Scott D. Snyder

Phylogenetic and biogeographical relationships among some holarctic frog lung flukes (Digenea: Haematoloechidae)

A phylogenetic study of 8 North American and European species of frog lung flukes belonging to Haematoloechus was conducted using approximately 850 to 1,000 bases of the internal transcribed spacer region (ITS 1 + 5.8S + ITS 2) and 1,250 bases of the large subunit (LSU) of the nuclear ribosomal DNA. Adequate phylogenetic resolution could not be obtained from 5.8S or ITS 2 data. Analysis of ITS 1 data produced 2 equally parsimonious trees that differed only in the position of Haematoloechus breviplexus relative to H. medioplexus and H. varioplexus. Single, identical trees were produced by analysis of both LSU sequence data and a data set comprised of all ITS and LSU data. All trees demonstrated 3 distinct evolutionary lineages within the Holarctic Haematoloechus examined. The results confirmed the taxonomic validity of H. abbreviatus and demonstrated that the presence or absence of extracecal uterine loops is not a character meaningful to the recognition of evolutionary lineages or differentiation of genera. Examination of ITS sequence data revealed almost no intraspecific variation within 5 species of Haematoloechus and demonstrated an approximately 150-base indel common to the North American H. longiplexus and the European H. asper. Two of 3 clades revealed by the phylogenetic analyses are comprised of both European and North American species, indicating that lineages of Haematoloechus arose before the breakup of Laurasia and radiated after Eurasia and North America split. Within each of 3 evolutionary lineages, members share similar patterns of arthropod host specificity distinct from patterns found in the other lineages. This suggests that second intermediate host specificity may be a trait that has been conserved through evolutionary time.

AN APPROACH TO REVEALING BLOOD FLUKE LIFE CYCLES, TAXONOMY, AND DIVERSITY: PROVISION OF KEY REFERENCE DATA INCLUDING DNA SEQUENCE FROM SINGLE LIFE CYCLE STAGES

Revealing diversity among extant blood flukes, and the patterns of relationships among them, has been hindered by the difficulty of determining if specimens described from different life cycle stages, hosts, geographic localities, and times represent the same or different species. Persistent collection of all available life cycle stages and provision of exact collection localities, host identification, reference DNA sequences for the parasite, and voucher specimens eventually will provide the framework needed to piece together individual life cycles and facilitate reconciliation with classical taxonomic descriptions, including those based on single life cycle stages. It also provides a means to document unique or rare species that might only ever be recovered from a single life cycle stage. With an emphasis on the value of new information from field collections of any available life cycle stages, here we provide data for several blood fluke cercariae from freshwater snails from Kenya, Uganda, and Australia. Similar data are provided for adult worms of Macrobilharzia macrobilharzia and miracidia of Bivitellobilharzia nairi. Some schistosome and sanguinicolid cercariae that we recovered have peculiar morphological features, and our phylogenetic analyses (18S and 28S rDNA and mtDNA CO1) suggest that 2 of the new schistosome specimens likely represent previously unknown lineages. Our results also provide new insights into 2 of the 4 remaining schistosome genera yet to be extensively characterized with respect to their position in molecular phylogenies, Macrobilharzia and Bivitellobilharzia. The accessibility of each life cycle stage is likely to vary dramatically from one parasite species to the next, and our examples validate the potential usefulness of information gleaned from even one such stage, whatever it might be.

The Nematode Genus Rhabdias (Nematoda: Rhabdiasidae) from Amphibians and Reptiles of the Nearctic

Abstract Data on the representatives of the nematode family Rhabdiasidae in the Nearctic are summarized from the literature, study of new material, and examination of museum specimens. A description of Rhabdias tarichae sp. nov. and redescriptions of Rhabdias joaquinensis, Rhabdias ambystomae, Rhabdias ranae, and Rhabdias americanus are given. New host and geographical records are provided for several Rhabdias species. Rhabdias sp., probably representing another species new to science, was found among museum specimens from the California newt Taricha torosa; however, the poor state of the specimens did not permit description. Keys for determination of Nearctic Rhabdias species are provided, and the applicability of some morphological characters for differentiation of Rhabdias species is discussed.

Evolutionary Constraints on Population Structure: The Parasites of Fundulus zebrinus (Pisces: Cyprinodontidae) in the South Platte River of Nebraska

Population and community descriptor values (parasites per host, prevalence per parasite species, variance/mean ratios, species density, and diversity indices) for the 7-species parasite community of 61 relatively homogeneous samples of Fundulus zebrinus (Pisces: Cyprinodontidae) in the South Platte River of Nebraska, U.S.A., taken over a 14-yr period, are reported. South Platte River streamflow fluctuates over 2 orders of magnitude on several time scales-monthly, annually, and over multiple year wet-dry cycles. Relatively homogeneous sampling of a single host species with several parasite species provided a system that allowed assessment of the contribution of evolved parasite life cycles to population structure in an everchanging environment. No significant negative species-to-species associations were observed. Species abundance, order of abundance, and diversity were affected most strongly by streamflow, with high water reducing prevalence and abundance of larval trematode parasites. Each parasite species had its characteristic long- and short-term patterns of variation in population descriptor values, with mostly long-term stability superimposed on sometimes extreme short-term fluctuations of descriptor values. The differences in these characteristic patterns were considered products primarily of the evolved life cycle traits and transmission mechanisms operating in the common fluctuating environment. The parasite community as a whole showed resilience, returning to preperturbation diversity following extended periods of high water.

Molecular insight into systematics, host associations, life cycles and geographic distribution of the nematode family Rhabdiasidae

Rhabdiasidae Railliet, 1915 is a globally distributed group of up to 100 known species of nematodes parasitic in amphibians and reptiles. This work presents the results of a molecular phylogenetic analysis of 36 species of Rhabdiasidae from reptiles and amphibians from six continents. New DNA sequences encompassing partial 18S rDNA, ITS1, 5.8S rDNA, ITS2 and partial 28S rDNA regions of nuclear ribosomal DNA were obtained from 27 species and pre-existing sequences for nine species were incorporated. The broad taxonomic, host and geographical coverage of the specimens allowed us to address long-standing questions in rhabdiasid systematics, evolution, geographic distribution, and patterns of host association. Our analysis demonstrated that rhabdiasids parasitic in snakes are an independent genus sister to the rest of the Rhabdiasidae, a status supported by life cycle data. Based on the combined evidence of molecular phylogeny, morphology and life cycle characteristics, a new genus Serpentirhabdias gen. nov. with the type species Serpentirhabdias elaphe (Sharpilo, 1976) comb. nov. is established. The phylogeny supports the monophyly of Entomelas Travassos, 1930, Pneumonema Johnston, 1916 and the largest genus of the family, Rhabdias Stiles and Hassall, 1905. DNA sequence comparisons demonstrate the presence of more than one species in the previously monotypic Pneumonema from Australian scincid lizards. The distribution of some morphological characters in the genus Rhabdias shows little consistency within the phylogenetic tree topology, in particular the apical structures widely used in rhabdiasid systematics. Our data suggest that some of the characters, while valuable for species differentiation, are not appropriate for differentiation among higher taxa and are of limited phylogenetic utility. Rhabdias is the only genus with a cosmopolitan distribution, but some of the lineages within Rhabdias are distributed on a single continent or a group of adjacent zoogeographical regions. Serpentirhabdias, Entomelas and Pneumonema show rather strict specificity to their host groups. The evolution of the Rhabdiasidae clearly included multiple host switching events among different orders and families of amphibians as well as switching between amphibians and squamatan reptiles. Only a few smaller lineages of Rhabdias demonstrate relatively strict associations with a certain group of hosts.

Behavioral Basis of Second Intermediate Host Specificity among Four Species of Haematoloechus (Digenea: Haematoloechidae)

Cercarial behavior patterns were examined in 4 species of frog lung flukes (Haematoloechus spp.). Cercariae of Haematoloechus complexus, Haematoloechus medioplexus, Haematoloechus longiplexus, and Haematoloechus varioplexus were exposed to 3 species of experimental arthropods and an inanimate control. The number of cercariae attached to an experimental host at 5 min postexposure was recorded. Haematoloechus longiplexus and H. complexus cercariae attached to experimental hosts at higher rates than cercariae of H. medioplexus and H. varioplexus. Cercariae of H. longiplexus attached to experimental hosts in approximately the same numbers as H. complexus, but H. longiplexus penetrated only damselfly naiads, and only at the base of the zygopteran caudal gills. Cercariae of H. complexus, a second intermediate host generalist, were able to penetrate and enter several arthropod species at the intersegmental membranes. Haematoloechus medioplexus and H. varioplexus are restricted to development in dragonfly naiads and cercariae rarely attached to and never penetrated experimental hosts. These behavioral patterns dictate the range of hosts suitable for metacercarial development of H. complexus, H. longiplexus, H. medioplexus, and H. varioplexus. The evolution of disparate patterns of behavior among the cercariae of these 4 congeners has directly affected subsequent patterns of transmission to the definitive host.

Alternative Life Cycle Strategies and Colonization of Young Anurans by Gorgoderina attenuata in Nebraska

Abstract Studies on life cycles and epizootiology of North American frog bladder flukes indicate that adult frogs become infected predominantly by ingesting tadpoles or other frogs that serve as second intermediate hosts for gorgoderid metacercariae. Other studies have indicated that newly metamorphosed frogs are rarely infected with these parasites because they are gape-limited predators that cannot feed on large intermediate hosts such as tadpoles and other frogs. We examined the role of potential intermediate hosts in the recruitment of the frog bladder fluke, Gorgoderina attenuata, to metamorphosed northern leopard frogs, Woodhouses toads, and bullfrogs from western Nebraska. We completed the life cycle of G. attenuata in the laboratory in 3 anuran species by experimentally infecting a variety of hosts. In addition, we generated and compared DNA sequence data from life cycle stages collected from a variety of naturally infected hosts. Our field and laboratory data indicate that in Nebraska G. attenuata has a truncated, 2-host life cycle that includes fingernail clams and anurans. Cercariae are ingested directly by tadpoles; unencysted juvenile worms then develop in the kidneys of tadpoles before moving to, and maturing in, the urinary bladder when tadpoles metamorphose. Additionally, G. attenuata can infect metamorphosed leopard frogs, bullfrogs, and toads when metacercariae in damselfly second intermediate hosts are ingested. These worms can also infect adult bullfrogs when they feed on other infected anurans possessing worms in their kidneys. Comparison of our material to published accounts of G. attenuata morphology and life cycles in Massachusetts suggests that previous work may have inadvertently involved 2 different species of gorgoderids. Our comparative approach to life cycle studies in different anuran life stages and multiple species of hosts suggests that tadpoles and metamorphosed anurans have favored alternative life cycle strategies in this trematode.

Second Intermediate Host-Specificity of Haematoloechus complexus and Haematoloechus medioplexus (Digenea: Haematoloechidae)

Second intermediate host-specificity was examined for 2 species of the frog lung fluke genus Haematolechus. Nine species of freshwater arthropods were exposed to cercariae of H. complexus and H. medioplexus. Metacercariae of H. complexus developed in all arthropod species used. Metacercariae of H. medioplexus developed only in anisopteran odonate naiads. This difference in host utilization may have epizootiological implications. The potential development of H. complexus in a greater number of arthropods than H. medioplexus may increase the chances of ingestion of H. complexus by an anuran host. The range of arthropods parasitized by H. complexus indicates that host-specificity of the metacercarial stage of this fluke is not restricted by the phylogeny of aquatic arthropods.

New Methods for the Collection and Preservation of Spirorchiid Trematodes and Polystomatid Monogeneans from Turtles

Abstract New techniques and protocols for the field collection and preservation of spirorchiid trematodes and polystomatid monogeneans are delineated for inclusion in existing postmortem examination protocols for the parasites of turtles. Spirorchiid trematodes are collected in body and organ washes of citrated saline solution concentrated by sedimentation in a separatory funnel. Polystomatid monogeneans are collected by precise dissection and examination of the urinary and accessory bladders, nasal and oral cavities, and the conjunctival sacs and nictitating membranes of the eye. Fixation by distilled water osmotony or heat followed by preservation and storage in 95% undenatured ethanol are recommended to produce specimens for subsequent morphological and molecular analysis.

New Genus and Species of Aporocotylidae (Digenea) from a Basal Actinopterygian, the American Paddlefish, Polyodon spathula , (Acipenseriformes: Polyodontidae) from the Mississippi Delta

Acipensericola petersoni n. gen., n. sp. (Digenea: Aporocotylidae) infects the heart of the American paddlefish Polyodon spathula (Walbaum, 1792) in the Mississippi Delta. It has robust, spike-like body spines arranged in ventrolateral transverse rows; a bowl-shaped anterior sucker centered on the mouth and having minute spines on the inner anteroventral surface only; a pharynx; an inverse U-shaped ceca extending to near the posterior body end; intercecal testes comprising a pre-ovarian testicular column plus a single testis posteriorly; an extensively lobed ovary located medially and immediately posterior to the testicular column; a spherical ootype that is intercecal and post-ovarian; a Laurers canal; and a common genital pore. The new species is the first-named aporocotylid collected from a basal actinopterygian. It resembles the chondrichthyan aporocotylids Chimaerohemecus trondheimensis, Orchispirium heterovitellatum, and Hyperandrotrema cetorhini in having an inverse U-shaped ceca, but it is morphologically most similar to the anguilliform aporocotylid Paracardicoloides yamagutii in having that feature plus a comparable anterior sucker, a single testis posteriorly, an intertesticular ovary, and a common genital pore. Sequence data for the complete small subunit ribosomal DNA (18S) do not refute its membership within Aporocotylidae nor its affinity to 1 of those aforementioned aporocotylids: A. petersoni was basal to the few teleost aporocotylids analyzed, and C. trondheimensis was the only taxon basal to A. petersoni. We regard the specimens of Spirorchis sp. previously reported from the shortnose sturgeon Acipenser brevirostrum Lesueur, 1818 as congeneric with the new species.