Thomas J. Fayton

Large Scale Screening of Digeneans for Neorickettsia Endosymbionts Using Real-Time PCR Reveals New Neorickettsia Genotypes, Host Associations and Geographic Records

Digeneans are endoparasitic flatworms with complex life cycles including one or two intermediate hosts (first of which is always a mollusk) and a vertebrate definitive host. Digeneans may harbor intracellular endosymbiotic bacteria belonging to the genus Neorickettsia (order Rickettsiales, family Anaplasmataceae). Some Neorickettsia are able to invade cells of the digeneans vertebrate host and are known to cause diseases of wildlife and humans. In this study we report the results of screening 771 digenean samples for Neorickettsia collected from various vertebrates in terrestrial, freshwater, brackish, and marine habitats in the United States, China and Australia. Neorickettsia were detected using a newly designed real-time PCR protocol targeting a 152 bp fragment of the heat shock protein coding gene, GroEL, and verified with nested PCR and sequencing of a 1371 bp long region of 16S rRNA. Eight isolates of Neorickettsia have been obtained. Sequence comparison and phylogenetic analysis demonstrated that 7 of these isolates, provisionally named Neorickettsia sp. 1–7 (obtained from allocreadiid Crepidostomum affine, haploporids Saccocoelioides beauforti and Saccocoelioides lizae, faustulid Bacciger sprenti, deropegid Deropegus aspina, a lecithodendriid, and a pleurogenid) represent new genotypes and one (obtained from Metagonimoides oregonensis) was identical to a published sequence of Neorickettsia known as SF agent. All digenean species reported in this study represent new host records. Three of the 6 digenean families (Haploporidae, Pleurogenidae, and Faustulidae) are also reported for the first time as hosts of Neorickettsia. We have detected Neorickettsia in digeneans from China and Australia for the first time based on PCR and sequencing evidence. Our findings suggest that further surveys from broader geographic regions and wider selection of digenean taxa are likely to reveal new Neorickettsia lineages as well as new digenean host associations.

A new species of Intromugil (Digenea: Haploporidae) and redescription of Intromugil mugilicolus.

Abstract: Intromugil alachuaensis n. sp. is described based on specimens collected from the flathead grey mullet (Mugil cephalus) from the Santa Fe River in Florida. The new species is the fourth recognized species in the genus and the second from North America, with the other 2 being confined to South America. Intromugil mugilicolus from Louisiana and Mississippi is redescribed based on the holotype and newly collected material that was not flattened prior to fixation. Two generic features not previously reported are apparent in the new material from I. mugilicolus and I. alachuaensis n. sp.: an armed oral sucker and a series of sacs containing glandular material arranged in symmetrical rows in the hermaphroditic duct. Intromugil alachuaensis differs from I. mugilicolus by having an oral sucker longer than wide, body spines smaller and lanceolate rather than longer and hastate, and smaller vitelline follicles. Intromugil alachuaensis n. sp. differs from Intromugil simonei by having a large elongated pharynx rather than a smaller subspherical one, a proportionately larger and longer oral sucker, and a longer prepharynx (greater than 1 pharyngeal length). Intromugil alachuaensis n. sp. differs from Intromugil annakohnae by having a longer than wide pharynx, a relatively large oral sucker, less extensive vitellarium, and smaller body spines. Comparison of more than 2,400 base-pair-long sequences of nuclear rDNA (partial 18S, complete ITS1, complete 5.8S, complete ITS2, and partial 28S) from I. mugilicolus and I. alachuaensis n. sp. reveals 110 pairwise differences, including gaps, thus supporting our proposal of a new species. These represent the first published sequences from species in this genus.

New species of Plagioporus Stafford, 1904 (Digenea: Opecoelidae) from California, with an amendment of the genus and a phylogeny of freshwater plagioporines of the Holarctic

Plagioporus hageli n. sp. is described from the intestine of Oncorhynchus mykiss (Walbaum) collected from the River Yuba, California, USA. Of the accepted, nominal species of Plagioporus Stafford, 1904 from the Nearctic, the new species is morphologically similar to three intestinal species from the western USA parasitising diadromous fishes, including Plagioporus shawi (McIntosh, 1939), Plagioporus kolipinskii Tracey, Choudhury, Cheng & Ghosh, 2009 and Plagioporus siliculus Sinitsin, 1931, and is also similar to Plagioporus serotinus Stafford, 1904 from catostomids from eastern Canada. Plagioporus hageli n. sp. is distinguished from the former three species in lacking a dorsal vitelline field and from the latter species in having a consistent interruption in the distribution of the vitellarium at the level of the ventral sucker. The new species is also morphologically similar to an unnamed species of Plagioporus and a species misidentified as ‘Plagioporus angusticolle’ that were collected from California, but it is easily distinguished from both in its shorter body length. To estimate the placement of the new species within Plagioporus and within the Opecoelidae Ozaki, 1925, we conducted a Bayesian inference (BI) analysis of partial 28S rDNA sequence data that included sequences from Plagioporus hageli n. sp., five other species of Plagioporus, three species of Neoplagioporus Shimazu, 1990, including the type-species, Neoplagioporus zacconis (Yamaguti, 1934), two species of Urorchis Ozaki, 1927 (including the type-species, Urorchis goro Ozaki, 1927) and sequences of 42 opecoelid species obtained from GenBank. Our phylogenetic analysis revealed (i) plagioporines parasitising freshwater hosts form a monophyletic group; (ii) Plagiocirrus loboides Curran, Overstreet & Tkach, 2007 nested within the rest of the members of Plagioporus; (iii) the new species was closer to Plagiocirrus loboides than to Plagioporus shawi, the other salmonid parasite included in our analysis; (iv) P. shawi was the poorly supported sister to its congeners; (v) Neoplagioporus elongatus (Goto & Ozaki, 1930) Shimazu, 1990 was closer to the two species of Urorchis than to the other two species of Neoplagioporus; and (vi) the paraphyly of the Plagioporinae Manter, 1947 was reinforced. Based on 28S rDNA sequence data and our BI analysis, we propose Plagioporus loboides (Curran, Overstreet & Tkach, 2007) n. comb., and amend Plagioporus accordingly. This analysis represents the first phylogenetic study of the opecoelids that estimates the interrelationships of the Plagioporinae that includes a member of Plagioporus.

Alloglossidium floridense n. sp. (Digenea: Macroderoididae) from a Spring Run in North Central Florida

Abstract:u2003 A new species of Alloglossidium is described from the intestines of 2 madtom species (Noturus leptacanthus and Noturus gyrinus) that were collected from the run of a small, unnamed spring system that drains into the Santa Fe River, Florida. Alloglossidium floridense n. sp. is morphologically very similar to other nonprecocious Alloglossidium spp. that use ictalurids as definitive hosts, but can be distinguished by a combination of its smaller overall size (length and width), large eggs in relation to its small body size, position of the vitellaria, ovary shape, and position of the ovary in relation to the cirrus sac. A comparison of nuclear rDNA sequences (spanning partial 18s, complete ITS1, 5.8s, ITS2, and partial 28s regions) showed that A. floridense n. sp. diverged by 0.70–3.17% from the other 4, nonprecocious species that infect ictalurids (Alloglossidium corti, Alloglossidium fonti, Alloglossidium geminum, and Alloglossidium kenti). The new species of Alloglossidium, described herein, is the first of the genus to be reported from Florida and the first to be recorded from N. leptacanthus. In light of the subtle morphological differences among the nonprecocious species that infect ictalurids, we discuss how previous descriptions of species traits that are not supported with genetic data are difficult to interpret because of the possible past nonrecognition of distinct species.

Real-time PCR detection and phylogenetic relationships of Neorickettsia spp. in digeneans from Egypt, Philippines, Thailand, Vietnam and the United States.

Neorickettsia (Rickettsiales, Anaplasmataceae) is a genus of obligate intracellular bacterial endosymbionts of digeneans (Platyhelminthes, Digenea). Some Neorickettsia are able to invade cells of the digeneans vertebrate host and are known to cause diseases of domestic animals, wildlife, and humans. In this study we report the results of screening digenean samples for Neorickettsia collected from bats in Egypt and Mindoro Island, Philippines, snails and fishes from Thailand, and fishes from Vietnam and the USA. Neorickettsia were detected using a real-time PCR protocol targeting a 152bp fragment of the heat shock protein coding gene, GroEL, and verified with nested PCR and sequencing of a 1853bp long region of the GroESL operon and a 1371bp long region of 16S rRNA. Eight unique genotypes of Neorickettsia were obtained from digenean samples. Neorickettsia sp. 8 obtained from Lecithodendrium sp. from Egypt; Neorickettsia sp. 9 and 10 obtained from two species of Paralecithodendrium from Mindoro, Philippines; Neorickettsia sp. 11 from Lecithodendrium sp. and Neorickettsia sp. 4 (previously identified from Saccocoelioides lizae, from China) from Thailand; Neorickettsia sp. 12 from Dicrogaster sp. Florida, USA; Neorickettsia sp. 13 and SF agent from Vietnam. Sequence comparison and phylogenetic analysis demonstrated that the forms, provisionally named Neorickettsia sp. 8-13, represent new genotypes. We have for the first time detected Neorickettsia in a digenean from Egypt (and the African continent as a whole), the Philippines, Thailand and Vietnam based on PCR and sequencing evidence. Our findings suggest that further surveys from the African continent, SE Asia, and island countries are likely to reveal new Neorickettsia lineages as well as new digenean host associations.

Trematode Parasites (Digenea) of the Slender Madtom Noturus exilis and Black River Madtom Noturus maydeni (Siluriformes: Ictaluridae) from Arkansas, U.S.A

ABSTRACT: Between June 2012 and July 2013, 43 slender madtoms (Noturus exilis) and 21 Black River madtoms (Noturus maydeni) were collected from 4 counties of northern Arkansas and examined for helminths. Eleven (26%) slender madtoms were infected with digenean trematodes, including 3 (7%) with metacercaria of Posthodiplostomum minimum, 3 (7%) with Alloglossidium sp., and 7 (16%) with Plagioporus sp.; a single (5%) Black River madtom was infected with Caecincola sp. We document new host records for each of these 4 helminths. Notable among these new host records are new distributional records for Caecincola sp. To our knowledge this represents the first report of any helminth from N. exilis and N. maydeni.

An additional genus and two additional species of Forticulcitinae (Digenea: Haploporidae).

Forticulcita platana sp. n. and Forticulcita apiensis sp. n. are described from Mugil liza Valenciennes in Argentina, and from Mugil cephalus Linnaeus in Salt Springs, Florida, USA, respectively. Supplemental material relating to the hermaphroditic sac of Forticulcita gibsoni Blasco-Costa, Montero, Balbuena, Raga et Kostadinova, 2009 is provided from a specimen isolated from M. cephalus off Crete, Greece. Forticulcita platana can be distinguished from all species of Forticulcita Overstreet, 1982 except F. gibsoni, based on possessing small pads or gland cells along the hermaphroditic duct. It can be differentiated from that species in possessing a hermaphroditic sac that is one and a half to two times longer than wide rather than one that is approximately three times longer than wide, longer eggs (44-52 μm rather than 34-44 μm long) and a shorter post-testicular space (< 45% of the body length). Forticulcita apiensis can be differentiated from the other species of Forticulcita in possessing a testis that is shorter than or equal to the pharynx rather than one that is longer than the pharynx. Xiha gen. n. is erected for Dicrogaster fastigatus Thatcher et Sparks, 1958 as Xiha fastigata (Thatcher et Sparks, 1958) comb. n., and we tentatively consider Dicrogaster fragilis Fernández Bargiela, 1987 to be Xiha fragilis (Fernández Bargiela, 1987) comb. n. The new genus fits within the concept of Forticulcitinae Blasco-Costa, Balbuena, Kostadinova et Olson, 2009 in having a vitellarium comprised of a single elongate to subspherical mass. Xiha can be differentiated from Forticulcita in having spines lining the hermaphroditic duct, or intromittent organ. A Bayesian inference analysis of partial 28S rDNA sequences of the two New World species of Forticulcita, Xiha fastigata and previously published haploporids places Xiha fastigata within the Forticulcitinae and sister to Forticulcita. Amended diagnoses for the subfamily and for Dicrogaster Looss, 1902 are provided.

Two New Species of Homalometron (Digenea: Apocreadiidae) from Nearctic Freshwater Fundulids, Elucidation of the Life Cycle of H. cupuloris, and Molecular Phylogenetic Analysis of Some Congeners

Abstract:u2003 Two species of digeneans belonging in Homalometron are described from Nearctic freshwater fundulid fishes: Homalometron robisoni n. sp. is described from the Blackstripe Topminnow, Fundulus notatus, from Oklahoma and Homalometron frocioneae n. sp. is described from the Banded Killifish, Fundulus diaphanus, from New York. Homalometron robisoni n. sp. differs from all congeners by having vitelline follicles that extend into the forebody, a feature that necessitates altering the generic diagnosis for the genus. Homalometron frocioneae n. sp. may be distinguished from North and Middle American congeners by the position of the intestinal bifurcation (relatively more posterior in the forebody than in other species) and tegumental spine coverage on the body (spines are absent from the posterior body extremity and on most of the dorsal surface). Comparison of ribosomal DNA (ITS 1 and 2 regions, 5.8S gene, and partial fragment of 28S gene) from the 2 new species and some congeners from the Western Hemisphere provided evidence for the validity of the 2 new species and affirmed a close relationship between H. robisoni n. sp. and Homalometron pallidum. Comparison of ribosomal DNA from newly collected Homalometron spp. and larval stages of an apocreadiid from brackish water hydrobiid snails (cercariae in rediae in Littoridinops palustris and metacercariae in L. palustris and Amnicola limosa) from a tidal river in Mississippi revealed that larval stages represented Homalometron cupuloris. A phylogeny based on Bayesian inference analysis using partial 28S rDNA gene fragments from 14 species of Homalometron (all from the Western Hemisphere) and 1 megaperine and rooted by a second megaperine was conducted and produced a strongly supported phylogram that estimates the interrelationships among species. The estimated phylogeny suggests that ecological factors such as salinity and food web interactions between species of Homalometron, intermediate hosts, and fishes drive coevolutionary forces influencing speciation within Homalometron.

Helminth Parasites of the Northern Studfish, Fundulus catenatus (Cypriniformes: Fundulidae) from the Ouachita and Ozark Mountains of Arkansas, U.S.A.

ABSTRACT: Between May 2014 and September 2014, 50 Northern Studfish, Fundulus catenatus, were collected from sites in the interior highlands of the Ouachita Mountains (n = 25) and Ozark Mountains (n = 25) of Arkansas and examined for helminths. Twenty-three (46%) of the northern studfish, including 7 (28%) from the Ouachitas and 23 (92%) from the Ozarks were infected with helminths. These included 8 (16%) with Neascus-type metacercariae (black grub), 4 (8%) with metacercaria of Clinostomum marginatum, 8 (16%) with Creptotrema sp., 1 (2%) with Posthodiplostomum minimum, 3 (6%) with immature Proteocephalus sp., 9 (18%) with larval Eustrongylides sp., 1 (2%) with Rhabdochona cascadilla, 2 (4%) with larval Spinitectus sp., and 17 (34%) with acanthocephalan cystacanths. In addition, 6 of 8 individuals (75%) from one site in the Ouachitas harbored the monogenean, Salsuginus fundulus. We document new host records for most of these helminths and new distributional records for Creptotrema sp., Eustrongylides ...

Acolpenteron willifordensis n. sp. (Monogenoidea: Dactylogyridae) Parasitic in the Kidney and Ureters of the Spotted Sucker Minytrema melanops (Rafinesque) (Cypriniformes: Catostomidae) from Econfina Creek, Florida

ABSTRACT:u2005 Acolpenteron willifordensis n. sp. (Monogenoidea: Dactylogyridae) is described from the ureters and kidney (presumably the renal tubules) of the spotted sucker Minytrema melanops (Cypriniformes: Catostomidae) collected from the Econfina Creek in the panhandle of northern Florida. The new species is similar to Acolpenteron catostomi Fischthal and Allison, 1942 in body shape and size, position of the vaginal pore, and the general shape of the posteriorly notched haptor. It differs from A. catostomi by having a broad tubular shaft of the male copulatory organ that lacks a proximal double recurve immediately distal to its base. Possible biogeographic explanations for the occurrences of species of Acolpenteron in primary freshwater fishes of North America, Eurasia and South America are presented.