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A novel avian trypanosome, Trypanosoma culicavium sp. nov., isolated from Culex mosquitoes, is described on the basis of naturally and experimentally infected vectors and bird hosts, localization in the vector, morphological characters and molecular data. This study provides the first comprehensive description of a trypanosome species transmitted by mosquitoes, in which parasites form plugs and rosettes on the stomodeal valve. Trypanosomes occurred as long epimastigotes and short trypomastigotes in vectors and culture and as long trypomastigotes in birds. Transmission of parasites to bird hosts was achieved exclusively by ingestion of experimentally infected Culex mosquito females by canaries (Serinus canaria), but not by Japanese quails (Coturnix japonica), nor by the bite of infected vectors, nor by ingestion of parasites from laboratory cultures. Transmission experiments and the identity of isolates from collared flycatchers (Ficedula albicollis) and Culex mosquitoes suggests that the natural hosts of T. culicavium are insectivorous songbirds (Passeriformes). Phylogenetic analyses of small-subunit rRNA and glycosomal glyceraldehyde-3-phosphate dehydrogenase gene sequences demonstrated that T. culicavium sp. nov. is more related to Trypanosoma corvi than to other avian trypanosomes (e.g. Trypanosoma avium and Trypanosoma bennetti).

Induction of penetration gland emptying by cercariae of the bird schistosomes Trichobilharzia szidati and T. regenti employing linoleic acid, linolenic acid, praziquantel and calcium ionophore A23187 showed that both postacetabular and circumacetabular cells released their content at chosen stimulant concentrations. The gland secretions consisted of soluble and insoluble parts. The former one adhering to the ground seemed to have different saccharide composition from the glands of Schistosoma mansoni. It bound labelled saccharides, thus exhibiting lectin-like activity. Protein profiles of the latter one were identical after stimulation by all four stimulants in T. szidati. The soluble secretions contained several proteolytic enzymes; 31 kDa and 33 kDa cysteine proteases were identified in E/S products of T. szidati and T. regenti, respectively. The circumacetabular glands contained a significant amount of calcium. Immunohistochemistry revealed that the origin of E/S products after in vitro stimulation is in both penetration glands and tegumental structures. No crossreactivity was observed between the bird schistosomes and a serum raised against S. mansoni elastase.

Three strains of a trypanosomatid protozoan were isolated from the midguts of two naturally infected species of biting midges [Culicoides (Oecacta) festivipennis and Culicoides (Oecacta) truncorum] and characterized by light and electron microscopy and by molecular techniques. Morphological characteristics and sequences of the 18S rRNA, 5S rRNA, spliced leader RNA and glycosomal glyceraldehyde-3-phosphate dehydrogenase genes indicate that the studied flagellates represent a novel phylogenetic lineage within the Trypanosomatidae. Based on phylogenetic analyses, the novel endosymbiont-free, monoxenous trypanosomatid was classified as Sergeia podlipaevi gen. nov., sp. nov. Interestingly, it is closely related to another trypanosomatid species that parasitizes the sand fly Lutzomyia evansi, a blood-sucking dipteran from South America. The type strain of S. podlipaevi sp. nov., ICUL/CZ/2000/CER3, was obtained from Malpighian tubes. Of 2518 females of seven species of biting midges trapped in the Czech Republic, more than 1.5 % were infected by trypanosomatid parasites. An unrelated insect species, Culicoides (Monoculicoides) nubeculosus, was experimentally infected with S. podlipaevi, demonstrating that its host range extends to different subgenera of biting midges.

BackgroundPhlebotomine sand flies are blood-sucking insects transmitting Leishmania parasites. In bitten hosts, sand fly saliva elicits specific immune response and the humoral immunity was shown to reflect the intensity of sand fly exposure. Thus, anti-saliva antibodies were suggested as the potential risk marker of Leishmania transmission. In this study, we examined the long-term kinetics and persistence of anti-Phlebotomus papatasi saliva antibody response in BALB/c and C57BL/6 mice. We also tested the reactivity of mice sera with P. papatasi salivary antigens and with the recombinant proteins.Methodology/Principal FindingsSera of BALB/c and C57BL/6 mice experimentally bitten by Phlebotomus papatasi were tested by ELISA for the presence of anti-saliva IgE, IgG and its subclasses. We detected a significant increase of specific IgG and IgG1 in both mice strains and IgG2b in BALB/c mice that positively correlated with the number of blood-fed P. papatasi females. Using western blot and mass spectrometry we identified the major P. papatasi antigens as Yellow-related proteins, D7-related proteins, antigen 5-related proteins and SP-15-like proteins. We therefore tested the reactivity of mice sera with four P. papatasi recombinant proteins coding for most of these potential antigens (PpSP44, PpSP42, PpSP30, and PpSP28). Each mouse serum reacted with at least one of the recombinant protein tested, although none of the recombinant proteins were recognized by all sera.ConclusionsOur data confirmed the concept of using anti-sand fly saliva antibodies as a marker of sand fly exposure in Phlebotomus papatasi–mice model. As screening of specific antibodies is limited by the availability of salivary gland homogenate, utilization of recombinant proteins in such studies would be beneficial. Our present work demonstrates the feasibility of this implementation. A combination of recombinant salivary proteins is recommended for evaluation of intensity of sand fly exposure in endemic areas and for estimation of risk of Leishmania transmission.

To explain the evolution of egg colouration in open cup nesting species, a number of functions have been suggested. Recent studies focus on the role of eggshell colour as a postmating sexually selected trait of females which manipulates male parental investment. A basic prediction of this hypothesis is that egg pigmentation reflects female quality. In this study we examine whether there is a relationship between eggshell colouration and either female quality or egg quality in reed warblers. This open cup nesting species has eggs that are heavily spotted with brownish marks on a bluish-green background. We used several parameters describing female and egg quality, and measured eggshell colouration at the blunt pole and the egg centre, deriving four colour variables from colour spectrometry. To determine egg quality parameters, the third egg of each clutch was sampled and analysed. To determine female quality, females were trapped shortly after egg laying, and several morphological and a single conditional variable were determined. Additionally, a blood sample was taken to determine blood parasites (avian malaria and Trypanosoma spp.) and a faecal sample to determine intestinal parasites (Isospora spp). Our results revealed that eggshell pigmentation appears to be independent of female condition and parasites, but reflects concentrations of egg compounds such as testosterone and lysozyme. Egg colouration is also related to yolk weight and egg size. Our results further suggested that the information about colour varies depending on the position on the egg (blunt pole or egg centre). The only relationship with females was between female size (tarsus length) and egg colouration, which suggests a genetic component. We discuss reasons for the absence of a relationship between egg colouration and female quality

Monoxenous trypanosomatid Herpetomonas trimorpha sp. nov. was isolated from the digestive tract of the biting midge Culicoides truncorum (Ceratopogonidae, Diptera). This species forms three distinct morphotypes in culture: the microflagellate promastigote, the small promastigote and the long promastigote. The last form is unique for the newly described species. Phylogenetic analyses of SSU rRNA and glycosomal glyceraldehyde phosphate dehydrogenase genes showed that H. trimorpha sp. nov. is the closest relative of Herpetomonas ztiplika, another monoxenous trypanosomatid isolated from biting midges. However, morphological and randomly amplified polymorphic DNA analyses confirmed that H. trimorpha sp. nov. is distinct from H. ztiplika. INTRODUCTIONThe genus Herpetomonas comprises monoxenous trypanosomatids, which mainly parasitize the digestive tract of muscid Diptera (Wallace, 1966;Molyneux, 1977;McGhee & Cosgrove, 1980;Podlipaev, 1990). However, herpetomonads have also been isolated from plants and even mammals (Morsy et al., 1988; Catarino et al., 2001;Fiorini et al., 2001;Podlipaev et al., 2004a;Marín et al., 2007). The classification of the genus Herpetomonas is problematic and, similar to most other monoxenous trypanosomatid genera, the genus is polyphyletic (Camargo et al., 1992;Teixeira et al., 1997;Hollar et al., 1998;Hughes & Piontkivska, 2003;Podlipaev et al., 2004a). Therefore, a major taxonomic revision of 'lower' trypanosomatids is needed (see Bulat et al., 1999;Podlipaev, 2000Podlipaev, , 2001Maslov et al., 2001;Merzlyak et al., 2001;Momen, 2001;Yurchenko et al., 2008Yurchenko et al., , 2009).Over 30 herpetomonad species have been described at the time of writing (McGhee & Cosgrove, 1980;Podlipaev, 1990) , 2008, 2009). Indeed, the classical diagnostic character, i.e. the presence of promastigotes and opisthomastigotes, is no longer considered to be sufficient for assigning a trypanosomatid to the genus Herpetomonas (Camargo et al., 1992;Podlipaev et al., 2004b). On the other hand, some trypanosomatids were described solely on the basis of their host specificity, following the 'one host -one parasite' paradigm. However, this approach was not found universally applicable as single trypanosomatid species have been shown to infect more host species and vice versa (Bulat et al., 1999;Podlipaev, 2003;Podlipaev et al., 2004a;Svobodová et al., 2007). Recently, the most promising approach for species description seems to be a combination of several methods including molecular phylogenetics (Batistoti et al., 2001;Yurchenko et al., 2006;Svobodová et al., 2007).Here, based on morphology, ultrastructure, phylogenetic analysis of SSU rRNA and glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) genes, and randomly amplified polymorphic DNA (RAPD) analysis, we describe Herpetomonas trimorpha sp. nov. as a parasite of the intestinal tract of biting midges (Ceratopogonidae), closely related to Herpetomonas ztiplika. The main feature of the novel species is the ability to form three distinct morphotypes in cu...