The Pharmacopea within Triatomine Salivary Glands.

Triatomines are blood-feeding insects that prey on vertebrate hosts. Their saliva is largely responsible for their feeding success. The triatomine salivary content has been studied over the past decades, revealing multifunctional bioactive proteins targeting the host´s hemostasis and immune system. Recently, sequencing of salivary-gland mRNA libraries revealed increasingly complex and complete transcript databases that have been used to validate the expression of deduced proteins through proteomics. This review provides an insight into the journey of discovery and characterization of novel molecules in triatomine saliva.

MALDI-TOF MS protein profiling for the rapid identification of Chagas disease triatomine vectors and application to the triatomine fauna of French Guiana.

Triatomines are haematophagous insects involved in the transmission of Trypanosoma cruzi, the aetiological agent of Chagas disease. The vector competence of these arthropods can be highly variable, depending on the species. A precise identification is therefore crucial for the epidemiological surveillance of T. cruzi and the determination of at-risk human populations. To circumvent the difficulties of morphological identification and the lack of comprehensiveness of the GenBank database, we hereby propose an alternative method for triatomine identification. The femurs of the median legs of triatomines from eight different species from French Guiana were subjected to matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis. Method evaluation was performed on fresh specimens and was applied to dry specimens collected between 1991 and 2003. Femur-derived protein extracts provided reproducible spectra within the same species along with significant interspecies heterogeneity. Validation of the study by blind test analysis provided 100% correct identification of the specimens in terms of the species, sex and developmental stage. MALDI-TOF mass spectrometry appears to be a powerful tool for triatomine identification, which is a major step forward in the fight against Chagas disease.

Use of noninvasive 'bug-eggs' to enable comparative inferences on genetic mating system with and without parental information: A study in a cattle egret colony.

Colonial waterbirds such as herons, egrets and spoonbills exhibit ecological characteristics that could have promoted the evolution of conspecific brood parasitism and extra-pair copulation. However, an adequate characterization of the genetic mating systems of this avian group has been hindered by the lack of samples of elusive candidate parents which precluded conducting conventional parentage allocation tests. Here, we investigate the genetic mating system of the invasive cattle egret using hematophagous insects contained in fake eggs to collect blood from incubating adults in a wild breeding colony. We tested a protocol with a previously unused Neotropical Triatominae, Panstrongylus megistus, obtained blood samples from males and females in 31 nests built on trees, drew blood from 89 nestlings at those nests, and genotyped all samples at 14 microsatellite loci, including six new species-specific loci. We comparatively addressed the performance of parentage allocation versus kinship classification of nestlings to infer the genetic mating system of cattle egrets. In line with previous behavioral observations, we found evidence in support of a non-monogamous genetic mating system, including extra-pair paternity (EPP) and conspecific brood parasitism (CBP). Parentage allocation tests detected a higher percentage of nests with alternative reproductive tactics (EPP: 61.7%; CBP: 64.5%) than the kinship classification method (EPP: 50.0%; CBP: 43.3%). Overall, these results indicate that rates of alternative reproductive tactics inferred in the absence of parental genetic information could be underestimated and should be interpreted with caution. This study highlights the importance of incorporating samples from candidate parents to adequately determine the genetic mating system of a species. We expand knowledge on the reproductive tactics of colonial waterbirds, contributing novel data on the genetic mating system of the cattle egret, valuable for the design of management strategies for this invasive bird.

[Diversity of Triatominae (Hemiptera: Reduviidae) in Santander, Colombia: Epidemiological implications].

INTRODUCTION: Domestic and wild triatomines in the department of Santander have an epidemiological impact, as recently they have been linked to outbreaks of acute Chagas disease. The analysis of their diversity and temporal variation contributes to the understanding of their biology and ecology in one of the most endemic areas of the country. OBJECTIVES: To analyze triatominae diversity in two regions of Santander. MATERIALS AND METHODS: We analyzed the triatomine records for Santander contained in the CINTROP-UIS entomology lab database. We grouped the information for two regions: the Middle Magdalena area and the Andean region, and for each one we designed species accumulation and range-abundance curves, we calculated diversity and equality indices, and we analyzed colonization and temporal variation or persistence of the community. RESULTS: Ninety five percent of triatomines came from the Andean area and 4.57% from Magdalena Medio, with nine and ten species each. The dominant species in the Andean area were Rhodnius prolixus and Triatoma dimidiata while in Magdalena Medio they were Rhodnius pallescens and Panstrongylus geniculatus. We found a greater diversity and richness in Middle Magdalena compared to the Andean area. The temporal variation showed persistence of communities over time. CONCLUSIONS: Results revealed differences in the diversity of the two regions and the potential of wild species to occupy artificial ecotopes. Triatomines intrusion and the recent involvement of wild species in the transmission of Trypanosoma cruzi emphasize the need to further investigate the ecology of these vectors in order to guide population control strategies.

[Risk of transmission of Chagas disease by intrusion of triatomines and wild mammals in Bucaramanga, Santander, Colombia].

INTRODUCTION: Notice of triatomines in dwellings of some neighborhoods in Bucaramanga motivated the realization of this study.Objetive: To evaluate the intrusion of triatomines and mammals, as well as some risk factors in urban dwellings. MATERIALS AND METHODS: Triatomines were collected in a neighborhood in Bucaramanga, Santander, on a monthly basis during one year with participation of the community. Collection included manual search in lamp posts, use of light traps, animal bait, and chemical attractants in nearby forests. Reservoirs were collected with bait traps. Insects and mammals were identified and examined in order to determine their natural infection. Risk factors in homes were assessed by means of a social-environmental survey. RESULTS: Eleven adult specimens of Pastrongylus geniculatus, as well as 63 of Rhodnius pallescens were collected in the forest, recreational peridomiciliary areas, and houses. Even two females and 21 nymphs of R. pallescens were found in bedrooms. Two specimens of Didelphis marsupialis were captured in neighboring forests. Out of the eleven P. geniculatus captured, nine were examined. Of these, five were positive for Trypanosoma cruzi. It was not possible to establish a significant risk factor;however, the dwellings with report of triatomines were located nearer to the adjacent forest. CONCLUSIONS: The finding of intrusive triatominae species and mammals with T. cruzi in intradomiciliary and peridomiciliary areas and periurban forests prove the potential risk to acquire infection from these populations that dwell in urban housing adjacent to these ecotopes where the sylvan cycle is kept.

Diversidad de Triatominae (Hemiptera: Reduviidae) en Santander, Colombia: implicaciones epidemiológicas / Diversity of Triatominae (Hemiptera: Reduviidae) in Santander, Colombia: Epidemiological implications

Resumen Introducción: Los triatominos domiciliados y silvestres constituyen un problema de impacto epidemiológico en el departamento de Santander, pues se han asociado recientemente con brotes agudos de la enfermedad de Chagas, por lo cual el análisis de su diversidad y variación temporal contribuye al conocimiento de su biología y ecología en una de las áreas más endémicas del país. Objetivo: Analizar la diversidad de triatominos en dos regiones de Santander. Materiales y métodos: Se analizó la información de la base de datos del Laboratorio de Entomología del Centro de Investigaciones en Enfermedades Tropicales de la Universidad Industrial de Santander (CINTROP-UIS), la cual contiene registros de triatominos en Santander. La información se separó en dos regiones, el Magdalena Medio y la zona andina, para cada una de las cuales se diseñaron curvas de acumulación de especies y de rango de abundancia, se calcularon los índices de diversidad y de igualdad, se analizó la colonización y se evaluó la variación temporal o persistencia de la comunidad. Resultados: El 95 % de los triatominos provenía de la zona andina y, el 4,57 %, del Magdalena Medio, con nueve y diez especies, respectivamente. Se encontró mayor diversidad y riqueza en el Magdalena Medio en comparación con la zona andina. Las especies dominantes en la zona andina fueron Rhodnius prolixus y Triatoma dimidiata, mientras que en Magdalena Medio fueron Rhodnius pallescens y Panstrongylus geniculatus. La variación temporal mostró persistencia de las comunidades en el tiempo. Conclusiones:. Los resultados evidenciaron diferencias en la diversidad de las dos regiones, además del potencial de las especies silvestres para ocupar ecótopos artificiales. La intrusión de triatominos y la reciente incriminación de especies silvestres en la transmisión de Trypanosoma cruzi, indican la necesidad de un mayor conocimiento de la ecología de estos vectores para orientar las estrategias de control.

Abstract Introduction: Domestic and wild triatomines in the department of Santander have an epidemiological impact, as recently they have been linked to outbreaks of acute Chagas disease. The analysis of their diversity and temporal variation contributes to the understanding of their biology and ecology in one of the most endemic areas of the country. Objectives: To analyze triatominae diversity in two regions of Santander. Materials and methods: We analyzed the triatomine records for Santander contained in the CINTROPUIS entomology lab database. We grouped the information for two regions: the Middle Magdalena area and the Andean region, and for each one we designed species accumulation and range-abundance curves, we calculated diversity and equality indices, and we analyzed colonization and temporal variation or persistence of the community. Results: Ninety five percent of triatomines came from the Andean area and 4.57% from Magdalena Medio, with nine and ten species each. The dominant species in the Andean area were Rhodnius prolixus and Triatoma dimidiata while in Magdalena Medio they were Rhodnius pallescens and Panstrongylus geniculatus. We found a greater diversity and richness in Middle Magdalena compared to the Andean area. The temporal variation showed persistence of communities over time. Conclusions: Results revealed differences in the diversity of the two regions and the potential of wild species to occupy artificial ecotopes. Triatomines intrusion and the recent involvement of wild species in the transmission of Trypanosoma cruzi emphasize the need to further investigate the ecology of these vectors in order to guide population control strategies.

Riesgo de transmisión de la enfermedad de Chagas por intrusión de triatominos y mamíferos silvestres en Bucaramanga, Santander, Colombia / Risk of transmission of Chagas disease by intrusion of triatomines and wild mammals in Bucaramanga, Santander, Colombia

Introducción: La notificación de triatominos en las viviendas de algunos barrios de Bucaramanga motivó la realización de este estudio. Objetivo: Evaluar la intrusión de triatominos y mamíferos, así como algunos factores de riesgo para la enfermedad de Chagas en viviendas urbanas. Materiales y métodos: En un barrio de Bucaramanga, Santander, se recolectaron triatominos mensualmente durante un año con participación comunitaria mediante búsqueda manual en el alumbrado público, y el uso de trampas de luz, cebo animal y atrayentes químicos en el bosque cercano. Los reservorios se recolectaron con trampas cebadas. Los insectos y mamíferos se determinaron y examinaron para establecer su infección natural. Los factores de riesgo de las viviendas se midieron mediante una encuesta sobre factores sociales y ambientales. Resultados: Se recolectaron 11 adultos de Panstrongylus geniculatus y 63 de Rhodnius pallescens en el bosque, en zonas de recreación en el peridomicilio y en el domicilio, incluidas dos hembras y 21 ninfas de R. pallescens en dormitorios. Se capturaron dos ejemplares de Didelphis marsupialis en el bosques adyacente. De los 11 individuos de P. geniculatus capturados, se examinaron nueve, de los cuales cinco fueron positivos para Trypanosoma cruzi (56 %); de los 63 individuos de R. pallescens capturados, se examinaron ocho, cuatro de los cuales fueron positivos para T. cruzi (50 %). De dos especímenes de D. marsupiales capturados, uno fue examinado y se encontró que era positivo para T. cruzi. No se pudo establecer un factor de riesgo significativo, sin embargo, las viviendas con reporte de triatominos se encontraban más cerca del bosque adyacente. Conclusiones: El hallazgo de especies de triatominos intrusivas y de mamíferos con T. cruzi en el domicilio y el peridomicilio, así como en los bosques periurbanos, demuestra el riesgo de infección en las poblaciones que habitan en viviendas urbanas adyacentes a los ecótopos donde se mantiene el ciclo silvestre.

Abstract Introduction: Notice of triatomines in dwellings of some neighborhoods in Bucaramanga motivated the realization of this study. Objetive: To evaluate the intrusion of triatomines and mammals, as well as some risk factors in urban dwellings. Materials and methods: Triatomines were collected in a neighborhood in Bucaramanga, Santander, on a monthly basis during one year with participation of the community. Collection included manual search in lamp posts, use of light traps, animal bait, and chemical attractants in nearby forests. Reservoirs were collected with bait traps. Insects and mammals were identified and examined in order to determine their natural infection. Risk factors in homes were assessed by means of a social-environmental survey. Results: Eleven adult specimens of Pastrongylus geniculatus, as well as 63 of Rhodnius pallescens were collected in the forest, recreational peridomiciliary areas, and houses. Even two females and 21 nymphs of R. pallescens were found in bedrooms. Two specimens of Didelphis marsupialis were captured in neighboring forests. Out of the eleven P. geniculatus captured, nine were examined. Of these, five were positive for Trypanosoma cruzi. It was not possible to establish a significant risk factor; however, the dwellings with report of triatomines were located nearer to the adjacent forest. Conclusions: The finding of intrusive triatominae species and mammals with T. cruzi in intradomiciliary and peridomiciliary areas and periurban forests prove the potential risk to acquire infection from these populations that dwell in urban housing adjacent to these ecotopes where the sylvan cycle is kept.

Combination of cytochrome b heteroduplex-assay and sequencing for identification of triatomine blood meals.

The identification of blood meals in vectors contributes greatly to the understanding of interactions between vectors, microorganisms and hosts. The aim of the current work was to complement the validation of cytochrome b (Cytb) heteroduplex assay (HDA) previously described, and to add the sequencing of the Cytb gene of some samples for the identification of blood meals in triatomines. Experimental feedings of reared triatomines helped to clarify the sensitivity of the HDA. Moreover, the sequencing coupled with the HDA, allowed the assessment of the technique's taxonomic level of discrimination. The primers used to produce DNA fragments of Cytb genes for HDA had a very high sensitivity for vertebrate DNAs, rather similar for mammals, birds and reptiles. However, the formation of heteroduplex depended on blood meal's quality rather than its quantity; a correlation was observed between blood meals' color and the positivity of HDA. HDA electrophoresis profiles were reproducible, and allowed the discrimination of blood origins at the species level. However, in some cases, intraspecific variability of Cytb gene generated different HDA profiles. The HDA based on comparison of electrophoresis profiles is a very useful tool for screening large samples to determine blood origins; the subsequent sequencing of PCR products of Cytb corresponding to different HDA profiles allowed the identification of species whatever the biotope in which the vectors were captured.

2-DE-based proteomic investigation of the saliva of the Amazonian triatomine vectors of Chagas disease: Rhodnius brethesi and Rhodnius robustus.

The triatomine bugs are obligatory haematophagous organisms that act as vectors of Chagas disease by transmitting the protozoan Trypanosoma cruzi. Their feeding success is strongly related to salivary proteins that allow these insects to access blood by counteracting host haemostatic mechanisms. Proteomic studies were performed on saliva from the Amazonian triatomine bugs: Rhodnius brethesi and R. robustus, species epidemiologically relevant in the transmission of T. cruzi. Initially, salivary proteins were separated by two-dimensional gel electrophoresis (2-DE). The average number of spots of the R. brethesi and R. robustus saliva samples were 129 and 135, respectively. The 2-DE profiles were very similar between the two species. Identification of spots by peptide mass fingerprinting afforded limited efficiency, since very few species-specific salivary protein sequences are available in public sequence databases. Therefore, peptide fragmentation and de novo sequencing using a MALDI-TOF/TOF mass spectrometer were applied for similarity-driven identifications which generated very positive results. The data revealed mainly lipocalin-like proteins which promote blood feeding of these insects. The redundancy of saliva sequence identification suggested multiple isoforms caused by gene duplication followed by gene modification and/or post-translational modifications. In the first experimental assay, these proteins were predominantly phosphorylated, suggesting functional phosphoregulation of the lipocalins.

(4S,5S)-2,2,4-Triethyl-5-methyl-1,3-dioxolane: a new volatile released by a triatomine bug.

Adults of the triatomine bug Triatoma brasiliensis release 2,2,4-triethyl-5-methyl-1,3-dioxolane (1) as a mixture of the (4S,5S)- and (4R,5R)-enantiomers in a ratio of 4:1. Among the volatile acetals identified from insects so far, this is the first example resulting from an intermolecular condensation of a carbonyl moiety and a diol substructure.

Immunogenic salivary proteins of Triatoma infestans: development of a recombinant antigen for the detection of low-level infestation of triatomines.

BACKGROUND: Triatomines are vectors of Trypanosoma cruzi, the etiological agent of Chagas disease in Latin America. The most effective vector, Triatoma infestans, has been controlled successfully in much of Latin America using insecticide spraying. Though rarely undertaken, surveillance programs are necessary in order to identify new infestations and estimate the intensity of triatomine bug infestations in domestic and peridomestic habitats. Since hosts exposed to triatomines develop immune responses to salivary antigens, these responses can be evaluated for their usefulness as epidemiological markers to detect infestations of T. infestans. METHODOLOGY/PRINCIPAL FINDINGS: T. infestans salivary proteins were separated by 2D-gel electrophoresis and tested for their immunogenicity by Western blotting using sera from chickens and guinea pigs experimentally exposed to T. infestans. From five highly immunogenic protein spots, eight salivary proteins were identified by nano liquid chromatography-electrospray ionization-tandem mass spectrometry (nanoLC-ESI-MS/MS) and comparison to the protein sequences of the National Center for Biotechnology Information (NCBI) database and expressed sequence tags of a unidirectionally cloned salivary gland cDNA library from T. infestans combined with the NCBI yeast protein sub-database. The 14.6 kDa salivary protein [gi|149689094] was produced as recombinant protein (rTiSP14.6) in a mammalian cell expression system and recognized by all animal sera. The specificity of rTiSP14.6 was confirmed by the lack of reactivity to anti-mosquito and anti-sand fly saliva antibodies. However, rTiSP14.6 was recognized by sera from chickens exposed to four other triatomine species, Triatoma brasiliensis, T. sordida, Rhodnius prolixus, and Panstrongylus megistus and by sera of chickens from an endemic area of T. infestans and Chagas disease in Bolivia. CONCLUSIONS/SIGNIFICANCE: The recombinant rTiSP14.6 is a suitable and promising epidemiological marker for detecting the presence of small numbers of different species of triatomines and could be developed for use as a new tool in surveillance programs, especially to corroborate vector elimination in Chagas disease vector control campaigns.

Cuticular hydrocarbons of triatomines.

Triatomine insects (Hemiptera) are the vectors of Chagas disease. Their cuticular surface is covered by a thin layer of lipids, mainly hydrocarbons, wax esters, fatty alcohols, and free or esterified fatty acids. These lipids play a major role in preventing a lethal desiccation, altering the absorption of chemicals and microorganism penetration, they also participate in chemical communication events. Lipid components are biosynthetically related, the synthesis of long chain and very long chain fatty acids was first shown in the integument of Triatoma infestans through the concerted action of fatty acid synthases (FAS's) and fatty acyl-CoA elongases. A final decarboxylation step produces the corresponding hydrocarbon. Capillary gas chromatography coupled to mass spectrometry analyses showed that cuticular hydrocarbons of Triatominae comprise saturated straight and methyl-branched chains, from 18 to more than 43 carbon atoms. Odd-chain hydrocarbons, mostly from 27 to 33 carbons, are the major straight chains. Different isomers of mono, di, tri, and tetramethylcomponents, mostly from 29 to 39 atoms in the carbon skeleton, account for the major methyl-branched hydrocarbons. The presence, absence, and relative quantities of these hydrocarbons represent characters for their chemical phenotype, and are useful for differentiating genera, species and populations. In this review, we will discuss the metabolic pathways involved in hydrocarbon formation, and their structure, together with their role in insect survival. We will also review the utility of cuticular hydrocarbon fingerprints in chemotaxonomy.

Chemical ecology of triatomine bugs: vectors of Chagas disease.

Knowledge of chemical ecology of haematophagous triatomine bugs (Hemiptera: Heteroptera: Reduviidae) could be of practical value because this polyphyletic subfamily includes species of medical importance, such as vectors of Trypanosoma cruzi Chagas (Kinetoplastida: Trypanosomatidae), the aetiological agent of Chagas disease. For particular species of Dipetalogaster, Panstrongylus, Rhodnius and Triatoma, therefore, we review information on exocrine glands and products, interpreting their apparent roles as semiochemicals in the aggregation, alarm, defence, host-finding and sexual behaviour of these Triatominae.

Presence of a fatty acid-binding protein and lipid stores in flight muscles of Dipetalogaster maximus (Hemiptera: Reduviidae).

A fatty acid-binding protein (FABP) from the cytosolic fraction of the triatomine Dipetalogaster maximus (Uhler) flight muscles was purified by a procedure based on gel filtration, reverse-phase high performance liquid chromatography, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein has an apparent molecular mass of 14 kDa, and its N-terminus is unblocked. Its N-terminal sequence was obtained by submitting an SDS-PAGE band blotted onto a polyvinylidene difluoride membrane to Edman degradation. The sequence obtained indicates that this FABP belongs to the heart type. This is the first time that a fatty acid-binding protein has been reported for a triatomine. The presence of said FABP, abundant mitochondria, and lipid stores in the flight muscles of D. maximus suggests that beta oxidation of fatty acids is used by the triatomine thoracic muscle as an energy source, and could be related to its dispersal capacity.

Isobutyric acid from the Brindley's glands of Triatoma lecticularia.

The contents of the Brindley's gland of Triatoma lecticularia (Stål) (Heteroptera: Reduviidae: Triatominae) have been identified by gas chromatography-mass spectrometry as isobutyric acid. This compound is found in similar secretions from certain other members of the Triatominae, and is suggested to play a role as a defensive pheromone and for intraspecies communication.

Mitochondrial DNA sequence variation among triatomine vectors of Chagas' disease.

Kissing bugs or triatomines (Reduviidae: Triatominae) are vectors of the Chagas' disease agent Trypanosoma cruzi. There is a current need for more sensitive tools for use in discrimination of different bug populations and species, thus allowing a better understanding of these insects as it relates to disease transmission and control. In a preliminary analysis of the mitochondrial large subunit ribosomal RNA (mtlsurRNA) and cytochrome B (mtCytB) genes, we used DNA sequencing to study species identification and phylogeny. In both examined gene regions, about 46% of nucleotide positions exhibited polymorphism. The examined region of mtCytB appears to have evolved more rapidly than the examined region of mtlsurRNA. Phylogenetic analysis of both gene fragments in the examined species produced similar results that were generally consistent with the accepted taxonomy of the subfamily. The two major tribes, Rhodniini and Triatomini, were supported, along with additional clades that corresponded to accepted species complexes within the Rhodnius and Triatoma genera. The one chief exception was that Psammolestes coreodes sorted into the Rhodnius prolixus-robustus-neglectus clade, with bootsrap values of 99% and 81%, respectively, for the mtlsurRNA and mtCytB fragments. All of the individual species examined could be distinguished at both genetic loci.

Biochemical characterization of a thrombin inhibitor from the bloodsucking bug Dipetalogaster maximus.

From the bloodsucking bug Dipetalogaster maximus, a protein with anticoagulant activity was isolated and biochemically characterized. The isolated protein, named dipetalogastin, possesses an average molecular mass of 11.8 kD. Its N-terminal sequence shows homology to rhodniin, a thrombin inhibitor isolated from the bug Rhodnius prolixus. The in vitro anticoagulant activity of dipetalogastin occurs via the inhibition of thrombin. The anticoagulant and thrombin inhibitory potency of dipetalogastin is comparable to that of recombinant hirudin. Its specific thrombin inhibitory activity is 9,300 antithrombin units/mg protein. Dipetalogastin forms only 1:1 molar complexes with thrombin. It is a tight-binding inhibitor of thrombin possessing a dissociation constant of 125 fM. It does not inhibit factor Xa or alpha-chymotrypsin and only weakly inhibits trypsin.