Pacific marine gregarines (Apicomplexa) shed light on biogeographic speciation patterns and novel diversity among early apicomplexans.

Gregarines are the most biodiverse group of apicomplexan parasites. This group specializes on invertebrate hosts (e.g., ascidians, crustaceans, and polychaetes). Marine gregarines are of particular interest because they are considered to be the earliest evolving apicomplexan lineage, having subsequently speciated (and radiated) through virtually all existing animal groups. Still, mechanisms governing the broad (global) distribution and speciation patterns of apicomplexans are not well understood. The present study examines Pacific lecudinids, one of the most species-rich and diverse groups of marine gregarines. Here, marine polychaetes were collected from intertidal zones. Single trophozoite cells were isolated for light and electron microscopy, as well as molecular phylogenetic analyses using the partial 18S rRNA gene. The cytochrome c oxidase subunit 1 gene was used to confirm morphology-based host identification. This study introduces Undularius glycerae n. gen., n. sp. and Lecudina kitase n. sp. (Hokkaido, Japan), as well as Difficilina fasoliformis n. sp. (California, USA). Occurrences of Lecudina cf. longissima and Lecudina cf. tuzetae (California, USA) are also reported. Phylogenetic analysis revealed a close relationship between L. pellucida, L. tuzetae, and L. kitase n. sp. Additionally, clustering among North Atlantic and Pacific L. tuzetae formed a species complex, likely influenced by biogeography.

Dispersal and invasive stages of Urospora eugregarines (Apicomplexa) from brown bodies of a polychaete host.

Urosporid eugregarines (Apicomplexa: Urosporidae) are unicellular eukaryotic parasites inhabiting the coelom or the intestine of marine invertebrates such as annelids, molluscs, nemerteans, and echinoderms. Despite the availability of published morphological and phylogenetical analyses of coelomic gregarines, their long-term survival in the host body cavity and dispersal routes into the marine environment remain unclear. Here, we focus on Urospora gametocysts and oocysts with sporozoites, which were found viable inside the so-called brown bodies floating in the body cavity of the polychaete Travisia forbesii. Brown bodies form as a result of host defence where coelomocytes encapsulate dead host cells and foreign objects including potential pathogens. We hypothesise the long-term persistence of Urospora eugregarines in brown bodies through evasion of the host immunity and outline possible pathways for their egress into the marine environment, applicable as dispersal routes for other parasites as well. Unique features revealed by detailed ultrastructural analysis of detected eugregarine stages include asynchronous sporogony, a massive sporozoite secretion apparatus, as well as the presence of free (possibly autoinfective) sporozoites within the gametocyst. The assignment to the genus Urospora and the complete identity with U. ovalis and U. travisiae were confirmed by analysing 18S rDNA sequences obtained from isolated gametocysts. The 18S rDNA phylogeny confirmed the affiliation of Urosporidae to Lecudinoidea and the grouping of all Urospora sequences with Difficilina from nemerteans and environmental sequences from the Artic region. We also enriched the Apicomplexa set by partial 28S rDNA sequences of two Urospora species enabling more complex phylogenetic analyses prospectively.

Molecular phylogeny and new light microscopic data of Metchnikovella spiralis (Microsporidia: Metchnikovellidae), a hyperparasite of eugregarine Polyrhabdina sp. from the polychaete Pygospio elegans.

Metchnikovellids are a deep-branching group of microsporidia, parasites of gregarines inhabiting the alimentary tract of polychaetes and some other invertebrates. The diversity and phylogeny of these hyperparasites remain poorly studied. Modern descriptions and molecular data are still lacking for many species. The results of a light microscopy study and molecular data for Metchnikovella spiralis Sokolova et al., 2014, a hyperparasite of the eugregarine Polyrhabdina sp., isolated from the polychaete Pygospio elegans, were obtained. The original description of M. spiralis was based primarily on the analysis of stained preparations and transmission electron microscopy images. Here, the species description was complemented with the results of in vivo observations and phylogenetic analysis based on the SSU rRNA gene. It was shown that in this species, free sporogony precedes sac-bound sporogony, as it occurs in the life cycle of most other metchnikovellids. Spore sacs are entwined with spirally wound cords, and possess only one polar plug. Phylogenetic analyses did not group M. spiralis with M. incurvata, another metchnikovellid from the same gregarine species, but placed it as a sister branch to Amphiacantha. The paraphyletic nature of the genus Metchnikovella was discussed. The taxonomic summary for M. spiralis was emended.

A new Bradophila Marchenkov, 2002 (Copepoda, Cyclopoida, Bradophilidae) parasitic on a flabelligerid polychaete from the Chukchi Sea.

The cyclopoid copepod family Bradophilidae includes a few species of mesoparasitic copepods infecting flabelligerid polychaetes. It contains two species of Bradophila Levinsen, 1878, the type genus: B. pygmaea Levinsen, 1878 and B. minuta Boxshall, O'Reilly, Sikorski & Summerfield, 2019, both known from North Europe. Two other genera (i.e., Trophoniphila M'Intosh, 1885 and Flabellicola Gravier, 1918) have some affinities with this family including their host preference. Mesoparasitic copepods are highly specialized, morphologically reduced forms. Part of their body (endosoma) is partially lodged in the host body and the other part is external (ectosoma); both parts are connected by an intersomital stalk. Infection by these copepods can be readily detected by the presence of the egg-carrying ectosoma on the host external surface. From the analysis of flabelligerid polychaetes collected in 2012 from the Chukchi Sea, two ovigerous female individuals of a bradophilid copepod were recorded. These specimens were recognized as representative of an undescribed species of Bradophila. The new species, B. susanae n. sp., shows the generic diagnostic characters and differs from its two other known congeners in several respects, including the cuticular ectosomal ornamentation, body proportions, size of the intersomital stalk, position of the genital pore, and shape and arrangement of egg sacs. Also, the new species ectosomal size range (0.440 - 0.450 µm) falls between the size range of its two known congeners. Our finding expands the known host range of bradophilid copepods to include a new flabelligerid host, Bradabyssa nuda (Annenkova-Chlopina) from the Russsian Arctic region.

Evolutionary relationships of Metchnikovella dogieli Paskerova et al., 2016 (Microsporidia: Metchnikovellidae) revealed by multigene phylogenetic analysis.

The species Metchnikovella dogieli (Paskerova et al. Protistology 10:148-157, 2016) belongs to one of the early diverging microsporidian groups, the metchnikovellids (Microsporidia: Metchnikovellidae). In relation to typical ('core') microsporidia, this group is considered primitive. The spores of metchnikovellids have no classical polar sac-anchoring disk complex, no coiled polar tube, no posterior vacuole, and no polaroplast. Instead, they possess a short thick manubrium that expands into a manubrial cistern. These organisms are hyperparasites; they infect gregarines that parasitise marine invertebrates. M. dogieli is a parasite of the archigregarine Selenidium pygospionis (Paskerova et al. Protist 169:826-852, 2018), which parasitises the polychaete Pygospio elegans. This species was discovered in samples collected in the silt littoral zone at the coast of the White Sea, North-West Russia, and was described based on light microscopy. No molecular data are available for this species, and the publicly accessible genomic data for metchnikovellids are limited to two species: M. incurvata Caullery & Mesnil, 1914 and Amphiamblys sp. WSBS2006. In the present study, we applied single-cell genomics methods with whole-genome amplification to perform next-generation sequencing of M. dogieli genomic DNA. We performed a phylogenetic analysis based on the SSU rRNA gene and reconstructed a multigene phylogeny using a concatenated alignment that included 46 conserved single-copy protein domains. The analyses recovered a fully supported clade of metchnikovellids as a basal group to the core microsporidia. Two members of the genus Metchnikovella did not form a clade in our tree. This may indicate that this genus is paraphyletic and requires revision.

In vivo cultivation of tuna blood fluke Cardicola orientalis in terebellid intermediate hosts.

Some fish blood flukes of the genus Cardicola (Aporocotylidae) are considered important pathogens of farmed/ranched tuna, Thunnus spp. Infections with Cardicola spp. might obstruct the blood flow in the gills via massive accumulations of eggs and often lead to mass mortalities in captive tuna. At present, oral administration of an anthelminthic drug, praziquantel is the most effective treatment, but the tuna farming industries are seeking non-drug control measures. Development of prophylactic and holistic measures have been difficult, owing to a lack of basic knowledge about these parasites. Unlike other trematodes which use molluscs, blood flukes of marine actinopterygian fish use terebellid polychaetes as intermediate hosts. However, information about the development of Cardicola spp. within intermediate hosts is very limited. Recent success in Cardicola opisthorchis sporocyst transplantation into the host polychaete has opened possibilities for the cultivation of Cardicola in the laboratory. Here, we conducted several transplantation trials with another tuna blood fluke, Cardicol orientalis, into its natural and surrogate polychaete hosts. Cardicola orientalis sporocysts were injected into a total of 195 Nicolea gracilibranchis, the natural host, and clear sporocyst development and reproduction was observed in 32 recipients (overall success rate 16.4%). The production of daughter sporocysts in the transplanted polychaete occurred within 14 days post injection, and one sporocystogenous cycle took approximately 4 weeks. Serial passage culture via transplantation of in vivo-cultured sporocysts was also achieved, but with limited sporocyst reproduction. In addition, sporocysts were successfully retrieved from six and one individuals of the surrogate hosts, Thelepus setosus (n = 10) and Thelepus japonicus (n = 5), respectively. These results indicate that the in vivo cultivation of C. orientalis sporocysts is possible, not only in its natural host but also in other terebellids, although the problems of high mortality and inconsistency in successful transplantation need to be resolved.

Morphology and molecular analyses of four epibiotic peritrichs on crustacean and polychaete hosts, including descriptions of two new species (Ciliophora, Peritrichia).

Four epibiotic sessilid peritrichs, i.e., Zoothamnium wilberti n. sp., Baikalonis microdiscus n. sp., Epistylis anastatica (Linnaeus, 1767) Ehrenberg, 1830, and Rhabdostyla commensalisMöbius, 1888, were isolated from one syllid polychaete and three crustacean hosts in Qingdao, China. For each species, specimens were observed both in vivo and following silver staining. Their SSU rDNA was also sequenced for phylogenetic analyses. Zoothamnium wilberti n. sp. is characterized by the appearance of its colony, which is up to 350 µm high, and usually has fewer than 16 zooids, and the dichotomously branched stalk with transverse wrinkles, the conspicuously conical peristomial disc, and infundibular polykinety 3 comprising three isometric ciliary rows. Baikalonis microdiscus n. sp. can be recognized by its barrel-shaped zooid, small peristomial disc, smooth and short stalk, and its unusual infundibular polykinety 3 comprising a long inner row and a short outer row. Two poorly known species, i.e., Epistylis anastatica and Rhabdostyla commensalis, are redescribed and redefined. Phylogenetic analyses reveal that: (i) R. commensalis is closely related to the family Astylozoidae rather than to the morphologically similar Epistylididae; (ii) B. microdiscus n. sp. is sister to the family Scyphidiidae; (iii) E. anastatica groups with vorticellids and ophrydiids, which further supports the polyphyly of the genus Epistylis; and (iv) Z. wilberti n. sp. is nested within the Zoothamniidae, as expected.

The intermediate hosts of Wardium cirrosa (Krabbe, 1869) Spassky, 1961 (Cestoda, Cyclophyllidea, Aploparaksidae) in Ukraine.

The metacestodes of aploparaksid cestode Wardium cirrosa Krabbe, 1869 parasitic in gulls were found in polychaetes of the family Nereidae collected off the Black Sea coast, Ukraine. Two species of polychaetes, Hediste diversicolor (prevalence 5.3%; intensity 1-3 specimens) and Neanthes succinea (prevalence 9.9%; intensity 1-39 specimens), were infected with cysticercoids that were observed either individually or in accumulations. The preliminary identification of the material based on morphological characteristics was later confirmed by experimental infection of the definitive host, Larus cachinnans (Charadriiformes: Laridae) with metacestodes, and by the identity of the partial 28S rDNA sequences of cysticercoids and experimentally obtained adults. Although previous studies suggested freshwater leeches as the intermediate host for W. cirrosa, our study provides the evidence for marine polychaetes to serve as intermediate hosts. This study is the first to present the morphological characteristics of metacestodes of W. cirrosa in addition to molecular data for this species, as well as reporting the possibility of several cysticercoids developing from a single oncosphere. Morphology of the adult specimens obtained in the experiment was compared with adults of W. cirrosa previously collected from L. cachinnans in Ukraine. The results of our study suggest that further research focused on the elucidation of the life cycles of cestodes within the genus Wardium should consider marine invertebrates as potential intermediate hosts.

First Myxozoan Infection (Cnidaria: Myxosporea) in a Marine Polychaete from North America and Erection of Actinospore Collective Group Saccimyxon.

In a survey of marine annelids for myxosporean infection in Charleston Harbor, South Carolina, we collected 3,214 polychaetes from 21 families and found infections in 6 spionid individuals. Based on gross morphology and COI sequencing, all infected spionids were identified as Streblospio benedicti. Infection prevalence was 0.8% (6/734) of that species of spionid, and 0.2% of all 3,214 polychaetes examined. Pansporocysts contained 8 actinospores and developed in the tegument of the annelid host. This is the first myxozoan infection recorded from this polychaete species, second in the family, and the first marine myxozoan found in the Americas. It is the first marine species found to develop in the tegument of its annelid host; a site of development observed only once before, in Ceratonova shasta infections of its freshwater sabellid polychaete host. Mature actinospores were morphologically simple, truncated ellipsoids, lacking processes or ornamentation, 9.0 ± 0.5 µm × 6.0 ± 0.4 µm. Their sack-like shape was similar to 9 of the 12 actinospores described previously from polychaetes; 10/12 had been and ascribed originally to the morphological collective group Tetractinomyxon despite 9 of these having few similarities to the original description of this group. We propose to name the simple, spherical to ellipsoidal spore morphotype Saccimyxon to encompass both our novel actinospore and the 9 other sack-like polychaete actinospore types in the literature. In the present study, 18S rDNA sequencing demonstrated that the myxozoans that infected the 6 spionids were genetically the same species (type sample 1,737 nucleotides, GenBank accession number MH791159) and was not >95% similar to any sequence in GenBank. Phylogenetic analysis showed that the myxozoan species we encountered is basal to the kudoids and thus likely to have a morphologically simple myxospore stage with fewer than 4 valves. However, without a genetic match, the presumptive vertebrate host remains unknown.

Fine structure and Molecular Phylogenetic Position of Two Marine Gregarines, Selenidium pygospionis sp. n. and S. pherusae sp. n., with Notes on the Phylogeny of Archigregarinida (Apicomplexa).

Archigregarines are a key group for understanding the early evolution of Apicomplexa. Here we report morphological, ultrastructural, and molecular phylogenetic evidence from two archigregarine species: Selenidium pygospionis sp. n. and S. pherusae sp. n. They exhibited typical features of archigregarines. Additionally, an axial row of vacuoles of a presumably nutrient distribution system was revealed in S. pygospionis. Intracellular stages of S. pygospionis found in the host intestinal epithelium may point to the initial intracellular localization in the course of parasite development. Available archigregarine SSU (18S) rDNA sequences formed four major lineages fitting the taxonomical affiliations of their hosts, but not the morphological or biological features used for the taxonomical revision by Levine (1971). Consequently, the genus Selenidioides Levine, 1971 should be abolished. The branching order of these lineages was unresolved; topology tests rejected neither para- nor monophyly of archigregarines. We provided phylogenies based on LSU (28S) rDNA and near-complete ribosomal operon (concatenated SSU, 5.8S, LSU rDNAs) sequences including S. pygospionis sequences. Although being preliminary, they nevertheless revealed the monophyly of gregarines previously challenged by many molecular phylogenetic studies. Despite their molecular-phylogenetic heterogeneity, archigregarines exhibit an extremely conservative plesiomorphic structure; their ultrastructural key features appear to be symplesiomorphies rather than synapomorphies.

First evidence of polychaete intermediate hosts for Neospirorchis spp. marine turtle blood flukes (Trematoda: Spirorchiidae).

Life cycles of spirorchiids that infect the vascular system of turtles are poorly understood. Few life cycles of these blood flukes have been elucidated and all intermediate hosts reported are gastropods (Mollusca), regardless of whether the definitive host is a freshwater or a marine turtle. During a recent survey of blood fluke larvae in polychaetes on the coast of South Carolina, USA, spirorchiid-like cercariae were found to infect the polychaetes Amphitrite ornata (Terebellidae) and Enoplobranchus sanguineus (Polycirridae). Cercariae were large, furcate, with a ventral acetabulum, but no eyespots were observed. Partial sequences of D1-D2 domains of the large ribosomal subunit, the internal transcribed spacer 2, and the mitochondrial cytochrome oxidase 1 genes allowed the identification of sporocysts and cercariae as belonging to two unidentified Neospirorchis species reported from the green turtle, Chelonia mydas, in Florida: Neospirorchis sp. (Neogen 13) in A. ornata and Neospirorchis sp. (Neogen 14) in E. sanguineus. Phylogenetic analysis suggests that infection of annelids by blood flukes evolved separately in aporocotylids and spirorchiids. Our results support the contention that the Spirorchiidae is not a valid family and suggest that Neospirorchis is a monophyletic clade within the paraphyletic Spirorchiidae. Since specificity of spirorchiids for their intermediate hosts is broader than it was thus far assumed, surveys of annelids in turtle habitats are necessary to further our understanding of the life history of these pathogenic parasites.

Morphology and molecular systematic of marine gregarines (Apicomplexa) from Southwestern Atlantic spionid polychaetes.

Gregarines are a common group of parasites that infect the intestines of marine invertebrates, and particularly polychaetes. Here, we describe for the first time four gregarine species that inhabit the intestines of three spionid species: Dipolydora cf. flava, Spio quadrisetosa and Boccardia proboscidea from the Patagonian coast, Argentina, using light and scanning electron microscopy and molecular phylogenetic analyses of small subunit (SSU) rDNA sequences. Even though the spionid species thrive in the same environments, our results showed a high host specificity of the gregarine species. Selenidium cf. axiferens and Polyrhabdina aff. polydorae were both identified from the intestine of D. cf. flava. The new species, Polyrhabdina madrynense sp. n. and Selenidium patagonica sp. n., were described from the intestines of S. quadrisetosa and the invasive species B. proboscidea, respectively. All specimens of D. cf. flava and S. quadrisetosa were infected by gregarines (P = 100%), recording the highest mean intensity values of infection (MI = 80; 60 respectively), in contrast to B. proboscidea (P = 60%; MI = 38). We associated this finding with the recent invasion of this host. It is expected that in the future, an increase of its population density might favour a rising intensity of this gregarine infection.

Evolutionary Genomics of Metchnikovella incurvata (Metchnikovellidae): An Early Branching Microsporidium.

Metchnikovellids are highly specialized hyperparasites, which infect and reproduce inside gregarines (Apicomplexa) inhabiting marine invertebrates. Their phylogenetic affiliation was under constant discussion until recently, when analysis of the first near-complete metchnikovellid genome, that of Amphiamblys sp., placed it in a basal position with respect to most Microsporidia. Microsporidia are a highly diversified lineage of extremely reduced parasites related to Rozellida (Rozellosporidia = Rozellomycota = Cryptomycota) within the Holomycota clade of Opisthokonta. By sequencing DNA from a single-isolated infected gregarine cell we obtained an almost complete genome of a second metchnikovellid species, and the first one of a taxonomically described and well-documented species, Metchnikovella incurvata. Our phylogenomic analyses show that, despite being considerably divergent from each other, M. incurvata forms a monophyletic group with Amphiamplys sp., and confirm that metchnikovellids are one of the deep branches of Microsporidia. Comparative genomic analysis demonstrates that, like most Microsporidia, metchnikovellids lack mitochondrial genes involved in energy transduction and are thus incapable of synthesizing their own ATP via mitochondrial oxidative phosphorylation. They also lack the horizontally acquired ATP transporters widespread in most Microsporidia. We hypothesize that a family of mitochondrial carrier proteins evolved to transport ATP from the host into the metchnikovellid cell. We observe the progressive reduction of genes involved in DNA repair pathways along the evolutionary path of Microsporidia, which might explain, at least partly, the extremely high evolutionary rate of the most derived species. Our data also suggest that genome reduction and acquisition of novel genes co-occurred during the adaptation of Microsporidia to their hosts.

Molecular Phylogeny and Morphology of Haplozoon ezoense n. sp. (Dinophyceae): A Parasitic Dinoflagellate with Ultrastructural Evidence of Remnant Non-photosynthetic Plastids.

This study describes a novel species of Haplozoon, H. ezoense n. sp., a dinoflagellate parasite isolated from the intestines of Praxillella pacifica (Polychaeta). Trophonts (feeding stages) of H. ezoense n. sp. were isolated and studied with scanning and transmission electron microscopy, and molecular phylogenetic analyses was performed using 18S rDNA and 28S rDNA. Trophonts had an average length of 120µm, and were linear, forming a single longitudinal row comprising a trophocyte with a stylet, an average of 14 gonocytes (width=10µm), and bulbous cells that we concluded were likely sporocytes. The surface of H. ezoense n. sp. was covered with projections of the amphiesma. Sections viewed under TEM revealed multiple triple membrane-bound organelles reminiscent of relic non-photosynthetic plastids within the gonocytes. Haplozoon ezoense n. sp., H. praxillellae, and H. axiothellae formed a well-supported clade in the 18S rDNA datasets. The sequences of H. ezoense n. sp. differed from H. praxillellae, a species of Haplozoon isolated from the same host species in the Northeast Pacific, at 88/1,748 bases; and 155/1,752 bases from H. axiothellae. Concatenated 18S rDNA and 28S rDNA datasets were unable to resolve the deeper relationships of Haplozoon in the context of dinoflagellates.

Molecular Phylogenetic Positions and Ultrastructure of Marine Gregarines (Apicomplexa) Cuspisella ishikariensis n. gen., n. sp. and Loxomorpha cf. harmothoe from Western Pacific scaleworms (Polynoidae).

Marine gregarines are unicellular parasites of invertebrates commonly found infecting the intestine and coelomic spaces of their hosts. Situated at the base of the apicomplexan tree, marine gregarines offer an opportunity to explore the earliest stages of apicomplexan evolution. Classification of marine gregarines is often based on the morphological traits of the conspicuous feeding stages (trophozoites) in combination with host affiliation and molecular phylogenetic data. Morphological characters of other life stages such as the spore are also used to inform taxonomy when such stages can be found. The reconstruction of gregarine evolutionary history is challenging, due to high levels of intraspecific variation of morphological characters combined with relatively few traits that are taxonomically unambiguous. The current study combined morphological data with a phylogenetic analysis of small subunit rDNA sequences to describe and establish a new genus and species (Cuspisella ishikariensis n. gen., n. sp.) of marine gregarine isolated from the intestine of a polynoid host (Lepidonotus helotypus) collected from Hokkaido, Japan. This new species possesses a set of unusual morphological traits including a spiked attachment apparatus and sits on a long branch on the molecular phylogeny. Furthermore, this study establishes a molecular phylogenetic position for Loxomorpha cf. harmothoe, a previously described marine gregarine, and reveals a new group of gregarines that infect polynoid hosts.

Molecular Phylogenetic Positions of Two New Marine Gregarines (Apicomplexa)-Paralecudina anankea n. sp. and Lecudina caspera n. sp.-from the Intestine of Lumbrineris inflata (Polychaeta) Show Patterns of Co-evolution.

Gregarine apicomplexans are unicellular parasites commonly found in the intestines and coeloms of invertebrate hosts. Traits associated with the conspicuous feeding stage of gregarines, known as the trophozoite, have been used in combination with molecular phylogenetic data for species delimitation and the reconstruction of evolutionary history. Trophozoite morphology alone is often inadequate for inferring phylogenetic relationships and delimiting species due to frequent cases of high intraspecific variation combined with relatively low interspecific variation. The current study combined morphological data with small subunit (SSU) rDNA sequences to describe and establish two novel marine gregarine species isolated from the intestine of a polychaete host Lumbrineris inflata collected in British Columbia (Canada): Paralecudina anankea n. sp. and Lecudina caspera n. sp. The sister species to the host is Lumbrineris japonica, which can be found on the opposite side of the Pacific Ocean (Japan) and contains two different species of gregarine parasites: Paralecudina polymorpha and Lecudina longissima. Molecular phylogenetic analyses placed P. anankea n. sp. as the sister species to P. polymorpha and L. caspera n. sp. as the sister species to L. longissima. This phylogenetic pattern demonstrates a co-evolutionary history whereby speciation of the host (Lumbrineris) corresponds with simultaneous speciation of the two different lineages of intestinal gregarines (Paralecudina and Lecudina).

Blood flukes Cardicola parvus and C. laruei (Trematoda: Aporocotylidae): life cycles and cryptic infection in spotted seatrout, Cynoscion nebulosus (Teleost: Sciaenidae).

Aporocotylidae comprises a diverse family of fish blood flukes, with adults found in blood or body cavity of marine, brackish, or freshwater fish. Aporocotylids are unique among the Digenea with many developing in polychaetes. The life cycle has been elucidated for only a few species that develop in polychaetes from marine/brackish environments and none for western Atlantic aporocotylids. The basis for this study was observations of blood fluke larvae in annelids from South Carolina, USA in 1982 prior to possible usage of molecular tools to specifically identify parasite larvae. Recent description of aporocotylid species and genotyping tools prompted revisiting original collection sites to examine polychaetes and fish as potential hosts. Polycirrid Enoplobranchus sanguineus and terebellids Amphitrite ornata, and Terebella lapidaria revealed infections with aporocotylid larvae. Adult blood flukes were also collected from fish commonly encountered in the same habitat: spotted seatrout (Cynoscion nebulosus), red drum (Sciaenops ocellatus), black drum (Pogonias cromis), and Atlantic croaker (Micropogonias undulatus). Sporocysts containing cercariae were found in individuals of each annelid species. Adult Cardicola parvus were found in spotted seatrout and Atlantic croaker, C. laruei in spotted seatrout, C. currani in red drum, and C. palmeri in black drum. Genotype analysis of ITS-2 and lsrDNA of all forms confirmed conspecific infections by C. parvus in E. sanguineus and A. ornata and C. laruei in T. lapidaria. This is the first description of complete life cycles of aporocotylids in the Western Atlantic and first evidence of cryptic infections of Cy. nebulosus with C. parvus.

Redescription and phylogenetic analyses of Durchoniella spp. (Ciliophora, Astomatida) associated with the polychaete Cirriformia tentaculata (Montagu, 1808).

Microscopic and phylogenetic analyses were performed on endocommensal astome ciliates retrieved from the middle intestine of a marine cirratulid polychaete, Cirriformia tentaculata, collected in the bay of Roscoff (English Channel, Northwest French coast) and on the Southwest English coast. Three morphotypes of the astome genus Durchoniella were identified, two corresponding to described species (the type species Durchoniella brasili (Léger and Duboscq, 1904) De Puytorac, 1954 and Durchoniella legeriduboscqui De Puytorac, 1954) while a third morphotype remains undescribed. Their small subunit (SSU) rRNA gene sequences showed at least 97.2% identity and phylogenetic analyses grouped them at the base of the subclass Scuticociliatia (Oligohymenophorea), as a sister lineage to all astomes from terrestrial oligochaete annelids. Ultrastructural examination by transmission electron microscopy and fluorescence in situ hybridization analyses revealed the presence of endocytoplasmic cocci and rod-shaped bacteria surrounded by a very thin membrane. These endocytoplasmic bacteria may play a role in the association between endocommensal astome ciliates and cirratulid polychaetes inhabiting in anoxic coastal sediments.

Evidence that blood flukes (Trematoda: Aporocotylidae) of chondrichthyans infect bivalves as intermediate hosts: indications of an ancient diversification of the Schistosomatoidea.

Blood flukes (Aporocotylidae) of actinopterygians (bony fishes) have been shown to infect freshwater gastropods and marine polychaetes as intermediate hosts. However, no life cycle is known for any aporocotylid of chondrichthyans (cartilaginous fishes) and no adult aporocotylid has been linked to a cercaria infecting a bivalve. Here we report two novel infections that fill these gaps. Cercariae consistent with the family Aporocotylidae were found developing in sporocysts in the gonad of the surf pipi, Donax deltoides Lamarck, 1818 (Bivalvia: Donacidae), from Stockton Beach, central New South Wales, Australia. Adult aporocotylids were found in the heart of the giant shovelnose ray, Glaucostegus typus (Anonymous [Bennett], 1830), from Moreton Bay, southeastern Queensland, Australia. Phylogenetic analyses of the 28S rDNA region generated from the new specimens resulted in phylograms in which the two parasites form a strongly supported clade with Chimaerohemecus trondheimensis van der Land, 1967, the only aporocotylid known from a holocephalan and the only other chondrichthyan-infecting aporocotylid for which sequence data are available. Most marine aporocotylids of actinopterygians also form a strongly supported clade. These findings lead us to hypothesise that the aporocotylids of chondrichthyans are distinct from all other blood flukes in infecting bivalves as intermediate hosts. Putative cophyly between three major blood fluke clades and both definitive and intermediate host groups is consistent with diversification of the Schistosomatoidea over 400million years ago.

Spatial and Temporal Changes in the Distribution of Blood Fluke Infection in Nicolea gracilibranchis (Polychaeta: Terebellidae), the Intermediate Host for Cardicola orientalis (Digenea: Aporocotylidae), at a Tuna Farming Site in Japan.

Fish blood flukes of the genus Cardicola (Digenea: Aporocotylidae) are important pathogens in tuna aquaculture. Recent advances in marine blood fluke research have led to the elucidation of the lifecycles of 3 Cardicola spp. infecting tuna; all 3 flukes utilize terebellid polychaetes as the intermediate host. In our survey, we obtained large numbers of Nicolea gracilibranchis infected by larval Cardicola orientalis at our tuna farming site. To determine the spatial and temporal changes in the distribution of N. gracilibranchis surrounding tuna culture cages and their infection by C. orientalis, we conducted monthly sampling for a period of 1 yr. Terebellids were most abundant on the floats and ropes of culture cages, but a significantly higher proportion of infected N. gracilibranchis was detected on ropes, particularly up to 4 m in depth. Cardicola orientalis infection in N. gracilibranchis was clearly seasonal, with a higher infection rate between April and July. Our findings indicate that the infected terebellids inhabit specific microhabitats, and both abiotic and biotic factors likely influence blood fluke infection in the intermediate terebellid host. This information is important to better understand the general biology of marine aporocotylids and may be useful to develop a control strategy for blood fluke infection in tuna aquaculture.