduction of these parasites into South American waters may have occurred due to the migratory behavior of snappers, which are well distributed along the Mexican coast, where the parasites are usually reported. Here, we also bring the first occurrence of M. incisa for Lutjanus analis and Lutjanus jocu and M. impudicus for Ocyurus chrysurus and Lutjanus synagris, helping in the mapping and distribution of these monogenetic species in the Americas.

First molecular identification of the trematode Pulmonicola cochleotrema (Platyhelminthes: Opisthotrematidae) in West Indian manatees (Trichechus manatus, Sirenia: Trichechidae) from Puerto Rico and Florida.

West Indian manatees (Trichechus manatus) harbor a variety of endoparasites, including the nasal trematode Pulmonicola cochleotrema, which infects the respiratory tract, especially the nasal passages. Previous studies have described and identified this digenean using morphological data only. This study presents the first molecular identification of P. cochleotrema in West Indian manatees from Puerto Rico and Florida. Samples of the trematode were collected from seven manatees found stranded dead at both locations. The small subunit ribosomal DNA (18S rDNA) was amplified from each sample using universal primers for different regions of the gene, resulting in a consensus sequence of 1871 base pairs. The phylogenetic reconstruction was carried out using DNA sequences of other species of digenean parasites from other hosts, including a trematode of the same taxonomic family from another sirenian species. Specimens collected from both locations show the same molecular identity using SSU rDNA sequence data. The identity of P. cochleotrema was confirmed using the Basic Local Alignment Search Tool from the National Center for Biotechnology Information database, yielding a high similarity of 98.8 % with Opisthotrema dujonis and 98.2 % with Lankatrema mannarense located in the same clade in our analysis. The latter two digeneans belong to the Opisthotrematidae as does P. cochleotrema and previous studies reported them infecting the Eustachian tubes, esophagus, and digestive tract in dugongs (Dugong dugon). These findings evidence that the nasal trematode of manatees in Florida and Puerto Rico and the dugong, all inhabiting and feeding in marine environments, will have a marine mollusk as an intermediate host, probably a gastropod. The question remains, which species of nasal trematode are harbored by lotic-dwelling manatees in other parts of their distribution like South America.

Convergent evolution of the sensory pits in and within flatworms.

BACKGROUND: Unlike most free-living platyhelminths, catenulids, the sister group to all remaining flatworms, do not have eyes. Instead, the most prominent sensory structures in their heads are statocysts or sensory pits. The latter, found in the family Stenostomidae, are concave depressions located laterally on the head that represent one of the taxonomically important traits of the family. In the past, the sensory pits of flatworms have been homologized with the cephalic organs of nemerteans, a clade that occupies a sister position to platyhelminths in some recent phylogenies. To test for this homology, we studied morphology and gene expression in the sensory pits of the catenulid Stenostomum brevipharyngium. RESULTS: We used confocal and electron microscopy to investigate the detailed morphology of the sensory pits, as well as their formation during regeneration and asexual reproduction. The most prevalent cell type within the organ is epidermally-derived neuron-like cells that have cell bodies embedded deeply in the brain lobes and long neurite-like processes extending to the bottom of the pit. Those elongated processes are adorned with extensive microvillar projections that fill up the cavity of the pit, but cilia are not associated with the sensory pit. We also studied the expression patterns of some of the transcription factors expressed in the nemertean cephalic organs during the development of the pits. Only a single gene, pax4/6, is expressed in both the cerebral organs of nemerteans and sensory pits of S. brevipharyngium, challenging the idea of their deep homology. CONCLUSIONS: Since there is no morphological or molecular correspondence between the sensory pits of Stenostomum and the cerebral organs of nemerteans, we reject their homology. Interestingly, the major cell type contributing to the sensory pits of stenostomids shows ultrastructural similarities to the rhabdomeric photoreceptors of other flatworms and expresses ortholog of the gene pax4/6, the pan-bilaterian master regulator of eye development. We suggest that the sensory pits of stenostomids might have evolved from the ancestral rhabdomeric photoreceptors that lost their photosensitivity and evolved secondary function. The mapping of head sensory structures on plathelminth phylogeny indicates that sensory pit-like organs evolved many times independently in flatworms.

Unraveling the Unusual Subgenomic Organization in the Neopolyploid Free-Living Flatworm Macrostomum lignano.

Whole genome duplication (WGD) is an evolutionary event resulting in a redundancy of genetic material. Different mechanisms of WGD, allo- or autopolyploidization, lead to distinct evolutionary trajectories of newly formed polyploids. Genome studies on such species are important for understanding the early stages of genome evolution. However, assembling neopolyploid is a challenging task due to the presence of 2 homologous (or homeologous) chromosome sets and therefore the existence of the extended paralogous regions in its genome. Post-WGD evolution of polyploids includes cytogenetic diploidization leading to the formation of species, whose polyploid origin might be hidden by disomic inheritance. Earlier we uncovered the hidden polyploid origin of the free-living flatworms of the genus Macrostomum (Macrostomum lignano, M. janickei, and M. mirumnovem). Cytogenetic diploidization in these species is accompanied by intensive chromosomal rearrangements including chromosomes fusions. In this study, we unravel the M. lignano genome organization through generation and sequencing of 2 sublines of the commonly used inbred line of M. lignano (called DV1) differing only in a copy number of the largest chromosome (MLI1). Using nontrivial assembly free comparative analysis of their genomes, we deciphered DNA sequences belonging to MLI1 and validated them by sequencing the pool of microdissected MLI1. Here we presented the uncommon mechanism of genome rediplodization of M. lignano, which consists of (i) presence of 3 subgenomes, which emerged via formation of large fused chromosomes and its variants, and (ii) sustaining their heterozygosity through inter- and intrachromosomal rearrangements.

Patterns of host-parasite associations between marine meiofaunal flatworms (Platyhelminthes) and rhytidocystids (Apicomplexa).

Microturbellarians are abundant and ubiquitous members of marine meiofaunal communities around the world. Because of their small body size, these microscopic animals are rarely considered as hosts for parasitic organisms. Indeed, many protists, both free-living and parasitic ones, equal or surpass meiofaunal animals in size. Despite several anecdotal records of "gregarines", "sporozoans", and "apicomplexans" parasitizing microturbellarians in the literature-some of them dating back to the nineteenth century-these single-celled parasites have never been identified and characterized. More recently, the sequencing of eukaryotic microbiomes in microscopic invertebrates have revealed a hidden diversity of protist parasites infecting microturbellarians and other meiofaunal animals. Here we show that apicomplexans isolated from twelve taxonomically diverse rhabdocoel taxa and one species of proseriate collected in four geographically distinct areas around the Pacific Ocean (Okinawa, Hokkaido, and British Columbia) and the Caribbean Sea (Curaçao) all belong to the apicomplexan genus Rhytidocystis. Based on comprehensive molecular phylogenies of Rhabdocoela and Proseriata inferred from both 18S and 28S rDNA sequences, as well as a molecular phylogeny of Marosporida inferred from 18S rDNA sequences, we determine the phylogenetic positions of the microturbellarian hosts and their parasites. Multiple lines of evidence, including morphological and molecular data, show that at least nine new species of Rhytidocystis infect the microturbellarian hosts collected in this study, more than doubling the number of previously recognized species of Rhytidocystis, all of which infect polychaete hosts. A cophylogenetic analysis examining patterns of phylosymbiosis between hosts and parasites suggests a complex picture of overall incongruence between host and parasite phylogenies, and varying degrees of geographic signals and taxon specificity.

Morphological, pathogenic and genetic diversity in Diplodia seriata associated with black rot canker of apple in India.

Apple cankers are extremely destructive diseases threatening the global apple industry through direct and indirect losses. The population structure of the pathogens is of paramount significance for the development of efficient management strategies. Therefore, phenotypic, pathogenic, and genetic diversity of Diplodia seriata causing black rot canker of apple was investigated in this study. All the isolates were included for investigating the in vitro mycelial growth, conidial dimensions, and pathogenic variability on two-year-old potted apple seedlings. The ISSR approach was used to investigate the molecular diversity of D. seriata. Mycelial growth rates were found to vary significantly amongst the isolates; however, there were no major variations seen between the different geographical groupings of isolates. Pathogenicity tests revealed variations in the size of cankers among the isolates indicating the presence of virulence variability. The isolates were segregated into three virulence groups based on canker length. The Bayesian analyses of ISSR data divided the isolates into two genetic clusters. The genetic clustering of the isolates revealed no relationship with geographical origin of the isolates. Furthermore, no direct relationship of genetic clustering was observed with morphological or pathogenic variability. The ISSR primers revealed very high level of variability in D. seriata; however, no distinct populations of the pathogen existed which is an indication of high level of gene flow between the diverse geographical populations. According to our knowledge, this is the first thorough investigation on the diversity of D. seriata associated with apple black rot canker in India.

Nearctic Dactylogyrus species (Platyhelminthes, Monogenea) parasitizing cypriniform fishes in the context of morphology and phylogeny, with descriptions of seven new species.

Ribosomal DNA sequences are currently available for 32 morphologically recognized species of Dactylogyrus parasitizing Nearctic cypriniforms, but only 16 of them are assigned to nominal species. Herein, morphological data on 28 of the 32 species are provided, together with comments on their phylogenetic relationships in the context of the morphology of taxonomically important structures. Seven new species of Dactylogyrus are described from five species of leuciscids and one species of catostomids, as follows: D. aduncus n. sp. from Campostoma spadiceum, D. cloutmani n. sp. from Luxilus chrysocephalus isolepis, D. cornifrons n. sp. from Cyprinella venusta, D. fimbratus n. sp. from Rhinichthys cataractae, D. mcallisteri n. sp. from Semotilus atromaculatus, and D. chieni n. sp. and D. haneki n. sp. from Hypentelium nigricans. Four previously described species of Dactylogyrus, D. atromaculatus from S. atromaculatus, D. eos from C. neogaeus, D. parvicirrus from Notemigonus crysoleucas and D. perlus from L. c. isolepis, are redescribed and/or figured. As for the remaining 17 species, only the male copulatory organs (MCOs) taken from the respective hologenophores are illustrated. On the basis of phylogenetic analyses, two main clades of Nearctic Dactylogyrus were recognized and supported by the different morphology of the MCO. The first one included 22 strictly Nearctic species sharing the same MCO type with Dactylogyrus spp. parasitizing cyprinids likely of Asian origin. The second clade encompassed Dactylogyrus spp. with diverse MCO morphology and was placed in the sister position to Dactylogyrus spp. parasitizing European leuciscids and North-West African cyprinids.

Title: Espèce néarctique de Dactylogyrus (Platyhelminthes, Monogenea) parasitant des poissons cypriniformes dans un contexte de morphologie et phylogénie, avec description de sept nouvelles espèces. Abstract: Des séquences d'ADN ribosomal sont actuellement disponibles pour 32 espèces morphologiquement reconnues de Dactylogyrus parasitant les cypriniformes néarctiques, mais seulement 16 d'entre elles sont attribuées à des espèces nominales. Ici, des données morphologiques sur 28 des 32 espèces sont fournies avec des commentaires sur leurs relations phylogénétiques dans le contexte de la morphologie des structures taxonomiquement importantes. Sept nouvelles espèces de Dactylogyrus sont décrites à partir de cinq espèces de leuciscidés et d'une espèce de catostomidés, comme suit : D. aduncus n. sp. de Campostoma spadiceum, D. cloutmani n. sp. de Luxilus chrysocephalus isolepis, D. cornifrons n. sp. de Cyprinella venusta, D. fimbratus n. sp. de Rhinichthys cataractae, D. mcallisteri n. sp. de Semotilus atromaculatus et D. chieni n. sp. et D. haneki n. sp. de Hypentelium nigricans. Quatre espèces de Dactylogyrus précédemment décrites, D. atromaculatus de S. atromaculatus, D. eos de C. neogaeus, D. parvicirrus de Notemigonus crysoleucas et D. perlus de L. c. isolepis, sont redécrites et/ou figurées. Comme pour les 17 espèces restantes, seuls les organes copulateurs mâles (OCM) prélevés sur les hologénophores respectifs sont illustrés. Sur la base d'analyses phylogénétiques, deux clades principaux de Dactylogyrus néarctiques ont été reconnus et soutenus par des morphologies différentes de l'OCM. Le premier comprenait 22 espèces strictement néarctiques partageant le même type d'OCM que les Dactylogyrus spp. parasitant des cyprinidés probablement d'origine asiatique. Le deuxième clade comprenait des Dactylogyrus spp. avec diverses morphologies d'OCM et était placé en position sœur des Dactylogyrus spp. parasitant les leuciscidés européens et les cyprinidés d'Afrique du Nord-Ouest.