Single and concomitant experimental infections by Endotrypanum spp. and Leishmania (Viannia) guyanensis (Kinetoplastida: Trypanosomatidae) in the neotropical sand fly Lutzomyia longipalpis (Diptera: Psychodidae)

Lutzomyia longipalpis females received single and mixed infections with Endotrypanum and Leishmania. Two biological parameters were analyzed: the percentage of infected females and the distribution of flagellates in the gut of the females. The principal comparisons were performed between (1) two strains of Endotrypanum, (2) cloned versus primary sample of one strain of Endotrypanum, (3) Endotrypanum versus Leishmania guyanensis, and (4) the pattern of flagellates behaviour by optical microscopy in females with single or mixed infection versus the identification of parasites isolated from digestive tracts by isoenzyme electrophoresis. Flagellates of Endotrypanum showed distinct patterns of infection suggesting that there is variation between and within strains. The distribution of Endotrypanum and L. guyanensis differed significantly in relation to the colonization of the stomodeal valve. In co-infection with L. guyanensis, a large number of flagellates were seen to be plentifully infecting the stomodeal valve in significantly more specimens than in females infected by Endotrypanum only. However, the electrophoretic profiles of isoenzymes of parasites recovered from all co-infected specimens corresponded to Endotrypanum. This suggests that the mere correlation sand fly infection-biochemical analysis of isolates may induce parasitological incorrect consideration.     

The Leishmania hertigi (Kinetoplastida; Trypanosomatidae) Complex and the Lizard Leishmania: Their Classification and Evidence for a Neotropical Origin of the Leishmania-Endotrypanum Clade

ABSTRACT. The relationships of the Leishmania hertigi complex and the lizard Leishmania species to the main groups of mammalian Leishmania and Endotrypanum parasites were examined. Restriction fragment length polymorphisms and sequences of small subunit ribosomal RNA genes and hybridization studies of kinetoplast DNA indicated that the L. hertigi complex was more closely related to the genus Endotrypanum than to the genus Leishmania . The lizard Leishmania species were found to be at the crown of the Leishmania tree. The data provides strong evidence for a Neotropical origin of the Endotrypanum/Leishmania clade since the parasites closest to the root of the tree are all found exclusively in the Neotropics. The evolution of the Leishmania/Endotrypanum clade in relation to the evolution of the known hosts of these parasites is discussed.     

Minicircle kDNA microheterogeneity in Endotrypanum indicate diversity within this genus

A comparison of kDNA restriction-endonuclease fragment patterns from strains representing selected Endotrypanum zymodemes was done by schizodeme analysis. As the degree of heterogeneity within mini-circles varied among species or strains of Endotrypanum, the fingerprint obtained with each of the restriction enzymes was unique for each of these parasites. The data have revealed that this trypanosomatid genus is much more complex than it was originally thought to be.     

Ubiquity of Cysteine- and Metalloproteinase Activities in a Wide Range of Trypanosomatids

We have analysed the proteinase profiles of 11 species from 7 different genera of trypanosomatids by in situ detection of enzyme activities on SDS-PAGE gels containing co-polymerized gelatin as substrate, and the use of specific proteinase inhibitors. Our survey indicates that while cysteine- and metalloproteinases are distributed ubiquitously among trypanosomatids, there are marked differences between the enzyme profiles from the monogenetic (Crithidia, Herpetomonas, Leptomonas) and digenetic (Trypanosoma, Endotrypanum, Phytomonas, Leishmania) species. The detected metalloproteinase activities, ranging in size from 50–100 kDa, partitioned into the detergent-phase after Triton X-114 extraction, while most of cysteine proteinases, of three distinct molecular mass ranges (30–50 kDa, 80–100 kDa and 116–205 kDa), partitioned into the aqueous phase. Thus, within this group of organisms, the metalloproteinase activities seem to be predominantly membrane-associated proteins. We also show that the plant parasites of the genus Phytomonas exhibit a distinctive cysteine proteinase profile that might be exploited further as a criterion for taxonomy of the genus.     

Monoclonal Antibodies Specific for Members of the Genus Endotrypanum

Twenty-six monoclonal antibodies were produced against membrane-enriched preparations of Endotrypanum schaudinni or Endotrypanum sp. promastigotes. Fifteen of these monoclonal antibodies (E1-E15) reacted only with the standard strain of E. schaudinni , M6159. Monoclonal antibodies E16-E26 were considered Endotrypanum specific; no cross reactivity was detected with any other genus of the family Trypanosomatidae (Leishmania, Trypanosoma, Leptomonas. Herpetomonas or Crithidia) by dot-blot radioimmune assay. By indirect immunofluorescence assay, the antigens recognized by Endotrypanum specific monoclonal antibodies appear to be associated with the surface of the parasite. Based on Western blot analysis, 4 antigenic molecules ranging in molecular weight from 24 kD to 160 kD were identified by monoclonal antibodies specific for the strain of E. schaudinni , M6159. Monoclonal antibodies specific for the genus Endotrypanum identified an antigen of molecular weight 48 kD as well as a diffuse component migrating with an apparent molecular weight of 64–200 kD.     

Monoclonal antibodies specific for members of the genus Endotrypanum

Twenty-six monoclonal antibodies were produced against membrane-enriched preparations of Endotrypanum schaudinni or Endotrypanum sp. promastigotes. Fifteen of these monoclonal antibodies (E1-E15) reacted only with the standard strain of E. schaudinni, M6159. Monoclonal antibodies E16-E26 were considered Endotrypanum specific; no cross reactivity was detected with any other genus of the family Trypanosomatidae (Leishmania, Trypanosoma, Leptomonas, Herpetomonas or Crithidia) by dot-blot radioimmune assay. By indirect immunofluorescence assay, the antigens recognized by Endotrypanum specific monoclonal antibodies appear to be associated with the surface of the parasite. Based on Western blot analysis, 4 antigenic molecules ranging in molecular weight from 24 kD to 160 kD were identified by monoclonal antibodies specific for the strain of E. schaudinni, M6159. Monoclonal antibodies specific for the genus Endotrypanum identified an antigen of molecular weight 48 kD as well as a diffuse component migrating with an apparent molecular weight of 64-200 kD.     

Multigene analysis of phylogenetic relationships and divergence times of primate sucking lice (Phthiraptera: Anoplura)

Cospeciation between hosts and parasites offers a unique opportunity to use information from parasites to infer events in host evolutionary history. Although lice (Insecta: Phthiraptera) are known to cospeciate with their hosts and have frequently served as important markers to infer host evolutionary history, most molecular studies are based on only one or two markers. Resulting phylogenies may, therefore, represent gene histories (rather than species histories), and analyses of multiple molecular markers are needed to increase confidence in the results of phylogenetic analyses. Herein, we phylogenetically examine nine molecular markers in primate sucking lice (Phthiraptera: Anoplura) and we use these markers to estimate divergence times among louse lineages. Individual and combined analyses of these nine markers are, for the most part, congruent, supporting relationships hypothesized in previous studies. Only one marker, the nuclear protein-coding gene Histone 3, has a significantly different tree topology compared to the other markers. The disparate evolutionary history of this marker, however, has no significant effect on topology or nodal support in the combined phylogenetic analyses. Therefore, phylogenetic results from the combined data set likely represent a solid hypothesis of species relationships. Additionally, we find that simultaneous use of multiple markers and calibration points provides the most reliable estimates of louse divergence times, in agreement with previous studies estimating divergences among species. Estimates of phylogenies and divergence times also allow us to verify the results of [Reed, D.L., Light, J.E., Allen, J.M., Kirchman, J.J., 2007. Pair of lice lost or parasites regained: the evolutionary history of anthropoid primate lice. BMC Biol. 5, 7.]; there was probable contact between gorilla and archaic hominids roughly 3 Ma resulting in a host switch of Pthirus lice from gorillas to archaic hominids. Thus, these results provide further evidence that data from cospeciating organisms can yield important information about the evolutionary history of their hosts.     

Molecular systematics of the bark lice infraorder Caeciliusetae (Insecta: Psocodea)

The phylogenetic relationships of bark lice and parasitic lice (Insecta: Psocodea) have been studied in a number of recent molecular phylogenetic analyses based on DNA sequences. Many of these studies have focused on the position of parasitic lice within the free‐living bark lice. However, fewer such studies have examined the relationships among major groups of free‐living bark lice and their implications for classification. In this study we focus on the infraorder Caeciliusetae, a large group of bark lice (?1000 species) within the suborder Psocomorpha. Using sequences of two mitochondrial and two nuclear genes, we estimated the phylogeny for relationships among the five recognized families within the infraorder Caeciliusetae. Based on the results, the sister‐group relationship and respective monophyly of Stenopsocidae and Dasydemellidae is strongly supported. Monophyly of the larger families Amphipsocidae and Caeciliusidae was not supported, although the causes of this were the placement of two distinct subfamilies (Paracaeciliinae and Calocaeciliinae). The monophyly of Asiopsocidae could not be tested because it was sampled only by one species. Based on these results and consideration of morphological characters, we propose a new classification for Caeciliusetae, recognizing six families: Amphipsocidae, Stenopsocidae, Dasydemellidae, Asiopsocidae, Paracaeciliidae and Caeciliusidae. We expect that this new classification will stabilize the higher‐level taxonomy of this group and help to identify groups in need of further work among these insects.     

Nematode parasites of fishes: recent advances and problems of their research

Although nematodes (Nematoda) belong to the most frequent and the most important parasites of fishes in the freshwater, brackish-water and marine environments throughout the world, the present knowledge of these parasites remains still incomplete, especially as to their biology and ecology, but also taxonomy, phylogeny, zoogeography, and the like. However, a certain progress in the research of fish nematodes has been achieved during recent years. An overview of some of the most important discoveries and results obtained is presented. As an example, existing problems in the taxonomy of these nematodes are shown in the dracunculoid family Philometridae (presently including 109 species in 9 genera), where they are associated mainly with some biological peculiarities of these mostly important tissue parasites. Nematodes of the Dracunculoidea as a whole remain poorly known; for example, of 139 valid species parasitizing fishes, 81 (58%) are known by females only and the males have not yet been described for members of 8 (27%) of genera. A taxonomic revision of this nematode group, based on detailed morphological, life history and molecular studies of individual species, is quite necessary; for the time being, Moravec (2006) has proposed a new classification system of dracunculoids, where, based on previous molecular studies, the Anguillicolidae is no longer listed in Dracunculoidea, but in an independent superfamily Anguillicoloidea. Important results have recently been achieved also in the taxonomy of fish nematodes belonging to other superfamilies, as well as in studies of their geographical distribution and diversity in different parts of the world and those of their biology. Opportunities for more detailed studies of fish nematodes have recently greatly improved with the use of some new methods, in particular SEM and DNA studies. There is a need to create a new classification system of these parasites reflecting phylogenetic relationships; a prerequisite for this is taxonomic revisions of different groups based on detailed studies of individual species, including mainly their morphology, biology and genetics. Further progress should concern studies on various aspects of biology, ecology and host-parasite relationships, because these data may have practical implications.     

Origin and evolution of African Polystoma (Monogenea: Polystomatidae) assessed by molecular methods

Among Polystomatidae (Monogenea), the genus Polystoma, which mainly infests neobatrachian hosts, is the most diverse and occurs principally in Africa, from where half the species have been reported. Previous molecular phylogenetic studies have shown that this genus originated in South America, and later colonised Eurasia and Africa. No mention was made on dispersal corridors between Europe and Africa or of the origin of the African Polystoma radiation. Therefore, a molecular phylogeny was inferred from ITS1 sequences of 21 taxa comprising two species from America, seven representatives from Europe and 12 from Africa. The topology of the phylogenetic tree reveals that a single event of colonisation took place from Europe to Africa and that the putative host carrying along the ancestral polystome is to be found among ancestral pelobatids. Percentage divergences estimates suggest that some presumably distinct vesicular species in unrelated South African anurans and some neotenic forms found in several distinct hosts in Ivory Coast, could, in fact, belong to two single polystome species parasitising divergent hosts. Two main factors are identified that may explain the diversity of African polystomes: (i), we propose that following some degree of generalism, at least during the juvenile stages of both hosts and parasites, distinctive larval behaviour of polystomes engenders isolation between parasite populations that precludes sympatric speciations; (ii), cospeciation events between Ptychadena hosts and their parasites are another factor of diversification of Polystoma on the African continent. Finally, we discuss the systematic status of the Madagascan parasite Metapolystoma, as well as the colonisation of Madagascar by the host Ptychadena mascareniensis.     

Molecular karyotype analysis and mapping of housekeeping genes to chromosomes of selected species complexes of Leishmania.

The molecular karyotypes for 20 reference strains of species complexes of Leishmania were determined by contour-clamped homogeneous electric field (CHEF) electrophoresis. Determination of number/position of chromosome-sized bands and chromosomal DNA locations of housekeeping genes were the two criteria used for differentiating and classifying the Leishmania species. We have established two gel running conditions for optimal separation of chromosomes, which resolved DNA molecules as large as 2,500 kilobase pairs (kb). Chromosomes were polymorphic in number (22-30) and size (200-2,500 kb) of bands among members of five complexes of Leishmania. Although each stock had a distinct karyotype, in general the differences found between strains and/or species within each complex were not clear enough for parasite identification. However, each group showed a specific number of size-concordant DNA molecules, which allowed distinction among the Leishmania complex parasites. Clear differences between the Old and New world groups of parasites or among some New World Leishmania species were also apparent in relation to the chromosome locations of beta-tubulin genes. Based on these results as well as data from other published studies the potential of using DNA karyotype for identifying and classifying leishmanial field isolates is discussed.     

Origin and higher-level relationships of psoroptidian mites (Acari: Astigmata: Psoroptidia): evidence from three nuclear genes

Phylogenic relationships of the Psoroptidia, a group of primarily parasitic mites of vertebrates, were investigated based on sequences from three nuclear genes (4.2 kb aligned) sampled from 126 taxa. Several morphological classification schemes and a recent molecular analysis, suggesting that the group may not be monophyletic were statistically rejected by newly generated molecular data, and the results are robust under a range of analytical and partition strategies. Six families Psoroptidae, Lobalgidae (mammalian parasites), Pyroglyphidae (house dust mites and parasites inside feather calamus), Turbinoptidae (upper respiratory track parasites of birds), Psoroptoididae (downy feather mites), and Epidermoptidae (skin parasites of birds) form a well-supported monophyletic group (the epidermoptid-psoroptid complex). These relationships, recovered by combined and separate analyses of all gene partitions, were previously suspected based on some morphological evidence, but evidence has been dismissed as resulting from convergence based on similar parasitic ecologies. The existence of the epidermoptid-psoroptid complex and the statistical rejection of Sarcoptoidea (the morphology-based group joining all mammal-associated mites) indicate that current classification criteria, influenced as they are by host preferences, need to be reassessed for non-pterolichoid superfamilies. However, two of our findings remain sensitive to analytical methods and assumptions: (i) the families Heterocoptidae and Hypoderatidae as the first and second closest outgroups of Psoroptidia, respectively, and (ii) the superfamily Pterolichoidea (including Freyanoidea) forming a sister clade to the remaining psoroptidian superfamilies. Our findings suggest that (i) house dust mites (Pyroglyphidae: Dermatophagoidinae) originated from a parasitic ancestor within the core of Psoroptidia, violating a basic principle of evolution that it is virtually impossible for a permanent parasite to become free-living, and (ii) there were at least two shifts from presumably avian to mammalian hosts.     

Intracellular protozoan parasites of humans: the role of molecular chaperones in development and pathogenesis

Certain kinetoplastid (Leishmania spp. and Tryapnosoma cruzi) and apicomplexan parasites (Plasmodium falciparum and Toxoplasma gondii) are capable of invading human cells as part of their pathology. These parasites appear to have evolved a relatively expanded or diverse complement of genes encoding molecular chaperones. The gene families encoding heat shock protein 90 (Hsp90) and heat shock protein 70 (Hsp70) chaperones show significant expansion and diversity (especially for Leishmania spp. and T. cruzi), and in particular the Hsp40 family appears to be an extreme example of phylogenetic radiation. In general, Hsp40 proteins act as co-chaperones of Hsp70 chaperones, forming protein folding pathways that integrate with Hsp90 to ensure proteostasis in the cell. It is tempting to speculate that the diverse environmental insults that these parasites endure have resulted in the evolutionary selection of a diverse and expanded chaperone network. Hsp90 is involved in development and growth of all of these intracellular parasites, and so far represents the strongest candidate as a target for chemotherapeutic interventions. While there have been some excellent studies on the molecular and cell biology of Hsp70 proteins, relatively little is known about the biological function of Hsp70-Hsp40 interactions in these intracellular parasites. This review focuses on intracellular protozoan parasites of humans, and provides a critique of the role of heat shock proteins in development and pathogenesis, especially the molecular chaperones Hsp90, Hsp70 and Hsp40.     

Complex interactions among a nematode parasite (Daubaylia potomaca), a commensalistic annelid (Chaetogaster limnaei limnaei), and trematode parasites in a snail host (Helisoma anceps)

Many biotic interactions can affect the prevalence and intensity of parasite infections in aquatic snails. Historically, these studies have centered on interactions between trematode parasites or between trematodes and other organisms. The present investigation focuses on the nematode parasite Daubaylia potomaca and its interactions with a commensal, Chaetogaster limnaei limnaei , and a variety of trematode species. It was found that the presence of C. l. limnaei indirectly increased the mean intensity of D. potomaca infections, apparently by acting as a restraint for various trematode parasites, particularly the rediae of Echinostoma sp. In turn, Echinostoma sp. rediae adversely affected the mean intensity of D. potomaca by their consumption of both juvenile and adult nematodes present in tissues of the snail. These organisms not only belong to 3 different phyla but occupy distinct trophic levels as well. The complex interactions among these 3 organisms in the snail host provide an excellent example of biotic interactions influencing the infection dynamics of parasites in aquatic snails.     

Biochemical differentiation in bile duct cestodes and their marsupial hosts

Isozyme electrophoresis was used to assess possible cospeciation of parasites (cestodes of the Progamotaenia festiva complex) and their hosts (Australian diprotodont marsupials) and to compare the extent of interspecific genetic diversity of the parasites and their hosts. On the basis of morphology, there are three species in the complex, although electrophoresis revealed 14 distinct genetic types, most of which were host specific, although there were three cases of apparent host switching. The evolutionary relationships among the parasites were only partially concordant with those among the hosts. Moreover, the extent of electrophoretic diversity among the parasites was much higher than that among hosts.      

Diptera vectors of avian Haemosporidian parasites: untangling parasite life cycles and their taxonomy

Haemosporida is a large group of vector-borne intracellular parasites that infect amphibians, reptiles, birds, and mammals. This group includes the different malaria parasites (Plasmodium spp.) that infect humans around the world. Our knowledge on the full life cycle of these parasites is most complete for those parasites that infect humans and, to some extent, birds. However, our current knowledge on haemosporidian life cycles is characterized by a paucity of information concerning the vector species responsible for their transmission among vertebrates. Moreover, our taxonomic and systematic knowledge of haemosporidians is far from complete, in particular because of insufficient sampling in wild vertebrates and in tropical regions. Detailed experimental studies to identify avian haemosporidian vectors are uncommon, with only a few published during the last 25 years. As such, little knowledge has accumulated on haemosporidian life cycles during the last three decades, hindering progress in ecology, evolution, and systematic studies of these avian parasites. Nonetheless, recently developed molecular tools have facilitated advances in haemosporidian research. DNA can now be extracted from vectors' blood meals and the vertebrate host identified; if the blood meal is infected by haemosporidians, the parasite's genetic lineage can also be identified. While this molecular tool should help to identify putative vector species, detailed experimental studies on vector competence are still needed. Furthermore, molecular tools have helped to refine our knowledge on Haemosporida taxonomy and systematics. Herein we review studies conducted on Diptera vectors transmitting avian haemosporidians from the late 1800s to the present. We also review work on Haemosporida taxonomy and systematics since the first application of molecular techniques and provide recommendations and suggest future research directions. Because human encroachment on natural environments brings human populations into contact with novel parasite sources, we stress that the best way to avoid emergent and reemergent diseases is through a program encompassing ecological restoration, environmental education, and enhanced understanding of the value of ecosystem services.     

Heterogeneous production of metallo-type peptidases in parasites belonging to the family Trypanosomatidae

Proteolytic enzymes play a central role in the physiology of all living organisms, participating in several metabolic pathways and in different phases of parasite-host interactions. We have identified cell-associated peptidase activities in 33 distinct flagellates, including representatives of almost all known trypanosomatid genera parasitizing insects (Herpetomonas, Crithidia, Leishmania, Trypanosoma, Leptomonas, Phytomonas, Blastocrithidia and Endotrypanum) as well as the biflagellate kinetoplastid Bodo, by using SDS-PAGE containing gelatin as co-polymerized substrate and proteolytic inhibitors. Under the alkaline pH (9.0) conditions employed, all the flagellates presented at least one peptidase, with the exception of Crithidia acanthocephali and Phytomonas serpens, which did not display any detectable proteolytic enzyme activity. All the proteolytic activities were completely inhibited by 1,10-phenanthroline, a zinc-chelating agent, putatively identifying these activities as metallo-type peptidases. EDTA and EGTA, two other metallopeptidase inhibitors, E-64 (a cysteine peptidase inhibitor), pepstatin A (an aspartyl peptidase inhibitor) and PMSF (a serine peptidase inhibitor) did not interfere with the metallopeptidase activities detected in the studied trypanosomatids. Conversely, Bodo-derived peptidases were resistant to 1,10-phenanthroline and only partially inhibited by EDTA, showing a distinct inhibition profile. Together, our data demonstrated great heterogeneity of expression of metallopeptidases in a wide range of parasites belonging to the family Trypanosomatidae.     

Phylogenetic position of the adeleorinid coccidia (Myzozoa, Apicomplexa, Coccidia, Eucoccidiorida, Adeleorina) inferred using 18S rDNA sequences

Investigating the evolutionary relationships of the major groups of Apicomplexa remains an important area of study. Morphological features and host-parasite relationships continue to be important in the systematics of the adeleorinid coccidia (suborder Adeleorina), but the systematics of these parasites have not been well-supported or have been constrained by data that were lacking or difficult to interpret. Previous phylogenetic studies of the Adeleorina have been based on morphological and developmental characters of several well-described species or based on nuclear 18S ribosomal DNA (rDNA) sequences from taxa of limited taxonomic diversity. Twelve new 18S rDNA sequences from adeleorinid coccidia were combined with published sequences to study the molecular phylogeny of taxa within the Adeleorina and to investigate the evolutionary relationships of adeleorinid parasites within the Apicomplexa. Three phylogenetic methods supported strongly that the suborder Adeleorina formed a monophyletic clade within the Apicomplexa. Most widely recognized families within the Adeleorina were hypothesized to be monophyletic in all analyses, although the single Hemolivia species included in the analyses was the sister taxon to a Hepatozoon sp. within a larger clade that contained all other Hepatozoon spp. making the family Hepatozoidae paraphyletic. There was an apparent relationship between the various clades generated by the analyses and the definitive (invertebrate) host parasitized and, to lesser extent, the type of intermediate (vertebrate) host exploited by the adeleorinid parasites. We conclude that additional taxon sampling and use of other genetic markers apart from 18S rDNA will be required to better resolve relationships among these parasites.     

Phylogenetic analysis of Hepatozoon species (Apicomplexa: Adeleorina) infecting frogs of Nova Scotia, Canada, determined by ITS-1 sequences

Species of Hepatozoon are apicomplexan parasites infecting tetrapod vertebrates and hematophagous arthropods. Two species, Hepatozoon catesbianae and Hepatozoon clamatae, have been described inhabiting the erythrocytes of bullfrogs and green frogs. A number of characteristics typically used to distinguish between members of this genus are shared between these 2 species, prompting speculation as to whether or not these organisms are in fact distinct species. To test the species distinction, bullfrogs and green frogs were captured at various sites across Nova Scotia, blood samples were collected, and DNA was extracted from samples containing parasites. The internal transcribed spacer 1 (ITS-1) from geographically diverse samples of both species was amplified by PCR, sequenced, and analyzed. ITS-1 sequences from the 2 species revealed single-nucleotide polymorphisms at 6 sites. Phylogenetic analysis of these molecular data and cytopathological features place isolates of each species in separate monophyletic groups. Comparison of the ITS-1 sequences between isolates from Nova Scotia and Ontario revealed that ITS-1 sequences of H. catesbianae from a previous study were mischaracterized as being those of H. clamatae. Phylogenetic data based on molecular variation and cytopathological features from this study provide the strongest evidence to date supporting the distinction between these 2 species.