Relationship between genetic diversity and morpho-functional characteristics of flight-related traits in Triatoma garciabesi (Hemiptera: Reduviidae).

BACKGROUND: Triatoma garciabesi, a potential vector of the parasitic protozoan Trypanosoma cruzi, which is the causative agent of Chagas disease, is common in peridomestic and wild environments and found throughout northwestern and central Argentina, western Paraguay and the Bolivian Chaco. Genetic differentiation of a species across its range can help to understand dispersal patterns and connectivity between habitats. Dispersal by flight is considered to be the main active dispersal strategy used by triatomines. In particular, the morphological structure of the hemelytra is associated with their function. The aim of this study was to understand how genetic diversity is structured, how morphological variation of dispersal-related traits varies with genetic diversity and how the morphological characteristics of dispersal-related traits may explain the current distribution of genetic lineages in this species. METHODS: Males from 24 populations of T. garciabesi across its distribution range were examined. The cytochrome c oxidase I gene (coI) was used for genetic diversity analyses. A geometric morphometric method based on landmarks was used for morpho-functional analysis of the hemelytra. Centroid size (CS) and shape of the forewing, and contour of both parts of the forewing, the head and the pronotum were characterised. Length and area of the forewing were measured to estimate the aspect ratio. RESULTS: The morphometric and phylogenetic analysis identified two distinct lineages, namely the Eastern and Western lineages, which coincide with different ecological regions. The Eastern lineage is found exclusively in the eastern region of Argentina (Chaco and Formosa provinces), whereas the Western lineage is prevalent in the rest of the geographical range of the species. CS, shape and aspect ratio of the hemelytra differed between lineages. The stiff portion of the forewing was more developed in the Eastern lineage. The shape of both portions of the hemelytra were significantly different between lineages, and the shape of the head and pronotum differed between lineages. CONCLUSIONS: The results provide preliminary insights into the evolution and diversification of T. garciabesi. Variation in the forewing, pronotum and head is congruent with genetic divergence. Consistent with genetic divergence, morphometry variation was clustered according to lineages, with congruent variation in the size and shape of the forewing, pronotum and head.

Near infrared spectroscopy accurately detects Trypanosoma cruzi non-destructively in midguts, rectum and excreta samples of Triatoma infestans.

Chagas disease is a neglected tropical disease caused by Trypanosoma cruzi parasite with an estimated 70 million people at risk. Traditionally, parasite presence in triatomine vectors is detected through optical microscopy which can be low in sensitivity or molecular techniques which can be costly in endemic countries. The aim of this study was to evaluate the ability of a reagent-free technique, the Near Infrared Spectroscopy (NIRS) for rapid and non-invasive detection of T. cruzi in Triatoma infestans body parts and in wet/dry excreta samples of the insect. NIRS was 100% accurate for predicting the presence of T. cruzi infection Dm28c strain (TcI) in either the midgut or the rectum and models developed from either body part could predict infection in the other part. Models developed to predict infection in excreta samples were 100% accurate for predicting infection in both wet and dry samples. However, models developed using dry excreta could not predict infection in wet samples and vice versa. This is the first study to report on the potential application of NIRS for rapid and non-invasive detection of T. cruzi infection in T. infestans in the laboratory. Future work should demonstrate the capacity of NIRS to detect T. cruzi in triatomines originating from the field.

Vector competence and feeding-excretion behavior of Triatoma rubrovaria (Blanchard, 1843) (Hemiptera: Reduviidae) infected with Trypanosoma cruzi TcVI.

BACKGROUND: Several studies addressed changes on the insect vector behavior due to parasite infection, but little is known for triatomine bugs, vectors of Trypanosoma cruzi, the etiological agent of Chagas disease. We assessed infection rates and metacyclogenesis of T. cruzi (TcVI) in fifth-instar nymphs of Triatoma rubrovaria comparing with the primary vector Triatoma infestans. Also, biological parameters related to feeding-excretion behavior were evaluated aiming to identify which variables are most influenced by T. cruzi infection. METHODOLOGY/PRINCIPAL FINDINGS: Fifth-instar nymphs of T. rubrovaria and T. infestans were fed on mice infected with T. cruzi (TcVI). We compared the presence and the number of parasite evolutive forms in excreta of both triatomine species at 30, 60 and 90 days post-infection (dpi) with traditional statistical analyses. Moreover, both species were analyzed through generalized linear models and multinomial logistic regression hypotheses for seven behavioral parameters related to host-seeking and feeding-excretion. Triatoma rubrovaria and T. infestans had similar overall infection and metacyclogenesis rates of T. cruzi TcVI in laboratory conditions. Regarding vector behavior, we confirmed that the triatomine's tendency is to move away from the bite region after a blood meal, probably to avoid being noticed by the vertebrate host. Interspecific differences were observed on the volume of blood ingested and on the proportion of individuals that excreted after the blood meal, revealing the higher feeding efficiency and dejection rates of T. infestans. The amount of ingested blood and the bite behavior of T. rubrovaria seems to be influenced by TcVI infection. Infected specimens tended to ingest ~25% more blood and to bite more the head of the host. Noteworthy, in two occasions, kleptohematophagy and coprophagy behaviors were also observed in T. rubrovaria. CONCLUSIONS/SIGNIFICANCE: Laboratory infections revealed similar rate of T. cruzi TcVI trypomatigotes in excreta of T. rubrovaria and T. infestans, one of the most epidemiological important vectors of T. cruzi. Therefore, TcVI DTU was able to complete its life cycle in T. rubrovaria under laboratory conditions, and this infection changed the feeding behavior of T. rubrovaria. Considering these results, T. rubrovaria must be kept under constant entomological surveillance in Rio Grande do Sul, Brazil.

Assessing the entomo-epidemiological situation of Chagas disease in rural communities in the state of Piauí, Brazilian semi-arid region.

BACKGROUND: In northeastern Brazil, the wild nature of Trypanosoma cruzi vectors has challenged control actions. This study aims to describe the entomological and epidemiological scenario of Chagas disease in rural communities in the state of Piauí. METHODS: A cross-sectional study (n=683 individuals/244 dwellings) was carried out to obtain serum samples, sociodemographic data and intra- and peridomestic triatomines. RESULTS: The overall seroprevalence rate was 8.1%, with no positive tests among subjects <30 y of age. Prevalence rates reached 34.3% and 39.1% among subjects 61-75 and >75 y of age, respectively; 1474 triatomines were collected, of which 90.3% were found in peridomiciliary structures and 9.7% inside houses; 87.2% were classified as Triatoma brasiliensis. T. cruzi infection rates in insects were 0.5% by light microscopy and 0.9% by culture in NNN/LIT medium. Five cultivated isolates were submitted to molecular genotyping, three of which were identified as T. cruzi I and two as T. cruzi II. CONCLUSIONS: Although no vector transmission currently occurs, prevalence rates are high in adults and the elderly. This disease should be targeted by primary healthcare providers. Insect surveillance and control activities should not be discontinued in an environment favourable to the perpetuation of house colonization by triatomines.

Development of conventional and real-time multiplex PCR-based assays for estimation of natural infection rates and Trypanosoma cruzi load in triatomine vectors.

BACKGROUND: Chagas disease is a complex anthropozoonosis with distinct domestic and sylvatic mammal species acting as potential reservoirs. The diversity of vector species and their habitats are among the factors that hinder the control of the disease. Control programs periodically monitor the prevalence of T. cruzi infection in insect bugs through microscopical observation of diluted feces. However, microscopy presents limited sensitivity in samples with low parasite numbers, difficulties in examining all evolutionary stages of the insect and may in turn be limited to differentiate T. cruzi from other morphologically similar trypanosomatids. Here, we report two highly sensitive and accurate methodologies to infer T. cruzi infection rates and to quantify parasite load in the gut of field-collected triatomines. METHODS: Triatomines were manually collected in the period 2011-2012 and 2014-2015, in domestic, peridomestic or sylvatic habitats in rural areas of 26 municipalities, encompassing three distinct Brazilian biomes: Caatinga, Cerrado and Atlantic Rainforest. Following morphological and taxonomical identification, the search for flagellated protozoa was performed by optical microscopy. A conventional PCR targeting T. cruzi kDNA and a TaqMan qPCR directed to the parasite nuclear satellite DNA (SAT) were developed, both in multiplex, with the triatomine 12S subunit ribosomal RNA gene, used as internal amplification control. Both methods were used for detection (kDNA-PCR) and parasite load quantification (SAT-DNA-qPCR), to investigate T. cruzi infection in captured triatomines. RESULTS: The combined methods were assayed on a panel of 205 field-collected triatomine samples. Diagnostic analysis revealed 21% positivity for the kDNA-PCR, whereas microscopic examination enabled identification of T. cruzi in only 7.0% of the PCR-positive samples. Negative PCR results were confirmed by the absence of T. cruzi flagellates using microscopy. Caatinga biome yielded the highest T. cruzi infection rate (60%), followed by the Atlantic Rainforest and Cerrado with 7.1 and 6.1%, respectively. In addition, a wide range distribution of parasite load, varying from 8.05 × 10-2 to 6.31 × 1010 was observed with a median of 2.29 × 103 T. cruzi/intestine units. When parasite load was analyzed by triatomine species, a significantly higher median was found for Panstrongylus lutzi in comparison with Triatoma brasiliensis. CONCLUSIONS: Our results demonstrate highly sensitive PCR-based methodologies to monitor T. cruzi infection in triatomines. In addition, the qPCR assay offers the possibility of further evaluation parasite load, as a promising biomarker of the vectorial capacity of triatomines in Chagas disease endemic areas.

Importância epidemiológica de espécies do subcomplexo Triatoma rubrovaria por meio da análise de competência vetorial e hábito alimentar

O controle da doença de Chagas, que é causada pelo protozoário Trypanosoma cruzi, concentra-se principalmente na eliminação de vetores com comprovada adaptação aos domicílios humanos. Ainda que insetos vetores domiciliados sejam os de maior importância epidemiológica, vetores autóctones de importância secundária são coletados constantemente em ambientes antrópicos, proporcionando risco de transmissão do parasito para humanos. Este estudo propôs identificar espécies do subcomplexo Triatoma rubrovaria por taxonomia molecular (Cyt b) e analisar seu potencial vetorial na transmissão do parasito no bioma Pampa. Diferentes aspectos relacionados à capacidade vetorial foram avaliados, como fonte alimentar, taxa de infecção e genotipagem parasitária. Em paralelo, parâmetros bionômicos de T. rubrovaria após infecção por T. cruzi foram analisados experimentalmente. Um total de 1.724 triatomíneos foi coletado no Rio Grande do Sul, dos quais 927 insetos tiveram o DNA de seus intestinos extraídos para as análises moleculares. A filogenia do subcomplexo agrupou as 92 amostras sequenciadas com sucesso para o fragmento de Cyt b em nove clados. Das amostras sequenciadas, 19 (20,7%) foram identificadas como T. carcavalloi, 17 (18,5%) como T. circummaculata e 12 (13,04%) como T. rubrovaria. As restantes foram agrupadas em cinco clados sem sequência-referência, sendo quatro no clado 1, três no clado 2, cinco no clado 3, quinze no clado 4 e dezessete no clado 5. Dada a baixa divergência entre as sequências do subcomplexo e a sobreposição das divergências intra e interclados (K2P = 0,0-2,6% e 0,7-5,0%), é provável que os membros do subcomplexo estejam em processo de especiação e que ainda haja fluxo gênico interespecífico. Foram observadas no campo uma taxa de infecção por T. cruzi de 2,8% (26/927), com variação de carga parasitária de 1,5 × 101 a 2,3 × 107 equivalentes de parasito/intestino e a presença de TcI, TcV e coinfecção por TcI + TcIV. A partir de iniciadores universais para vertebrados, doze espécies de mamíferos foram identificadas, além de aves e insetos, sendo Homo sapiens a fonte alimentar detectada com maior frequência (73,5%), seguida de Gallus gallus (33,1%). Para análises de competência vetorial, ninfas N5 de T. rubrovaria e T. infestans foram alimentadas em camundongos infectados com T. cruzi (TcVI), em condições de laboratório. Comparamos a presença e o número de formas evolutivas do parasito nas excretas de ambas as espécies de triatomíneos aos 30, 60 e 90 dias pós-infecção. Triatoma rubrovaria e T. infestans apresentaram resultados semelhantes nas taxas de infecção e metaciclogênese de T. cruzi TcVI. Em relação ao comportamento vetorial, confirmamos que o triatomíneo tende a se afastar do local de picada após o repasto sanguíneo. Diferenças interespecíficas foram observadas quanto ao volume de sangue ingerido e proporção de indivíduos que excretaram após alimentação de sangue, revelando a maior eficiência alimentar e taxa de dejeção de T. infestans. O volume de sangue ingerido e o comportamento de picada de T. rubrovaria parecem ser influenciados pela infecção por TcVI. Espécimes infectados tendem a ingerir ~25% a mais de sangue e a picar mais a cabeça do hospedeiro. Comportamentos de cleptohematofagia e de coprofagia também foram observados em T. rubrovaria. Os resultados obtidos aqui sugerem a necessidade urgente de uma revisão taxonômica do grupo. Ademais, nossas análises evidenciam que T. rubrovaria é um vetor potencial de T. cruzi, apresentando parâmetros bionômicos associados à sua capacidade vetorial semelhantes ao do vetor T. infestans, principalmente quando infectados, alertando para a importância da vigilância entomológica constante nas áreas estudadas.

Avaliação de taxas de infecção, carga parasitária e tipagem molecular de Trypanosoma cruzi em triatomíneos provenientes do Ceará (Caatinga) e Rio Grande do Sul (Pampa)

Embora o Brasil mantenha constante a operação de vigilância e controle dos principais vetores da doença de Chagas, o risco de transmissão do Trypanosoma cruzi ainda persiste. Estimativas de taxas de infecção natural por T. cruzi em triatomíneos são importantes para avaliar um possível risco de transmissão vetorial. Deste modo, o objetivo do presente estudo é estimar taxas de infecção natural, carga parasitária e caracterização de linhagens de T. cruzi diretamente de triatomíneos capturados em dois biomas brasileiros: Caatinga e Pampa. A técnica de PCR convencional multiplex, por apresentar maior sensibilidade, reprodutibilidade e fidedignidade, comparada à observação microscópica direta do parasito, foi utilizada para a detecção simultânea do DNA cinetoplástico de T. cruzi e da subunidade 12S do gene ribossomal de triatomíneos. Para a estimativa da carga parasitária por qPCR, foram realizados ensaios de quantificação absoluta utilizando o sistema TaqMan multiplex, com o mesmo alvo para triatomíneos e empregando o DNA nuclear satélite de T. cruzi. A caracterização molecular do parasito foi realizada a partir de adaptações dos algoritmos de PCR preconizados previamente. A PCR convencional multiplex demonstrou sensibilidade superior à microscopia para o diagnóstico de triatomíneos coletados. No Ceará o percentual de positividade para T. cruzi, por PCR, foi maior em P. lutzi (6/7; 85,7%), seguido de T. brasiliensis (29/158; 18,4%) e T. pseudomaculata (28/230; 12,2%). As maiores cargas parasitárias observadas foram em T. pseudomaculata e T. brasiliensis (9,1 × 107 e 1,9 × 107 equivalentes de parasito, respectivamente), e a menor em P. lutzi (1,2 × 10-2 equivalentes de T. cruzi). O genótipo TcI foi detectado em T. brasiliensis, T. pseudomaculata e P. lutzi; e as DTUs TcV, TcII e TcVI em T. brasiliensis. Coinfecções por TcIII+V e TcI+II/V/VI foram observadas em P. lutzi e em T. brasiliensis, respectivamente. T. brasiliensis infectados com TcII e TcVI apresentaram cargas parasitárias mais altas (1,8 × 107 e 1,9 × 107 equivalentes de parasito, respectivamente). No Rio Grande do Sul a taxa de infecção natural foi superior em T. rubrovaria (4/8; 50%), seguido de T. circummaculata (3/9; 33,3%), T. infestans (10/123; 8,1%) e T. carcavalloi (2/33; 6,1%). Entre os Triatoma sp., 4,8% (11/228) foram positivas para T. cruzi. A maior carga parasitária foi observada em T. rubrovaria (3 × 107 equivalentes de parasito) e as mais baixas em Triatoma sp. e T. infestans (1,5 × 101 e 2,1 × 101 equivalentes de T. cruzi, respectivamente). TcI foi encontrada em T. rubrovaria, T. infestans e Triatoma sp.. T. circummaculata continha coinfecção por TcI+IV. A presença de triatomíneos infectados por T. cruzi nos ambientes domiciliar e peridomiciliar sugerem um possível risco de transmissão em humanos nas regiões estudadas. Além disso, os dados apresentados demonstram que o ciclo de transmissão de T. cruzi nos diferentes ecótopos é altamente complexo e que a distribuição geográfica dos genótipos do parasito é subestimada no Brasil, sendo necessárias novas investigações para uma melhor compreensão acerca da interação parasito-vetor-hospedeiro-reservatório. O presente estudo confirmou a fidedignidade das técnicas moleculares empregadas para a avaliação de triatomíneos naturalmente infectados por T. cruzi.