The enigmatic RNase MRP of kinetoplastids.

The ribonucleoprotein RNase MRP is responsible for the processing of ribosomal RNA precursors. It is found in virtually all eukaryotes that have been examined. In the Euglenozoa, including the genera Euglena, Diplonema and kinetoplastids, MRP RNA and protein subunits have so far escaped detection using bioinformatic methods. However, we now demonstrate that the RNA component is widespread among the Euglenozoa and that these RNAs have secondary structures that conform to the structure of all other phylogenetic groups. In Euglena, we identified the same set of P/MRP protein subunits as in many other protists. However, we failed to identify any of these proteins in the kinetoplastids. This finding poses interesting questions regarding the structure and function of RNase MRP in these species.

Differences in growth of Trypanoplasma borreli in carp serum is dependent on transferrin genotype.

Kinetoplastid parasites require transferrin (Tf), being the main source of iron, for growth and multiplication. This group of parasites developed a unique receptor-mediated system for acquiring host Tf which bears no structural homology with the host transferrin receptor. Trypanoplasma borreli, a blood parasite of common carp, probably uses a similar mechanism to sequester iron from host transferrin. In this study, we demonstrate a critical role of Tf for parasite growth. For in vitro studies we isolated and purified Tf from carp homozygous for the D or G allele of Tf. We obtained Tf-depleted serum using specific antibodies to carp Tf and studied gene expression in vivo during T. borreli infection with Real Time-quantitative PCR. We demonstrate that T. borreli cannot survive in medium supplemented with Tf-depleted serum while reconstitution with Tf restores normal growth. The critical role of Tf for parasite survival was shown in incomplete medium (medium without serum): addition of purified Tf significantly increased parasite survival. We also demonstrate that Tf polymorphism has a significant impact on T. borreli multiplication. Cultured parasites die more quickly in an environment containing D-typed Tf, as compared to medium with G-typed Tf. Gene expression during T. borreli infection in carp did not show an acute phase response. We could, however, observe an increased transcription of Tf in the head kidney, which may be associated with an immunological function of the Tf protein.

Response of Anaerobic Protozoa to Oxygen Tension in Anaerobic System.

The effect of oxygen on anaerobic protozoa was studied in anaerobic batch reactors inoculated with sludge and protozoa cultures. Among the protozoa genera, Metopus, Brachonella, Plagiopyla, Trepomonas, and Vanella were more sensitive to oxygen compared to other genera. Protozoa genera Menoidium, Rhynchomonas, Cyclidium, Spathidium, and Amoeba were found to survive under aerobic conditions, and the growth rate was slightly higher or similar to anaerobic condition. O2 tension resulted in the loss of free and endosymbiotic methanogens in anaerobic system, while methanogens were observed inside the protozoan cysts. Survival of anaerobic protozoa declined considerably when the O2 tension exceeded 1% atm. sat. and showed chemosensory behavior in response to O2 exposure. Superoxide dismutase activity was detected in survived protozoa cells under O2 tension. Facultative anaerobic protozoa with SOD activity can provide a mechanism to overcome possible occurrence of oxygen toxicity in the treatment of wastewater in anaerobic reactor.

Lipid metabolism in insect disease vectors.

More than a third of the world population is at constant risk of contracting some insect-transmitted disease, such as Dengue fever, Zika virus disease, malaria, Chagas' disease, African trypanosomiasis, and others. Independent of the life cycle of the pathogen causing the disease, the insect vector hematophagous habit is a common and crucial trait for the transmission of all these diseases. This lifestyle is unique, as hematophagous insects feed on blood, a diet that is rich in protein but relatively poor in lipids and carbohydrates, in huge amounts and low frequency. Another unique feature of these insects is that blood meal triggers essential metabolic processes, as molting and oogenesis and, in this way, regulates the expression of various genes that are involved in these events. In this paper, we review current knowledge of the physiology and biochemistry of lipid metabolism in insect disease vectors, comparing with classical models whenever possible. We address lipid digestion and absorption, hemolymphatic transport, and lipid storage by the fat body and ovary. In this context, both de novo fatty acid and triacylglycerol synthesis are discussed, including the related fatty acid activation process and the intracellular lipid binding proteins. As lipids are stored in order to be mobilized later on, e.g. for flight activity or survivorship, lipolysis and ß-oxidation are also considered. All these events need to be finely regulated, and the role of hormones in this control is summarized. Finally, we also review information about infection, when vector insect physiology is affected, and there is a crosstalk between its immune system and lipid metabolism. There is not abundant information about lipid metabolism in vector insects, and significant current gaps in the field are indicated, as well as questions to be answered in the future.

Genome sequencing reveals metabolic and cellular interdependence in an amoeba-kinetoplastid symbiosis.

Endosymbiotic relationships between eukaryotic and prokaryotic cells are common in nature. Endosymbioses between two eukaryotes are also known; cyanobacterium-derived plastids have spread horizontally when one eukaryote assimilated another. A unique instance of a non-photosynthetic, eukaryotic endosymbiont involves members of the genus Paramoeba, amoebozoans that infect marine animals such as farmed fish and sea urchins. Paramoeba species harbor endosymbionts belonging to the Kinetoplastea, a diverse group of flagellate protists including some that cause devastating diseases. To elucidate the nature of this eukaryote-eukaryote association, we sequenced the genomes and transcriptomes of Paramoeba pemaquidensis and its endosymbiont Perkinsela sp. The endosymbiont nuclear genome is ~9.5 Mbp in size, the smallest of a kinetoplastid thus far discovered. Genomic analyses show that Perkinsela sp. has lost the ability to make a flagellum but retains hallmark features of kinetoplastid biology, including polycistronic transcription, trans-splicing, and a glycosome-like organelle. Mosaic biochemical pathways suggest extensive 'cross-talk' between the two organisms, and electron microscopy shows that the endosymbiont ingests amoeba cytoplasm, a novel form of endosymbiont-host communication. Our data reveal the cell biological and biochemical basis of the obligate relationship between Perkinsela sp. and its amoeba host, and provide a foundation for understanding pathogenicity determinants in economically important Paramoeba.

Selenoproteins of African trypanosomes are dispensable for parasite survival in a mammalian host.

The trace element selenium is found in polypeptides as selenocysteine, the 21(st) amino acid that is co-translationally inserted into proteins at a UGA codon. In proteins, selenocysteine usually plays a role as an efficient redox catalyst. Trypanosomatids previously examined harbor a full set of genes encoding the machinery needed for selenocysteine biosynthesis and incorporation into three selenoproteins: SelK, SelT and, the parasite-specific, Seltryp. We investigated the selenoproteome of kinetoplastid species in recently sequenced genomes and assessed the in vivo relevance of selenoproteins for African trypanosomes. Database mining revealed that SelK, SelT and Seltryp genes are present in most kinetoplastids, including the free-living species Bodo saltans, and Seltryp was lost in the subgenus Viannia from the New World Leishmania. Homology and sinteny with bacterial sulfur dioxygenases and sulfur transferases suggest a putative role for Seltryp in sulfur metabolism. A Trypanosoma brucei selenocysteine synthase (SepSecS) null-mutant, in which selenoprotein synthesis is abolished, displayed similar sensitivity to oxidative stress induced by a short-term exposure to high concentrations of methylglyoxal or H2O2 to that of the parental wild-type cell line. Importantly, the infectivity of the SepSecS knockout cell line was not impaired when tested in a mouse infection model and compensatory effects via up-regulation of proteins involved in thiol-redox metabolism were not observed. Collectively, our data show that selenoproteins are not required for survival of African trypanosomes in a mammalian host and exclude a role for selenoproteins in parasite antioxidant defense and/or virulence. On this basis, selenoproteins can be disregarded as drug target candidates.

HPLC-DAD phenolic profile, cytotoxic and anti-kinetoplastidae activity of Melissa officinalis.

Context Melissa officinalis subsp. inodora Bornm. (Lamiaceae) has been used since ancient times in folk medicine against various diseases, but it has not been investigated against protozoa. Objective To evaluate the activities of M. officinalis against Leishmania braziliensis, Leishmania infantum and Trypanosoma cruzi as well as its cytotoxicity in fibroblast cell line. Materials and methods The fresh leaves were chopped into 1 cm(2) pieces, washed and macerated with 99.9% of ethanol for 72 h at room temperature. Antiparasitic activity of M. officinalis was accessed by direct counting of cells after serial dilution, while the cytotoxicity of M. officinalis was evaluated in fibroblast cell line (NCTC929) by measuring the reduction of resazurin. The test duration was 24 h. High-performance liquid chromatography (HPLC) was used to characterise the extract. Results The extract at concentrations of 250 and 125 µg/mL inhibited 80.39 and 54.27% of promastigote (LC50 value = 105.78 µg/mL) form of L. infantum, 80.59 and 68.61% of L. brasiliensis (LC50 value = 110.69 µg/mL) and against epimastigote (LC50 value = 245.23 µg/mL) forms of T. cruzi with an inhibition of 54.45 and 22.26%, respectively, was observed. The maximum toxicity was noted at 500 µg/mL with 95.41% (LC50 value = 141.01 µg/mL). The HPLC analysis identified caffeic acid and rutin as the major compounds. Discussion The inhibition of the parasites is considered clinically relevant (< 500 µg/mL). Rutin and caffeic acids may be responsible for the antiprotozoal effect of the extract. Conclusion The ethanol extract of M. officinalis can be considered a potential alternative source of natural products with antileishmania and antitrypanosoma activities.

Gene Loss and Error-Prone RNA Editing in the Mitochondrion of Perkinsela, an Endosymbiotic Kinetoplastid.

UNLABELLED: Perkinsela is an enigmatic early-branching kinetoplastid protist that lives as an obligate endosymbiont inside Paramoeba (Amoebozoa). We have sequenced the highly reduced mitochondrial genome of Perkinsela, which possesses only six protein-coding genes (cox1, cox2, cox3, cob, atp6, and rps12), despite the fact that the organelle itself contains more DNA than is present in either the host or endosymbiont nuclear genomes. An in silico analysis of two Perkinsela strains showed that mitochondrial RNA editing and processing machineries typical of kinetoplastid flagellates are generally conserved, and all mitochondrial transcripts undergo U-insertion/deletion editing. Canonical kinetoplastid mitochondrial ribosomes are also present. We have developed software tools for accurate and exhaustive mapping of transcriptome sequencing (RNA-seq) reads with extensive U-insertions/deletions, which allows detailed investigation of RNA editing via deep sequencing. With these methods, we show that up to 50% of reads for a given edited region contain errors of the editing system or, less likely, correspond to alternatively edited transcripts. IMPORTANCE: Uridine insertion/deletion-type RNA editing, which occurs in the mitochondrion of kinetoplastid protists, has been well-studied in the model parasite genera Trypanosoma, Leishmania, and Crithidia. Perkinsela provides a unique opportunity to broaden our knowledge of RNA editing machinery from an evolutionary perspective, as it represents the earliest kinetoplastid branch and is an obligatory endosymbiont with extensive reductive trends. Interestingly, up to 50% of mitochondrial transcripts in Perkinsela contain errors. Our study was complemented by use of newly developed software designed for accurate mapping of extensively edited RNA-seq reads obtained by deep sequencing.

Rethinking niche evolution: experiments with natural communities of Protozoa in pitcher plants.

Classic niche theory predicts that competing species will evolve to use different resources and interact less, whereas recent niche-converge ideas predict that species evolve to use similar resources and interact more. Most data supporting niche evolution are based on observations of contemporary niche use, whereas experimental support is quite sparse. We followed the evolution of four species of Protozoa during succession in the water-filled leaves of the pitcher plant, Sarracenia purpurea, and found that evolution in multispecies systems follows a surprising pattern. Over several hundred generations, weak competitors evolved to be stronger, while strong competitors evolved to become weaker, which does not conform to expectations of either niche divergence or convergence. Evolution in this system appears to occur in response to characteristics of a suite of several competitors in the community, rather than pairwise interactions. Ecologists may need to rethink the roles of competition and evolution in structuring communities.

Protozoan growth rates on secondary-metabolite-producing Pseudomonas spp. correlate with high-level protozoan taxonomy.

Different features can protect bacteria against protozoan grazing, for example large size, rapid movement, and production of secondary metabolites. Most papers dealing with these matters focus on bacteria. Here, we describe protozoan features that affect their ability to grow on secondary-metabolite-producing bacteria, and examine whether different bacterial secondary metabolites affect protozoa similarly. We investigated the growth of nine different soil protozoa on six different Pseudomonas strains, including the four secondary-metabolite-producing Pseudomonas fluorescens DR54 and CHA0, Pseudomonas chlororaphis MA342 and Pseudomonas sp. DSS73, as well as the two nonproducers P. fluorescens DSM50090(T) and P. chlororaphis ATCC43928. Secondary metabolite producers affected protozoan growth differently. In particular, bacteria with extracellular secondary metabolites seemed more inhibiting than bacteria with membrane-bound metabolites. Interestingly, protozoan response seemed to correlate with high-level protozoan taxonomy, and amoeboid taxa tolerated a broader range of Pseudomonas strains than did the non-amoeboid taxa. This stresses the importance of studying both protozoan and bacterial characteristics in order to understand bacterial defence mechanisms and potentially improve survival of bacteria introduced into the environment, for example for biocontrol purposes.

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.

Chemosensory response of marine flagellate towards L- and D- dissolved free amino acids generated during heavy grazing on bacteria.

This study investigated the generation of dissolved free amino acids (DFAA) by the bacterivorous flagellate Rhynchomonas nasuta when feeding on abundant prey. Specifically, it examined whether this flagellate protist exhibits a chemosensory response towards those amino acids. The concentrations of glycine and the L- and D-enantiomers of glutamate, serine, threonine, alanine, and leucine were determined in co-cultures of the flagellate and bacteria. Glycine, L- and D-alanine, and L-serine were found to accumulate under these conditions in amounts that correlated positively with flagellate abundance, suggesting that protists are involved in their generation. Investigations of the chemotactic response of young and old foraging protists to the same amino acids, offered in concentrations similar to those previously generated, showed that glycine elicited the strongest attraction in both age groups. Young protists were strongly attracted to all the assayed amino acids, whereas older protists maintained a high level of attraction only for glycine. These results suggest that glycine generated by protists actively grazing in bacterially enriched patches functions as an infochemical, signaling to foraging protists the presence of available prey in the aquatic environment.

Genetic resistance of carp (Cyprinus carpio L.) to Trypanoplasma borreli: influence of transferrin polymorphisms.

In serum most of the iron molecules are bound to transferrin (Tf), which is a highly polymorphic protein in fish. Tf is an essential growth factor for mammalian trypanosomes. We performed a series of experiments with Trypanoplasma borreli to detect putative correlations between different Tf genotypes of common carp (Cyprinus carpio L.) and susceptibility to this blood parasite. Five genetically different, commercially exploited carp lines (Israelian 'D', Polish 'R2' and 'K', Ukrainian 'Ur', Hungarian 'R0') and a reference laboratory cross ('R3xR8') were challenged with T. borreli and parasitaemia measured to determine susceptibility to the parasite. Among the commercial carp lines, Israelian 'D' carp were identified as most and Polish 'R2' carp as least susceptible, and used to produce a next generation and reciprocal crosses. These progenies were challenged with T. borreli and parasitaemia measured. We demonstrated significant effects of genetic background of the carp lines on susceptibility to T. borreli. This genetic effect was preserved in a next generation. We also observed a significant male effect on susceptibility to T. borreli in the reciprocal crosses. Serum samples from a representative number of fish from two infection experiments were used for Tf genotyping by polyacrylamide gel electrophoresis (PAGE), identifying DD, DG and DF as most frequent Tf genotypes. We could detect a significant association of the homozygous DD genotype with low parasitaemia in the least susceptible 'R2' (and 'K') carp lines and the lack of a such an association in the most susceptible 'D' carp line. Upon examination of parasite growth in vitro in culture media supplemented with 3% serum taken from fish with different Tf genotypes, we could show a faster decrease in number of parasites in culture media with serum from DD-typed animals.

[Occurrence of the flagellated protozoan, Cryptobia helicis Leidy, 1846 (Kinetoplasta: Bodonea: Cryptobiidae) in the garden snail, Helix aspersa]. / Bahçe Salyangozu Helix aspersa'da Kamçili Protozoon Cryptobia helicis Leidy, 1846 (Kinetoplasta: Bodonea: Cryptobiidae)'in Bulunusu.

In this survey, the prevalence and cytological features of the flagellated protozoan, Cryptobia helicis living in the bursa copulatrix of the garden snail, Helix aspersa Müller 1774 found in the vicinity of Izmir, Turkey was investigated. The prevalence of Cryptobia helicis in garden snails collected in the spring of 2005 was found to be 68.65%. This study is the first record of the occurrence of Cryptobia helicis in the garden snail Helix aspersa found in Turkey.

Comparison of tank treatments with copper sulfate and potassium permanganate for sunshine bass with ichthyobodosis.

The biflagellated, single-celled parasite Ichthyobodo necator can cause significant losses among fish populations, particularly those cultured in tanks. Treatments of KMnO4 and CuSO4 were evaluated against a naturally occurring I. necator infestation on sunshine bass (female white bass Morone chrysops x male striped bass M. saxatilis) raised in tanks. Four-hour static treatments with 3 mg of KMnO4/L of water (2.5 mg/L above the determined KMnO4 demand) or 2 mg of CuSO4/L of water (total alkalinity = 207 mg/L; total hardness = 95 mg/L) were randomly applied to 4 tanks/treatment (23 fish/tank); the same treatments were reapplied 2 d later. Four tanks were used as positive controls. By 2 d posttreatment (after the second treatment), only 17.4% of the untreated control fish survived, and a sample of the remaining fish was heavily infested with I. necator. All remaining control fish were dead by 5 d posttreatment. The KMnO4 treatment significantly curtailed the initial mortality (survival = 92.4%) and slightly reduced the high parasite loads at 2 d posttreatment. However, fish mortalities increased dramatically over the next 3 d (survival at 5 d posttreatment = 37.5%), and parasite loads from sampled fish remained high. The CuSO4 treatment was effective in significantly lowering the parasite load (almost eliminating I. necator) and maintaining a high fish survival (87.5%) by 5 d posttreatment. The findings in this study clearly demonstrate that CuSO4 is a viable treatment for ichthyobodosis in tanks.

Hemoflagellates of Oregon marine fishes with the description of new species of Trypanosoma and Trypanoplasma.

Of 2,122 marine fishes representing 36 species collected in the northeastern Pacific Ocean in the vicinity of Newport, Oregon from 1971 to 1973, 541 individuals (25.5%) representing 8 species (22.2%) were infected with hemoflagellates. Four morphologically distinct trypanosomes and 3 distinct trypanoplasms were found in fishes collected offshore, but no hemoflagellates were observed in fishes from Yaquina Bay estuary. Trypanosoma pacifica was found in English sole Parophrys vetulus, Pacific sanddab Citharichthys sordidus, and slender sole Lyopsetta exilis, and survived in 5 other species after intraperitoneal injection. Trypanosoma gargantua was found in big skate Raja binoculata, and the leech Orientobdella confluens was able to transmit the trypanosome in experimental conditions. Trypanosoma khani n. sp. occurred in P. vetulus, petrale sole Eopsetta jordani, and Dover sole Microstomus pacificus. Trypanosoma murmanense was found in L. exilis collected from 200 m, but not in L. exilis collected from 80 m. Trypanoplasma beckeri parasitized the cabezon Scorpaenichthys marmoratus. Trypanoplasma bobolsoni n. sp. was found in E. jordani, L. exilis, and P. vetulus, and survived in 2 other species after intraperitoneal injection. A distinct, but unnamed trypanoplasm, was found in P. vetulus.

Protective immunity in fish against protozoan diseases.

The demand for and costs of producing land-based animal protein continues to escalate as the world population increases. Fish is an excellent protein, but the catch-fishery is stagnant or in decline. Intensive cage culture of fish is a viable option especially in countries with lakes/rivers and/or a long coastline; however, disease outbreaks will likely occur more frequently with cage culture. Hence protective strategies are needed, and one approach is to exploit the piscine immune system. This discussion highlights immunity (innate/natural and adaptive/acquired) in fish against three pathogenic protozoa (Amyloodinium ocellatum, Ichthyophthirius multifiliis and Cryptobia salmositica). Histone-like proteins in the mucus and skin of naturally resistant fish kill trophonts of A. ocellatum, and also may cause abnormal development of tomonts. Breeding of Cryptobia-resistant brook charrs is possible as resistance is controlled by a dominant Mendelian locus, and the parasite is lysed via the Alternative Pathway of Complement Activation. Production of transgenic Cryptobia-tolerant salmon is an option. Recovered fish are protected from the three diseases (acquired immunity). Live I. multifiliis theronts injected intraperitoneally into fish elicit protection. Also, a recombinant immoblizing-antigen vaccine against ichthyophthirosis has been developed but further evaluations are necessary. The live Cryptobia vaccine protects salmonids from infections while the DNA-vaccine stimulates production of antibodies to neutralize the disease causing factor (metalloprotease) in cryptobiosis; hence infected fish recover more rapidly.

Strains of the heterotrophic flagellate Bodo designis from different environments vary considerably with respect to salinity preference and SSU rRNA gene composition.

The morpho species Bodo designis is widespread and abundant globally in highly contrasting terrestrial and aquatic ecosystems. Whether the forms of Bodo designis from contrasting environments are conspecific, i.e. largely genetically identical, or whether they merely share the external morphology is presently not known. We examined the ability of different strains of Bodo designis isolated from different environments at different geographical sites to survive and grow at a salinity range of 0.5-45%. The Bodo designis strains from marine, freshwater, and terrestrial environments showed a different ability to cope with altered physiological conditions. Most of the tested strains were only able to tolerate a small salinity range, whereas others were able to withstand all tested salinity levels. We further examined the phylogenetic relationship between the different strains by sequencing the small subunit (SSU) rRNA gene. The resulting phylogenetic analyses suggest a huge genetic variation within Bodo designis, and also imply that Dimastigella and Rhyncomonas are developed inside Bodo designis. If the biological species concept is used, the genetic differences as well as the physiological barriers between the different strains of Bodo designis, would suggest that they should be assigned to different species.

The origin of dihydroorotate dehydrogenase genes of kinetoplastids, with special reference to their biological significance and adaptation to anaerobic, parasitic conditions.

Trypanosoma cruzi dihydroorotate dehydrogenase (DHOD), the fourth enzyme of the de novo pyrimidine biosynthetic pathway, is localized in the cytosol and utilizes fumarate as electron acceptor (fumarate reductase activity), while the enzyme from other various eukaryotes is mitochondrial membrane-linked. Here we report that DHOD-knockout T. cruzi did not express the enzyme protein and could not survive even in the presence of pyrimidine nucleosides, substrates for the potentially active salvage pathway, suggesting a vital role of fumarate reductase activity in the regulation of cellular redox balance. Cloning and phylogenetic analysis of euglenozoan DHOD genes showed that the euglenoid Euglena gracilis had a mitochondrial DHOD and that biflagellated bodonids, a sister group of trypanosomatids within kinetoplastids, harbor the cytosolic DHOD. Further, Bodo saliens, a bodonid, had an ACT/DHOD gene fusion encoding aspartate carbamoyltransferase (ACT), the second enzyme of the de novo pyrimidine pathway, and DHOD. This is the first report of this novel gene structure. These results are consistent with suggestions that an ancient common ancestor of Euglenozoa had a mitochondrial DHOD whose descendant exists in E. gracilis and that a common ancestor of kinetoplastids (bodonids and trypanosomatids) subsequently acquired a cytosolic DHOD by horizontal gene transfer. The cytosolic DHOD gene thus acquired may have contributed to adaptation to anaerobiosis in the kinetoplastid lineage and further contributed to the subsequent establishment of parasitism in a trypanosomatid ancestor. Different molecular strategies for anaerobic adaptation in pyrimidine biosynthesis, used by kinetoplastids and by euglenoids, are discussed. Evolutionary implications of the ACT/DHOD gene fusion are also discussed.

The haemoflagellate Trypanoplasma borreli induces the production of nitric oxide, which is associated with modulation of carp (Cyprinus carpio L.) leucocyte functions.

In an attempt to characterise the role of nitric oxide (NO) in immune responses of carp, carp leucocytes obtained during an acute T. borreli infection were examined, for their capacity to generate NO. In a second set of experiments the impact NO on viability of the parasite and on the modulation of functional carp leucocyte responses were tested in vitro. Both in carp head-kidneys and in the peripheral blood, the fractions of lymphoblasts among separated leucocytes were increased. However, the relative proportions of granulocytes among head-kidney leucocytes (HKL) significantly decreased during infection, whereas granulocytes appeared among peripheral blood leucocytes (PBL). The cellular dynamics of HKL and PBL of infected carp were paralleled by an enhanced spontaneous NO release in vitro. NO production was further increased after addition of viable parasites to these cultures. The hypothesis that NO had a possible role in granulocyte activation and lymphocyte proliferation in carp was supported by the reduction of mitogen-induced proliferative responses of PBL from healthy carp in the presence of NO donor substances. The negative effects of NO on lymphocyte proliferation were contrasted by enhancing effects on granulocyte functions: the inhibition of NO generation in T. borreli-stimulated HKL cultures by the l-arginine analogue L-NMMA reduced the viability of granulocytes and their phagocytic activity. Even massive amounts of nitric oxide produced by donor substances (up to 600 micromol l(-1) NO(-)(2)) caused no reduction in the numbers of viable T. borreli flagellates in vitro. Thus, in carp, T. borreli seems to induce high amounts of NO in vivo which are apparently not harmful for the parasite but which may interfere with co-ordinated interactions of activated cells aiming at the defence of the parasite.