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Trypanosomatids are obligatory parasites, some of which are responsible for important human and animal diseases, but the vast majority of trypanosomatids are restricted to invertebrate hosts. Isolation and in vitro cultivation of trypanosomatids from insect hosts enable their description, characterization, and subsequently genetic and genomic studies. However, exact nutritional requirements are still unknown for most trypanosomatids and thus very few defined media are available. This mini review provides information about the role of different ingredients, recommendations and advice on essential supplements and important physicochemical parameters of culture media with the aim of facilitating first attempts to cultivate insect-infesting trypanosomatids, with a focus on monoxenous trypanosomatids., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Background: Trypanosomatid parasites are widely distributed in nature and can have a monoxenous or dixenous life-cycle. These parasites thrive in a wide number of insect orders, some of which have an important economic and environmental value, such as bees. The objective of this study was to develop a robust and sensitive real-time quantitative PCR (qPCR) assay for detecting trypanosomatid parasites in any type of parasitized insect sample., Methods: A TaqMan qPCR assay based on a trypanosomatid-conserved region of the α-tubulin gene was standardized and evaluated. The limits of detection, sensitivity and versatility of the α-tubulin TaqMan assay were tested and validated using field samples of honeybee workers, wild bees, bumblebees and grasshoppers, as well as in the human infective trypanosomatid Leishmania major., Results: The assay showed a detection limit of 1 parasite equivalent/µl and successfully detected trypanosomatids in 10 different hosts belonging to the insect orders Hymenoptera and Orthoptera. The methodology was also tested using honeybee samples from four apiaries (n = 224 worker honeybees) located in the Alpujarra region (Granada, Spain). Trypanosomatids were detected in 2.7% of the honeybees, with an intra-colony prevalence of 0% to 13%. Parasite loads in the four different classes of insects ranged from 40.6 up to 1.1 × 10 8 cell equivalents per host., Conclusions: These results show that the α-tubulin TaqMan qPCR assay described here is a versatile diagnostic tool for the accurate detection and quantification of trypanosomatids in a wide range of environmental settings., (© 2023. The Author(s).)

In this review, we summarize the current data on development of monoxenous trypanosomatids and phytomonads in various insects. Of these, Diptera and Hemiptera are the main host groups, and, consequently, most available information concerns their parasites. Within the insect body, the midgut and hindgut are the predominant colonization sites; in addition, some trypanosomatids can invade the foregut, Malpighian tubules, hemolymph, and/or salivary glands. Differences in the intestinal structure and biology of the host determine the variety of parasites' developmental and transmission strategies. Meanwhile, similar mechanisms are used by unrelated trypanosomatids, reflecting the limited range of options to achieve the same goal., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Monoxenous (one host) trypanosomatids from insects and other invertebrates can be introduced into axenic culture relatively easily and efficiently, allowing for their transfer from the field into the laboratory. Here we describe simple methods and alternative cultivation protocols, the wider application of which will allow substantial expansion of trypanosomatids available for research.

The extreme biological diversity of Oceanian archipelagos has long stimulated research in ecology and evolution. However, parasitic protists in this geographic area remained neglected and no molecular analyses have been carried out to understand the evolutionary patterns and relationships with their hosts. Papua New Guinea (PNG) is a biodiversity hotspot containing over 5% of the world's biodiversity in less than 0.5% of the total land area. In the current work, we examined insect heteropteran hosts collected in PNG for the presence of trypanosomatid parasites. The diversity of insect flagellates was analysed, to our knowledge for the first time, east of Wallace's Line, one of the most distinct biogeographic boundaries of the world. Out of 907 investigated specimens from 138 species and 23 families of the true bugs collected in eight localities, 135 (15%) were infected by at least one trypanosomatid species. High species diversity of captured hosts correlated with high diversity of detected trypanosomatids. Of 46 trypanosomatid Typing Units documented in PNG, only eight were known from other geographic locations, while 38 TUs (~83%) have not been previously encountered. The widespread trypanosomatid TUs were found in both widely distributed and endemic/sub-endemic insects. Approximately one-third of the endemic trypanosomatid TUs were found in widely distributed hosts, while the remaining species were confined to endemic and sub-endemic insects. The TUs from PNG form clades with conspicuous host-parasite coevolutionary patterns, as well as those with a remarkable lack of this trait. In addition, our analysis revealed new members of the subfamilies Leishmaniinae and Strigomonadinae, potentially representing new genera of trypanosomatids., (Copyright © 2019 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Current understanding of flagellum/cilium length regulation focuses on a few model organisms with flagella of uniform length. Leptomonas pyrrhocoris is a monoxenous trypanosomatid parasite of firebugs. When cultivated in vitro , L. pyrrhocoris duplicates every 4.2 ± 0.2 h, representing the shortest doubling time reported for trypanosomatids so far. Each L. pyrrhocoris cell starts its cell cycle with a single flagellum. A new flagellum is assembled de novo , while the old flagellum persists throughout the cell cycle. The flagella in an asynchronous L. pyrrhocoris population exhibited a vast length variation of ∼3 to 24 μm, casting doubt on the presence of a length regulation mechanism based on a single balance point between the assembly and disassembly rate in these cells. Through imaging of live L. pyrrhocoris cells, a rapid, partial disassembly of the existing, old flagellum is observed upon, if not prior to, the initial assembly of a new flagellum. Mathematical modeling demonstrated an inverse correlation between the flagellar growth rate and flagellar length and inferred the presence of distinct, cell cycle-dependent disassembly mechanisms with different rates. On the basis of these observations, we proposed a min-max model that could account for the vast flagellar length range observed for asynchronous L. pyrrhocoris. This model may also apply to other flagellated organisms with flagellar length variation. IMPORTANCE Current understanding of flagellum biogenesis during the cell cycle in trypanosomatids is limited to a few pathogenic species, including Trypanosoma brucei , Trypanosoma cruzi , and Leishmania spp. The most notable characteristics of trypanosomatid flagella studied so far are the extreme stability and lack of ciliary disassembly/absorption during the cell cycle. This is different from cilia in Chlamydomonas and mammalian cells, which undergo complete absorption prior to cell cycle initiation. In this study, we examined flagellum duplication during the cell cycle of Leptomonas pyrrhocoris With the shortest duplication time documented for all Trypanosomatidae and its amenability to culture on agarose gel with limited mobility, we were able to image these cells through the cell cycle. Rapid, cell cycle-specific flagellum disassembly different from turnover was observed for the first time in trypanosomatids. Given the observed length-dependent growth rate and the presence of different disassembly mechanisms, we proposed a min-max model that can account for the flagellar length variation observed in L. pyrrhocoris ., (Copyright © 2019 He et al.)

Trypanosomes and leishmanias are widely known parasites of humans. However, they are just two out of several phylogenetic lineages that constitute the family Trypanosomatidae. Although dixeny - the ability to infect two hosts - is a derived trait of vertebrate-infecting parasites, the majority of trypanosomatids are monoxenous. Like their common ancestor, the monoxenous Trypanosomatidae are mostly parasites or commensals of insects. This review covers recent advances in the study of insect trypanosomatids, highlighting their diversity as well as genetic, morphological and biochemical complexity, which, until recently, was underappreciated. The investigation of insect trypanosomatids is providing an important foundation for understanding the origin and evolution of parasitism, including colonization of vertebrates and the appearance of human pathogens., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Lipid uptake and metabolism by trypanosomatid parasites from vertebrate host blood have been well established in the literature. However, there is a lack of knowledge regarding the same aspects concerning the parasites that cross the hemolymph of their invertebrate hosts. We have investigated the lipid composition and metabolism of the insect trypanosomatid Herpetomonas muscarum by 3 H- palmitic acid and phosphate ( 32 Pi) and the parasite interaction with Lipophorin (Lp) the main lipid carrying protein of insect hemolymph. Gas chromatography-mass spectrometry (GC-MS) analyses were used to identify the fatty acids and sterols composition of H.muscarum. Furthermore, we investigated the Lp binding site in the plasma membrane of parasite by Immunolocalization. We showed that H. muscarum incorporated 3H-palmitic acid and inorganic phosphate (32Pi) which were readily used as precursor molecules of lipid biosynthetic pathways. Furthermore, H. muscarum was able to take up both protein and lipid moieties of Lp which could be used as nutrient sources. Moreover, we have also demonstrated for the first time the presence of a Lp binding site in the membrane of a parasite. Such results point out the role of describing the metabolic pathways of trypanosomatids in order to provide a better understanding of parasite-host interaction peculiarities. Such studies may enhance the potential form the identification of novel chemotherapeutic targets in harmful parasites., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Insect trypanosomatids are inhabitants of the insect digestive tract. These parasites can be either monoxenous or dixenous. Plant trypanosomatids are known as insect trypanosomatids once they and are transmitted by phytophagous insects. Such parasites can be found in latex, phloem, fruits and seeds of many plant families. Infections caused by these pathogens are a major cause of serious economic losses. Studies by independent groups have demonstrated the metabolic flow of lipids from the vertebrate host to trypanosomatids. This mechanism is usually present when parasites possess an incomplete de novo lipid biosynthesis pathway. Here, we show that both insect trypanosomatids Phytomonas françai and Leptomonas wallacei incorporate (3)H-palmitic acid and inorganic phosphate. These molecules are used for lipid biosynthesis. Moreover, we have isolated the main hemolymphatic lipoprotein, Lipophorin (Lp) from Oncopeltus fasciatus, the natural insect vector of such parasites. Both parasites were able to incorporate Lp to be utilized both as a lipid and protein source for their metabolism. Also, we have observed the presence of Lp binding sites in the membrane of a parasite. In conclusion, we believe that the elucidation of trypanosomatid metabolic pathways will lead to a better understanding of parasite-host interactions and the identification of novel potential chemotherapy targets., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Several new species of trypanosomatids (Euglenozoa, Kinetoplastea, Trypanosomatidae), isolated from the intestines of Neotropical insects (Heteroptera), were genotyped on the basis of spliced leader RNA, and also defined phylogenetically using gene sequences of small subunit ribosomal RNA and glycosomal glyceraldehyde phosphate dehydrogenase. The taxonomic descriptions also included characterization using morphometry and electron microscopy. Our phylogenetic analyses placed the new species within the clade, previously designated "SE" for "Slowly Evolving" sequences of ribosomal RNA genes, a clade that also includes numerous monoxenous parasites of insects from the genera Crithidia, Leptomonas, and Wallaceina, as well as the dixenous genus Leishmania. Based on the high phylogenetic support for this clade, which is consistently recovered in all recent phylogenetic reconstructions, a proposal is put forward to recognize this natural taxon as a new subfamily, Leishmaniinae, within the family Trypanosomatidae., (© 2012 The Author(s) Journal of Eukaryotic Microbiology © 2012 International Society of Protistologists.)

Mitochondrial ND8 gene is pan-edited in most known trypanosomatid species and 5'-edited only in a few trypanosomatid species. In this work, the ND8 nucleotide sequences from three species of insect trypanosomatids ("Wallaceina" sp. Wsd, Leptomonas rigidus and Leptomonas collosoma) were obtained and compared with previously known ND8 cryptogene sequences and mature mRNA sequences from other trypanosomatid species. We found a new pattern of editing: only 18 U residues are inserted in the 5' region of the primary transcript in all investigated species and the potential start codon is encoded on the DNA level. This is the first case when a pre-edited ND8 sequence is found to contain a putative ORF. Previously, a 5'-edited ND8 cryptogene was known only from Strigomonas oncopelti, but it contained only about half of the ORF and no start codon. Here we present the ND8 gene sequence from Angomonas deanei which has similar cryptogene structure. We also show that according to 18S rRNA phylogenetic analysis, the species with the encoded ORF group together and so do species with the 5'-edited and pan-edited forms of the gene. This result sheds light on the evolution of cryptogene structure which involved multiple events of editing domain length reduction., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Three hundred and eighty-six heteropteran specimens belonging to more than 90 species captured in Ghana, Kenya and Ethiopia were examined for the presence of trypanosomatid flagellates. Of those, 100 (26%) specimens were positive for trypanosomatids and the spliced leader RNA gene sequence was obtained from 81 (80%) of the infected bugs. Its sequence-based analysis placed all examined flagellates in 28 typing units. Among 19 newly described typing units, 16 are restricted to sub-Saharan Africa, three belong to previously described species and six to typing units found on other continents. This result was corroborated by the analysis of the ssrRNA gene, sequenced for at least one representative of each major spliced leader RNA-based clade. In all trees obtained, flagellates originating from sub-Saharan Africa were intermingled with those isolated from American, Asian and European hosts, revealing a lack of geographic correlation. They are dispersed throughout most of the known diversity of monoxenous trypanosomatids. However, a complex picture emerged when co-evolution with their heteropteran hosts was taken into account, since some clades are specific for a single host clade, family or even species, whereas other flagellates display a very low host specificity, with a capacity to parasitise heteropteran bugs belonging to different genera/families. The family Reduviidae contains the widest spectrum of trypanosomatids, most likely a consequence of their predatory feeding behaviour, leading to an accumulation of a variety of flagellates from their prey. The plant pathogenic genus Phytomonas is reported here from Africa, to our knowledge for the first time. Finding the same typing units in hosts belonging to different heteropteran families and coming from different continents strongly indicates that the global diversity of the insect trypanosomatids is most likely lower than was predicted on the basis of the "one host-one parasite" paradigm. The analysis presented significantly extends the known diversity of monoxenous insect trypanosomatids and will be instrumental in building a new taxonomy that reflects their true phylogenetic relationships., (Copyright © 2012. Published by Elsevier Ltd.)

Antimicrobial peptides (AMPs) are multifunctional components of the innate systems of both insect and mammalian hosts of the pathogenic trypanosomatids Leishmania and Trypanosoma species. Structurally diverse AMPs from a wide range of organisms have in vitro activity against these parasites acting mainly to disrupt surface-membranes. In some cases AMPs also localize intracellularly to affect calcium levels, mitochondrial function and induce autophagy, necrosis and apoptosis. In this review we discuss the work done in the area of AMP interactions with trypanosomatid protozoa, propose potential targets of AMP activity at the cellular level and discuss how AMPs might influence parasite growth and differentiation in their hosts to determine the outcome of natural infection., ((c) 2010 Elsevier Inc. All rights reserved.)

Flagellates of the class Kinetoplastea are known to frequently parasitize insects. We have collected 67 isolates from 407 Heteroptera hosts captured in several locations of South-West China. Their splice leader (SL) RNA gene repeats and small subunit (SSU) rRNA genes were PCR amplified from the infected tissue samples. In most cases, parasites were found in the midgut, rarely the infection was confined to the Malpighian tubes. Phylogenetic analysis of the obtained sequences has significantly expanded the known diversity of these monoxenous parasites. Fifteen typing units were found among these isolates including 11 potentially new species. Four typing units matched the previously known typing units from the Neotropics indicating a global distribution of the respective parasite species. At the same time, new clades appeared, testifying for a certain level of endemism. The host record of the parasites found indicated a variable specificity level of the host-parasite association including several cases of a very broad host range. Our results disprove the "one host - one parasite" paradigm and show that although the global diversity of monoxenous parasites is high, it is not as enormous as suggested earlier. Moreover, phylogenetic analysis revealed the presence, among the isolated strains, of a new Phytomonas species, which is the first documentation of this potentially pathogenic dixenous parasite of plants in China.

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.

The genes for the mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase were identified in Trypanosoma brucei and Leishmania major genomes. We have expressed the L. major gene in Saccharomyces cerevisiae and confirmed the subcellular localization and activity of the produced enzyme. Using cultured T. brucei procyclic and Leishmania mexicana promastigote cells with a permeabilized plasma membrane and containing intact glycosomes, it was shown that dihydroxyacetone phosphate is converted into pyruvate, and stimulates oxygen consumption, indicating that all components of the glycerol 3-phosphate/dihydoxyacetone phosphate shuttle between glycosomes and mitochondrion are present in these insect stages of both organisms. A computer model has been prepared for the energy and carbohydrate metabolism of these cells. It was used in an elementary mode analysis to get insight into the metabolic role of the shuttle in these insect-stage parasites. Our analysis suggests that the shuttle fulfils important roles for these organisms, albeit different from its well-known function in the T. brucei bloodstream form. It allows (1) a high yield of further metabolizable glycolytic products by decreasing the need to produce a secreted end product of glycosomal metabolism, succinate; (2) the consumption of glycerol and glycerol 3-phosphate derived from lipids; and (3) to keep the redox balance of the glycosome finely tuned due to a highly flexible and redundant system.

We have determined the sequences of 5S rRNA and spliced leader (SL) RNA genes, and adjacent intergenic regions for representatives of all known trypanosomatid genera parasitizing insects. The genetic loci have been analyzed separately as well as by a combined approach. Several isolates, assigned by morphology to different genera (Leptomonas spp., Blastocrithidia spp.), seem to belong to a single species with an unexpectedly wide host and geographical range. An unnamed trypanosomatid isolated from rats in Egypt was found to belong to the genus Herpetomonas, so far associated with insect hosts only. It is closely related to Herpetomonas ztiplika, a parasite of a blood-sucking biting midge. Apparently several different trypanosomatid species can infect one insect species, as exemplified by Leptomonas sp. PL and Wallaceina sp. Wsd, which were isolated from different specimens of Salda littoralis on the same locality and day. However, since the same species of Leptomonas was obtained from insect hosts belonging to different genera, some insect trypanosomatids may have low host specificity. Our data revealed additional discrepancies between molecular phylogenetic data and cell morphology, rendering current trypanosomatid taxonomy unreliable.

Protease activity was found in spent culture medium collected from Leishmania donovani, L. mexicana, L. major, as well as the insect trypanosomatids, Crithidia luciliae and Leptomonas seymouri. Released protease activity increased linearly over time and was correlated to promastigote density. In SDS-PAGE, zymogram gels showed that the protease's molecular weight ranged from 43-100 kDa. Spent culture medium proteases were blocked by the metallo-protease inhibitors, 1,10-phenanthroline and Z-Tyr-Leu-NHOH, but not by bestatin, leupeptin, ABESF, pepstatin A, E-64 or aprotinin. Monoclonal and/or polyclonal antibodies to the leishmanial gp63 reacted with the released Crithidia, Leptomonas, L. major and L. donovani proteases. Cell surface biotinylation and immune precipitation using gp63-specific antibodies showed that >34% of the released protease originated from the surface. Antibodies against the Trypanosoma brucei variable surface glycoprotein cross-reactive determinant (CRD) did not recognize this activity, suggesting that the gp63 is not cleaved from the cell surface by a parasite phospholipase, but is released by an alternative mechanism.

From 10 trypanosomatids genera six comprise monogenetic parasites of insects and for the rest of four genera insects may serve as vectors. The invertebrate host is an essential element of trypanosomatids life cycle, but from more than 900 recognised vertebrate hosts only about 500 species of insects have been discovered to be the hosts of homoxenous trypanosomatids. Nothing or very little is known about insect trypanosomatids in many extensive areas such as South East Asia, Australia, Japan and some others. Each new region explored brings many new findings. Recently flagellates were found in new insect species and families. The border of parasites distribution was expanded till Central Asia, Far East and North over the Polar Circle. As paleogeographical events are now under contemplating in trypanosomatids phylogeny researches so northern insect trypanosomatids may attract some attention as the elements of postglacial fauna which is definitely young. Very broad host specificity of insect trypanosomatids and high probability to isolate non-specific parasite show causes that only the investigation of a culture may solve the question 'what parasite was really isolated?'. Examination of cell morphotypes in the host has clearly demonstrated that they are not sufficient for classification and may lead us to be mistaken. The number of insect trypanosomatid cultures is inadequate for characterisation of the diversity of insects trypanosomatids. Trypanosoma is actually the only trypanosomatid genus which is out of questions. Insect trypanosomatids comprise the most diversified part of trypanosomatids evolutionary tree. Recent ssrRNA phylogenetic analysis and morphological data show that three insect isolates represent new lineages on trypanosomatid evolutionary tree, as well as dendrograms derived from PCR data demonstrated some new groups of isolates. Therefore, the more insect trypanosomatids are involved in laboratory investigations--the more new clusters or/and new lineages are appearing on the tree.

Electrophoretic karyotypes of homoxenous trypanosomatids Leptomonas peterhoffi, L. mycophilus, L. nabiculae and Leptomonas sp. have been studied by transverse alternating-field electrophoresis under varying electrophoretic conditions. From 12 to 17 chromosomal DNA bands, ranging from 370 to more than 1500 kb were detected in the karyograms of the species compared. In each pattern, some intensely stained bands could represent more than one chromosome. Taking into account the number of intensely stained bands, the karyotype of L. peterhoffi was estimated to contain at least 18 chromosomes, the karyotypes of L. mycophilus and L. nabiculae, at least 21 chromosome each, and the karyotype of Leptomonas sp. up to 20 chromosomes. Interclonal variations of electrophoretic karyotypes of 10 clones of Leptomonas sp. (cfmI-cfmX) were studied. Seven of ten clones had identical electrophoretic patterns. In the karyograms of three clones (cfmI, cfmVI, cfmVII), additional chromosomal DNA bands were observed. The obtained results suggest, that electrophoretic karyotypes cannot be used as reliable markers of species of homoxenous trypanosomatids, since intraspecies variability does occur in these parasites.

Of ten recognized trypanosomatid genera, only two - pathogenic Trypanosoma and Leishmania - have been actively investigated for any length of time while the plant flagellates - Phytomonas - have recently begun to attract attention due to their role as agricultural parasites. The remaining genera that comprise parasites associated with insects have been largely neglected except for two or three containing popular isolates. This publication reviews current knowledge of trypanosomatids from insects.

Assessing the diversity of the Trypanosomatidae is difficult because of the relatively small number of species that can be cultured. This same problem thwarts efforts to identify the hosts and insect vectors of Phytomonas, a genus of parasites of plants that includes species responsible for devastating epiphytotics of economically important plantations. Here, Myrna Serrano, Marta Teixeira and Erney Camargo review the studies that have led to the development of a PCR-based technique for processing insect and plant juices fixed on glass slides. The method overcomes the need for cultivation, facilitates field collections and also permits the molecular examination of archival smears of Phytomonas. In principle, the method can be adapted to any trypanosomatid as well as to any fastidious parasitic or free-living organism.

The results of comparative analysis of two phylogenetic trees of the trypanosomatids based on morphological and molecular characters are discussed. The morphological dendrogram was based on 33 ultrastructural characters, 6 light microscope characteristics and 8 biological characters. Molecular UPGMA dendrogram depicting differences (Dice distance) between examined trypanosomatids is based on the universally primed PCR polymorphisms. The general topology of both dendrograms are similar, with the Trypanosoma at the base. The genus Wallaceina appears to be monophyletic. In a contrary, the genera Leptomonas, Crithidia and Herpetomonas look like artificial groups according to both methods used. The cyst-forming homoxenous trypanosomatids from insects represent a monophyletic clade, which seems to be a separate genus. Two species of within genus Wallaceina are arranged as a separate subgenus.

The random amplification of polymorphic DNA was used for easy, quick and sensitive assessment of genetic polymorphism within Phytomonas to discriminate isolates and determine genetic relationships within the genus. We examined 48 Phytomonas spp., 31 isolates from plants and 17 from insects, from different geographic regions. Topology of the dendrogram based on randomly amplified polymorphic DNA fingerprints segregated the Phytomonas spp. into 5 main clusters, despite the high genetic variability within this genus. Similar clustering could also be obtained by both visual and cross-hybridization analysis of randomly amplified synapomorphic DNA fragments. There was some concordance between the genetic relationship of isolates and their plant tissue tropism. Moreover, Phytomonas spp. from plants and insects were grouped according to geographic origin, thus revealing a complex structure of this taxon comprising several clusters of very closely related organisms.

A PCR-based method was adapted for the amplification of DNA from methanol-fixed smears of insects and plants parasitized by trypanosomatids. The PCR target was the multicopy spliced leader (SL) gene. Amplicons were hybridized with an oligonucleotide probe (SL3') specific for Phytomonas. The method has the advantage of dispensing with the cultivation of parasites, many of which are very fastidious or non-cultivable. The technique was applied to archival glass slides and to newly collected material. It proved to specific for Phytomonas spp., enabling their detection in plants and insects. Sequence comparison of the amplicons obtained revealed the existence of different strains/species of Phytomonas circulating among diseased palsms and fruit.

In this paper we describe a method for the detection of Phytomonas spp. from plants and phytophagous insects using the PCR technique by targeting a genus-specific sequence of the spliced leader (SL) gene. PCR amplification of DNA from 48 plant and insect isolates previously classified as Phytomonas by morphological, biochemical, and molecular criteria resulted in all cases in a 100-bp fragment that hybridized with the Phytomonas-specific spliced leader-derived probe SL3'. Moreover, this Phytomonas-specific PCR could also detect Phytomonas spp. in crude preparations of naturally infected plants and insects. This method shows no reaction with any other trypanosomatid genera or with plant and insect host DNA, revealing it to be able to detect Phytomonas spp. from fruit, latex, or phloem of various host plants as well as from salivary glands and digestive tubes of several species of insect hosts. Results demonstrated that SLPCR is a simple, fast, specific, and sensitive method that can be applied to the diagnosis of Phytomonas among cultured trypanosomatids and directly in plants and putative vector insects. Therefore, the method was shown to be a very specific and sensitive tool for diagnosis of Phytomonas without the need for isolation, culture, and DNA extraction of flagellates, a feature that is very convenient for practical and epidemiological purposes., (Copyright 1999 Academic Press.)