Wolbachia strains wMel and wAlbB differentially affect Aedes aegypti traits related to fecundity.

Two Wolbachia strains, wMel and wAlbB, have been transinfected into Aedes aegypti mosquitoes for population replacement with the aim of reducing dengue transmission. Epidemiological data from various endemic sites suggest a pronounced decrease in dengue transmission after implementing this strategy. In this study, we investigated the impact of the Wolbachia strains wMel and wAlbB on Ae. aegypti fitness in a common genetic background. We found that Ae. aegypti females infected with the wMel strain exhibited several significant differences compared with those infected with the wAlbB strain. Specifically, wMel-infected females laid significantly fewer eggs, ingested a lower amount of blood, had a reduced egg production rate, and exhibited a decreased Wolbachia density at a later age compared with mosquitoes infected with the wAlbB strain. Conversely, the wAlbB strain showed only mild negative effects when compared with Wolbachia-uninfected specimens. These differential effects on Ae. aegypti fitness following infection with either wMel or wAlbB may have important implications for the success of population replacement strategies in invading native Ae. aegypti populations in endemic settings. Further research is needed to better understand the underlying mechanisms responsible for these differences in fitness effects and their potential impact on the long-term efficacy of Wolbachia-based dengue control programs.IMPORTANCEThe transmission of arboviruses such as dengue, Zika, and chikungunya is on the rise globally. Among the most promising strategies to reduce arbovirus burden is the release of one out of two strains of Wolbachia-infected Aedes aegypti: wMel and wAlbB. One critical aspect of whether this approach will succeed involves the fitness cost of either Wolbachia strains on mosquito life history traits. For instance, we found that wMel-infected Ae. aegypti females laid significantly fewer eggs, ingested a lower amount of blood, had a reduced egg production rate, and exhibited a decreased Wolbachia density at a later age compared with mosquitoes infected with the wAlbB strain. Conversely, the wAlbB strain showed only mild negative effects when compared with Wolbachia-uninfected specimens. These differential effects on mosquito fitness following infection with either wMel or wAlbB may have important implications for the success of population replacement strategies in invading native Ae. aegypti populations.

Gut Bacterial Diversity of Field and Laboratory-Reared Aedes albopictus Populations of Rio de Janeiro, Brazil.

BACKGROUND: The mosquito microbiota impacts different parameters in host biology, such as development, metabolism, immune response and vector competence to pathogens. As the environment is an important source of acquisition of host associate microbes, we described the microbiota and the vector competence to Zika virus (ZIKV) of Aedes albopictus from three areas with distinct landscapes. METHODS: Adult females were collected during two different seasons, while eggs were used to rear F1 colonies. Midgut bacterial communities were described in field and F1 mosquitoes as well as in insects from a laboratory colony (>30 generations, LAB) using 16S rRNA gene sequencing. F1 mosquitoes were infected with ZIKV to determine virus infection rates (IRs) and dissemination rates (DRs). Collection season significantly affected the bacterial microbiota diversity and composition, e.g., diversity levels decreased from the wet to the dry season. Field-collected and LAB mosquitoes' microbiota had similar diversity levels, which were higher compared to F1 mosquitoes. However, the gut microbiota composition of field mosquitoes was distinct from that of laboratory-reared mosquitoes (LAB and F1), regardless of the collection season and location. A possible negative correlation was detected between Acetobacteraceae and Wolbachia, with the former dominating the gut microbiota of F1 Ae. albopictus, while the latter was absent/undetectable. Furthermore, we detected significant differences in infection and dissemination rates (but not in the viral load) between the mosquito populations, but it does not seem to be related to gut microbiota composition, as it was similar between F1 mosquitoes regardless of their population. CONCLUSIONS: Our results indicate that the environment and the collection season play a significant role in shaping mosquitoes' bacterial microbiota.

Genomic analysis of two phlebotomine sand fly vectors of Leishmania from the New and Old World.

Phlebotomine sand flies are of global significance as important vectors of human disease, transmitting bacterial, viral, and protozoan pathogens, including the kinetoplastid parasites of the genus Leishmania, the causative agents of devastating diseases collectively termed leishmaniasis. More than 40 pathogenic Leishmania species are transmitted to humans by approximately 35 sand fly species in 98 countries with hundreds of millions of people at risk around the world. No approved efficacious vaccine exists for leishmaniasis and available therapeutic drugs are either toxic and/or expensive, or the parasites are becoming resistant to the more recently developed drugs. Therefore, sand fly and/or reservoir control are currently the most effective strategies to break transmission. To better understand the biology of sand flies, including the mechanisms involved in their vectorial capacity, insecticide resistance, and population structures we sequenced the genomes of two geographically widespread and important sand fly vector species: Phlebotomus papatasi, a vector of Leishmania parasites that cause cutaneous leishmaniasis, (distributed in Europe, the Middle East and North Africa) and Lutzomyia longipalpis, a vector of Leishmania parasites that cause visceral leishmaniasis (distributed across Central and South America). We categorized and curated genes involved in processes important to their roles as disease vectors, including chemosensation, blood feeding, circadian rhythm, immunity, and detoxification, as well as mobile genetic elements. We also defined gene orthology and observed micro-synteny among the genomes. Finally, we present the genetic diversity and population structure of these species in their respective geographical areas. These genomes will be a foundation on which to base future efforts to prevent vector-borne transmission of Leishmania parasites.

Molecular and cytogenetic evidence for sibling species in the Chagas disease vector Triatoma maculata.

Triatoma maculata (Hemiptera, Reduviidae, Triatominae) occurs across dry-to-semiarid ecoregions of northern South America, where it transmits Trypanosoma cruzi, causative agent of Chagas disease. Using 207 field-caught specimens from throughout the species' range, mitochondrial(mt) DNA sequence data, and cytogenetics, we investigated inter-population genetic diversity and the phylogenetic affinities of T. maculata. Mitochondrial DNA sequence analyses (cytb and nd4) disclosed a monophyletic T. maculata clade encompassing three distinct geographic groups: Roraima formation (Guiana shield), Orinoco basin, and Magdalena basin (trans-Andean). Between-group cytb distances (11.0-12.8%) were larger than the ~7.5% expected for sister Triatoma species; the most recent common ancestor of these T. maculata groups may date back to the late Miocene. C-heterochromatin distribution and the sex-chromosome location of 45S ribosomal DNA clusters both distinguished Roraima bugs from Orinoco and Magdalena specimens. Cytb genealogies reinforced that T. maculata is not sister to Triatoma pseudomaculata and probably represents an early (middle-late Miocene) offshoot of the 'South American Triatomini lineage'. In sum, we report extensive genetic diversity and deep phylogeographic structuring in T. maculata, suggesting that it may consist of a complex of at least three sibling taxa. These findings have implications for the systematics, population biology, and perhaps medical relevance of T. maculata sensu lato.

Rapid and Non-Invasive Detection of Aedes aegypti Co-Infected with Zika and Dengue Viruses Using Near Infrared Spectroscopy.

The transmission of dengue (DENV) and Zika (ZIKV) has been continuously increasing worldwide. An efficient arbovirus surveillance system is critical to designing early-warning systems to increase preparedness of future outbreaks in endemic countries. The Near Infrared Spectroscopy (NIRS) is a promising high throughput technique to detect arbovirus infection in Ae. aegypti with remarkable advantages such as cost and time effectiveness, reagent-free, and non-invasive nature over existing molecular tools for similar purposes, enabling timely decision making through rapid detection of potential disease. Our aim was to determine whether NIRS can differentiate Ae. aegypti females infected with either ZIKV or DENV single infection, and those coinfected with ZIKV/DENV from uninfected ones. Using 200 Ae. aegypti females reared and infected in laboratory conditions, the training model differentiated mosquitoes into the four treatments with 100% accuracy. DENV-, ZIKV-, and ZIKV/DENV-coinfected mosquitoes that were used to validate the model could be correctly classified into their actual infection group with a predictive accuracy of 100%, 84%, and 80%, respectively. When compared with mosquitoes from the uninfected group, the three infected groups were predicted as belonging to the infected group with 100%, 97%, and 100% accuracy for DENV-infected, ZIKV-infected, and the co-infected group, respectively. Preliminary lab-based results are encouraging and indicate that NIRS should be tested in field settings to evaluate its potential role to monitor natural infection in field-caught mosquitoes.

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.

Under pressure: phenotypic divergence and convergence associated with microhabitat adaptations in Triatominae.

BACKGROUND: Triatomine bugs, the vectors of Chagas disease, associate with vertebrate hosts in highly diverse ecotopes. It has been proposed that occupation of new microhabitats may trigger selection for distinct phenotypic variants in these blood-sucking bugs. Although understanding phenotypic variation is key to the study of adaptive evolution and central to phenotype-based taxonomy, the drivers of phenotypic change and diversity in triatomines remain poorly understood. METHODS/RESULTS: We combined a detailed phenotypic appraisal (including morphology and morphometrics) with mitochondrial cytb and nuclear ITS2 DNA sequence analyses to study Rhodnius ecuadoriensis populations from across the species' range. We found three major, naked-eye phenotypic variants. Southern-Andean bugs primarily from vertebrate-nest microhabitats (Ecuador/Peru) are typical, light-colored, small bugs with short heads/wings. Northern-Andean bugs from wet-forest palms (Ecuador) are dark, large bugs with long heads/wings. Finally, northern-lowland bugs primarily from dry-forest palms (Ecuador) are light-colored and medium-sized. Wing and (size-free) head shapes are similar across Ecuadorian populations, regardless of habitat or phenotype, but distinct in Peruvian bugs. Bayesian phylogenetic and multispecies-coalescent DNA sequence analyses strongly suggest that Ecuadorian and Peruvian populations are two independently evolving lineages, with little within-lineage phylogeographic structuring or differentiation. CONCLUSIONS: We report sharp naked-eye phenotypic divergence of genetically similar Ecuadorian R. ecuadoriensis (nest-dwelling southern-Andean vs palm-dwelling northern bugs; and palm-dwelling Andean vs lowland), and sharp naked-eye phenotypic similarity of typical, yet genetically distinct, southern-Andean bugs primarily from vertebrate-nest (but not palm) microhabitats. This remarkable phenotypic diversity within a single nominal species likely stems from microhabitat adaptations possibly involving predator-driven selection (yielding substrate-matching camouflage coloration) and a shift from palm-crown to vertebrate-nest microhabitats (yielding smaller bodies and shorter and stouter heads). These findings shed new light on the origins of phenotypic diversity in triatomines, warn against excess reliance on phenotype-based triatomine-bug taxonomy, and confirm the Triatominae as an informative model system for the study of phenotypic change under ecological pressure .

High throughput estimates of Wolbachia, Zika and chikungunya infection in Aedes aegypti by near-infrared spectroscopy to improve arbovirus surveillance.

Deployment of Wolbachia to mitigate dengue (DENV), Zika (ZIKV) and chikungunya (CHIKV) transmission is ongoing in 12 countries. One way to assess the efficacy of Wolbachia releases is to determine invasion rates within the wild population of Aedes aegypti following their release. Herein we evaluated the accuracy, sensitivity and specificity of the Near Infrared Spectroscopy (NIRS) in estimating the time post death, ZIKV-, CHIKV-, and Wolbachia-infection in trapped dead female Ae. aegypti mosquitoes over a period of 7 days. Regardless of the infection type, time post-death of mosquitoes was accurately predicted into four categories (fresh, 1 day old, 2-4 days old and 5-7 days old). Overall accuracies of 93.2, 97 and 90.3% were observed when NIRS was used to detect ZIKV, CHIKV and Wolbachia in dead Ae. aegypti female mosquitoes indicating NIRS could be potentially applied as a rapid and cost-effective arbovirus surveillance tool. However, field data is required to demonstrate the full capacity of NIRS for detecting these infections under field conditions.

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.

Limited risk of Zika virus transmission by five Aedes albopictus populations from Spain.

BACKGROUND: Aedes albopictus, the Asian tiger mosquito, is an exotic invasive species in Europe. It has substantial public health relevance due to its potential role in transmitting several human pathogens. Out of the European countries, Spain has one of the highest risk levels of autochthonous arbovirus transmission due to both the high density of Ae. albopictus and the extensive tourist influx from vector-endemic areas. This study aims to investigate the susceptibility of five Ae. albopictus populations from mainland Spain and the Balearic Islands to a Brazilian Zika virus (ZIKV) strain. METHODS: The F1 generation of each Ae. albopictus population was orally challenged with a ZIKV-infected blood meal (1.8 × 106 PFU/ml). At 7 and 14 days post-infection (dpi), mosquito bodies (thorax and abdomen) and heads were individually analysed through RT-qPCR to determine the infection rate (IR) and dissemination rate (DR), respectively. The saliva of infected mosquitoes was inoculated in Vero cells and the transmission rate was assessed by plaque assay or RT-qPCR on ~33 individuals per population. RESULTS: The IR and DR ranged between 12-88%, and 0-60%, respectively, suggesting that ZIKV is capable of crossing the midgut barrier. Remarkably, no infectious viral particle was found in saliva samples, indicating a low ability of ZIKV to overcome the salivary gland barrier. A subsequent assay revealed that a second non-infective blood meal 48 h after ZIKV exposure did not influence Ae. albopictus vector competence. CONCLUSIONS: The oral experimental ZIKV infections performed here indicate that Ae. albopictus from Spain become infected and disseminate the virus through the body but has a limited ability to transmit the Brazilian ZIKV strain through biting. Therefore, the results suggest a limited risk of autochthonous ZIKV transmission in Spain by Ae. albopictus.

Phylogeography and demographic history of the Chagas disease vector Rhodnius nasutus (Hemiptera: Reduviidae) in the Brazilian Caatinga biome.

BACKGROUND: Rhodnius nasutus, a vector of the etiological agent Trypanosoma cruzi, is one of the epidemiologically most relevant triatomine species of the Brazilian Caatinga, where it often colonizes rural peridomestic structures such as chicken coops and occasionally invades houses. Historical colonization and determination of its genetic diversity and population structure may provide new information towards the improvement of vector control in the region. In this paper we present thoughtful analyses considering the phylogeography and demographic history of R. nasutus in the Caatinga. METHODOLOGY/PRINCIPAL FINDINGS: A total of 157 R. nasutus specimens were collected from Copernicia prunifera palm trees in eight geographic localities within the Brazilian Caatinga biome, sequenced for 595-bp fragment of the mitochondrial cytochrome b gene (cyt b) and genotyped for eight microsatellite loci. Sixteen haplotypes were detected in the cyt b sequences, two of which were shared among different localities. Molecular diversity indices exhibited low diversity levels and a haplotype network revealed low divergence among R. nasutus sequences, with two central haplotypes shared by five of the eight populations analyzed. The demographic model that better represented R. nasutus population dynamics was the exponential growth model. Results of the microsatellite data analyses indicated that the entire population is comprised of four highly differentiated groups, with no obvious contemporary geographic barriers that could explain the population substructure detected. A complex pattern of migration was observed, in which a western Caatinga population seems to be the source of emigrants to the eastern populations. CONCLUSIONS/SIGNIFICANCE: R. nasutus that inhabit C. prunifera palms do not comprise a species complex. The species went through a population expansion at 12-10 ka, during the Holocene, which coincides with end of the largest dry season in South America. It colonized the Caatinga in a process that occurred from west to east in the region. R. nasutus is presently facing an important ecological impact caused by the continuous deforestation of C. prunifera palms in northeast Brazil. We hypothesize that this ecological disturbance might contribute to an increase in the events of invasion and colonization of human habitations.

Dissecting the phyloepidemiology of Trypanosoma cruzi I (TcI) in Brazil by the use of high resolution genetic markers.

BACKGROUND: Trypanosoma cruzi, the causal agent of Chagas disease, is monophyletic but genetically heterogeneous. It is currently represented by six genetic lineages (Discrete Typing Units, DTUs) designated TcI-TcVI. TcI is the most geographically widespread and genetically heterogeneous lineage, this as is evidenced by a wide range of genetic markers applied to isolates spanning a vast geographic range in Latin America. METHODOLOGY/PRINCIPAL FINDINGS: In total, 78 TcI isolated from hosts and vectors distributed in 5 different biomes of Brazil, were analyzed using 6 nuclear housekeeping genes, 25 microsatellite loci and one mitochondrial marker. Nuclear markers reveal substantial genetic diversity, significant gene flow between biomes, incongruence in phylogenies, and haplotypic analysis indicative of intra-DTU genetic exchange. Phylogenetic reconstructions based on mitochondrial and nuclear loci were incongruent, and consistent with introgression. Structure analysis of microsatellite data reveals that, amongst biomes, the Amazon is the most genetically diverse and experiences the lowest level of gene flow. Investigation of population structure based on the host species/genus, indicated that Didelphis marsupialis might play a role as the main disperser of TcI. CONCLUSIONS/SIGNIFICANCE: The present work considers a large TcI sample from different hosts and vectors spanning multiple ecologically diverse biomes in Brazil. Importantly, we combine fast and slow evolving markers to contribute to the epizootiological understanding of TcI in five distinct Brazilian biomes. This constitutes the first instance in which MLST analysis was combined with the use of MLMT and maxicircle markers to evaluate the genetic diversity of TcI isolates in Brazil. Our results demonstrate the existence of substantial genetic diversity and the occurrence of introgression events. We provide evidence of genetic exchange in TcI isolates from Brazil and of the relative isolation of TcI in the Amazon biome. We observe the absence of strict associations with TcI genotypes to geographic areas and/or host species.

Effects of Light and Temperature on Daily Activity and Clock Gene Expression in Two Mosquito Disease Vectors.

Most organisms feature an endogenous circadian clock capable of synchronization with their environment. The most well-known synchronizing agents are light and temperature. The circadian clock of mosquitoes, vectors of many pathogens, drives important behaviors related to vectoral capacity, including oviposition, host seeking, and hematophagy. Main clock gene expression, as well as locomotor activity patterns, has been identified in Aedes aegypti and Culex quinquefasciatus under artificial light-dark cycles. Given that these mosquito species thrive in tropical areas, it is reasonable to speculate that temperature plays an important role in the circadian clock. Here, we provide data supporting a different hierarchy of light and temperature as zeitgebers of two mosquito species. We recorded their locomotor activity and quantified mRNA expression of the main clock genes in several combinations of light and temperature cycles. We observed that A. aegypti is more sensitive to temperature, while C. quinquefasciatus is more responsive to light. These variations in clock gene expression and locomotor activity may have affected the mosquito species' metabolism, energy expenditure, fitness cost, and pathogen transmission efficiency. Our findings are relevant to chronobiology studies and also have epidemiological implications.

Rapid, noninvasive detection of Zika virus in Aedes aegypti mosquitoes by near-infrared spectroscopy.

The accelerating global spread of arboviruses, such as Zika virus (ZIKV), highlights the need for more proactive mosquito surveillance. However, a major challenge during arbovirus outbreaks has been the lack of rapid and affordable tests for pathogen detection in mosquitoes. We show for the first time that near-infrared spectroscopy (NIRS) is a rapid, reagent-free, and cost-effective tool that can be used to noninvasively detect ZIKV in heads and thoraces of intact Aedes aegypti mosquitoes with prediction accuracies of 94.2 to 99.3% relative to quantitative reverse transcription polymerase chain reaction (RT-qPCR). NIRS involves simply shining a beam of light on a mosquito to collect a diagnostic spectrum. We estimated in this study that NIRS is 18 times faster and 110 times cheaper than RT-qPCR. We anticipate that NIRS will be expanded upon for identifying potential arbovirus hotspots to guide the spatial prioritization of vector control.

Spatial and Molecular Epidemiology of Giardia intestinalis Deep in the Amazon, Brazil.

BACKGROUND: Current control policies for intestinal parasitosis focuses on soil-transmitted helminths, being ineffective against Giardia intestinalis, a highly prevalent protozoon that impacts children's nutritional status in developing countries. The objective of this study was to explore spatial and molecular epidemiology of Giardia intestinalis in children of Amerindian descent in the Brazilian Amazon. METHODOLOGY/PRINCIPAL FINDINGS: A cross sectional survey was performed in the Brazilian Amazon with 433 children aged 1 to 14 years. Fecal samples were processed through parasitological techniques and molecular characterization. Prevalence of G. intestinalis infection was 16.9% (73/433), reaching 22.2% (35/158) among children aged 2-5 years, and a wide distribution throughout the city with some hot spots. Positivity-rate was similar among children living in distinct socioeconomic strata (48/280 [17.1%] and 19/116 [16.4%] below and above the poverty line, respectively). Sequencing of the ß-giardin gene revealed 52.2% (n = 12) of assemblage A and 47.8% (n = 11) of assemblage B with high haplotype diversity for the latter. The isolates clustered into two well-supported G. intestinalis clades. A total of 38 haplotypes were obtained, with the following subassemblages distribution: 5.3% (n = 2) AII, 26.3% (n = 10) AIII, 7.9% (n = 3) BIII, and 60.5% (n = 23) new B genotypes not previously described. CONCLUSIONS/SIGNIFICANCE: Giardia intestinalis infection presents a high prevalence rate among Amerindian descended children living in Santa Isabel do Rio Negro/Amazon. The wide distribution observed in a small city suggests the presence of multiple sources of infection, which could be related to environmental contamination with feces, possibly of human and animal origin, highlighting the need of improving sanitation, safe water supply and access to diagnosis and adequate treatment of infections.

Rhodnius prolixus and R. robustus (Hemiptera: Reduviidae) nymphs show different locomotor patterns on an automated recording system.

BACKGROUND: Circadian rhythms of triatomines, vectors of the etiological agent Trypanosoma cruzi responsible for Chagas disease, have been extensively studied in adults of the two most epidemiologically relevant vector species, Rhodnius prolixus and Triatoma infestans. However, little attention has been dedicated to the activity patterns in earlier developmental stages, even though triatomine nymphs are equally capable of transmitting T. cruzi to humans. Because circadian rhythms may differ even between closely related species, studies that focus on this behavioral trait can also be used to shed light on the taxonomy of controversial taxa, which becomes especially relevant regarding vector species. METHODS: We compared the daily locomotor activity patterns of second- and third-instar nymphs of Rhodnius prolixus and Rhodnius robustus in order to unveil possible behavioral differences between these cryptic species. Mitochondrial and nuclear markers were sequenced to confirm species identification. RESULTS: Nymphs of both species had a bimodal pattern of locomotion and similar daily activity patterns, but R. prolixus is more active under light/dark cycles and depicts a more pronounced activity rhythm under constant darkness conditions. CONCLUSIONS: We describe the implementation of an often-used automated method for the recording of individual locomotor activity to differentiate sibling species of Rhodnius with distinct epidemiological relevance. The higher levels of activity observed in the nymphs of R. prolixus could potentially contribute to increased vector capacity.

Rhodnius barretti, a new species of Triatominae (Hemiptera: Reduviidae) from western Amazonia.

Rhodnius barretti, a new triatomine species, is described based on adult specimens collected in rainforest environments within the Napo ecoregion of western Amazonia (Colombia and Ecuador). R. barretti resembles Rhodnius robustus s.l., but mitochondrial cytochrome b gene sequences reveal that it is a strongly divergent member of the "robustus lineage", i.e., basal to the clade encompassing Rhodnius nasutus, Rhodnius neglectus, Rhodnius prolixus and five members of the R. robustus species complex. Morphometric analyses also reveal consistent divergence from R. robustus s.l., including head and, as previously shown, wing shape and the length ratios of some anatomical structures. R. barretti occurs, often at high densities, in Attalea butyracea and Oenocarpus bataua palms. It is strikingly aggressive and adults may invade houses flying from peridomestic palms. R. barretti must therefore be regarded as a potential Trypanosoma cruzi vector in the Napo ecoregion, where Chagas disease is endemic.

A nuclear single-nucleotide polymorphism (SNP) potentially useful for the separation of Rhodnius prolixus from members of the Rhodnius robustus cryptic species complex (Hemiptera: Reduviidae).

The design and application of rational strategies that rely on accurate species identification are pivotal for effective vector control. When morphological identification of the target vector species is impractical, the use of molecular markers is required. Here we describe a non-coding, single-copy nuclear DNA fragment that contains a single-nucleotide polymorphism (SNP) with the potential to distinguish the important domestic Chagas disease vector, Rhodnius prolixus, from members of the four sylvatic Rhodnius robustus cryptic species complex. A total of 96 primer pairs obtained from whole genome shotgun sequencing of the R. prolixus genome (12,626 random reads) were tested on 43 R. prolixus and R. robustus s.l. samples. One of the seven amplicons selected (AmpG) presented a SNP, potentially diagnostic for R. prolixus, on the 280th site. The diagnostic nature of this SNP was then confirmed based on the analysis of 154 R. prolixus and R. robustus s.l. samples representing the widest possible geographic coverage. The results of a 60% majority-rule Bayesian consensus tree and a median-joining network constructed based on the genetic variability observed reveal the paraphyletic nature of the R. robustus species complex, with respect to R. prolixus. The AmpG region is located in the fourth intron of the Transmembrane protein 165 gene, which seems to be in the R. prolixus X chromosome. Other possible chromosomal locations of the AmpG region in the R. prolixus genome are also presented and discussed.