Misidentification of two Brazilian triatomes, Triatoma arthurneivai and Triatoma wygodzinskyi, revealed by geometric morphometrics.

Triatoma arthurneivai Lent & Martins and Triatoma wygodzinskyi Lent (Hemiptera: Reduviidae) are two Brazilian species found in the sylvatic environment. Several authors may have misidentified T. arthurneivai and consequently published erroneous information. This work reports the use of geometric morphometric analysis on wings in order to differentiate T. arthurneivai and T. wygodzinskyi, and thus to detect possible misidentifications. Triatomines collected from the field in the states of Minas Gerais and São Paulo, and from laboratory colonies, were used. Analyses show a clear differentiation between specimens of T. arthurneivai and T. wygodzinskyi. This indicates that T. arthurneivai populations from São Paulo state were misidentified and should be considered as T. wygodzinskyi. This study also suggests that T. arthurneivai is an endemic species from Serra do Cipó, Minas Gerais state.

Infestation of peridomestic Attalea phalerata palms by Rhodnius stali, a vector of Trypanosoma cruzi in the Alto Beni, Bolivia.

OBJECTIVES: To determine (i) whether peridomestic Attalea phalerata palms in fragmented human-occupied areas of the Alto Beni, Bolivia, are infested by triatomines; (ii) the specific status of triatomines captured in the area; and (iii) the rate of natural Trypanosoma cruzi infection among those triatomines. METHODS: One hundred and twenty-five live-bait traps were used to sample 47 A. phalerata palms in three Alto Beni localities. Active search for vectors was also performed in 10 chicken coops and three rice storage units. Only Rhodnius specimens were found. As nymphs of closely related Rhodnius species are morphologically undistinguishable, and because of controversy in the literature regarding which Rhodnius species occur in Bolivia, collected insects were identified through molecular taxonomy. Phylogenetic analyses of DNA sequences obtained for a fragment of the mitochondrial cytochrome b gene and for the nuclear ITS-2 ribosomal region were used as molecular markers. Natural infection rates were determined using a pair of primers that PCR-amplify a 330-bp fragment of the parasite's kDNA. RESULTS: Twelve nymphs were captured in five A. phalerata palms (from two of the three localities studied), and an adult was collected from a chicken coop in Iniqua (and morphologically identified as Rhodnius stali). All nymphs (as well as the adult) were molecularly identified as R. stali based on the two molecular markers used. A single nymph was found to be infected with T. cruzi. CONCLUSIONS: Attalea phalerata palms represent an important sylvatic ecotope occupied by R. stali in the Alto Beni region of Bolivia, where there are signs of T. cruzi transmission to humans, despite the preliminary indication of low level of natural infection of the vectors.

The association between the geographic distribution of Triatoma pseudomaculata and Triatoma wygodzinskyi (Hemiptera: Reduviidae) with environmental variables recorded by remote sensors.

In this study, predictive models of geographic distribution patterns of Triatoma pseudomaculata (Tps) and T. wygodzinskyi (Twy) were carried out. They were based on biophysical variables estimated from information provided by the satellite remote sensors AVHRR (Advanced Very High Resolution Radiometer) and MODIS (MODerate-resolution Imaging Spectroradiometer). Our goal was to analyze the potential geographic distribution of Tps and Twy and to assess the performance of three predictive models (one for each species and one for both species together) based on temperature, vapour pressure deficit, vegetation and altitude. The geographic distribution analysis shows that all models performed well (>85.7% of overall correct classification of presence and absence point data). The MODIS-based models showed lower correct classifications than the AVHRR-based models. The results strongly suggest that environmental information provided by remote sensors can be successfully used in studies on the geographic distribution of poorly understood Chagas disease vector species.

Molecular population genetics and phylogeography of the Chagas disease vector Triatoma infestans in South America.

Knowledge of the genetic variability, population structure, and evolutionary history of Triatoma infestans may be useful for developing rational vector control strategies. A 661-bp fragment of the mitochondrial gene cytochrome oxidase I (COI) was sequenced and analyzed in bugs from Argentina, Uruguay, Peru, and Bolivia, including peridomestic, domestic, Andean, and Chaco sylvatic bugs. A total of 48 polymorphic sites among 37 haplotypes were described. Nucleotide variation fluctuated among samples, with the highest nucleotide diversity observed in seven Argentinean provinces. Within this group, some populations showed patterns of variability compatible with population expansions and/or fine-scale population structure, whereas others suggested population bottlenecks and/or population admixture processes. A maximum parsimony analysis of the haplotypes showed the presence of a Bolivian/Peruvian and an Argentinean/Uruguayan clade. Bolivian sequences were further divided in Chaco sylvatic and Andean domestic and sylvatic. Two different nested clades were found within the Argentinean/Uruguayan cluster. Analysis of molecular variance (AMOVA) and K(ST)* analysis supported a strong population structure in Argentina, where genetic differentiation was correlated with geographic distance. Departures from neutrality expectations and a nested cladistic analysis suggest a recent population expansion of T. infestans in Argentina, followed by restricted gene flow and patterns of isolation by distance. This expansion could have taken place as a two-wave process, as was shown by the phylogenetic analysis and signatures of population admixture in the southern most Argentinean populations.

Microsatellite markers in Triatoma pseudomaculata (Hemiptera, Reduviidae, Triatominae), Chagas' disease vector in Brazil.

Six polymorphic microsatellite loci were isolated and characterized using a microsatellite-enriched genomic library from the Chagas' disease vector Triatoma pseudomaculata. This species is found in Brasil in Caatinga areas and predominantly in peridomestic habitats. All the microsatellites tested on a population of T. pseudomaculata sampled in the Bahia State, Brazil, were polymorphic (2-15 alleles). Markers amplification was also tested on six Triatoma species and some loci successfully amplified in the most phylogenetically related species, in particular Triatoma brasiliensis.

Microsatellite markers from the Chagas disease vector, Rhodnius prolixus (Hemiptera, Reduviidae), and their applicability to Rhodnius species.

Ten microsatellites were isolated and characterized from a partial genomic library of Rhodnius prolixus, the principal Chagas disease vector in Venezuela, Colombia and Central America. These polymorphic molecular markers could be particularly useful in Chagas disease control initiatives. A wider applicability of the primer-pairs isolated was shown, from 6 to 10 loci being amplifiable in five out of the ten Rhodnius species tested, namely R. domesticus, R. nasutus, R. neglectus, R. neivai and R. robustus. Interestingly, all the loci were amplified in the latter. These markers may be of interest to trace the colonization of human dwellings from triatomine sylvatic populations in order to better define epidemiological risk patterns.

Evolutionary relationships based on genetic and phenetic characters between Triatoma maculata, Triatoma pseudomaculata and morphologically related species (Reduviidae: Triatominae).

The maculata group currently comprises two species of Triatominae, Triatoma maculata and Triatoma pseudomaculata, which share morphologic and chromatic characteristics. In order to clarify the systematic status of these two vectors of Trypanosoma cruzi and to infer their evolutionary relationships, we performed an enzymatic, morphometric and cytogenetic comparison of them, also taking into account two sister species not included in the group (T. arthurneivai and T. wygodzinskyi). According to our results, T. maculata and T. pseudomaculata belong to distinct evolutionary lineages. Similarly, T. arthurneivai topotypes from Minas Gerais form an independent isolated group by morphometrics. Our results also support the specific status of the Triatoma population from São Paulo State (formerly referred to T. arthurneivai), and suggest the possibility that it is T. wygodzinskyi. Finally, we suggest that only the arboricolous T. pseudomaculata from northeast Brazil and the rupicolous sister species originated from São Paulo State should be classified together in the same group.

Origin and phylogeography of the Chagas disease main vector Triatoma infestans based on nuclear rDNA sequences and genome size.

For about half of all Chagas disease cases T. infestans has been the responsible vector. Contributing to its genetic knowledge will increase our understanding of the capacity of geographic expansion and domiciliation of triatomines. Populations of all infestans subcomplex species, T. infestans, T. delpontei, T. platensis and T. melanosoma and the so-called T. infestans "dark morph", from many South American countries were studied. A total of 10 and 7 different ITS-2 and ITS-1 haplotypes, respectively, were found. The total intraspecific ITS-2 nucleotide variability detected in T. infestans is the highest hitherto known in triatomines. ITS-1 minisatellites, detected for the first time in triatomines, proved to be homologous and thus become useful markers. Calculations show that ITS-1 evolves 1.12-2.60 times faster than ITS-2. Despite all species analyzed presenting the same n=22 chromosome number, a large variation of the haploid DNA content was found, including a strikingly high DNA content difference between Andean and non-Andean specimens of T. infestans (mean reduction of 30%, with a maximum of up to 40%) and a correlation between presence/absence of minisatellites and larger/smaller genome size. Population genetics analysis of the eight composite haplotypes of T. infestans and net differences corroborate that there are clear differences between western and eastern populations (60%), and little genetic variation among populations (1.3%) and within populations (40%) within these two groups with migration rates larger than one individual per generation corresponding only to pairs of populations one from each of these groups. These values are indicative either of a large enough gene flow to prevent population differentiation by drift within each geographic area or a very recent spread, the latter hypothesis fitting available data better. Phylogenetic trees support a common ancestor for T. infestans and T. platensis, an origin of T. infestans in Bolivian highlands and two different dispersal lines, one throughout Andean regions of Bolivia and Peru and another in non-Andean lowlands of Chile, Paraguay, Argentina, Uruguay and Brazil.

Peridomestic structure, farming activity and triatomine infestation.

The role of peridomestic structure and farming activity on triatomine infestation was studied on two vector species of Chagas disease (Triatoma pseudomaculata and T. brasiliensis) in Bahia State, northeastern Brazil. A randomly selected population issued from 136 farms was divided into four categories according to the householder activity. At regional scale, the dwellings of farmers working on degraded land of irrigated farms are less exposed to T. pseudomaculata infestation. At premises scale, the farmers and casual workers, who have smaller peridomiciles and less cattle, are also less exposed to T. pseudomaculata. The association of T. brosiliensis with the most mobile populations (casual workers and young breeders) suggests a passive transport of this competitive species. Finally, the retired farmers that own large premises and cattle, but have more sedentary behavior, are the most exposed to T. pseudomaculata infestation.

Nuclear rDNA ITS-2 sequences reveal polyphyly of Panstrongylus species (Hemiptera: Reduviidae: Triatominae), vectors of Trypanosoma cruzi.

Panstrongylus species are widely distributed throughout the Americas, where they act as vectors of Trypanosoma cruzi, agent of Chagas disease. Their intraspecific relationships, taxonomic position and phylogeny in relation to other Triatomini were explored using ribosomal DNA (rDNA) internal transcribed spacer 2 (ITS-2) sequence polymorphisms and maximum parsimony, distance and maximum likelihood analyses of 10 populations representing six species of the genus (P. megistus, P. geniculatus, P. rufotuberculatus, P. lignarius, P. herreri and P. chinai). At the subspecific level, P. megistus appeared more homogeneous than P. rufotuberculatus and P. geniculatus (both with broader distribution). Several dinucleotide microsatellites were detected in the sequences of given species. Many of these microsatellites (GC, TA, GT and AT) showed different number of repeats in different populations and thus, may be very useful for population differentiation and dynamics analyses in future studies. The sequences of P. lignarius (considered sylvatic) and P. herreri (a major disease vector in Peru) were identical, suggesting that these species should be synonymised. Intrageneric analysis showed a clear separation of P. rufotuberculatus, with closest relationships between P. geniculatus and P. chinai, and P. megistus occupying a separate branch. Genetic distances between Panstrongylus species (0.11585-0.22131) were higher than those between Panstrongylus and other Triatomini (16 species from central and North America and South America) (0.08617-0.11039). The distance between P. megistus and P. lignarius/herreri (0.22131) was the largest so far recorded in the tribe. The pronounced differences in length and nucleotide composition suggest a relatively old divergence of Panstrongylus species. P. rufotuberculatus was closer to Mesoamerican Triatoma, Meccus and Dipetalogaster species than to other Panstrongylus. All Panstrongylus clustered with the Mesoamerican clade; P. rufotuberculatus clustered with the phyllosoma complex and T. dimidiata, with D. maxima and T. barberi in a basal position. The rest of Panstrongylus appeared paraphyletically in the tree. This is evidence suggesting polyphyly within the genus Panstrongylus, whose species may be related to the ancestors giving rise to central and North American Triatomini.

Population dynamics of Loa loa and Mansonella perstans infections in individuals living in an endemic area of the Congo.

A follow-up of Loa loa and Mansonella perstans microfilaremia was carried out in an adult population living in a highly endemic area of the Congo. Infection rates and parasite loads were found to be stable in the general population, both in the short-term (two months) and long-term (3-4 years) followup. The microfilarial status of most of the subjects examined did not change between tests. At the individual level, the microfilarial densities of L. loa and M. perstans also remained remarkably constant over time. This results in a qualitative and quantitative stability of the parasitic material available for the vectors.

Transmission indices of Loa loa in the Chaillu Mountains, Congo.

A longitudinal entomological survey of the vectors of loiasis was conducted in the Missama area (Lekoumou region) in the Congo from September 1987 to August 1989. The principal catching site was a palm grove surrounded by forest 3 km from the village. Landing/biting densities of Chrysops were measured by standardized fly catches lasting 11 hr carried out twice a month. Vector landing densities were also assessed in the Bantu and Pygmy villages and in the fields. Populations of Chrysops from the palm grove were examined 6 times a month for infection with the infective stage of Loa loa. Chrysops silacea was the predominate vector except at the beginning of the rainy season, when C. dimidiata was the prevailing species. Chrysops were caught throughout rainy season, from October to June. The host-seeking activity of C. silacea was greatest in the middle of this season (February), but occurred sooner (October) for C. dimidiata. The following variables associated with transmission were calculated from our observations in the palm grove (the first figure corresponds to the first year of the study and the figure in parentheses corresponds to the second year). It was calculated that 2.658 (2.185) C. silacea and 1.412 (1.182) C. dimidiata could bite a person in the palm grove per year, including an average of 14.4 (12.7) infective C. silacea and 9.8 (7.2) infective C. dimidiata. The percentage of all dissected flies with third stage larvae in the head and the mean number of larvae in the head/infective fly were 0.57% and 10.1 +/- 6.8 for C. silacea and 0.66% and 11.2 +/- 6.5 for C. dimidiata, respectively. The estimated annual transmission potentials were 171.1 (102.9) for C. silacea and 116.1 (73.8) for C. dimidiata. In the palm grove, transmission was ensured by 2 effective vectors during the rainy season (October to May). Although the annual biting rate for both species was twice as low in the village as in the forest, our data suggest that effective transmission occurs there also.

Clinical and biological study of Loa loa filariasis in Congolese.

Clinical and biological evaluations were carried out on 84 Congolese patients with parasitologically confirmed Loa loa filariasis (without concurrent infection with other filariae) and on 98 controls without filariasis. On the patients, 72 presented with microfilaremia; another 12 with negative blood tests were seen towards the end of an episode of subconjunctival migration of the adult worm. The incidence and severity of the clinical signs depended upon the method of recruitment. The 3 most common signs were pruritus and edema (both occurring in successive acute episodes affecting mainly the hands and forearms) and subconjunctival migration of adult filariae. Papulovesicular eruptions were located mainly on the arms. Headaches and arthralgia were noted more frequently than in the controls. No relation was found between the ABO blood groups and loiasis. Eosinophilia (higher in patients with symptoms) and raised serum IgE levels were found in nearly all patients and were strongly marked in approximately 66%. A positive correlation was observed between these 2 parameters. Fluorescent antibody levels (adult filaria Dipetalonema viteae antigen) were comparatively low in patients with microfilaremia.

Field application of polymerase chain reaction diagnosis and strain typing of Trypanosoma cruzi in Bolivian triatomines.

A new approach for direct identification and characterization of Trypanosoma cruzi stocks in biological samples was tested for field applicability on an extensive sample of feces collected from triatomine vectors from four different species found in Bolivia. The first step of the technique is polymerase chain reaction (PCR) amplification of the hypervariable region of kinetoplast DNA minicircles of T. cruzi parasites. In this report, 345 fecal samples were analyzed and the PCR results were compared with microscopic examination. For Triatoma infestans, the principal Bolivian vector, both techniques were in concordance 85.3% of the time. For the three other species, Rhodnius pictipes, Eratyrus mucronatus, and Triatoma sordida, the fecal samples were all negative by microscopic examination whereas PCR results showed several T. cruzi-infected insects in each species. The second step of the procedure is the characterization of the T. cruzi clones by means of hybridization of the PCR products with clone-specific probes generated by the PCR. We used two probes corresponding to major clones circulating in high frequency in Bolivia (as shown by previous population genetic studies using isoenzyme characterization). We obtained four primary results: 1) we confirm the importance of two major clones in Bolivia in two distinct regions; 2) we report high rates of mixed infections (multiple clones in a single vector) in Triatoma infestans, up to 22% and 35% in Cochabamba and La Paz departments, respectively; 3) the results favor the absence of interaction between different clones; and 4) we find, for the first time, evidence of the major clones circulating in three species of triatomines that are known as mainly sylvatic species.(ABSTRACT TRUNCATED AT 250 WORDS)

Chrysops silacea and C. dimidiata: fly densities and infection rates with Loa loa in the Chaillu mountains, Congo Republic.

The densities, infection rates with Loa loa, and parous rates of Chrysops silacea and C. dimidiata were studied in various ecological zones throughout the Chaillu mountains in the People's Republic of the Congo. In the rainy season, C. dimidiata was the major vector in the forest, whereas C. silacea was predominant in the cleared forest zones. Fly densities were higher in the forest (natural forest or palmgrove) than in the villages. Parous and infection rates varied according to the ecological zone. The infection rate of parous females was related to the microfilarial rate in the human population, indicating that the Chrysops populations were extremely stable in the rainy season.

Loa loa and Mansonella perstans filariasis in the Chaillu mountains, Congo: parasitological prevalence.

1934 Bantus and 379 Pygmies were investigated for Loa loa and Mansonella perstans filariasis in 7 villages in the Chaillu forest of the Congo. Bantus were more frequently infected with L. loa than Pygmies (18.9% of microfilariae carriers compared with 10.6%). In individuals over 30 years of age, males were more frequently infected than females. Microfilarial densities increased until the age of 20 years and then remained stable. Parasite load was not significantly different in the two ethnic groups. For mansonelliasis, the microfilarial rate was higher in the Pygmies (67.5% compared with 22.0%) and males of the 2 groups were more frequently infected than females. Microfilarial load was also higher in Pygmies than in Bantus (mean microfilarial densities (MfD 50) 13 and 2 respectively). In the Pygmy group, MfD 50 for M. perstans increased with age whereas it remained stable in the Bantus. 53.8% of the 249 questioned persons had experienced worm migration under the conjunctiva. Both ethnic groups were equally exposed to the vectors of L. loa and reasons for the difference in prevalence of microfilaria carriers are discussed. For mansonelliasis increased contact with vectors may explain the higher degree of infestation observed in Pygmies. Other filariases were infrequent in (Mansonella streptocerca), or absent from (Onchocerca volvulus and Wuchereria bancrofti), the study area.

Natural ecotopes of Triatoma infestans dark morph and other sylvatic triatomines in the Bolivian Chaco.

A survey of natural ecotopes of Triatoma infestans dark morph and other triatomine sylvatic species was performed in an uninhabited area of the Bolivian Chaco. Among the 321 triatomines collected by light trapping, only 4 T. infestans dark morph specimens were identified. Predominant flying species were T. guasayana and T. sordida group 2 (51.7% and 37.1% of capture, respectively). The same species prevailed in terrestrial and epiphytic bromeliads where scarce T. infestans dark morph nymphal instars were also detected. In parrot nests T. delpontei prevailed broadly over other species (90.2% of the capture) and only 4 T. infestans dark morph adults were collected. In contrast, T. infestans dark morph was the predominant species captured in hollow trees (46.0% of the total collected). The abundance of immature forms (88.2% of the collection) shows that hollow trees constitute a favourable ecotope for this species. Of the 421 trees investigated, 33.7% were positive for triatomines. T. infestans dark morph, found inside 15.0% of them, also had higher apparent density than other species (average number of T. infestans in positive trees, 2.0 +/- 1.6 vs 1.3 +/- 0.6 for other species). Light trapping seems to be an efficient method to sample the T. sordida-T. guasayana complex in that it shows a similar distribution to that observed in natural ecotopes; however, this method is ineffective for the assessment of the local abundance of T. infestans dark morph.

Serodiagnosis of sleeping sickness in the Republic of the Congo: comparison of indirect immunofluorescent antibody test and card agglutination test.

The card agglutination test for trypanosomiasis (CATT) was evaluated and compared to the classical immunofluorescent antibody test (IFAT) in the immunological diagnosis of Gambian trypanosomiasis. Tests were performed on serum and whole blood. Cross-reactions were found in the CATT with sera from patients suffering from parasitic infections other than sleeping sickness, but could be largely overcome by selecting 1/10 as the specific threshold dilution. At 1/40 dilution no false positive result was observed in the IFAT. At the specific threshold dilution, the sensitivity of IFAT was 94.7%, compared with 91.6% for the CATT. On whole blood, a more convenient sample in the field, IFAT specificity (100%) was greater than that of the CATT (94.3%), as was its sensitivity (92% compared with 82.5%). In view of its simplicity and rapidity of execution, the CATT is an efficient serological test to detect new foci. When greater sensitivity is required, IFAT should be preferred to CATT.

Serum lipid and lipoprotein abnormalities in human African trypanosomiasis.

We have studied the serum lipoprotein system in human African trypanosomiasis (Trypanosoma brucei gambiense infection). The study was carried out on 74 Congolense patients suffering from sleeping sickness and 34 Congolense control subjects living in the endemic region of Boko Songho. We have determined the serum concentrations of lipids (triglycerides, cholesterol, phospholipids) and apolipoproteins (apolipoprotein A-I and B), and the separation of serum lipoproteins by electrophoresis. For the patients infected with T. b. gambiense, in comparison with control subjects, the results have shown (i) a significant increase in triglyceride concentration and a decrease in cholesterol concentration; (ii) a significant rise in apolipoprotein B concentration and a significant reduction in apolipoprotein A-I concentration; and (iii) an increase in low density lipoproteins and a decrease in high density lipoproteins. We conclude, therefore, that human African trypanosomiasis is associated with marked alterations in the composition and levels of host lipoproteins.

Low probability of transmission of Trypanosoma cruzi to humans by domiciliary Triatoma sordida in Bolivia.

The role of Triatoma sordida in the domestic transmission of Trypanosoma cruzi was assessed in 7 rural localities in Velasco Province, Department of Santa Cruz, Bolivia. Tri. sordida, the only triatomine species identified in these localities, was found inside 58.0% of houses but not in large numbers (3.1 bugs per infested house on average). A total of 220 faecal samples from domiciliary bugs was examined microscopically and by the polymerase chain reaction for the presence of trypanosomes: 21.4% were infected. Analysis of blood meals of domiciliary Tri. sordida showed that humans were the commonest host (70.4%), followed by chickens and dogs. Four of 418 persons tested were seropositive for Tryp. cruzi. Only 2 of a second group of 62 persons living in dwellings infested by Tri. sordida were seropositive. Tryp. cruzi infection was demonstrated in dogs and domestic rats. Three other species of small mammals were found to be infected with trypanosomes. In our study area, domestic Tri. sordida are mainly incriminated in the transmission of Tryp. cruzi to synanthropic animals, whereas transmission to humans is very rare. The presence in houses of small populations of Tri. sordida infected with Tryp. cruzi is therefore currently insufficient for this insect to constitute a major epidemiological risk factor.