Diet of the sylvatic triatomine Mepraia spinolai: Association with Trypanosoma cruzi infection near human settlements.

The proximity between infectious disease vector populations and human settlements, and the infection prevalence of vector populations can determine the rate of encounters between vectors and humans and hence infection risk. The diet of sylvatic triatomine vectors (kissing bugs) provides evidence about the host species involved in the maintenance of the protozoan Trypanosoma cruzi, the etiological agent of Chagas disease. Here, we characterized the diet of the Chilean endemic triatomine Mepraia spinolai using Next Generation Sequencing (NGS), and evaluated the relation between T. cruzi infection status and proximity to human settlements, with the proportion of human and human-associated (domestic and synanthropic) vertebrates in the diet. We sampled 28 M. spinolai populations, covering a latitudinal range of ∼800 km in Chile. For each population, genomic DNA was obtained from M. spinolai intestinal content. We assessed T. cruzi infection individually, and sequenced vertebrate cytochrome b to characterize the diet from infected and uninfected pooled samples. Human and human-associated animals were present in the diet of both T. cruzi-infected (13.50 %) and uninfected (10.43 %) kissing bugs. The proportion of human and human-associated vertebrates in the diet of infected M. spinolai was negatively associated with the distance from surrounding human settlements, but no relationship was detected for uninfected kissing bugs. This pattern could be related to alterations of kissing bug feeding behavior when infected by the protozoan. Our results highlight the relevance of developing a deeper knowledge of the wild transmission cycle of T. cruzi, thus advancing in the surveillance of vectors present in the natural environment near human settlements.

Humans as blood-feeding sources in sylvatic triatomines of Chile unveiled by next-generation sequencing.

BACKGROUND: Triatomines are blood-sucking insects capable of transmitting Trypanosoma cruzi, the parasite that causes Chagas disease in humans. Vectorial transmission entails an infected triatomine feeding on a vertebrate host, release of triatomine infective dejections, and host infection by the entry of parasites through mucous membranes, skin abrasions, or the biting site; therefore, transmission to humans is related to the triatomine-human contact. In this cross-sectional study, we evaluated whether humans were detected in the diet of three sylvatic triatomine species (Mepraia parapatrica, Mepraia spinolai, and Triatoma infestans) present in the semiarid-Mediterranean ecosystem of Chile. METHODS: We used triatomines collected from 32 sites across 1100 km, with an overall T. cruzi infection frequency of 47.1% (N = 4287 total specimens) by conventional PCR or qPCR. First, we amplified the vertebrate cytochrome b gene (cytb) from all DNA samples obtained from triatomine intestinal contents. Then, we sequenced cytb-positive PCR products in pools of 10-20 triatomines each, grouped by site. The filtered sequences were grouped into amplicon sequence variants (ASVs) with a minimum abundance of 100 reads. ASVs were identified by selecting the best BLASTn match against the NCBI nucleotide database. RESULTS: Overall, 16 mammal (including human), 14 bird, and seven reptile species were identified in the diet of sylvatic triatomines. Humans were part of the diet of all analyzed triatomine species, and it was detected in 19 sites representing 12.19% of the sequences. CONCLUSIONS: Sylvatic triatomine species from Chile feed on a variety of vertebrate species; many of them are detected here for the first time in their diet. Our results highlight that the sylvatic triatomine-human contact is noteworthy. Education must be enforced for local inhabitants, workers, and tourists arriving in endemic areas to avoid or minimize the risk of exposure to Chagas disease vectors.

Natural Trypanosoma cruzi Infection and Climatic Season Influence the Developmental Capacity in Field-Caught Mepraia spinolai Nymphs.

In this study, we evaluated the effect of the climatic season and infection by Trypanosoma cruzi, etiological agent of Chagas disease, on the molting capacity of the triatomine vector Mepraia spinolai endemic to Chile. We used wild-caught first-to-fourth instar nymphs during cooling (fall and winter) and warming (spring) periods. After capturing, nymphs were fed at the laboratory, and maintained under optimal rearing conditions. Feeding was repeated 40 days later. We followed-up the molting events on 709 nymphs, recording one, two or the absence of molts after two feeding opportunities. Within the same climatic period, only infected second- and fourth-instar nymphs from the warming period showed a larger proportion of double molting compared to uninfected nymphs. Regarding the climatic period, infected and uninfected first- and fourth-instar nymphs exhibited a larger proportion of double molting in the warming and cooling periods, respectively. The pattern of non-molting nymph occurrence suggests they probably reach diapause by environmental stochasticity. The effect of the climatic period and T. cruzi infection on the development of M. spinolai is an instar-dependent phenomenon, highlighting the occurrence of finely synchronized processes at different moments of the life cycle of such an hemimetabolous insect as triatomines.

Opportunistic or selective? Stage-dependent feeding behavior in a wild vector of Chagas disease.

The composition and contribution of different host species in the dynamics of vector-borne zoonotic parasites are particularly relevant for public health. Hence, the study of host selection by vectors is fundamental. Developmental stage and infection status are factors that may modulate vector feeding behavior. In the semi-arid Mediterranean ecosystem of South America, the transmission of Trypanosoma cruzi, the protozoan causing Chagas disease, includes the triatomine vector Mepraia spinolai and several vertebrate species. In this field study, we examined whether M. spinolai exhibits an opportunistic feeding behavior dependent upon developmental stage and/or infection status. We found that M. spinolai does not feed according to the relative availability of vertebrate species. In addition, early stage nymphs (first/second instars) fed on twice as many different species as middle (third/fourth instars) and late (fifth instars and adults) M. spinolai, with the former feeding on native rodents and lizards and the latter mostly on rabbits. Infected and uninfected M. spinolai showed similar feeding profiles. Wild triatomine species might be described as stage-dependent selective blood feeders, as a consequence of the temporal and spatial scale at which host-vector interactions occur, highlighting that all developmental stages might be infected and capable of transmitting T. cruzi.

Genetic diversity in a restricted-dispersal kissing bug: The centre-periphery hypothesis halfway.

The centre-periphery hypothesis (CPH) postulates that populations close to the centre of a species distribution will exhibit higher genetic diversity and lower genetic differentiation than populations located at the edge of the distribution. The centre of a species' distribution might represent an optimum for the environmental factors influencing the species absolute fitness and, therefore, genetic diversity. In species with wide distribution, the geographical variation of biotic and abiotic variables is crucial to understand the underlying mechanisms of the CPH. We evaluated the CPH and specifically tested which environmental variables better explained the patterns of genetic diversity in the kissing bug Mepraia spinolai, one of the main wild vectors of Chagas disease in southern South America, distributed across three Mediterranean climatic ecoregions in Chile. We analysed 2380 neutral single nucleotide polymorphisms to estimate genetic diversity. Mean winter temperature, mean summer temperature, vegetation cover, population abundance, proportion of winged individuals and female abdomen area were measured for each kissing bug population to construct a model. Lower genetic diversity was detected in populations at the edge of the distribution compared to those in the centre. However, genetic differentiation was not higher in the periphery. Genetic diversity was related to climatic and biological variables; there was a positive relationship with mean winter temperature and a negative association with mean summer temperature and body size. These results partially support the CPH and identify biotic (abdomen area) and abiotic (winter/summer temperatures) factors that would affect genetic diversity in this restricted-dispersal species of epidemiological relevance.

Trypanosoma cruzi could affect wild triatomine approaching behaviour to humans by altering vector nutritional status: A field test.

Hematophagous insects exhibit complex behaviour when searching for blood-meals, responding to several host stimuli. The hematophagous insect Mepraia spinolai is a wild vector of Trypanosoma cruzi, causative agent of Chagas disease in humans, in the semiarid-Mediterranean ecosystem of Chile. In this study, we evaluated the association between the approaching behaviour to a human host, with T. cruzi infection status and nutritional condition of M. spinolai. To this end, we captured 501 individuals in six consecutive 10 min-timespan, using a human as bait. Captured vectors were weighed, photographed and measured to calculate their nutritional status by means of a Standardized Body Mass Index. Trypanosoma cruzi infection was assessed in the intestinal content by using a real-time PCR assay. Ordinal logistic regressions were performed separately for infected and uninfected groups to evaluate if the nutritional status was associated with the approaching behaviour to a human host, recorded as the time-span of capture. Nutritional status of uninfected triatomines was higher than that from infected ones (p < 0.005). Among the infected, those with higher nutritional status approached first (p < 0.01); there was no effect of nutritional status in the uninfected group. Trypanosoma cruzi infection might affect the foraging behaviour of M. spinolai under natural conditions, probably deteriorating nutritional status and/or altering vector detection abilities.

Prevalence, infected density or individual probability of infection? Assessing vector infection risk in the wild transmission of Chagas disease.

Vector-borne infectious disease dynamics result mainly from the intertwined effect of the diversity, abundance, and behaviour of hosts and vectors. Most studies, however, have analysed the relationship between host-species diversity and infection risk, focusing on vector population instead of individuals, probably dismissing the level at which the transmission process occurs. In this paper, we examine the importance of the host community in accounting for infection risk, at both population and individual levels, using the wild transmission of the protozoan that causes Chagas disease as a vector-borne disease model. Chagas disease is caused by Trypanosoma cruzi, transmitted by triatomine insects to mammals. We assessed if T. cruzi infection in vectors is explained by small mammal diversity and their densities (total and infected), when infection risk is measured at population level as infection prevalence (under a frequency-dependent transmission approach) and as density of infected vectors (density-dependent transmission approach), and when measured at individual level as vector infection probability. We analysed the infection status of 1974 vectors and co-occurring small mammal hosts in a semiarid-Mediterranean ecosystem. Results revealed that regardless of the level of analysis, only one host rodent species accounted for most variation in vector infection risk, suggesting a key role in the transmission cycle. To determine the factors explaining vector-borne disease dynamics, infection risk should be assessed at different scales, reflecting the factors meaningful from the vector's perspective and considering vector class-specific features.

Survivorship of wild caught Mepraia spinolai nymphs: The effect of seasonality and Trypanosoma cruzi infection after feeding and fasting in the laboratory.

Chagas disease is caused by Trypanosoma cruzi. Vector survival is an important variable affecting vectorial capacity to determine parasite transmission risk. The aims of this study are to evaluate vector survival under fasting/starvation conditions of wild-caught Mepraia spinolai after feeding and fasting, the pathogenicity of T. cruzi infection, the parasite burden and seasonal variation in parasite discrete typing units (DTU). The survivorship of M. spinolai nymphs after two continuous artificial feedings was evaluated, assessing their infection with microscopic observation of fecal samples and PCR. Later, insects were fasted/starved until death. We performed qPCR analyses of parasite load in the fecal samples and dead specimens. T. cruzi genotyping was performed using conventional PCR amplicons and hybridization tests. Infection rate was higher in M. spinolai nymphs in summer and spring than in fall. Parasite burden varied from 3 to 250,000 parasites/drop. Survival rate for starved nymph stage II was lower in insects collected in the spring compared to summer and fall. TcII was the most frequent DTU. Mainly metacyclic trypomastigotes were excreted. We conclude that M. spinolai infection rate in nymphs varies among seasons, suggesting higher transmission risk in warmer seasons. However, nymphs stage II collected in spring are more sensitive to starvation compared to other seasons. TcII in single or mixed infection does not seem relevant to determine vector pathogenicity. These results of vector survivorship after fasting/starvation are important to determine the competence of M. spinolai as a vector of T. cruzi, since they excrete metacyclic trypomastigotes and the parasitism with T. cruzi seems to be poorly pathogenic to the vector under a severe fasting/starvation condition.

Wing Polymorphism and Trypanosoma cruzi Infection in Wild, Peridomestic, and Domestic Collections of Mepraia spinolai (Hemiptera: Reduviidae) From Chile.

Mepraia spinolai (Porter) is a vector of Trypanosoma cruzi that causes Chagas disease. Females are always wingless, but males may be winged or wingless. We determined by PCR the infection percentage with T. cruzi of M. spinolai adults and nymphs in domestic, peridomestic, and wild collections, in different regions of Chile. In all regions, winged males were more abundant than females and wingless males. Winged males collected inside houses were less parasitized than were those from peridomestic and wild environments. Although winged males of M. spinolai have comparatively low levels of infection, this segment may still represent the greatest vector threat in this species for transmission of T. cruzi to humans and other vertebrates in domestic, wild, and peridomestic habitats. Winged males represent the dispersive form of this species that invades human dwellings. Feeding deprivation resulting from the time required to find a food source and to search for reproductive females could explain the lower infection rates (negatives) of winged males collected from inside houses in comparison with winged males collected from peridomestic and wild habitats.

Can body traits, other than wings, reflect the flight ability of Triatominae bugs?

Abstract: INTRODUCTION : Insects of the subfamily Triatominae are vectors of Trypanosoma cruzi , the Chagas disease parasite, and their flying behavior has epidemiological importance. The flying capacity is strikingly different across and within Triatominae species, as well as between sexes or individuals. Many Triatoma infestans individuals have wings but no flying muscles. In other Triatominae species, no clear relationships were found between wing length and flying behavior. If wing presence or size is not reflective of the flying behavior, which other parts of the body could be considered as reliable markers of this important function? METHODS : The genus Mepraia has exceptional characteristics with invariably wingless females and wingless or winged males. We calculated the porous surface exposed to odorant molecules to estimate the olfactory capacity of Mepraia spinolai . The head shape and thorax size were estimated using the geometric morphometric approach and traditional morphometric techniques, respectively. RESULTS : Alary polymorphism in M. spinolai was significantly associated with consistent modification of the thorax size, head shape, and notable change in the estimated olfactory capacity. The macropterous individuals had a larger olfactory surface and thorax size and significantly different head shape compared to those of the micropterous individuals. CONCLUSIONS: We concluded that these structural changes could be associated with the flying potential of Triatominae. Thus, morphological attributes not found on wings could help determine the likely flying potential of the bugs.

Effects of mammal host diversity and density on the infection level of Trypanosoma cruzi in sylvatic kissing bugs.

Several reports have described host species diversity and identity as the most important factors influencing disease risk, producing either dilution or amplification of the pathogen in a host community. Triatomine vectors, mammals and the protozoan Trypanosoma cruzi (Trypanosomatida: Trypanosomatidae) Chagas are involved in the wild cycle of Chagas disease, in which infection of mammals occurs by contamination of mucous membranes or skin abrasions with insect-infected faeces. We examined the extent to which host diversity and identity determine the infection level observed in vector populations (i.e. disease risk in humans). We recorded infection in triatomine colonies and on the coexisting host mammalian species in semi-arid Chile. Host diversity, and total and infected host species densities are used as predictor variables for disease risk. Disease risk did not correlate with host diversity changes. However, the densities of each infected rodent species were positively associated with disease risk. We suggest that the infected host density surrounding the vector colonies is a relevant variable for disease risk and should be considered to understand disease dynamics. It is crucial to pay attention on the spatial scale of analysis, considering the pattern of vector dispersal, when the relationship between host diversity and disease risk is studied.

High phylogeographic structure in sylvatic vectors of Chagas disease of the genus Mepraia (Hemiptera: Reduviidae).

The hematophagous Hemiptera of the subfamily Triatominae are a very diverse group with a variety of morphs, behaviors and distributions. They have great epidemiological importance because many of its members are vectors of the protozoan Trypanosoma cruzi, the agent of Chagas disease. Mepraia is a genus of Triatominae endemic to Chile responsible for transmitting T. cruzi in the sylvatic cycle. Mepraia includes three species, M. gajardoi (18° 30'-26° 30' S) M. spinolai (26° 30'-34° 20' S) and the recently described M. parapatrica in intermediate zones (24° 36'-26° 51' S). Using mitochondrial DNA sequences, we inferred historical processes that led to the current structure of populations. Phylogeographic analyses identified three lineages, congruent with current taxonomy, and populations were highly structured. The times to the most recent common ancestor suggest that M. spinolai is the oldest lineage. We discuss the taxonomic and biogeographic implications of our results.