The white grunt, Haemulon plumierii (Lacepède, 1801) as paratenic and definitive host of two acanthocephalan species, with the description of a new species of Dollfusentis (Palaeacanthocephala: Leptohynchoididae) from the Yucatán Peninsula, Mexico.

Acanthocephalans are a group of obligate endoparasites that alternate between vertebrates and invertebrates to complete their life cycles. Occasionally, the same individual host acts as a definitive or paratenic host for different acanthocephalan species. In this study, acanthocephalans were sampled in marine fish in three localities of the Yucatán Peninsula; adults and cystacanths were recovered from the intestine and body cavity, respectively, of Haemulon plumierii from off the coast of Sisal, Yucatán. Ribosomal DNA sequences (small and large subunits) were used to test the phylogenetic position of the species of the genus Dollfusentis, whereas the mtDNA gene cox 1 was used for assessing species delimitation. The cox 1 analysis revealed an independent genetic lineage, which is recognized herein as a new species, Dollfusentis mayae n. sp. The new species is morphologically distinguished from the other six congeners by having a cylindrical proboscis armed with 22-25 longitudinal rows bearing 12 hooks each. The cystacanths were morphologically identified as Gorgorhynchus medius by having a cylindrical trunk covered with tiny irregular spines on the anterior region, and a cylindrical proboscis armed with 17-18 longitudinal rows of 21 hooks each; small and large subunit phylogenetic analyses yielded G. medius within the family Isthomosacanthidae, suggesting that Gorgorhynchus should be transferred to this family from Rhadinorhynchidae where it is currently allocated.

A molecular and ecological study of Macracanthorhynchus ingens (von Linstow, 1879) (Acanthocephala: Archiacanthocephala), in its paratenic and definitive hosts in southeastern Mexico and the Eastern USA.

The acanthocephalan Macracanthorhynchus ingens (von Linstow 1879) (Acanthocephala: Archiacanthocephala) is a parasite that infects the gut of carnivores (racoons, coyotes, wolves, foxes, badgers, skunks, opossum, mink and bears) as an adult and the body cavity of lizards, snakes, and frogs as a cystacanth in the Americas. In this study, adults and cystacanths of M. ingens from southeastern Mexico and southern Florida, USA, were identified morphologically by having a cylindrical proboscis armed with 6 rows of hooks each with 6 hooks. Hologenophores were used to sequence the small (SSU) and large (LSU) subunits of ribosomal DNA and cytochrome c oxidase subunit 1 (cox 1) from mitochondrial DNA. Phylogenetic analysis of the new SSU and LSU sequences of M. ingens placed them in a clade with other sequences available in GenBank identified as M. ingens. The cox 1 tree showed that the nine new sequences and six previously published sequences of M. ingens from the USA form a clade with other sequences previously identified as M. ingens from GenBank. The intraspecific genetic divergence among isolates from the Americas ranged from 0 to 2%, and in combination with the phylogenetic trees confirmed that the isolates belonged to the same species. The cox 1 haplotype network inferred with 15 sequences revealed 10 haplotypes separated from each other by a few substitutions. Rio Grande Leopard Frogs and Vaillant´s Frogs harbored cystacanths with low prevalence, 28% and 37% respectively, in Mexico. Brown Basilisks, an invasive lizard in Florida, USA, had high values of prevalence, 92% and 93% in males and females, respectively. Females harbored more cystacanths than males (0-39 vs 0-21) for unknown reasons that may, however, be related to ecological differences.

Molecular identification of a PGRMC-2 receptor in maturing oocytes of the zoonotic nematode parasite Trichinella spiralis.

We previously reported that the Trichinella nematode showed higher parasite loads in one gender than another, but also the parasite molting rate decreased when it was cultivated in the presence of progesterone. In this study we explored the hypothesis that the direct effect of progesterone on Trichinella spiralis could be mediated by a steroid-binding parasite protein. We sequenced, cloned and amplified the Cyt-domain of the progesterone receptor membrane component-2 of Trichinella spiralis (PGRMC2-Ts). Furthermore, we expressed the protein and developed an antibody to perform confocal microscopy and flow cytometry. The expression of the PGRMC2-Ts protein was exclusively detected at the oocyte and the parasite's cuticle in cross-sections of the parasite, and this expression was confirmed by western blot and flow cytometry. Molecular modeling studies and computer docking for the PGRMC2-Ts protein showed that it is potentially able to bind to progesterone, estradiol, testosterone, and dihydrotestosterone with different affinities. Furthermore, phylogenetic analysis demonstrated that T. spiralis PGRMC2 is related to a steroid-binding protein of another platyhelminth. Progesterone probably acts upon Trichinella spiralis oocytes by binding to PGRMC2-Ts. Our data showed that the PGRMC2-Ts protein is present in the parasite's oocytes, a development step that is crucial for the life cycle of the parasite. Indeed, this research might have implications in the field of host-parasite co-evolution and the sex-associated susceptibility to this infection. In a more practical matter, these results may contribute to the design of new drugs with anti-parasite effects.

Identification and functional characterization of the siRNA pathway in Taenia crassiceps by silencing Enolase A.

A gene silencing procedure on cysticerci of the taeniid cestode Taenia crassiceps is described. This is the first time this technique is reported in this species that is widely used as an animal model for human cysticercosis. Genome database searches were performed in order to find out if relevant genes involved in gene silencing and non-coding RNA processing, Argonaute and Dicer (AGO and Dcr) are present in T. crassiceps. We found three AGO and two Dcr orthologues that were designed TcAGO1, Tc2 and Tc3, as well as TcDcr1 and TcDcr2. In order to elucidate the evolutionary relationships of T. crassiceps TcAGO and TcDcr genes, separate phylogenetic analyses were carried out for each, including AGO and Dcr orthologues of other 20 platyhelminthes. Our findings showed a close phylogenetic relationship of TcAGO and TcDcr with those previously described for Echinococcus spp. Our RT-PCR studies demonstrated expression of all TcAGO and TcDcr orthologues. Our results show that the gene silencing machinery in T. crassiceps is functionally active by inducing silencing of TcEnoA (∼90%). These results clearly show that gene silencing using siRNAs can be used as a molecular methodology to study gene function in taeniid cestodes.

Morphological, molecular and histopathological characterization of Plagiorhynchus crassicollis (Acanthocephala: Plagiorhynchidae) from a neotropical shorebird in Patagonia, Argentina.

In this work we report Plagiorhynchus (Plagiorhynchus) crassicollis from Patagonia, Argentina. Specimens were collected from the small intestine of a charadriid shorebird with Neotropical distribution, the Two-banded Plover (Charadrius falklandicus). Both morphological and molecular characterization, plus pathological aspect of this species is provided. Plagiorhynchus (Plagiorhynchus) crassicollis is characterized by having a proboscis with 18-20 longitudinal rows of hooks and 11-14 hooks per row. Sequences of the small subunit (SSU) and large subunit (LSU) of the nuclear ribosomal DNA were obtained and compared with other sequences available in GenBank. Phylogenetic analyses inferred with each molecular marker consistently showed that P. (P.) crassicollis is sister taxa to Plagiorhynchus (Plagiorhynchus) aznari, a parasite of the long-billed curlew (Numenius americanus) from northern Mexico. Pathologic findings associated with the parasites include ulcerative enteritis, granulomatous inflammation, diffuse lymphocytic infiltration, serositis, and peritonitis. This record expands the host and geographical record of P. (P.) crassicollis, provide baseline information on its pathological aspects, and represents the first molecular characterization of P. crassicollis in the Neotropics.

First steps to understand the systematics of Echinorhynchidae Cobbold, 1876 (Acanthocephala), inferred through nuclear gene sequences.

Acanthocephalans of the order Echinorhynchida are one of the most diverse groups in their phylum, with approximately 470 species classified into 11 families that largely consist of parasites of freshwater, brackish and marine fishes and, sporadically, reptiles and amphibians distributed worldwide. Previous phylogenies inferred with molecular data have supported the paraphyly or polyphyly of some families, suggesting that most of them have been diagnosed based on unique combinations of characters, rather than shared derivative features. We expand the taxonomic sampling of several genera such as Acanthocephalus, Echinorhynchus and Pseudoacanthocephalus of Echinorhynchidae from diverse biogeographical zones in the Americas, Europe and Asia with the aim of testing the monophyly of the family by using two molecular markers. Sequences from small (SSU) and large (LSU) subunits of ribosomal DNA were obtained for six species representing the genera Acanthocephalus and Echinorhynchus from the Neotropical, Nearctic, Palearctic and Oriental regions. These sequences were aligned with other sequences available in the GenBank dataset from Echinorhynchidae. Phylogenetic trees inferred with the combined (SSU + LSU) and the individual data sets consistently placed the genera Acanthocephalus, Pseudoacanthocephalus and Echinorhynchus into three independent lineages. Two families, Paracanthocephalidae Golvan, 1960, and Pseudoacanthocephalidae Petrochenko, 1956, were resurrected to accommodate the genera Acanthocephalus and Pseudoacanthocephalus, respectively. The species of the genus Acanthocephalus from the Nearctic, Palearctic and Oriental biogeographic regions formed a clade that was well supported. However, Acanthocephalus amini from the Neotropical region was nested inside Arhythmacanthidae. Therefore, the genus Calakmulrhynchus was created to accommodate A. amini and resolve the paraphyly of Acanthocephalus. Finally, the diagnoses of the families Echinorhynchidae and Arhythmacanthidae were amended. The molecular phylogenies should be used as a taxonomic framework to find shared derived characters (synapomorphies) and build a more robust classification scheme that reflects the evolutionary history of the acanthocephalans.

Transmission of Corynosoma australe (Acanthocephala: Polymorphidae) from fishes to South American sea lions Otaria flavescens in Patagonia, Argentina.

Acanthocephalans display a two-host life cycle that involves arthropods as intermediate hosts and vertebrates as definitive hosts. Some species also use paratenic hosts to bridge the trophic gap between both obligatory hosts. However, the relative role of these paratenic hosts in the transmission to definitive hosts has seldom been assessed quantitatively. We report on infection patterns of cystacanths of Corynosoma australe Johnston, 1937 in 20 common teleost species and the Argentine shortfin squid Illex argentinus (Castellanos) from the Patagonian shelf of Argentina. We also explore the role of different fish species in the transmission of C. australe to the most important definitive host in the area, i.e. the South American sea lion Otaria flavescens Shaw. Cystacanths of C. australe were found in all host species except Heliconus lahillei Norman, Merluccius hubbsi Marini and I. argentinus. In eight fish species, the prevalence of C. australe was > 50% and mean intensity > 4, i.e. Acanthistius patachonicus (Jenyns), Nemadactylus bergi (Norman), Paralichthys isosceles Jordan, Percophis brasiliensis Quoy & Gaimard, Prionotus nudigula Ginsburg, Scomber colias Gmelin, Raneya brasiliensis (Kaup) and Xystreurys rasile (Jordan). Two surveys on the trophic ecology of South American sea lions in the study area consistently found a generalist diet dominated by M. hubbsi, and data on the frequency of occurrence and number of other fish and cephalopod species in stomach contents strongly suggest that only R. brasiliensis may play a prominent role in the transmission of C. australe. This result raises interesting questions on the costs of paratenicity.

A novel progesterone receptor membrane component (PGRMC) in the human and swine parasite Taenia solium: implications to the host-parasite relationship.

BACKGROUND: We have previously reported that progesterone (P4) has a direct in vitro effect on the scolex evagination and growth of Taenia solium cysticerci. Here, we explored the hypothesis that the P4 direct effect on T. solium might be mediated by a novel steroid-binding parasite protein. METHODS: By way of using immunofluorescent confocal microscopy, flow cytometry analysis, double-dimension electrophoresis analysis, and sequencing the corresponding protein spot, we detected a novel PGRMC in T. solium. Molecular modeling studies accompanied by computer docking using the sequenced protein, together with phylogenetic analysis and sequence alignment clearly demonstrated that T. solium PGRMC is from parasite origin. RESULTS: Our results show that P4 in vitro increases parasite evagination and scolex size. Using immunofluorescent confocal microscopy, we detected that parasite cells showed expression of a P4-binding like protein exclusively located at the cysticercus subtegumental tissue. Presence of the P4-binding protein in cyst cells was also confirmed by flow cytometry. Double-dimension electrophoresis analysis, followed by sequencing the corresponding protein spot, revealed a protein that was previously reported in the T. solium genome belonging to a membrane-associated progesterone receptor component (PGRMC). Molecular modeling studies accompanied by computer docking using the sequenced protein showed that PGRMC is potentially able to bind steroid hormones such as progesterone, estradiol, testosterone and dihydrodrotestosterone with different affinities. Phylogenetic analysis and sequence alignment clearly demonstrated that T. solium PGRMC is related to a steroid-binding protein of Echinoccocus granulosus, both of them being nested within a cluster including similar proteins present in platyhelminths such as Schistocephalus solidus and Schistosoma haematobium. CONCLUSION: Progesterone may directly act upon T. solium cysticerci probably by binding to PGRMC. This research has implications in the field of host-parasite co-evolution as well as the sex-associated susceptibility to this infection. In a more practical matter, present results may contribute to the molecular design of new drugs with anti-parasite actions.

Identification and characterization of Taenia solium enolase as a plasminogen-binding protein.

The larval stage of Taenia solium (cysticerci) is the causal agent of human and swine cysticercosis. When ingested by the host, T. solium eggs are activated and hatch in the intestine, releasing oncospheres that migrate to various tissues and evolve into cysticerci. Plasminogen (Plg) receptor proteins have been reported to play a role in migration processes for several pathogens. This work is aimed to identify Plg-binding proteins in T. solium cysticerci and determine whether T. solium recombinant enolase (rTsEnoA) is capable of specifically binding and activating human Plg. To identify Plg-binding proteins, a 2D-SDS-PAGE ligand blotting was performed, and recognized spots were identified by MS/MS. Seven proteins from T. solium cysticerci were found capable of binding Plg: fascicilin-1, fasciclin-2, enolase, MAPK, annexin, actin, and cytosolic malate dehydrogenase. To determine whether rTsEnoA binds human Plg, a ligand blotting was performed and the results were confirmed by ELISA both in the presence and absence of εACA, a competitive Plg inhibitor. Finally, rTsEnoA-bound Plg was activated to plasmin in the presence of tPA. To better understand the evolution of enolase isoforms in T. solium, a phylogenetic inference analysis including 75 enolase amino acid sequences was conducted. The origin of flatworm enolase isoforms, except for Eno4, is independent of their vertebrate counterparts. Therefore, herein we propose to designate tapeworm protein isoforms as A, B, C, and 4. In conclusion, recombinant enolase showed a strong plasminogen binding and activating activity in vitro. T. solium enolase could play a role in parasite invasion along with other plasminogen-binding proteins.

Experimental and Theoretical Approaches To Investigate the Immunogenicity of Taenia solium-Derived KE7 Antigen.

Taenia solium cysticercosis, a parasitic disease that affects human health in various regions of the world, is preventable by vaccination. Both the 97-amino-acid-long KETc7 peptide and its carboxyl-terminal, 18-amino-acid-long sequence (GK-1) are found in Taenia crassiceps Both peptides have proven protective capacity against cysticercosis and are part of the highly conserved, cestode-native, 264-amino-acid long protein KE7. KE7 belongs to a ubiquitously distributed family of proteins associated with membrane processes and may participate in several vital cell pathways. The aim of this study was to identify the T. solium KE7 (TsKE7) full-length protein and to determine its immunogenic properties. Recombinant TsKE7 (rTsKE7) was expressed in Escherichia coli Rosetta2 cells and used to obtain mouse polyclonal antibodies. Anti-rTsKE7 antibodies detected the expected native protein among the 350 spots developed from T. solium cyst vesicular fluid in a mass spectrometry-coupled immune proteomic analysis. These antibodies were then used to screen a phage-displayed 7-random-peptide library to map B-cell epitopes. The recognized phages displayed 9 peptides, with the consensus motif Y(F/Y)PS sequence, which includes YYYPS (named GK-1M, for being a GK-1 mimotope), exactly matching a part of GK-1. GK-1M was recognized by 58% of serum samples from cysticercotic pigs with 100% specificity but induced weak protection against murine cysticercosis. In silico analysis revealed a universal T-cell epitope(s) in native TsKE7 potentially capable of stimulating cytotoxic T lymphocytes and helper T lymphocytes under different major histocompatibility complex class I and class II mouse haplotypes. Altogether, these results provide a rationale for the efficacy of the KETc7, rTsKE7, and GK-1 peptides as vaccines.

Novel morphological and molecular data for Corynosoma hannae Zdzitowiecki, 1984 (Acanthocephala: Polymorphidae) from teleosts, fish-eating birds and pinnipeds from New Zealand.

The polymorphid acanthocephalan, Corynosoma hannae Zdzitowiecki, 1984 is characterised on the basis of newly collected material from a New Zealand sea lion, Phocarctos hookeri (Gray), and long-nosed fur seal, Arctophoca forsteri (Lesson) (definitive hosts), and from Stewart Island shags, Leucocarbo chalconotus (Gray), spotted shags, Phalacrocorax punctatus (Sparrman) and yellow-eyed penguins, Megadyptes antipodes (Hombron & Jacquinot) (non-definitive hosts) from New Zealand. Specimens are described in detail and scanning electron micrographs for C. hannae are provided. Additionally, cystacanths of C. hannae are reported and described for the first time from the body cavity and mesenteries of New Zealand brill, Colistium guntheri (Hutton) and from New Zealand sole, Peltorhamphus novaezeelandiae Günther from Kaka Point, Otago in New Zealand. Partial sequence data for the mitochondrial cytochrome c oxidase 1 gene (cox1) for adults, immature specimens and cystacanths of C. hannae were obtained. Phylogenetic analyses of the newly-generated sequences and for available cox1 sequences of Corynosoma spp. revealed a close relationship between C. hannae and C. australe Johnston, 1937, both species infecting pinnipeds in the Southern Hemisphere. However, a morphological comparison of the species suggests that C. hannae mostly closely resembles C. evae Zdzitowiecki, 1984 and C. semerme (Forssell, 1904), the latter of which occurs in pinnipeds in the Northern Hemisphere.

Evolution, molecular epidemiology and perspectives on the research of taeniid parasites with special emphasis on Taenia solium.

Human cysticercosis is known since old historical times in Greece and China; however, human infections by tapeworms have accompanied human beings for more that hundred thousand years. The disease is tightly bound to poverty and lack of hygiene, and has been eradicated in developed countries, but continues being a public health problem in developing countries of Latin-American, Sub-Saharan Africa and Asia, and is also remerging in a number of non endemic countries. It is considered a neglected disease. Here we revise a number of key scientific contributions on taeniid biology that open new avenues for more effective approaches to the control of cysticercosis. The evolution of flatworms and class Cestoda is analyzed, with special emphasis on the emergence of taeniid parasites and the colonization of the human species by tapeworms. The complex molecular host-parasite interplay in this relationship as result of co-evolution between two distantly related organisms. The relevant host and parasite's factors, in the prospect of identifying species-specific molecular markers useful in epidemiological studies carried out in endemic countries. The new possibilities arising with the characterization of the genomes for several species of tapeworms, including a deeper understanding of these organisms, as well as improved tools for diagnosis, vaccination and drug treatment. The need to revise the current control and management strategies for this tropical neglected disease.

A helminth cestode parasite express an estrogen-binding protein resembling a classic nuclear estrogen receptor.

The role of an estrogen-binding protein similar to a known mammalian estrogen receptor (ER) is described in the estradiol-dependent reproduction of the helminth parasite Taenia crassiceps. Previous results have shown that 17-ß-estradiol induces a concentration-dependent increase in bud number of in vitro cultured cysticerci. This effect is inhibited when parasites are also incubated in the presence of an ER binding-inhibitor (tamoxifen). RT-PCR assays using specific oligonucleotides of the most conserved ER sequences, showed expression by the parasite of a mRNA band of molecular weight and sequence corresponding to an ER. Western blot assays revealed reactivity with a 66 kDa protein corresponding to the parasite ER protein. Tamoxifen treatment strongly reduced the production of the T. crassiceps ER-like protein. Antibody specificity was demonstrated by immunoprecipitating the total parasite protein extract with anti-ER-antibodies. Cross-contamination by host cells was discarded by flow cytometry analysis. ER was specifically detected on cells expressing paramyosin, a specific helminth cell marker. Parasite cells expressing the ER-like protein were located by confocal microscopy in the subtegumental tissue exclusively. Analysis of the ER-like protein by bidimensional electrophoresis and immunoblot identified a specific protein of molecular weight and isoelectric point similar to a vertebrates ER. Sequencing of the spot produced a small fragment of protein similar to the mammalian nuclear ER. Together these results show that T. crassiceps expresses an ER-like protein which activates the budding of T. crassiceps cysticerci in vitro. To the best of our knowledge, this is the first report of an ER-like protein in parasites. This finding may have strong implications in the fields of host-parasite co-evolution as well as in sex-associated susceptibility to this infection, and could be an important target for the design of new drugs.

Progesterone induces scolex evagination of the human parasite Taenia solium: evolutionary implications to the host-parasite relationship.

Taenia solium cysticercosis is a health problem in underdeveloped and developed countries. Sex hormones are involved in cysticercosis prevalence in female and male pigs. Here, we evaluated the effects of progesterone and its antagonist RU486 on scolex evagination, which is the initial step in the development of the adult worm. Interestingly, progesterone increased T. solium scolex evagination and worm growth, in a concentration-independent pattern. Progesterone effects could be mediated by a novel T. solium progesterone receptor (TsPR), since RU486 inhibits both scolex evagination and worm development induced by progesterone. Using RT-PCR and western blot, sequences related to progesterone receptor were detected in the parasite. A phylogenetic analysis reveals that TsPR is highly related to fish and amphibian progesterone receptors, whereas it has a distant relation with birds and mammals. Conclusively, progesterone directly acts upon T. solium cysticerci, possibly through its binding to a progesterone receptor synthesized by the parasite.

Streptomyces bangladeshensis sp. nov., isolated from soil, which produces bis-(2-ethylhexyl)phthalate.

The taxonomic position of an actinomycete strain isolated from soil from Natore, Bangladesh, was examined by using a polyphasic approach. The strain, designated AAB-4(T), was assigned to the genus Streptomyces on the basis of chemical and morphological criteria. It formed Rectiflexibiles aerial hyphae that carried long chains of rounded spores. The 16S rRNA gene of strain AAB-4(T) was sequenced directly and then compared with those of previously studied streptomycetes following the generation of two phylogenetic trees by using maximum-likelihood and neighbour-joining algorithms. This confirmed the assignment of the novel strain to the genus Streptomyces. This strain showed a high level of 16S rRNA gene sequence similarity to Streptomyces thermoviolaceus, Streptomyces thermodiastaticus and Streptomyces longisporus, among others, but could be distinguished from them by phenotypic and physiological traits. This micro-organism produces bis-(2-ethylhexyl)phthalate, an antibacterial and antifungal agent. It is proposed that strain AAB-4(T) be classified as a novel species within the genus Streptomyces, as Streptomyces bangladeshensis sp. nov. (type strain, AAB-4(T)=LMG 22738(T)=NRRL B-24326(T)).

Streptomyces mexicanus sp. nov., a xylanolytic micro-organism isolated from soil.

The taxonomic position of a thermophilic actinomycete strain isolated from soil was examined using a polyphasic approach. The strain, designated CH-M-1035T, was assigned to the genus Streptomyces on the basis of chemical and morphological criteria. It formed Rectiflexibiles aerial hyphae that carried long chains of rounded, smooth spores. The almost complete nucleotide sequence of the 16S rRNA gene of strain CH-M-1035T was determined and its comparison with the 16S rDNA sequences of previously studied streptomycetes confirmed the assignment of the novel strain to the genus Streptomyces. Strain CH-M-1035T clustered with species belonging to the Streptomyces thermodiastaticus clade in the 1 6S-rDNA-based phylogenetic tree. However, the phenotypic properties of strain CH-M-1035T differed from those of the recognized species within this clade. Therefore, it is proposed that strain CH-M-1035T be classified as a novel species within the genus Streptomyces, as Streptomyces mexicanus (type strain CH-M-1035T =DSM 41796T =BM-B-384T =NRRL B-24196T).