Eric E. Pulis

A NEW SPECIES OF RHABDIAS FROM LUNGS OF THE WOOD FROG, RANA SYLVATICA, IN NORTH AMERICA: THE LAST SIBLING OF RHABDIAS RANAE ?

Rhabdias bakeri n. sp. is described from specimens found in lungs of the wood frog, Rana sylvatica, from North Dakota. The new species has previously been mistakenly identified as Rhabdias ranae Walton, 1929, a common parasite of the leopard frog, Rana pipiens. The new species differs from R. ranae and Rhabdias joaquinensis Ingles, 1935 by the shape and size of pseudolabia, shape and size of buccal capsule, and wider esophageal bulb. Molecular analysis based on the partial sequences of nuclear 18S rDNA gene, complete sequences of internal transcribed spacer region, and partial sequences of 28S gene demonstrates clear differences between Rhabdias from Ra. sylvatica and Ra. pipiens, and supports the status of R. bakeri as a new species.

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.

Molecular characterization of two opecoelid trematodes from fishes in the Gulf of Mexico, with a description of a new species of Helicometra

The plagioporine opecoelids Helicometra fasciata (Rudolphi, 1819) Odhner, 1902, and Macvicaria crassigula (Linton, 1910) Bartoli, Bray, and Gibson, 1989 have been reported from fishes in expansive geographic regions, disjointed from their type localities. New material of M. crassigula was collected from near its type locality as well as specimens resembling Helicometrafasciata sensu lato from three triglids in the Gulf of Mexico. Comparisons of the ribosomal DNA (rDNA) sequences, comprising the partial 18S rDNA, internal transcribed spacer region (= ITS1, 5.8S, and ITS2), and partial 28S rDNA gene, from M. crassigula and Helicometra fasciata sensu lato in the Gulf of Mexico were made with sequences deposited in GenBank from those species from the Mediterranean Sea. Results reveal that M. crassigula sensu stricto from the Gulf of Mexico is distinct from the two cryptic species of M. crassigula sensu lato from the Mediterranean Sea and Helicometra fasciata sensu lato in this study differs from H. fasciata sequences from the Mediterranean Sea, thus Helicometra manteri sp. nov. is described.

Hirudinella ventricosa (Pallas, 1774) Baird, 1853 represents a species complex based on ribosomal DNA

Digeneans in the genus Hirudinella de Blainville, 1828 (Hirudinellidae) from three species of pelagic fishes, Acanthocybiumsolandri (Cuvier), Makairanigricans Lacépède and Thunnusalbacares (Bonnaterre), and one benthic fish, Mulloidichthysmartinicus (Cuvier), from the Gulf of Mexico are investigated using comparison of ribosomal DNA. Four species are identified based on molecular differences: Hirudinella ventricosa (Pallas, 1774) Baird, 1853 from A. solandri, Hirudinellaahi Yamaguti, 1970 from T. albacares, and two unidentified but distinct species of Hirudinella, herein referred to as Hirudinella sp. A (from both M. nigricans and M. martinicus) and Hirudinella sp. B from M. nigricans. Additionally, H. ahi, based tentatively on morphological identification, is reported from Thunnusthynnus (Linnaeus). This represents the first record of a hirudinellid from M.martinicus and the first record of H. ahi from T. thynnus. A phylogeny of some Hemiurata Skrjabin & Guschanskaja, 1954 using partial fragments of the 28S rDNA sequences is consistent with earlier phylogenies and the position of the Hirudinellidae Dollfus, 1932 is well-supported as a derived group most closely related to the Syncoeliidae Looss, 1899.

A new species of Drepanocephalus Dietz, 1909 (Digenea: Echinostomatidae) from the double-crested cormorant Phalacrocorax auritus (Lesson) (Aves: Phalacrocoracidae) in North America

Drepanocephalus auritus n. sp. is described based on specimens from the double-crested cormorant Phalacrocorax auritus (Lesson) in North America. The new species differs from its congeners in its very narrow, elongate body, long uterine field and widely separated testes. Sequences of the nuclear rRNA gene cluster, spanning the 3′ end of the nuclear ribosomal 18S rRNA gene, internal transcribed spacer region (ITS1+5.8S gene+ITS2) and partial 28S gene (2,345 bp), were identical in specimens collected from North Dakota, Minnesota and Mississippi, USA. Sequences of the 651 bp long fragment of the mitochondrial cox1 gene exhibited very low intraspecific variability (< 1%). Comparisons of the newly-generated sequences with those available in the GenBank indicate that the sequences from North America published under the name D. spathans Dietz, 1909 in fact represent D. auritus n. sp.

A New Paramacroderoides Species (Digenea: Macroderoididae) From Two Species of Gar in the Southeastern United States

Abstract Paramacroderoides kinsellai n. sp. is described based on specimens collected from the spotted gar (Lepisosteus oculatus) from the Pascagoula River, Jackson County, Mississippi, and the Florida gar (Lepisosteus platyrhincus) from Cross Creek, Alachua County, Florida. The new species is the third recognized species of Paramacroderoides. Two Indian species of Paramacroderoides, originally described within Pseudoparamacroderoides, are transferred into Macroderoides. Paramacroderoides kinsellai n. sp. differs from Paramacroderoides echinus in having a much longer body, greater body width/length ratio, ovary situated at a significant distance from the cirrus sac rather than immediately posterior to it, and anterior margin of vitellaria at a significant distance posterior to ventral sucker. The new species differs from Paramacroderoides pseudoechinus in having a much longer body, greater body width/length ratio, ovary situated at a significant distance from the cirrus sac rather than immediately posterior to it, anterior margin of vitellaria at a significant distance anterior to ovary, vitellaria extending further posteriorly, and relative position of the intestinal bifurcation. The generic diagnosis of Paramacroderoides is amended to incorporate features found in the new species. This is the first record of a species of Paramacroderoides from the spotted gar and the first record from any host in Mississippi. Comparison of more than 2,300 base-pair sequences of nuclear rDNA (partial 18S, complete ITS region, and partial 28S) of 3 specimens from Florida and 2 specimens from Mississippi revealed only 1 base-pair difference between specimens collected in the 2 areas from the 2 host species. Sequence comparison between P. kinsellai n. sp. and previously published sequences of 3 Macroderoides species revealed that the levels of divergence between members of Paramacroderoides and Macroderoides are not much higher than levels of divergence among some species of Macroderoides.

Phylogenetic Position of Creptotrema funduli in the Allocreadiidae Based on Partial 28S rDNA Sequences

Abstract: The infrequently reported allocreadiid digenean Creptotrema funduli Mueller, 1934 is documented from the blackstripe topminnow, Fundulus notatus (Cyprinodontiformes: Fundulidae), in the headwaters of the Biloxi River, Harrison County, Mississippi. Specimens from Mississippi were compared with the type material from Fundulus diaphanus menona from Oneida Lake, New York, and no substantial difference was found. A fragment of ribosomal DNA, comprising a short portion of the 3′ end of 18S nuclear rDNA gene, internal transcribed spacer (ITS) genes (including ITS1, 5.8S, and ITS2), and the 5′ end of the 28S gene including variable domains D1–D3 was sequenced for the species. A portion of the 28S rDNA gene from C. funduli, plus similar fragments from 8 other allocreadiids and the callodistomatid Prosthenhystera sp., were aligned and subjected to maximum likelihood and Bayesian inference analyses. Resulting phylogenetic trees were derived from the analyses and used to estimate the relationship of Creptotrema Travassos, Artigas, and Pereira, 1928 with other allocreadiids. Creptotrema was found to be closely related to Megalogonia Surber, 1928 and 3 Neotropical genera, i.e., Wallinia Pearse, 1920, Creptotrematina Yamaguti, 1954, and Auriculostoma Scholz, Aguirre-Macedo, and Choudhury, 2004. No molecular data were available for species in Creptotrema prior to this study, so the ITS1, 5.8S, and ITS2 genes have been made available for comparative studies involving neotropical species in the genus.

Redescription and phylogenetic relationships of Euparyphium capitaneum Dietz, 1909, the type-species of Euparyphium Dietz, 1909 (Digenea: Echinostomatidae)

Euparyphium capitaneum Dietz, 1909, the type-species of the genus Euparyphium Dietz, 1909, is described on the basis of material collected from the type-host Anhinga anhinga (L.) from Pascagoula River, which drains into the northern coast of the Gulf of Mexico. Combination of light and scanning electron microscopy observations of freshly collected and properly fixed specimens in our study has allowed us to provide novel information on the morphology and topology of the reproductive systems and other morphological features of the species. A Bayesian inference analysis based on the newly-obtained partial sequence of the nuclear 28S rRNA gene for E. capitaneum and 24 previously published sequences from the superfamily Echinostomatoidea Looss, 1899 provided evidence supporting the distinct status of the genera Euparyphium and Isthmiophora Lühe, 1909.

Erection of the haploporid genus Litosaccus n. g. and its phylogenetic relationship within the Haploporidae Nicoll, 1914

Litosaccus n. g. is erected for Paralecithobotrys brisbanensis Martin, 1974 n. comb. for which an amended description is given. The new genus is morphologically similar to the haploporine Lecithobotrys Looss, 1902 but with a more elongate and cylindrical body; an infundibuliform oral sucker; a thin-walled hermaphroditic sac; a shallow genital atrium; and unequal, cylindrical and elongated caeca. It also resembles Pseudolecithobotrys Blasco-Costa, Gibson, Balbuena, Raga & Kostadinova, 2009, but the only member of that genus has a hermaphroditic sac that is twice the length of the ventral sucker, a hermaphroditic duct with intensely-staining cuboidal cells, an elongate testis, and single or paired caeca. A Bayesian inference analysis of partial 28S rDNA sequences of L. brisbanensis and 24 other haploporoids revealed that L. brisbanensis grouped with other haploporines and placed Intromugil Overstreet & Curran, 2005 in a clade with the chalcinotrematine Saccocoelioides Szidat, 1954 rather than the other seven tested waretrematine species. This analysis represents the first phylogenetic study of the Haploporidae Nicoll, 1914 that incorporates a haploporine from outside of the Mediterranean Sea.