Structure and function of the stylets of hematophagous Triatominae (Hemiptera: Reduviidae), with special reference to Dipetalogaster maxima.

Kissing bugs (Hemiptera: Reduviidae: Triatominae) are able to bend their rod-like maxillae while searching for blood vessels in the tissue of their vertebrate hosts. Little is known about the working mechanisms of these bending movements and the distal opening of the food channel. We compared the morphological structure of the stylets (mandibles and maxillae) of four triatomine species and analyzed the feeding process of Dipetalogaster maxima (Uhler, 1894). The maxillae of triatomine bugs are interlocked by a tongue-and-groove system, allowing longitudinal sliding. While penetrating the host tissue, the animals perform rapid alternate back and forth movements of the maxillae. The resistance of the surrounding tissue pushes the asymmetric apex of the maxillae away from its straight path, i.e., if one individual maxilla is protracted alone, its tip curves inwards, and the other maxilla follows. Once a blood vessel is tapped, the spine-like tip of the left maxilla splays outwards. Apically, each of the maxillae features an abutment, the left one exhibiting a notch that presumably facilitates splaying. The mechanical interaction of the two maxillary abutments enables the distal opening of the food channel but might also support the movements of the maxillary bundle attributable to different bending moment distributions.

The Fat Body of the Hematophagous Insect, Panstrongylus megistus (Hemiptera: Reduviidae): Histological Features and Participation of the ß-Chain of ATP Synthase in the Lipophorin-Mediated Lipid Transfer.

In insects, lipid transfer to the tissues is mediated by lipophorin, the major circulating lipoprotein, mainly through a nonendocytic pathway involving docking receptors. Currently, the role of such receptors in lipid metabolism remains poorly understood. In this work, we performed a histological characterization of the fat body of the Chagas' disease vector, Panstrongylus megistus (Burmeister), subjected to different nutritional conditions. In addition, we addressed the role of the ß-chain of ATP synthase (ß-ATPase) in the process of lipid transfer from lipophorin to the fat body. Fifth-instar nymphs in either fasting or fed condition were employed in the assays. Histological examination revealed that the fat body was composed by diverse trophocyte phenotypes. In the fasting condition, the cells were smaller and presented a homogeneous cytoplasmic content. The fat body of fed insects increased in size mainly due to the enlargement of lipid stores. In this condition, trophocytes contained abundant lipid droplets, and the rough endoplasmic reticulum was highly developed and mitochondria appeared elongated. Immunofluorescence assays showed that the ß-ATPase, a putative lipophorin receptor, was located on the surface of fat body cells colocalizing partially with lipophorin, which suggests their interaction. No changes in ß-ATPase expression were found in fasting and fed insects. Blocking the lipophorin-ß-ATPase interaction impaired the lipophorin-mediated lipid transfer to the fat body. The results showed that the nutritional status of the insect influenced the morphohistological features of the tissue. Besides, these findings suggest that ß-ATPase functions as a lipophorin docking receptor in the fat body.

Spatial prediction of risk areas for vector transmission of Trypanosoma cruzi in the State of Paraná, southern Brazil.

After obtaining certification of the absence of transmission of the Trypanosoma cruzi by Triatoma infestans in 2006, other native species of protozoan vectors have been found in human dwellings within municipalities of the State of Paraná, Southern Brazil. However, the spatial distribution of T. cruzi vectors and how climatic and landscape combined variables explain the distribution are still poorly understood. The goal of this study was to predict the potential distribution of T. cruzi vectors as a proxy for Chagas disease transmission risk using Ecological Niche Models (ENMs) based on climatic and landscape variables. We hypothesize that ENM based on both climate and landscape variables are more powerful than climate-only or landscape-only models, and that this will be true independent of vector species. A total of 2,662 records of triatomines of five species were obtained by community-based entomological surveillance from 2007 to 2013. The species with the highest number of specimens was Panstrongylus megistus (73%; n = 1,943), followed by Panstrongylus geniculatus (15.4%; 411), Rhodnius neglectus (6.0%; 159), Triatoma sordida (4.5%; 119) and Rhodnius prolixus (1.1%; 30). Of the total, 71.9% were captured at the intradomicile. T. cruzi infection was observed in 19.7% of the 2,472 examined insects. ENMs were generated based on selected climate and landscape variables with 1 km2 spatial resolution. Zonal statistics were used for classifying the municipalities as to the risk of occurrence of synanthropic triatomines. The integrated analysis of the climate and landscape suitability on triatomines geographical distribution was powerful on generating good predictive models. Moreover, this showed that some municipalities in the northwest, north and northeast of the Paraná state have a higher risk of T. cruzi vector transmission. This occurs because those regions present high climatic and landscape suitability values for occurrence of their vectors. The frequent invasion of houses by infected triatomines clearly indicates a greater risk of transmission of T. cruzi to the inhabitants. More public health attention should be given in the northern areas of the State of Paraná, which presents high climate and landscape suitabilities for the disease vectors. In conclusion, our results-through spatial analysis and predictive maps-showed to be effective in identifying areas of potential distribution and, consequently, in the definition of strategic areas and actions to prevent new cases of Chagas' disease, reinforcing the need for continuous and robust surveillance in these areas.

First report of Panstrongylus megistus (Hemiptera, Reduviidae, Triatominae) in the State of Acre and Rondônia, Amazon, Brazil.

INTRODUCTION: This article reports, for the first time, the occurrence of Panstrongylus megistus in the Brazilian Western Amazon. METHODS: Specimens of P. megistus were collected in the cities of Rio Branco, Acre and Extrema, Rondônia. RESULTS: The number of triatomine species in the State of Acre increased from eight to nine and in Rondônia from seven to eight. This was also the first report of P. megistus in the Brazilian Western Amazon. CONCLUSION: The occurrence of P. megistus in the Western Amazon evidences an epidemiological alert, since it is an important vector of T. cruzi.

Life Cycle, Feeding, and Defecation Patterns of Panstrongylus chinai (Hemiptera: Reduviidae: Triatominae) Under Laboratory Conditions.

Chagas disease is caused by the protozoan Trypanosoma cruzi Panstrongylus chinai (Del Ponte) is highly domiciliated in the Peruvian and Ecuadorian Andes and has been found naturally infected with T. cruzi The objective of this study was to describe the life cycle, feeding, and defecation patterns of P. chinai in the Loja province within southern Ecuador. To characterize its life cycle, a cohort of 70 individuals was followed from egg to adult. At each stage of development, prefeeding time, feeding time, weight of ingested meal, proportional weight increase, and the time to the first defecation were recorded. Panstrongylus chinai completed its development in 371.4 ± 22.3 d, (95% CI 355.4-387.4), which means that it is likely a univoltine species. Prefeeding time, feeding time, and weight of ingested meal increased as individuals developed through nymphal stages. Moreover, time to first defecation was shortest in the early nymphal stages, suggesting higher vector potential in the early developmental stages. Data obtained in this study represent an important advance in our knowledge of the biology of P. chinai, which should be considered as a secondary Chagas disease vector species in the Andean valleys of Loja (Ecuador) and in the north of Peru, and included in entomological surveillance programs.

Trypanosoma cruzi-infected Panstrongylus geniculatus and Rhodnius robustus adults invade households in the Tropics of Cochabamba region of Bolivia.

BACKGROUND: There are hardly any data available on the relationships between the parasite and the vector or regarding potential reservoirs involved in the natural transmission cycle of Trypanosoma cruzi in the Tropics of Cochabamba, Bolivia. Local families from communities were responsible for the capture of triatomine specimens, following a strategic methodology based on entomological surveillance with community participation developed by the National Chagas Programme of the Ministry of Health of Bolivia. FINDINGS: We describe the collection of adult Panstrongylus geniculatus and Rhodnius robustus naturally infected with Trypanosoma cruzi from houses and from the hospital of Villa Tunari municipality. The flagellates found in the digestive tract of P. geniculatus belong to genetic lineages or DTUs TcI and TcIII, whereas only lineage DTU TcI was found in R. robustus. The detection of these vectors infected with T. cruzi reveals the vulnerability of local communities. CONCLUSION: The results presented here highlight the risk of Chagas disease transmission in a region previously thought not to be endemic, indicating that the Tropics of Cochabamba should be placed under permanent entomological and epidemiological surveillance.

Comprehensive Survey of Domiciliary Triatomine Species Capable of Transmitting Chagas Disease in Southern Ecuador.

BACKGROUND: Chagas disease is endemic to the southern Andean region of Ecuador, an area with one of the highest poverty rates in the country. However, few studies have looked into the epidemiology, vectors and transmission risks in this region. In this study we describe the triatomine household infestation in Loja province, determine the rate of Trypanosoma cruzi infection in triatomines and study the risk factors associated with infestation. METHODOLOGY/PRINCIPAL FINDINGS: An entomological survey found four triatomine species (Rhodnius ecuadoriensis, Triatoma carrioni, Panstrongylus chinai, and P. rufotuberculatus) infesting domiciles in 68% of the 92 rural communities examined. Nine percent of domiciles were infested, and nymphs were observed in 80% of the infested domiciles. Triatomines were found in all ecological regions below 2,200 masl. We found R. ecuadoriensis (275 to 1948 masl) and T. carrioni (831 to 2242 masl) mostly in bedrooms within the domicile, and they were abundant in chicken coops near the domicile. Established colonies of P. chinai (175 to 2003 masl) and P. rufotuberculatus (404 to 1613 masl) also were found in the domicile. Triatomine infestation was associated with surrogate poverty indicators, such as poor sanitary infrastructure (lack of latrine/toilet [w = 0.95], sewage to environment [w = 1.0]). Vegetation type was a determinant of infestation [w = 1.0] and vector control program insecticide spraying was a protective factor [w = 1.0]. Of the 754 triatomines analyzed, 11% were infected with Trypanosoma cruzi and 2% were infected with T. rangeli. CONCLUSIONS/SIGNIFICANCE: To date, only limited vector control efforts have been implemented. Together with recent reports of widespread sylvatic triatomine infestation and frequent post-intervention reinfestation, these results show that an estimated 100,000 people living in rural areas of southern Ecuador are at high risk for T. cruzi infection. Therefore, there is a need for a systematic, sustained, and monitored vector control intervention that is coupled with improvement of socio-economic conditions.

Vital Statistics of Triatominae (Hemiptera: Reduviidae) Under Laboratory Conditions: IV. Panstrongylus geniculatus.

A cohort of 100 eggs of Panstrongylus geniculatus (Latreille) was reared in the laboratory under constant conditions (temperature 26 ± 1°C, 60 ± 10% RH), with mortality and fecundity data recorded weekly. We calculated stage-specific development times, age-specific mortality and fecundity (18.4 eggs/♀/wk), and stage-specific and total preadult mortality (31.6%), and the weekly intrinsic rate of natural increase (r(o) = 0.096), the finite population growth rate (λ = 1.109), the net reproductive rate (R(0) = 60.45), and the generation time (T = 46.34 wk). Elasticity analysis showed that the dominant life-history trait determining λ was survival (particularly the adult female's survival). Adult females dominated the stage-specific reproductive value, and the egg stage dominated the stable stage distribution (SSD). The damping ratio (ρ = 1.096) suggests a relatively rapid period of recovery to a disturbed SSD. Results were compared with one previous study and conform relatively well, considering that environmental conditions were not the same. We estimated the colonizing ability of P. geniculatus, using as a criterion the ro/b index, and obtained the value of 0.74, an indicator of a good colonizer, and similar to well-known invasive species such as Rhodnius prolixus and Triatoma infestans. The life history traits and demographic parameters here presented for P. geniculatus are discussed in terms of their usefulness for evolutionary studies and vector control activities.

A Deep Insight Into the Sialotranscriptome of the Chagas Disease Vector, Panstrongylus megistus (Hemiptera: Heteroptera).

Saliva of blood-sucking arthropods contains a complex cocktail of pharmacologically active compounds that assists feeding by counteracting their hosts' hemostatic and inflammatory reactions. Panstrongylus megistus (Burmeister) is an important vector of Chagas disease in South America, but despite its importance there is only one salivary protein sequence publicly deposited in GenBank. In the present work, we used Illumina technology to disclose and publicly deposit 3,703 coding sequences obtained from the assembly of >70 million reads. These sequences should assist proteomic experiments aimed at identifying pharmacologically active proteins and immunological markers of vector exposure. A supplemental file of the transcriptome and deducted protein sequences can be obtained from http://exon.niaid.nih.gov/transcriptome/P_megistus/Pmeg-web.xlsx.

Amines from vertebrates guide triatomine bugs to resources.

Most triatomine bugs (Heteroptera: Reduviidae) are nest-living insects that require vertebrate blood or invertebrate haemolymph to complete their life cycle. Vertebrates accumulate excretory products in or near their nesting sites and we hypothesize that triatomines use emanations from such host wastes when searching for resources. Here we recount how triatomine bugs increase upwind locomotion on a servosphere in response to volatile amine constituents of vertebrate excretions. Fresh chicken faeces is strongly attractive to Rhodnius prolixus nymphs. Ammonia induces attraction and an increase in both speed and total path length by R. prolixus on the servosphere. Whereas ethylamine and dimethylamine attract R. prolixus, Triatoma infestans and Panstrongylus geniculatus, other amine constituents of vertebrate excretions such as isobutylamine and hexylamine induce R. prolixus nymphs to walk faster and for a longer period. These amines are derived from generally occurring metabolites of vertebrates and from gut flora metabolism. We conclude that amines and other products associated with nesting hosts serve as signals for foraging triatomines.

Limitations of selective deltamethrin application for triatomine control in central coastal Ecuador.

BACKGROUND: This year-long study evaluated the effectiveness of a strategy involving selective deltamethrin spraying and community education for control of Chagas disease vectors in domestic units located in rural communities of coastal Ecuador. RESULTS: Surveys for triatomines revealed peridomestic infestation with Rhodnius ecuadoriensis and Panstrongylus howardi, with infestation indices remaining high during the study (13%, 17%, and 10%, at initial, 6-month, and 12-month visits, respectively), which indicates a limitation of this strategy for triatomine population control. Infestation was found 6 and 12 months after spraying with deltamethrin. In addition, a large number of previously vector-free domestic units also were found infested at the 6- and 12-month surveys, which indicates new infestations by sylvatic triatomines. The predominance of young nymphs and adults suggests new infestation events, likely from sylvatic foci. In addition, infection with Trypanosoma cruzi was found in 65%, 21% and 29% at initial, 6-month and 12-month visits, respectively. All parasites isolated (n = 20) were identified as TcI. CONCLUSION: New vector control strategies need to be devised and evaluated for reduction of T. cruzi transmission in this region.

The storage of nutritional resources during vitellogenesis of Panstrongylus megistus (Hemiptera: Reduviidae): the pathways of lipophorin in lipid delivery to developing oocytes.

In this work, we have analyzed the pathways by which lipophorin (Lp) delivers its lipid cargo to developing oocytes of Panstrongylus megistus, a hematophagous vector of Chagas' disease. Lp, vitellin, total lipids and proteins were measured in ovarian tissues at different stages of the reproductive cycle. Localization of Lp in developing oocytes, mainly at their cortical area, was demonstrated by immunofluorescence assays using an anti-Lp antibody labeled with FITC. In vivo approaches injecting fluorescently labeled Lp to follow the course of the entire particle (Lp-DiI or Lp-Oregon Green) or its lipid cargo (Lp-Bodipy-FA) were monitored by laser scanning confocal microscopy. Significant increases in the amounts of lipids, proteins and vitellin were observed in ovarian tissue with the progress of vitellogenesis. Unexpectedly, an increase in the amount of Lp was also observed. The experiments in vivo demonstrated that the uptake of fluorescent Lp labeled on its protein or lipid moiety by developing oocytes occurred very fast, being impaired at low temperatures. The co-injection of fluorescent Lp and vitellogenin (Vg) showed that both particles co-localized inside yolk bodies, confirming the endocytic pathway for Lp. When the fate of lipids transferred to oocytes was evaluated in vitellogenic females by co-injecting Lp-Bodipy-FA and Lp-DiI, the signal for Bodipy-FA was found in both lipid droplets and yolk bodies. In contrast, in injected females kept at 4°C the fluorescence was reduced, being observed exclusively in lipid droplets, implying that lipid transfer to the oocyte was diminished but not abolished. Taken together, the results demonstrate that in the hematophagous P. megistus, the storage of lipid resources by developing oocytes occurs by the convergence of different pathways by which Lp maximizes the delivery of its lipid cargo. In addition, it was also shown that, to some extent, lipids stored in the oocyte lipid droplets can also originate from endocytosed Vg. The relevance of these events in the context of the physiology of reproduction in P. megistus is discussed.

High household infestation rates by synanthropic vectors of Chagas disease in southern Ecuador.

Entomological surveys were conducted in five rural communities (138 domiciliary units [DUs]) in the southern Andes of Ecuador. Adobe walls and ceramic tile roofs were predominant construction materials. A 35% house infestation rate with Panstrongylus chinai (Del Ponte, 1929) (0.7%), Panstrongylus rufotuberculatus (Champion, 1899) (0.7%), Rhodnius ecuadoriensis (Lent & León, 1958) (27%), and/or Triatoma carrioni (Larrousse, 1926) (7%) was found. Adults and nymphs of R. ecuadoriensis and T. carrioni were found in intradomiciliary and peridomiciliary areas. Breeding triatomine colonies were present in 85% of infested DUs, and the average insect crowding was 52+/-113 triatomine bugs per infested house. T. cruzi-like organisms were found by microscopic examination in the feces or hindgut but not the salivary glands of 4% of examined R. ecuadoriensis and 12% T. carrioni. Serological tests detected a general anti-T. cruzi antibody seroprevalence of 3.9% (n = 1136). Only 2% of individuals had heard of Chagas disease, and although triatomines were reported as a major nuisance by the population they were not considered vectors of disease. Additional baseline field research is needed for the design and implementation of a Chagas disease control program in the region.

Development cycle of Panstrongylus rufotuberculatus (Hemiptera: Reduviidae) under laboratory conditions.

Determinant aspects of the development cycle of Panstrongylus rufotuberculatus (Champion, 1899) were evaluated under laboratory conditions. Fertility, duration of life cycle, mortality rate, feeding time, defecation time, thermal reaction time to food source, and volume of blood ingested were studied. From an initial cohort of fifth instars, nine adult pairs were formed, the females of which oviposited 2,338 eggs over 231 d. The eclosion rate was 84.4%; female and male progeny lived 223.56 (14.41) and 180.78 (14.41) d, respectively. The average total life cycle of immature stages (138.82+/-51.42 d) was distributed as egg, 24.22+/-0.42 d; nymph I, 18.94+/-5.15 d; nymph II, 15.80+/-3.29 d; nymph III, 16.55+/-4.36 d; nymph IV, 25.50+/-7.32 d; and nymph V, 36.71+/-2.75 d. Given these values, 2.6 generations per year are possible. Because of its longevity, rapid response to the presence of a host, large volume of blood ingested, and frequent defecation during feeding, this species constitutes a potentially important vector of Chagas' disease in nonendemic rural areas.

Mixed domestic infestation by Rhodnius prolixus Stal, 1859 and Panstrongylus geniculatus Latreille, 1811, vector incrimination, and seroprevalence for Trypanosoma cruzi among inhabitants in El Guamito, Lara State, Venezuela.

Mixed infestation of nymphs and adults of Rhodnius prolixus Stal, 1859 and Panstrongylus geniculatus Latreille, 1811 was detected in 3 (15%) of 20 dwellings in El Guamito, an endemic focus of Chagas disease in Lara State, Venezuela. In one of the houses, both species were positive for Trypanosoma cruzi: 14.3% (R. prolixus) and 20% (P. geniculatus ). The overall infection rate in 143 of 352 R. prolixus was 16.1%. Parasites isolated from R. prolixus were identified as T. cruzi I by random amplified polymorphic DNA analysis. Dot-enzyme-linked immunosorbent assays of 36 R. prolixus showed that 58.3% of the R. prolixus had fed on humans. The gut contents of one fifth-instar nymph of P. geniculatus that was positive for T. cruzi also reacted with anti-human serum. A questionnaire was used to gather data on the demographic and socioeconomic characteristics of the population. An indirect immunofluorescent test, an indirect hemaglutination test, and an ELISA were used to detect the presence of antibodies against T. cruzi in 84 of 86 inhabitants and in 15.5% of people more than 20 years old. The relative risk (RR) of infection was greater in men than in women (RR = 1.61, 95% confidence interval = 0.54-4.80). Of the people more than 15 years old, 36.6% had no formal education. All respondents recognized triatomine bugs, but they did not relate them to Chagas disease transmission. A total of 85.7% of the houses were "ranchos" suitable for the colonization of triatomine bugs. The possible domiciliation of P. geniculatus and the implications of competition with R. prolixus for resources are discussed. Since there is no clear separation of food sources, abiotic factors such as microclimatic variation within houses may be critical to predict the outcome of the process of competition and potential domestication of this generally sylvatic species.

Metabolic pathways for dietary lipids in the midgut of hematophagous Panstrongylus megistus (Hemiptera: Reduviidae).

The metabolism of dietary lipids in the anterior midgut of Panstrongylus megistus during blood digestion was studied. Fifth instar nymphs were fed a blood meal containing 7.1 +/- 0.4 mg of lipids, consisting mainly of triacylglycerol (TAG), and completed the overall process of digestion in about 20 days. Lipolysis of TAG and pathways for diacylglycerol (DAG) biosynthesis in the midgut were investigated by feeding the insects with [9,10-3H]-oleic acid-labeled triolein. Lumenal [3H]-triacylglycerol was hydrolyzed, generating mainly fatty acids (FA) and glycerol and to lesser extent, DAG. Almost no radioactivity associated with monoacylglycerol was found at any time. In midgut tissue, labeled fatty acids were incorporated into phosphatidic acid, DAG and TAG, whereas no significantly labeled monoacylglycerol was observed. In addition, the activities of enzymes related to DAG metabolism were assayed in non-blood fed midgut homogenates and at different times after feeding on a blood meal. Significant changes in the activities of phosphatidate phosphohydrolase (EC 3.1.3.4) and triacylglycerol lipase (EC 3.1.1.3) were observed during blood digestion, suggesting that these enzymes are important in regulating intracellular DAG synthesis and mobilization in midgut cells. Finally, the histological changes of lipid stores observed in anterior midgut confirmed the active process of uptake and trafficking of lipids performed by the enterocytes during blood digestion.

Geometric morphometric differences between Panstrongylus geniculatus from field and laboratory.

The finding of Panstrongylus geniculatus nymphs inside a house in northeastern Antioquia, Colombia, and the reports related to their increasing presence in homes suggest the need for surveillance methods for monitoring the invasion processes. We analyzed the morphological differences between a wild population and its laboratory descendants, using the techniques of geometric morphometry, with the idea that such differences might parallel those between sylvatic and synanthropic populations. The analyses over five generations showed differences in size but not in shape. Head size and wing size were both reduced from sylvatic to laboratory populations, but the decrease in head size occurred only up to the second generation while the decrease in wing size proceeded up to the fifth generation. In contrast, although a decrease in sexual size dimorphism has been proposed as a marker of colonization in human dwellings, we did not detect any significant loss of dimorphism between sexes of P. geniculatus over the five generations studied. We conclude that size changes may have a physiological origin in response to a change of ecotopes, but more than five generations may be required for the expression of permanent morphological markers of human dwellings colonization.

Experimental evidence for a demographic cline in Panstrongylus megistus populations.

The population biology of three populations of Panstrongylus megistus was compared to determine possible influence on the behaviour and epidemiological importance of this species. The results demonstrated differences in terms of egg eclosion time, nymphal mortality and development rates, and feeding and defaecation rates. These differences appeared to follow a geographical cline, primarily reflecting different degrees of adaptation to domestic habitats.