Transmission and Ecology of Trypanosomatid Flagellates of Water Striders (Hemiptera: Gerridae)

An investigation of transmission and ecology of the monogenetic trypanosomatids, Blastocrithidia gerridis and Crithidia flexonema , in Gerris is described. Motile free-living flagellates of both species were found in the faeces of Gerris and in the water on which the bugs inhabited. Transmission of both trypanosomatid species occurred from naturally infected wild-caught bugs to flagellate-free laboratory-bred bugs via water. Crithidia flexonema was also transmitted to laboratory-bred bugs after being isolated in culture. Observations of experimentally infected bugs indicate that C. flexonema flagellates are imbibed and pass through the fore- and midgut to the hindgut where they become attached and multiply. There was no evidence to suggest transovarial transmission. In a 3-yr investigation into the prevalence of trypanosomatids in a natural population of adult Gerris odontogaster , it was found that the infection rate varied between 19% and 100%. There was no significant difference in infection rates between females and males. The infection rate peaked for each year in late spring or early summer. The significance of these results is discussed in relation to the ecology and behaviour of Gerris . The results indicate that the infections are maintained in hibernating bugs over winter.     

Trypanosomatidae Isolated from Zelus leucogrammus (Perty, 1834) (Hemiptera,Reduviidae), with a Discussion on Flagellates of Insectivorous Bugs*

The insectivorous bug Zelus leucogrammus (Perty, 1834) (Hemiptera, Reduviidae) was found to harbor several species of trypanosomatids of the genera Leptomonas, Crithidia, Herpetomonas and Blastocrithidia. However, laboratory bred nymphs given sterile food were infected only by Blastocrithidia, the other flagellates appearing in their intestinal tract when they were fed on other insects. The Blastocrithidia was thought to belong to a single species and to be a parasite proper of Z. leucogrammus (and possibly other Hemiptera), producing luxuriant infections in the intestinal tract of the bugs captured in nature and spreading easily among isolated populations through cannibalism and ingestion of cysts; it was also abundant in the genital tract of gravid females but transovarian transmission was not proved. This Blastocrithidia experimentally infected Triatoma infestans and is very close to B. triatomae Cerisola, del Prado, Rohwedder and Bozzini, 1971. The other trypanosomatids, never numerous in naturally infected Z. leucogrammus, are acquired by the bug from many different insects, chiefly Diptera, on which it was seen to feed. Herpetomonas muscarum and C. luciliae, the common trypanosomatids among several muscoid Diptera, were isolated both from flies and from the bugs fed on them.     

Infection rates and pathogenicity of trypanosomatid gut parasites in the water strider Gerris odontogaster (Zett.) (Heteroptera: Gerridae)

Summary Trypanosomatid flagellates are common protozoan gut parasites of a wide range of insect species. Water striders (Gerridae) harbour the trypanosomatid Blastocrithidia gerridis. Three different populations of the water strider Gerris odontogaster in northern Sweden were sampled to assess the infection rate dynamics of trypanosomatids. The initially very low infection rates (0%–15%) early in the season were followed by a rapid increase during the reproductive period of the water striders, reaching very high levels (80%–90%). The pathogenic effects of trypanosomatids on G. odontogaster adults were studied in laboratory experiments. The parasites caused a general reduction of host vigour. Male skating endurance was negatively correlated with the intensity of the trypanosomatid infection. However, infection of trypanosomatids increased the mortality among adults only when the water striders were subjected to food stress. The trypanosomatids did not reduce the fecundity of females provided with food. We suggest that trypanosomatid gut parasites may be an important mortality factor in water strider populations. Since the pathogenicity of the parasites is enhanced by stress, parasitism by trypanosomatids may contribute to the regulation of host populations.     

Phylogenetic relationships of trypanosomatids parasitising true bugs (Insecta: Heteroptera) in sub-Saharan Africa

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.     

Trypanosomatids, Other Than Phytomonas spp., Isolated and Cultured from Fruit

Trypanosomatids were isolated from edible fruit. One of the isolates (from tangerine) presented a set of enzymes for the metabolism of arginine-ornithine similar to that of Leptomonas spp., and failed to be recognized by monoclonal antibodies specific for Phytomonas spp. The possibility that trypanosomatids other than Phytomonas spp. could infect fruit was further examined by inoculating tomatoes with species of Crithidia, Leptomonas and Herpetomonas. Some of these flagellates multiplied in tomatoes. Besides, house flies became infected with Crithidia sp. when fed on tomatoes experimentally inoculated with this flagellate. Therefore, isolation of a trypanosomatid from a plant should not constitute an absolute criterion for placing it in the genus Phytomonas.     

Classification of trypanosomatids from fruits and seeds using morphological, biochemical and molecular markers revealed several genera among fruit isolates

Trypanosomatids are widespread in several plant families and although most isolates have been classified as Phytomonas, other trypanosomatid genera can also infect plants. In order to assess the natural occurrence of non-Phytomonas trypanosomatids in plants we characterized 21 new trypanosomatid cultures, 18 from fruits and three from seeds of 17 plant species. The trypanosomatids from fruit and seeds were compared in terms of morphological, growth, biochemical and molecular features. The high diversity among the isolates permitted the classification of the new flagellates into the genera Crithidia and Leptomonas as well as Phytomonas. The data showed that natural fruit infection with non-Phytomonas trypanosomatids is more common than usually thought, being detected in 43% of the fruit isolates.     

Growth and Reproduction of Leptomonas oncopelti in the Milkweed Bug, Oncopeltus fasciatus

SYNOPSIS. A laboratory colony of Oncopeltus fasciatus was found to be infected with Leptomonas oncopelti. Inasmuch as the parasite is transmitted from parent to offspring an opportunity presented itself to study the biology and transmission of this parasite under controlled laboratory conditions. An apparatus for observing individual bugs was designed and the presence or absence of flagellates in the feces determined. Flagellates were not shed until the bugs became adults after which they appeared in every defecation. Dissection of infected bugs revealed that flagellates were not present in the rectum until adulthood. Further studies indicated that in the midgut of the insect there is a departure from binary fission to budding. The nucleus divides and one of the newly formed nuclei migrates toward a newly formed kinetoplast. Rarely there is still another kinetoplast/nucleus division. In the event the new axoneme grows within the cytoplasmic sheath of the parent flagellum, smaller organisms produced by unequal cytokinesis remain attached. If the axoneme grows free, the smaller daughter organisms become free-swimming. Passage into the rectum of the adult bugs causes a rounding up of all parasites although the leishmaniform organisms continue to divide. It is presumed that infection of clean bugs is accomplished by the ingestion of leishmaniform organisms through a common water source. The reason for the presence of flagellates in the rectum of the adult but not in the nymphal insect and the mechanism responsible for the change from binary to unequal fission are not known.     

Insect trypanosomatids in Papua New Guinea: high endemism and diversity

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.     

Influence of gut parasites on growth performance in the water strider Gerris buenoi (Hemiptera: Gerridae)

Water striders harbor a diverse assemblage of symbionts in their digestive tract We used a field experiment, in which water striders were reared in enclosures in their natural pond habitat, to assess the effects of gut symbionts on growth Trypanosomatid flagellates had significant adverse effects on both development time and adult size, and therefore are clearly parasitic Yet because of their low prevalences (2% or less), trypanosomatids cannot be a major factor in the dynamics of our study population Gregannes occurred in 36% of the water striders, often in high numbers, and filling the entire midgut of some bugs Nevertheless, infected and uninfected gerrids did not differ in their growth, and gregarine loads were uncorrelated with development time and adult size attained We also did not find effects of gregarines in a second experiment with different rearing conditions, including a treatment with food stress We used a quantitative genetic approach to test if resistance against gregarines has a heritable component There was no evidence for any genetic variation, suggesting that variability in gregarine loads is the result of environmental heterogeneity Comparison with published reports from water striders shows that there is a great variability in the diversity and prevalence of symbionts among different populations and species, of gerrids     

Experimental Infection of Triatoma infestans and Rhodnius prolixus with Trypanosomatidae of the Genera Crithidia and Blastocrithidia*

SYNOPSIS. Dissections of Triatoma infestans and Rhodnius prolixus at various intervals after experimental infection with insect Trypanosomatidae of the genera Blastocrithidia and Crithidia (B. euschisti, C. luciliae, C. acanthocephali and C. mellificae) disclosed the protozoa to be surviving and in most instances undergoing reproduction in the experimental bugs for as long as 2 months following infection. Epimastigotes and amastigotes were seen in bugs infected with B. euschisti and choanomastigotes and amastigotes were observed in insects experimentally infected with the Crithidia. Since C. acanthocephali and C. luciliae were from clones, this is the 1st finding of these 2 structural types in this genus using cultures originating from a single choanomastigote. In view of the loose host specificity of the insect Trypanosomatidae as further emphasized by this work, it would appear that the necessity is increased for field workers in trypanosomiasis and leishmaniasis areas of the world to become well acquainted with the infective potentialities of the insect trypanosomatids of their areas for the vectors of Trypanosoma and Leishmania along with the possible association in nature of these vectors with insects which might harbor insect-limited Trypanosomatidae.     

Phylogenetic validation of the genera Angomonas and Strigomonas of trypanosomatids harboring bacterial endosymbionts with the description of new species of trypanosomatids and of proteobacterial symbionts

We comparatively examined the nutritional, molecular and optical and electron microscopical characteristics of reference species and new isolates of trypanosomatids harboring bacterial endosymbionts. Sequencing of the V7V8 region of the small subunit of the ribosomal RNA (SSU rRNA) gene distinguished six major genotypes among the 13 isolates examined. The entire sequences of the SSU rRNA and glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) genes were obtained for phylogenetic analyses. In the resulting phylogenetic trees, the symbiont-harboring species clustered as a major clade comprising two subclades that corresponded to the proposed genera Angomonas and Strigomonas. The genus Angomonas comprised 10 flagellates including former Crithidia deanei and C. desouzai plus a new species. The genus Strigomonas included former Crithidia oncopelti and Blastocrithidia culicis plus a new species. Sequences from the internal transcribed spacer of ribosomal DNA (ITS rDNA) and size polymorphism of kinetoplast DNA (kDNA) minicircles revealed considerable genetic heterogeneity within the genera Angomonas and Strigomonas. Phylogenetic analyses based on 16S rDNA and ITS rDNA sequences demonstrated that all of the endosymbionts belonged to the Betaproteobacteria and revealed three new species. The congruence of the phylogenetic trees of trypanosomatids and their symbionts support a co-divergent host-symbiont evolutionary history.     

Identification of trypanosomes isolated from reduviidae from north Chile

Examination of 54 Triatoma infestans from a village near the European Southern Observatory La Silla in Chile and of 9 Triatoma spinolai from the territory of the observatory showed that 10 T. infestans were infected with trypanosomatids. Mice were infected with in vitro cultures initiated with five different trypanosomatid isolates and treated with the immunosuppressive drug cyclophosphamide to increase the parasitemia of the flagellates. Evidence of the presence of T. cruzi was provided by a comparative biometrical analysis of blood trypomastigotes and the occurrence of intracellular amastigotes. Three methods for further identification were used: examination of kDNA ultrastructure, disc electrophoresis of soluble proteins and the Aaptos papillata II lectin induced agglutination. We obtained the following results for all isolates: (1) presence of a central band of the kDNA; (2) T. cruzi specific double bands of the protein patterns; (3) positive reaction with Aaptos papillata II. No differences between the five isolates from Chile and T. cruzi or T. cruzi-like strains from other countries could be observed. Based on these results an infection of the bugs with T. rangeli and T. conorhini could be excluded.     

Insights into the role of gp63-like proteins in lower trypanosomatids

Any actual understanding of trypanosomatids in general requires a comprehensive analysis of the less-specialized species as thorough as our knowledge of the more specialized Leishmania and Trypanosoma. In this context, we have shown by antibody cross-reactivity that purified extracellular metallopeptidases from Phytomonas fran?ai, Crithidia deanei (cured strain) and Crithidia guilhermei share common epitopes with the leishmanial gp63. Flow cytometry and fluorescence microscopy analyses indicated the presence of gp63-like molecules on the cell surface of these lower trypanosomatids. Binding assays with explanted guts of Aedes aegypti incubated with purified gp63 and the pretreatment of trypanosomatids with anti-gp63 antibodies indicated that the gp63-like molecules are involved in the adhesive process of these trypanosomatids to the A. aegypti gut wall. In addition, our results indicate for the first time that the gp63-like molecule binds to a polypeptide of 50 kDa on the A. aegypti gut epithelium extract.     

Prevalence of a trypanosomatid in the southern green stink bug, Nezara viridula

The southern green stink bug, Nezara viridula (L.), and certain of its host plants were examined to determine the prevalence and biological characteristics of an intestinal trypanosomatid. Promastigotes with short (< or = 17.5 microm excluding flagellum) and long forms (> or = 25.0 microm) usually infected < 50% of the bugs before August and > 50% (maximum 95%) during August-October, but prevalence was not host-density dependent. The flagellate was detected in adults and in all nymphal instars, at all sampling sites where at least 10 bugs were captured, and in bugs from all host plants sampled (soybean, red clover, vetch). Of bugs with flagellates, 27% were heavily infected (> 20 flagellates per 160X microscope field). Weights of infected and uninfected adults did not differ. Live flagellates were detected in bug feces and in one stem of red clover. When bugs were fed soybean pods, tomatoes, or snap beans in the laboratory, only once were flagellates detected in plant tissue (snap beans). The flagellate was cultured in modified Medium 199. This flagellate is prevalent in N. viridula populations in Louisiana and apparently does not cause significant pathological effects in N. viridula or its host plants, including soybean.     

Ecology and Feeding Habits Drive Infection of Water Bugs with <Emphasis Type="Italic">Mycobacterium ulcerans</Emphasis>

Mycobacterium ulcerans (MU), the causative agent of Buruli ulcer, is present in a wide spectrum of environments, including terrestrial and aquatic ecosystems in tropical regions. The most promising studies on the epidemiological risk of this disease suggest that some ecological settings may favor infection of animals with MU including human. A species’ needs and impacts on resources and the environment, i.e., its ecological niche, may influence its susceptibility to be infected by this microbial form. For example, some Naucoridae may dive in fresh waters to prey upon infected animals and thus may get infected with MU. However, these studies have rarely considered that inference on the ecological settings favoring infection and transmission may be confounded because host carrier sister species have similar ecological niches, and potentially the same host–microbe interactions. Hence, a relationship between the ecological niche of Naucoridae and its infection with MU may be due to a symbiotic relationship between the host and the pathogen, rather than its ecological niche. To account for this confounding effect, we investigated the relationships between surrogates of the ecological niche of water bug species and their susceptibility to MU, by performing phylogenetic comparative analyses on a large dataset of 11 families of water bugs collected in 10 different sites across Cameroon, central Africa. Our results indicate that MU circulates and infects a couple of host taxa, i.e., Belostomatidae, Naucoridae, living both in the aquatic vegetation and as predators inside the trophic network and sister species of water bugs have indeed similar host–microbe interactions with MU.     

Seasonal and Regional Dynamics of M. ulcerans Transmission in Environmental Context: Deciphering the Role of Water Bugs as Hosts and Vectors

Background

Buruli ulcer, the third mycobacterial disease after tuberculosis and leprosy, is caused by the environmental mycobacterium M. ulcerans. Various modes of transmission have been suspected for this disease, with no general consensus acceptance for any of them up to now. Since laboratory models demonstrated the ability of water bugs to transmit M. ulcerans, a particular attention is focused on the transmission of the bacilli by water bugs as hosts and vectors. However, it is only through detailed knowledge of the biodiversity and ecology of water bugs that the importance of this mode of transmission can be fully assessed. It is the objective of the work here to decipher the role of water bugs in M. ulcerans ecology and transmission, based on large-scale field studies.

Methodology/Principal Findings

The distribution of M. ulcerans-hosting water bugs was monitored on previously unprecedented time and space scales: a total of 7,407 water bugs, belonging to large number of different families, were collected over one year, in Buruli ulcer endemic and non endemic areas in central Cameroon. This study demonstrated the presence of M. ulcerans in insect saliva. In addition, the field results provided a full picture of the ecology of transmission in terms of biodiversity and detailed specification of seasonal and regional dynamics, with large temporal heterogeneity in the insect tissue colonization rate and detection of M. ulcerans only in water bug tissues collected in Buruli ulcer endemic areas.

Conclusion/Significance

The large-scale detection of bacilli in saliva of biting water bugs gives enhanced weight to their role in M. ulcerans transmission. On practical grounds, beyond the ecological interest, the results concerning seasonal and regional dynamics can provide an efficient tool in the hands of sanitary authorities to monitor environmental risks associated with Buruli ulcer.     

Inference of cowpox virus transmission rates between wild rodent host classes using space-time interaction

There have been virtually no studies of 'who acquires infection from whom' in wildlife populations, but patterns of transmission within and between different classes of host are likely to be reflected in the spatiotemporal distribution of infection among those host classes. Here, we use a modified form of K-function analysis to test for space-time interaction among bank voles and wood mice infectious with cowpox virus. There was no evidence for transmission between the two host species, supporting previous evidence that they act as separate reservoirs for cowpox. Among wood mice, results suggested that transmission took place primarily between individuals of the opposite sex, raising the possibility that cowpox is sexually transmitted in this species. Results for bank voles indicated that infected females might be a more important source of infection to either sex than are males. The suggestion of different modes of transmission in the two species is itself consistent with the apparent absence of transmission between species.     

High Prevalence and Endemism of Trypanosomatids on a Small Caribbean Island

We describe the monoxenous trypanosomatids parasitizing true bugs and flies on the island of Curaçao. Out of 248 examined true bugs belonging to 17 species, 93 individuals were found to be infected (overall 38% prevalence) by at least one trypanosomatid species (referred to as typing units; TUs). Out of 80 flies, six were infected. All detected trypanosomatids were compared based on their 18S rRNA sequences with TUs parasitizing bugs and flies described from mainland South America, allowing us to assess their diversity and distribution. Besides Leptomonas pyrrhocoris and Leptomonas seymouri, two known species of the subfamily Leishmaniinae, our analysis revealed six new TUs falling into the groups ‘jaculum’, Blastocrithidia and Herpetomonas. Moreover, two new members of the genus Phytomonas and three new TUs belonging to the monophyletic group designated as ‘new clade II’ sensu Mol. Phylogenet. Evol, 69, 255 (2013) were isolated. The detected trypanosomatids were characterized by moderate diversity (13 TUs) species richness. Out of nine and four TUs from the heteropteran and dipteran hosts, respectively, 11 TUs have not been encountered before. Although a sampling bias may partially affect the comparison between trypanosomatid communities on Curaçao and the mainland, the high proportion of unique TUs from the former location suggests that the prominent role of islands in increasing the global diversity of macroscopic organisms may also extend to their protistan parasites.     

Endosymbiosis in protozoa of the Trypanosomatidae family

A small number of trypanosomatids present bacterium endosymbionts in the cytoplasm, which divide synchronously with the host cell. Crithidia oncopleti, Crithidia deanei. Crithidia desouzai, Blastocrithidia culicis and Herpetomonas roitmani are the best characterized species. The endosymbiont is surrounded by two membranes separated from each other by an electron-lucent space. The presence of the endosymbiont led to the appearance of morphological changes which include the lack of the paraflagellar rod associated to the axoneme, the morphology of the kinetoplast and the association of the sub-pellicular microtubules with portions of the protozoan plasma membrane. Aposymbiotic strains could be obtained by antibiotic treatment, opening the possibility to make comparative analysis of endosymbiont-containing an endosymbiont-free populations of the same species. It is clear that metabolic cycles are established between the prokaryiont and the host cell. The results obtained show that endosymbiont-containing species of trypanosomatids constitute an excellent model to study basic processes on the endosymbiont-host cell relationship and the origin of new organelles.     

Ectoparasite and hemoparasite infection in a diverse temperate lizard assemblage at Macraes Flat, South Island, New Zealand

A lizard assemblage at Macraes Flat, New Zealand, comprising the common skinks Oligosoma maccanni and Oligosoma nigriplantare polychroma, the endangered species Oligosoma grande and Oligosoma otagense, and the common gecko Hoplodactylus maculatus, was studied to determine the prevalence of ectoparasites and hemoparasites. The mites Odontacarus lygosomae and Ophionyssus scincorum infected all Oligosoma spp. only, and the mite Neotrombicula naultini infected only H. maculatus. The hemoparasite Hepatozoon lygosomarum infected all Oligosoma skinks, except O. n. polychroma. Oligosoma otagense had the highest infection levels of all parasites by several orders of magnitude. For all lizard species, there was no difference in mite prevalence between adult males and adult females, but juveniles were less often infected. For all skink species, there was a significant relationship between presence of the hemoparasite He. lygosomarum and infection intensity of the supposed vector, O. scincorum. It is unclear if patterns of parasite infection reflect species-specific susceptibility, host-parasite species-specific spatial ecology, or environmentally induced host physiological impairment. Considering the threatened nature of O. otagense, evidence of high parasitemia should stimulate further investigation.