From the Worm in a Bottle of Mezcal: iDNA Confirmation of a Leech Parasitizing the Antillean Manatee.

Invertebrate-derived ingested DNA (iDNA) is quickly proving to be a valuable, non-invasive tool for monitoring vertebrate species of conservation concern. Using the DNA barcoding locus, we successfully identified both the blood-feeding leech Haementeria acuecueyetzin and its blood meal-the latter is shown to be derived from the Caribbean manatee, Trichechus manatus . DNA amplification was successful despite the fact that the specimen was fixed in Mezcal (a beverage distilled from agave). We report the first confirmed case of a leech feeding on a manatee, the first record of H. acuecueyetzin for the State of Chiapas and, to our knowledge, the first case of successful DNA amplification of a biological sample fixed in Mezcal other than the caterpillar "worms" more commonly found in that beverage.

Validating Livanow: molecular data agree that leeches, Branchiobdellidans, and Acanthobdella peledina form a monophyletic group of oligochaetes.

To investigate the phylogenetic relationships of leeches, branchiobdellidans, and acanthobdellidans, whole nuclear 18S rDNA and over 650 bp of mitochondrial cytochrome c oxidase subunit I were acquired from 101 annelids, including 36 leeches, 18 branchiobdellidans, Acanthobdella peledina, as well as 28 oligochaetes and combined with homologous data for 17 polychaete outgroup taxa. Parsimony analysis of the combined aligned dataset supported monophyly of leeches, branchiobdellidans, and acanthobdellidans in 100% of jackknife replicates. Monophyly of the oligochaete order Lumbriculida with Acanthobdellida, Branchiobdellida, and Hirudinea was supported in 84% of jackknife replicates. These results provide support for the hypotheses that leeches and branchiobdellidans are sister groups, that acanthobdellidans are sister to them, and that together with the family Lumbriculidae they all constitute a clade within Oligochaeta. Results support synonymy of the classes Clitellata and the more commonly used Oligochaeta. Leeches branchiobdellidans, and acanthobdellidans should be regarded as orders equal to their closest relatives, the order Lumbriculida.

Transmission of Haemogregarina balli from painted turtles to snapping turtles through the leech Placobdella ornata.

Six leeches (Placobdella ornata) were allowed to feed on a painted turtle (Chrysemys picta marginata) infected with Haemogregarina balli and subjected to a period of diapause before being allowed to feed on 2 laboratory-reared snapping turtles (Chelydra serpentina). Weekly examination of thin blood films revealed infections of the turtles at 130 days postfeeding. These observations provide support for broad host specificity of hemogregarine parasites of chelonians.

Recent advances in our knowledge of the Myxozoa.

In the last few years two factors have helped to significantly advance our understanding of the Myxozoa. First, the phenomenal increase in fin fish aquaculture in the 1990s has lead to the increased importance of these parasites; in turn this has lead to intensified research efforts, which have increased knowledge of the development, diagnosis. and pathogenesis of myxozoans. The hallmark discovery in the 1980s that the life cycle of Myxobolus cerebralis requires development of an actinosporean stage in the oligochaete. Tubifex tubifex, led to the elucidation of the life cycles of several other myxozoans. Also, the life cycle and taxonomy of the enigmatic PKX myxozoan has been resolved: it is the alternate stage of the unusual myxozoan, Tetracapsula bryosalmonae, from bryozoans. The 18S rDNA gene of many species has been sequenced, and here we add 22 new sequences to the data set. Phylogenetic analyses using all these sequences indicate that: 1) the Myxozoa are closely related to Cnidaria (also supported by morphological data); 2) marine taxa at the genus level branch separately from genera that usually infect freshwater fishes; 3) taxa cluster more by development and tissue location than by spore morphology; 4) the tetracapsulids branched off early in myxozoan evolution, perhaps reflected by their having bryozoan, rather than annelid hosts; 5) the morphology of actinosporeans offers little information for determining their myxosporean counterparts (assuming that they exist); and 6) the marine actinosporeans from Australia appear to form a clade within the platysporinid myxosporeans. Ribosomal DNA sequences have also enabled development of diagnostic tests for myxozoans. PCR and in situ hybridisation tests based on rDNA sequences have been developed for Myxobolus cerebralis, Ceratomyxa shasta, Kudoa spp., and Tetracapsula bryosalmonae (PKX). Lectin-based and antibody tests have also been developed for certain myxozoans, such as PKX and C. shasta. We also review important diseases caused by myxozoans, which are emerging or re-emerging. Epizootics of whirling disease in wild rainbow trout (Oncorhynchus mykiss) have recently been reported throughout the Rocky Mountain states of the USA. With a dramatic increase in aquaculture of fishes using marine netpens, several marine myxozoans have been recognized or elevated in status as pathological agents. Kudoa thyrsites infections have caused severe post-harvest myoliquefaction in pen-reared Atlantic salmon (Salmo salar), and Ceratomyxa spp., Sphaerospora spp., and Myxidium leei cause disease in pen-reared sea bass (Dicentrarchus labrax) and sea bream species (family Sparidae) in Mediterranean countries.

Molecular phylogeny of the other tissue coccidia: Lankesterella and Caryospora.

Nearly complete sequences were obtained from the 18S rDNA genes of Eimeria falciformis (the type species of the genus), Caryospora bigenetica, and Lankesterella minima. Two clones of the rDNA gene from C. higenetica varied slightly in primary structure. Parsimony-based and maximum likelihood phylogenetic reconstructions with a number of other apicomplexan taxa support 2 major clades within the Eucoccidiorida, i.e., the isosporoid coccidia (consisting of Toxoplasma, Neospora, Isospora [in part], and Sarcocystis spp.) and a second clade containing Lankesterella and Caryospora spp., as well as the eimeriid coccidia (Cyclospora, Isospora [in part], and Eimeria spp.). Our observations suggest that Caryospora spp. may not belong in the family Eimeriidae but rather may be allied with the family Lankesterellidae with which they share molecular and life history similarities. This may be a third lineage of coccidian parasites that has independently evolved a unique heteroxenous transmission strategy.

Phylogeny of the leech family Glossiphoniidae based on mitochondrial gene sequences and morphological data.

The phylogenetic relationships of the Glossiphoniidae (Rhynchobdellida) were investigated using morphological characters and the mitochondrial genes cytochrome c oxidase subunit I and nicotinamide adenine dinucleotide dehydrogenase subunit 1. Thirty-five taxa representing 10 of the 23 currently recognized glossiphoniid genera were sampled, including more than 70% of known North American species, as well as others from Europe, South America, Africa, and a species endemic to Lake Baikal. Outgroup taxa included species from the Piscicolidae and Ozobranchidae. Cladistic analysis resulted in 1 most-parsimonious tree. Subfamily distinctions, i.e., Haementeriinae, Theromyzinae, and Glossiphoniinae, that have been based on eye morphology and reproductive biology are not corroborated. Results also provide insights into several problematic genus-level classifications. For example, relationships of Placobdella and Haementeria are clarified and elimination of Desserobdella may be necessary. Bloodfeeding from vertebrates is seen to be a primitive characteristic that has been lost twice within the clade. The hypothesis that the biannulate leech, Oligobdella biannulata, represents an important transitional form is re-evaluated in a phylogenetic context.

Higher level relationships of leeches (Annelida: Clitellata: Euhirudinea) based on morphology and gene sequences.

The evolutionary patterns of divergence of seven euhirudinean families were investigated by cladistic analysis of 33 euhirudinean species. Oligochaetes, Acanthobdella peledina, and branchiobdellidans were included as outgroup taxa. Cladistic analysis employed 1.8 kb of nuclear 18S ribosomal DNA and 651 bp of mitochondrial cytochrome c oxidase subunit I in addition to morphological data. The use of two molecular data sets, one nuclear gene and one mitochondrial gene, as well as morphological data combined historical information evolving under a variety of different constraints and therefore was less susceptible to the biases that could confound the use of only one type of data. Results suggest that the nuclear 18S rDNA gene yields a meaningful historical signal for determining higher level relationships. The more rapidly evolving CO-I gene was informative for recent or local areas of the evolutionary hypothesis, such as within-family relationships. Analyses combining all data from the three character sets yielded one most-parsimonious tree. Most of the higher taxa in recent leech systematics were well corroborated in the resulting topology. However, these results suggested paraphyly of the order Rhynchobdellida, which contradicts the presence of a proboscis as a synapomorphy. The medicinal leech family Hirudinidae was polyphyletic because Haemadipsidae and Haemopidae each have a hirudinid ancestor. In addition, all but one of the genera within the family Erpobdellidae must be either abandoned or renamed. Unusual findings included compelling evidence of historical plasticity in bloodfeeding behavior, having been lost at least four times in the course of euhirudinean evolution. Biogeographic patterns supported a New World origin for Arhynchobdellida.

Phylogeny of leeches (Hirudinea) based on mitochondrial cytochrome c oxidase subunit I.

The phylogenetic relationships of leeches were investigated for the first time using molecular data. Twenty-one species were examined representing 7 of the 10 conventionally recognized euhirudinean families. In addition, Acanthobdella peledina, a branchiobdellid, four oligochaetes, and two polychaetes were included. Cladistic analysis of the mitochondrial cytochrome c oxidase subunit I gene yielded one most-parsimonious tree. Contemporary taxonomic groupings of leeches into higher categories were found to be largely consistent with monophyletic groups identified in the analysis. Unusual relationships for which there is some precedent include a sister-group relationship between the piscicolids and Arhynchobdellida, as well as the grouping of the haemopids within Hirudinidae.

'Total evidence' refutes the inclusion of Perkinsus species in the phylum Apicomplexa.

The phylogenetic affinities of the oyster pathogen Perkinsus marinus were investigated with morphology, 18S-like rDNA data and actin sequence data. Morphological investigations revealed that Perkinsus species do not have a conoid and that other criteria which have been used to place them in the Apicomplexa are general to alveolates. When considered separately, 18S-like rDNA and actin data sets each support a closer affinity for Perkinsus marinus with the dinoflagellates. However, each of these separate analyses possess their own biases and weaknesses. Use of the phylogenetic principle of 'total evidence' in which data sets are combined in simultaneous analysis yielded a more robust hypothesis that is stable both to character and taxonomic sampling. The resulting cladogram strongly corroborates the placement of Perkinsus species with the Dinoflagellida and not with the Apicomplexa.

Phylogenetic analysis of Perkinsus based on actin gene sequences.

Perkinsus species presently are classified within the phylum Apicomplexa. This placement, however, is controversial. Based upon morphological observations and phylogenetic analyses of the small subunit ribosomal RNA gene, it has been suggested that Perkinsus may be more closely related to dinoflagellates. To reevaluate the phylogenetic position of Perkinsus, we obtained nucleotide sequence data for actin genes from Perkinsus marinus and 2 dinoflagellates, Prorocentrum minimum and Amphidinium carterae. Results indicated that there are 2 closely related actin genes in the genome of P. marinus. Phylogenetic comparisons of these actin gene fragments of P. marinus to available actin gene sequences for several ciliates and apicomplexans and to the 2 actin gene sequences from dinoflagellates obtained in this study supported a closer affinity of P. marinus to dinoflagellates than to apicomplexans.

Phylogeny of the Haplosporidia (Eukaryota: Alveolata) based on small subunit ribosomal RNA gene sequence.

The phylogenetic position of the phylum Haplosporidia was investigated with the complete small subunit rRNA gene sequences from 5 species in the phylum: Haplosporidium nelsoni and Haplosporidium costale, parasites of the eastern oyster Crassostrea virginica; Haplosporidium louisiana, a parasite of the mudcrab Panopeus herbstii; Minchinia teredinis, a parasite of shipworms (Teredo spp.) and Urosporidium crescens, a hyperparasite found in metacercariae of the trematode Megalophallus sp. in the blue crab, Callinectes sapidus. Multiple alignments of small subunit rRNA gene sequences included the 5 haplosporidian taxa and 14 taxa in the alveolate phyla Ciliophora, Dinoflagellida, and Apicomplexa. Maximum parsimony analysis placed the phylum Haplosporidia as a monophyletic group within the alveolate clade, as a taxon of equal rank with the other 3 alveolate phyla, and as a sister taxon to the clade composed of the phyla Dinoflagellida and Apicomplexa. Transversionally weighted parsimony placed the haplosporidians as a sister taxon to the ciliates. A separate analysis focused on the relationships of species in the genus Haplosporidium. Analyses were conducted with the haplosporidians as a functional ingroup, using each of the alveolate phyla individually as functional outgroups. The results indicated that species in the genus Haplosporidium do not form a monophyletic assemblage. As such, the present morphological criteria for distinguishing the genera Haplosporidium and Minchinia are insufficient.

The demise of a phylum of protists: phylogeny of Myxozoa and other parasitic cnidaria.

The notion that members of the phylum Myxozoa Grassé, 1970 do not properly belong in classifications of protists has frequently been suggested because the infective spores of these parasites are not unicellular. Systematists have failed to be decisive about myxozoan phylogenetic affinities, either finding the suggestion of a cnidarian connection to be preposterous or considering the recent suggestion of a relationship with nematodes to be an obvious failure of molecular phylogenetics. Thus, the group has remained in classifications as a protistan phylum in its own right. The ultrastructure of the development of myxozoans was critically re-examined in order to more fully explore the possibility of morphological synapomorphies with metazoan taxa. These morphological characters, in combination with small ribosomal subunit gene sequences, were used in a phylogenetic analysis in order to assess myxozoan origins. The results unequivocally support the inclusion of myxozoans as a clade of highly derived parasitic cnidarians, and as sister taxon to the narcomedusan Polypodium hydriforme. Reassessment of myxozoans as metazoans reveals terminal differentiation, typical metazoan cellular junctions, and collagen production. Their "polar capsules" are redescribed as typical nematocysts bearing atrichous isorhiza. Insofar as taxa cannot be contained within other taxa of equal rank, the phylum Myxozoa is abandoned and it is recommended that the group as a whole be removed from all protistan classifications and placed in a more comprehensive cnidarian system.

Molecular phylogenetic evidence that the phylum Haplosporidia has an alveolate ancestry.

The phylogenetic position of the phylum Haplosporidia among other protists was investigated with the complete 16S-like rRNA gene sequences from two species in the phylum: Haplosporidium nelsoni, a parasite of oysters, and Minchinia teredinis, a parasite of shipworms. Because the lack of obvious morphological homologies with other protists hampered decisions regarding taxonomic composition for sequence alignment and phylogenetic analysis, the complete sequences for these two haplosporidians were directed as search queries to the blast/ncbi.nlm.nih.gov electronic mail server. The results of this heuristic similarity search provided a basis for constructing a preliminary higher-taxonomic-level analysis comparing the haplosporidians with species from the slime molds, fungi, algae, amoebae, ciliates, dinoflagellates, and apicomplexans. Maximum parsimony yielded equivocal results, whereas transversionally weighted parsimony suggested an affinity with the alveolates (i.e., the ciliates, dinoflagellates, and apicomplexans). Multiple alignment of the two haplosporidian sequences against 17 taxa in a secondary analysis focusing on the alveolates and subsequent parsimony analysis placed the phylum Haplosporidia as a monophyletic group within the Alveolata and as a taxon of equal rank with the other three alveolate phyla. The precise placement within the Alveolata was sensitive to weighting.

Small subunit ribosomal RNA gene sequence of Minchinia teredinis (Haplosporidia: Haplosporidiidae) and a specific DNA probe and PCR primers for its detection.

Minchinia teredinis is a pathogen of wood-boring molluscs (shipworms), Teredo spp., along the middle Atlantic coast of the U.S. Genomic DNA was extracted from M. teredinis spores and small subunit (SSU) rDNA was amplified by PCR, cloned, and sequenced. The sequence of M. teredinis SSU rDNA was aligned with that of Haplosporidium nelsoni and various protists in GenBank. A 22-base oligonucleotide probe unique to M. teredinis, designated MIN702, was commercially synthesized and tested for sensitivity and specificity. In dot-blot hybridizations the probe detected 500 pg of cloned M. teredinis rDNA. The probe did not hybridize with cloned SSU rDNA of Teredo spp. or H. nelsoni. The probe was further tested for specificity with in situ hybridizations on AFA-fixed, paraffin-embedded tissue sections. The probe hybridized well with M. teredinis plasmodia and immature spores, but poorly with mature spores. The probe did not hybridize with shipworm tissue or with the haplosporidians Haplosporidium louisiana from mud crabs (Panopeus spp.) or H. nelsoni and H. costale from Crassostrea virginica. The probe and a second 18-base oligonucleotide, when used as PCR primers, amplified a 536-bp fragment of the M. teredinis SSU rRNA gene. The PCR assay was able to detect 10 fg of the cloned gene and also detected the presence of M. teredinis DNA in shipworms in which infections were observed microscopically. The 536-bp amplification product was not obtained in one Teredo sp. or in one Bankia gouldi, both categorized as uninfected after microscopic inspection. The DNA probe and PCR primers appear to be specific for M. teredinis and should be useful as diagnostic tools and for life cycle investigations.

Phylogeny of adeleid blood parasites with a partial systematic revision of the haemogregarine complex.

The phylogenetic relationships of taxa representative of blood parasitic adeleids were investigated in a cladistic analysis. Two phylogenetic analyses were performed. Monophyly of species of Haemogregarina (sensu lato) of some marine fishes with species of Haemogregarina (sensu stricto) was not supported in either analysis. A new genus, Desseria n.g., was created to accommodate these species. The historical burden placed on the genus Haemogregarina Danilewsky, 1885 as a repository for poorly known and inadequately described species is partially relieved through taxonomic revisions involving the genera Haemogregarina, and Desseria n.g.

Light and electron microscopic examination of so-called piroplasms of fishes from Atlantic Canada and systematic revision of the Haemohormiidae (Incertae sedis).

Haemohormidium terranovae infections in American plaice and infections with a similar parasite in oceanpout were examined by light and electron microscopy. All plaice were infected at the time of capture and remained infected for over 2 yr. Prevalence in oceanpout varied seasonally between 0% and 80%. Uninucleate, binucleate, and tetranucleate forms were found in both species. In addition, octonucleate stages were observed in some erythrocytes of infected plaice. The presence of DNA in parasite nuclei was confirmed. There was no evidence of any ultrastructural feature characteristic of the phylum Apicomplexa. It is proposed that the genus Haemohormidium Henry, 1910 be considered incertae sedis and the senior synonym to Haematractidium Henry, 1910.

The development of a hemogregarine of Lycodes raridens from Alaska in its definitive leech host.

Piscicolid leeches from the Bering Sea that had been removed from Lycodes raridens in 1977 were examined histologically. Developmental stages of a haemogregarine blood parasite of fish were found in the intestinal ceca and in salivary tissues. The developmental sequence, including gametogenesis and sporogony followed by intestinal merogonic development and anteriad migration, was the same as is known for Haemogregarina (sensu lato) myoxocephali of Atlantic sculpins. Merozoites with perinuclear crystalloid inclusions were found in the dorsal blood vessel, interstitially between the blood vessel and salivary cells and within both the somata and ductules of salivary cells. This confirms the importance of rhynchobdellid leech circulatory anatomy in facilitating the transmission of apicomplexan parasites. The lack of distinguishing characteristics precludes assigning a specific name to this parasite and may be indicative of frustrations that will be encountered in future revisions of the systematics of the genus Haemogregarina.

Cytopathological changes induced by Haemogregarina myoxocephali in its fish host and leech vector.

The cytopathological effects of infection with Haemogregarina myoxocephali were investigated by light and electron microscopy in its fish host, Myoxocephalus octodecemspinosus, and in its leech vector, Malmiana scorpii. Gamont-infected erythrocytes in fish appeared vacuolated and exhibited shrinkage and altered shape consistent with loss of cytoskeletal integrity. Infected intestinal epithelial cells in leeches showed changes suggestive of recruitment of host metabolic functions by the developing parasite, including hypertrophied mitochondria and accumulations of endoplasmic reticulum and secretory vesicles next to the parasitophorous vacuole.

An ultrastructural study of Brugerolleia algonquinensis gen. nov., sp. nov. (Diplomonadina; Diplomonadida), a flagellate parasite in the blood of frogs from Ontario, Canada.

A diplomonad flagellate found in the blood of frogs in Algonquin Park, Ontario is described by light and electron microscopy. Based on comparisons to earlier ultrastructural descriptions of various diplomonads, this flagellate warrants separate generic status and is consequently named Brugerolleia algonquinensis gen. nov., sp. nov. This organism measures 8.4 × 3.2 µm with the following features: paired reniform nuclei with kinetids arranged in binary axial symmetry; 3 pairs of flagella emerging from the anteriad and a pair of posteriorly directed recurrent flagella which emerge through fibrillar funnel-like structures; dimpled terminus; direct fibres posterior to the nuclei that recurve near the posterior end of the organism; concentric layers of endoplasmic reticulum; absence of mitochondria, Golgi bodies and caudal spike. The systematic position of this new genus is discussed with respect to the other diplomonads. Brugerolleia gen. nov. appears to have arisen between Octomitus spp. and Giardia spp. in the evolutionary scheme proposed by Brugerolle.