Differential activity on trypanosomatid parasites of a novel recombinant defensin type 1 from the insect Triatoma (Meccus) pallidipennis.

Defensins are one of the major families of antimicrobial peptides (AMPs) that are widely distributed in insects. In Triatomines (Hemiptera: Reduviidae) vectors of Trypanosoma cruzi the causative agent of Chagas disease, two large groups of defensin isoforms have been described: type 1 and type 4. The aim of this study was to analyze the trypanocidal activity of a type 1 recombinant defensin (rDef1.3) identified in Triatoma (Meccus) pallidipennis, an endemic specie from México. The trypanocidal activity of this defensin was evaluated in vitro, against the parasites T. cruzi, T. rangeli, and two species of Leishmania (L. mexicana and L. major) both causative agents of cutaneous leishmaniasis. Our data demonstrated that the defensin was active against all the parasites although in different degrees. The defensin altered the morphology, reduced the viability and inhibited the growth of T.cruzi. When tested against T. rangeli (a parasite that infects a variety of mammalian species), stronger morphological effects where observed. Surprisingly the greatest effects were observed against the two Leishmania species, of which L. major was the parasite most affected with 50% of dead cells or with damaged membranes, in addition of a reduction in its proliferative capacity in culture. These results suggest that rDef1.3 has an important antimicrobial effect against trypanosomatids which cause some of the more important neglected tropical diseases transmitted by insect vectors.

Salivary gland transcriptome of the Asiatic Triatoma rubrofasciata.

Salivary gland transcriptome analysis of the Asiatic Triatoma rubrofasciata was performed by high-throughput RNA sequencing. This analysis showed that the majority of reads accounting for 85.38% FPKM (fragments per kilobase of exon per million mapped fragments) were mapped with a secreted class. Of these, the most abundant subclass accounting for 89.27% FPKM was the lipocalin family. In the lipocalin family, the most dominant molecules making up 70.49% FPKM were homologues of procalin, a major allergen identified from T. protracta saliva, suggesting an important role in blood-sucking of T. rubrofasciata. Other lipocalins showed similarities to pallidipin and triplatin, inhibitors of collagen-induced platelet aggregation identified from T. pallidipennis and T. infestans, respectively, Td38 from T. dimidiata with unknown function, triatin-like lipocalin with unknown function, and triafestin, an inhibitor of the activation of the kallikrein-kinin system, identified from T. infestans saliva. Other than lipocalin family proteins, homologues of antigen-5 (3.38% FPKM), Kazal-type serine protease inhibitor (1.36% FPKM), inositol polyphosphate 5-phosphatase (1.32% FPKM), and apyrase/5'-nucleotidase (0.64% FPKM) were identified as abundant molecules in T. rubrofasciata saliva. Through this study, de novo assembly of 42,580,822 trimmed reads generated 35,781 trinity transcripts, and a total of 1,272 coding sequences for the secreted class were deposited in GenBank. The results provide further insights into the evolution of salivary components in blood-sucking arthropods.

Human Antimicrobial Peptide Isolated From Triatoma infestans Haemolymph, Trypanosoma cruzi-Transmitting Vector.

The importance of antimicrobial peptides (AMPs) in relation to the survival of invertebrates is well known. The source and the mode of action on the insects' immune system of these molecules have been described from different perspectives. Insects produce their own AMPs as well as obtain these molecules from various sources, for example by absorption through the intestinal tract, as previously described for Boophilus microplus. Blood-sucking barber bug Triatoma infestans attracts social, economic and medical interest owing to its role in the transmission of Chagas disease. Despite new studies, descriptions of AMPs from this insect have remained elusive. Thus, the aims of this work were to characterize the antimicrobial potential of human fibrinopeptide A (FbPA) obtained from the T. infestans haemolymph and identify its natural source. Therefore, FbPA was isolated from the T. infestans haemolymph through liquid chromatography and identified by mass spectrometry. This peptide exhibited antimicrobial activity against Micrococcus luteus. Native FbPA from human blood and the synthetic FbPA also exhibited antimicrobial activity. The synthetic FbPA was conjugated with fluorescein isothiocyanate and offered to the insects. The haemolymph collected after 72 h exhibited fluorescence at the same wavelength as fluorescein isothiocyanate. Our experiments show that beyond intrinsic AMP production, T. infestans is able to co-opt molecules via internalization and may use them as AMPs for protection.

Salivary Gland Extract of Kissing Bug, Triatoma lecticularia, Reduces the Severity of Intestinal Inflammation through the Modulation of the Local IL-6/IL-10 Axis.

Triatomines are known for their role as vectors of the causative agent of Chagas disease. The occurrence of an arsenal of molecules in their saliva is able to suppress vertebrate immune responses. Thus, it is reasonable to assume that the presence of molecules with therapeutic potential in their saliva is able to constrain inflammation in immune-mediated diseases. Thus, mice were exposed to dextran sulfate sodium (DSS) in drinking water uninterruptedly during 6 consecutive days and treated with T. lecticularia salivary gland extract (SGE) (3, 10, or 30 µg) or vehicle (saline) (n = 6/group). At the highest dose (30 µg), an improvement in clinical outcome and macroscopic aspects of the intestine were observed. This observation was followed by amelioration in histopathological aspects in the colon especially when the doses of 10 and 30 µg were used. Regardless of the concentration used, treatment with T. lecticularia SGE significantly reduced the levels of the inflammatory cytokine IL-6 in the intestine. The production of the anti-inflammatory cytokine IL-10 was positively impacted by the concentrations of 3 and 30 µg. Our results suggest that the presence of molecules in the T. lecticularia SGE is able to attenuate clinical outcome and colon shortening and improve intestinal architecture besides reducing the production of IL-6 and inducing a local production of IL-10 in the intestine.

Interaction between two aggregation chemical signals in Triatoma infestans (Hemiptera: Reduviidae).

The nymphs and adults of Triatoma infestans spend much of their time aggregated among themselves within narrow and dark shelters. The search for a suitable shelter depends in part on the recognition of chemical signals coming from the feces and the cuticle of the other individuals who use the refuge. The aim of this study was determine the possible interaction between the chemical signals associated to the feces and to the cuticle of T. infestans. The results showed that the insects remained significantly more time on the feces that had contact with legs and the feces plus footprints than feces or footprints alone, demonstrating the interaction between evaluated signals. These results demonstrates also that feces extracted a chemical stimulus from the legs. Understanding the interaction feces-legs as an interaction feces-cuticle of legs, the results suggest that the feces could extract some cuticular compound with activity on the behavior of the insects. This is the first report of the interaction between the two aggregation signals recognized in T. infestans and of the increase in the behavioral response of insects exposed to feces that had contact with a cuticular structure.

"Sweeter than a rose", at least to Triatoma phyllosoma complex males (Triatominae: Reduviidae).

BACKGROUND: The Triatoma phyllosoma complex of Trypanosoma cruzi vectors (Triatominae: Reduviidae) is distributed in both Neotropical and Nearctic bioregions of Mexico. METHODS: Volatile organic compounds emitted by disturbed Triatoma longipennis, Triatoma pallidipennis and Triatoma phyllosoma, and from their Brindley's and metasternal glands, were identified using solid-phase microextraction coupled with gas chromatography-mass spectrometry. RESULTS: Disturbed bugs and the metasternal glands from T. phyllosoma released or had significantly fewer compounds than T. longipennis and T. pallidipennis. Isobutyric acid was the most abundant compound secreted by disturbed bugs of the three species, while Brindley's glands of all species produced another four compounds: propanoic acid, isobutyric acid, pentyl butanoate, and 2-methyl hexanoic acid. Two novel compounds, both rose oxide isomers, were produced in MGs and released only by disturbed females of all three species, making this the first report in Triatominae of these monoterpenes. The principal compound in MGs of both sexes of T. longipennis and T. phyllosoma was 3-methyl-2-hexanone, while cis-rose oxide was the principal compound in T. pallidipennis females. The major components in male effluvia of T. pallidipennis were 2-decanol and 3-methyl-2-hexanone. CONCLUSION: Discriminant analysis of volatile organic compounds was significant, separating the three species and was consistent with morphological and genetic evidence for species distinctions within the complex.

Exploring the molecular complexity of Triatoma dimidiata sialome.

Triatoma dimidiata, a Chagas disease vector widely distributed along Central America, has great capability for domestic adaptation as the majority of specimens caught inside human dwellings or in peridomestic areas fed human blood. Exploring the salivary compounds that overcome host haemostatic and immune responses is of great scientific interest. Here, we provide a deeper insight into its salivary gland molecules. We used high-throughput RNA sequencing to examine in depth the T. dimidiata salivary gland transcriptome. From >51 million reads assembled, 92.21% are related to putative secreted proteins. Lipocalin is the most abundant gene family, confirming it is an expanded family in Triatoma genus salivary repertoire. Other putatively secreted members include phosphatases, odorant binding protein, hemolysin, proteases, protease inhibitors, antigen-5 and antimicrobial peptides. This work expands the previous set of functionally annotated sequences from T. dimidiata salivary glands available in NCBI from 388 to 3815. Additionally, we complemented the salivary analysis through proteomics (available data via ProteomeXchange with identifier PXD008510), disclosing the set complexity of 119 secreted proteins and validating the transcriptomic results. Our large-scale approach enriches the pharmacologically active molecules database and improves our knowledge about the complexity of salivary compounds from haematophagous vectors and their biological interactions. SIGNIFICANCE: Several haematophagous triatomine species can transmit Trypanosoma cruzi, the etiological agent of Chagas disease. Due to the reemergence of this disease, new drugs for its prevention and treatment are considered priorities. For this reason, the knowledge of vector saliva emerges as relevant biological finding, contributing to the design of different strategies for vector control and disease transmission. Here we report the transcriptomic and proteomic compositions of the salivary glands (sialome) of the reduviid bug Triatoma dimidiata, a relevant Chagas disease vector in Central America. Our results are robust and disclosed unprecedented insights into the notable diversity of its salivary glands content, revealing relevant anti-haemostatic salivary gene families. Our work expands almost ten times the previous set of functionally annotated sequences from T. dimidiata salivary glands available in NCBI. Moreover, using an integrated transcriptomic and proteomic approach, we showed a correlation pattern of transcription and translation processes for the main gene families found, an important contribution to the research of triatomine sialomes. Furthermore, data generated here reinforces the secreted proteins encountered can greatly contribute for haematophagic habit, Trypanosoma cruzi transmission and development of therapeutic agent studies.

[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.

Proteomic and transcriptomic analysis of saliva components from the hematophagous reduviid Triatoma pallidipennis.

Species belonging to the Triatominae subfamily are commonly associated with Chagas disease, as they are potential vectors of the parasite Trypanosoma cruzi. However, their saliva contains a cocktail of diverse anti-hemostatic proteins that prevent blood coagulation, vasodilation and platelet aggregation of blood; components with indisputable therapeutic potential. We performed a transcriptomic and proteomic analyses of salivary glands and protein spots from 2DE gels of milked saliva, respectively, from the Mexican Triatoma pallidipennis. Massive sequencing techniques were used to reveal this protein diversity. A total of 78 out of 233 transcripts were identified as proteins in the saliva, divided among 43 of 55 spots from 2DE gels of saliva, identified by LC-MS/MS analysis. Some of the annotated transcripts putatively code for anti-hemostatic proteins, which share sequence similarities with proteins previously described for South American triatomines. The most abundant as well as diverse transcripts and proteins in the saliva were the anti-hemostatic triabins. For the first time, a transcriptomic analysis uncovered other unrelated but relevant components in triatomines, including antimicrobial and thrombolytic polypeptides. Likewise, unique proteins such as the angiotensin-converting enzyme were identified not just in the salivary gland transcriptome but also at saliva proteome of this North American bloodsucking insect. BIOLOGICAL SIGNIFICANCE: This manuscript is the first report of the correlation between proteome and transcriptome of Triatoma pallidipennis, which shows for the first time the presence of proteins in this insect that have not been characterized in other species of this family. This information contributes to a better understanding of the multiple host defense mechanisms that are being affected at the moment of blood ingestion by the insect. Furthermore, this report gives a repertoire of possible therapeutic proteins.

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.

Comparative proteomic analysis of the saliva of the Rhodnius prolixus, Triatoma lecticularia and Panstrongylus herreri triatomines reveals a high interespecific functional biodiversity.

Triatomines are hematophagous arthropods that transmit Trypanosoma cruzi and Trypanosoma rangeli. Feeding behavior and pathogen transmission is known to vary between the different species, and this characteristic is directly or indirectly dependent on the bioactive molecules of the saliva that facilitate the vector-host-parasite interaction. Here, we identify, characterize and compare the sialoproteomic (from the Greek sialo: saliva) repertoire of important species of the main triatomine genera in the Americas (Rhodnius prolixus, Triatoma lecticularia and Panstrongylus herreri) to better explain this interaction through two-dimensional electrophoresis and mass spectrometry. We identified 221 proteins, 69 from R. prolixus, 100 from T. lecticularia and 52 from P. herreri. We identified high abundance molecules with a great potential to modulate host defenses and homeostasis, highlighting Nitrophorin-4 (28.7%), Salivary lipocalin-5 (65.2%) and Putative triabin (20.5%) in R. prolixus, T. lecticularia and P. herreri, respectively. We also observed that only a single hypothetical protein is shared among three species, which was not functionally categorized. This study corroborates previous findings with R. prolixus, increasing the knowledge about this species with relevant proteomic information and comparisons with the other two targets of the study, T. lecticularia and P. herreri, for which no studies are available from a proteomics perspective.

A new antimicrobial protein from the anterior midgut of Triatoma infestans mediates Trypanosoma cruzi establishment by controlling the microbiota.

The Reduviid Triatoma infestans is a vector for the protozoan Trypanosoma cruzi, the etiological agent of Chagas disease. The parasite must address the defense molecules and microbiota that colonize the anterior midgut of T. infestans. To obtain insight into T. cruzi - microbiota interactions in triatomine insects, we characterized a new antimicrobial product from the anterior midgut of T. infestans (TiAP) that may be involved in these relationships. The TiAP DNA fragment was cloned and expressed in a bacterial system, and the effect of the protein on bacteria and T. cruzi was evaluated by RNAi, qPCR and antimicrobial experiments. The number of T. cruzi in T. infestans anterior midguts was significantly lower in TiAP knockdown insects than in unsilenced groups. We also verified that the amount of bacteria in silenced T. infestans is approximately 600-fold higher than in unsilenced insects by qPCR. The 327-bp cDNA fragment that encodes mature TiAP was cloned into the pET-14b vector and expressed fused to a His-tag in Escherichia coli C43. The recombinant protein (rTiAP) was purified using an Ni-NTA column, followed by a HiTrap SP column. According to a trypanocidal assay, rTiAP did not interfere with the viability of T. cruzi trypomastigotes. Moreover, in antimicrobial experiments using E. coli and Micrococcus luteus, the protein was only bacteriostatic for Gram-negative bacteria. The data indicate that infection by T. cruzi increases the expression of TiAP to modulate the microbiota. The inhibition of microbiota growth by TiAP is important for parasite establishment in the T. infestans anterior midgut.

Deltamethrin Binding to Triatoma infestans (Hemiptera: Reduviidae) Lipoproteins. Analysis by Solvent Bar Microextraction Coupled to Gas Chromatography.

The binding of deltamethrin (DLM) to the hemipteran Triatoma infestans (Klug) hemolymph lipoproteins was evaluated in vitro. After DLM incubation with the insect hemolymph, lipoproteins were fractioned by ultracentrifugation. DLM binding was analyzed by a microextractive technique-solvent bar microextraction-a solventless methodology to extract DLM from each lipoprotein fraction. This is a novel use of the technique applied to extract an insecticide from an insect fluid. Capillary gas chromatography with microelectron capture detection was used to detect DLM bound by the T. infestans hemolymph lipoproteins and to identify the preferred DLM carrier. We show that Lp and VHDLp I lipoproteins are mainly responsible for DLM transport in T. infestans, both in DLM-resistant and DLM-susceptible bugs. Our results also indicate that DLM amounts transported are not related to DLM susceptibility.

Volatile compound diversity and conserved alarm behaviour in Triatoma dimidiata.

BACKGROUND: Triatoma dimidiata (Latreille) is a key vector complex of Trypanosoma cruzi, etiologic agent of Chagas disease, as it spans North, Central, and South America. Although morphological and genetic studies clearly indicate existence of at least five clades within the species, there has been no robust or systematic revision, or appropriate nomenclature change for species within the complex. Three of the clades (haplogroups) are distributed in Mexico, and recent evidence attests to dispersal of clades across previously "presumed" monotypic geographic regions. Evidence of niche conservatism among sister species of this complex suggests that geographic dispersal is possible for non-sympatric populations, although no information is available on the behavioural aspects of potential interclade interactions, for instance whether differentiation of chemical signaling or response to these signals could impede communication among the haplogroups. METHODS: Volatiles emitted by disturbed bugs, Brindley's (BGs), and metasternal (MGs) glands were identified using solid-phase micro-extraction (SPME) and gas chromatography coupled mass spectrometry (GC-MS). Volatile compounds emitted by BGs and MGs, and those secreted by disturbed nymphs and adults, of the three Mexican T. dimidiata haplogroups were tested for avoidance behaviour by conspecific nymphs and adults using an olfactometer. RESULTS: Triatoma dimidiata haplogroups all have three age-related alarm responses: absence of response by early stage nymphs, stage-specific response by 4-5th stage nymphs, and a shared 4-5th nymph and adult response to adult compounds. Disturbed bugs released 15 to 24 compounds depending on the haplogroup, among which were three pyrazines, the first report of these organoleptics in Triatominae. Isobutyric acid from BGs was the most abundant molecule in the response in all haplogroups, in addition to 15 (h1) to 21 (h2 and h3) MG compounds. Avoidance behaviour of disturbed bugs and volatiles emitted by BGs were haplogroup specific, while those from the MG were not. CONCLUSIONS: Discriminant and cluster analysis of BG + MG compounds indicate significant separation among the three haplogroups, while alarm response compounds were similar between h2 and h3, both distinct from h1. This latter haplogroup is ancestral phylogenetically to the other two. Our results suggest that alarm responses are a conserved behaviour in the Triatoma dimidiata complex.

Development and glycoprotein composition of the perimicrovillar membrane in Triatoma (Meccus) pallidipennis (Hemiptera: Reduviidae).

Hemipterans and thysanopterans (Paneoptera: Condylognatha) differ from other insects by having an intestinal perimicrovillar membrane (PMM) which extends from the base of the microvilli to the intestinal lumen. The development and composition of the PMM in hematophagous Reduviidae depend on factors related to diet. The PMM may also allow the human parasite Trypanosoma cruzi, the etiological agent of human Chagas Disease, to establish and develop in this insect vector. We studied the PMM development in the Mexican vector of Chagas Disease, Triatoma (Meccus) pallidipennis. We describe changes in the midgut epithelial cells of insects in response to starvation, and at different times (10, 15 and 20 days) after bloodfeeding. In starved insects, the midguts showed epithelial cells closely connected to each other but apparently free of PMM with some regions being periodic acid-Schiff (PAS-Schiff) positive. In contrast, the PMM was evident and fully developed in the midgut region of insects 15 days after feeding. After this time, the PMM completely covered the microvilli and reached the midgut lumen. At 15 days following feeding the labeled PAS-Schiff increased in the epithelial apex, suggesting an increase in carbohydrates. Lectins as histochemical reagents show the presence of a variety of glycoconjugates including mannose, glucose, galactosamine, N-acetyl-galactosamine. Also present were N-acetyl-glucosamine and sialic acid which contribute to the successful establishment and replication or T. cruzi in its insect vectors. By means of scanning electron microscopy (SEM) and transmission electron microscopy (TEM), the formation and structure of the PMM is confirmed at 15 days post feeding. Our results confirmed the importance of the feeding processes in the formation of the PMM and showed the nature of the biochemical composition of the vectors' intestine in this important Mexican vector of Chagas disease.

The cuticular hydrocarbons of the Triatoma sordida species subcomplex (Hemiptera: Reduviidae).

The cuticular hydrocarbons of the Triatoma sordida subcomplex (Hemiptera: Reduviidae: Triatominae) were ana-lysed by gas chromatography and their structures identified by mass spectrometry. They comprised mostly n-alkanes and methyl-branched alkanes with one-four methyl substitutions. n-alkanes consisted of a homologous series from C21-C33 and represented 33-45% of the hydrocarbon fraction; n-C29 was the major component. Methyl-branched alkanes showed alkyl chains from C24-C43. High molecular weight dimethyl and trimethylalkanes (from C35-C39) represented most of the methyl-branched fraction. A few tetramethylalkanes were also detected, comprising mostly even-numbered chains. Several components such as odd-numbered 3-methylalkanes, dimethylalkanes and trimethylalkanes of C37 and C39 showed patterns of variation that allowed the differentiation of the species and populations studied. Triatoma guasayana and Triatoma patagonica showed the most distinct hydrocarbon patterns within the subcomplex. The T. sordida populations from Brazil and Argentina showed significantly different hydrocarbon profiles that posed concerns regarding the homogeneity of the species. Triatoma garciabesi had a more complex hydrocarbon pattern, but it shared some similarity with T. sordida. The quantitative and qualitative variations in the cuticular hydrocarbons may help to elucidate the relationships between species and populations of this insect group.

The cuticular hydrocarbons of the Triatoma sordida species subcomplex (Hemiptera: Reduviidae)

The cuticular hydrocarbons of the Triatoma sordida subcomplex (Hemiptera: Reduviidae: Triatominae) were ana-lysed by gas chromatography and their structures identified by mass spectrometry. They comprised mostly n-alkanes and methyl-branched alkanes with one-four methyl substitutions. n-alkanes consisted of a homologous series from C21-C33 and represented 33-45% of the hydrocarbon fraction; n-C29 was the major component. Methyl-branched alkanes showed alkyl chains from C24-C43. High molecular weight dimethyl and trimethylalkanes (from C35-C39) represented most of the methyl-branched fraction. A few tetramethylalkanes were also detected, comprising mostly even-numbered chains. Several components such as odd-numbered 3-methylalkanes, dimethylalkanes and trimethylalkanes of C37 and C39 showed patterns of variation that allowed the differentiation of the species and populations studied. Triatoma guasayana and Triatoma patagonica showed the most distinct hydrocarbon patterns within the subcomplex. The T. sordida populations from Brazil and Argentina showed significantly different hydrocarbon profiles that posed concerns regarding the homogeneity of the species. Triatoma garciabesi had a more complex hydrocarbon pattern, but it shared some similarity with T. sordida. The quantitative and qualitative variations in the cuticular hydrocarbons may help to elucidate the relationships between species and populations of this insect group.

Risk of transmission of Trypanosoma cruzi by wild Triatoma infestans (Hemiptera: Reduviidae) in Bolivia supported by the detection of human blood meals.

We analyzed the food sources of Bolivian wild Triatoma infestans (the main vector of Chagas disease in this country), to assess the role of these populations in the epidemiological context of Chagas disease. Ninety-eight blood meals were identified by heteroduplex assay and sequencing. Most of them were from wild mammals but surprisingly 27 were from humans. This brings to light the occurrence of human-vector contacts at risk of Trypanosoma cruzi transmission in the wild environment by highly infected insects.

The Kazal-type inhibitors infestins 1 and 4 differ in specificity but are similar in three-dimensional structure.

Blood coagulation is an important process in haemostasis, and disorders of blood coagulation can lead to an increased risk of haemorrhage and thrombosis. Coagulation is highly conserved in mammals and has been comprehensively studied in humans in the investigation of bleeding or thrombotic diseases. Some substances can act as inhibitors of blood coagulation and may affect one or multiple enzymes throughout the process. A specific thrombin inhibitor called infestin has been isolated from the midgut of the haematophagous insect Triatoma infestans. Infestin is a member of the nonclassical Kazal-type serine protease inhibitors and is composed of four domains, all of which have a short central α-helix and a small antiparallel ß-sheet. Domains 1 and 4 of infestin (infestins 1 and 4) possess specific inhibitory activities. Infestin 1 inhibits thrombin, while infestin 4 is an inhibitor of factor XIIa, plasmin and factor Xa. Here, the structure determination and structural analysis of infestin 1 complexed with trypsin and of infestin 4 alone are reported. Through molecular modelling and docking, it is suggested that the protein-protein binding site is conserved in the infestin 1-thrombin complex compared with other Kazal-type inhibitors. Infestin 4 is able to bind factor XIIa, and the F9N and N11R mutants selected by phage display were shown to be more selective for factor XIIa in comparison to the wild type.

Structure elucidation and synthesis of dioxolanes emitted by two Triatoma species (Hemiptera: Reduviidae).

Volatiles from the metasternal glands of two species of true bugs of the Triatominae subfamily, Triatoma brasiliensis and Triatoma infestans, were analyzed by SPME-GC/MS. Two sets of new natural products were found: (4S,5S)- and (4R,5R)-2,2,4-triethyl-5-methyl-1,3-dioxolane (1) (major component) and (4S*,5S*)-2,4-diethyl-2,5-dimethyl-1,3-dioxolane (2) (trace component), (2R/S,4S,5S)- as well as (2R/S,4R,5R)-4-ethyl-5-methyl-2-(1-methylethyl)-1,3-dioxolane (3) (minor component), (2R/S,4S*,5S*)-4-ethyl-5-methyl-2-(1-methylpropyl)-1,3-dioxolane (4) (trace component), and (2R/S,4S*,5S*)-4-ethyl-5-methyl-2-(2-methylpropyl)-1,3-dioxolane (5) (trace component). Syntheses of optically active 1 and 3 were carried out by reacting pure enantiomers of 2,3-pentanediol with 3-pentanone or 2-methylpropanal. The preparation of pure stereoisomers of 2,3-pentanediol involved a novel key step for the synthesis of secondary alcohols: the reduction of a carboxylic ester by means of DIBAH and in situ alkylation of the intermediate by Grignard reaction at low temperature. Starting from the pure enantiomers of methyl lactate, all four stereoisomers of 2,3-pentanediol were synthesized and transformed to the corresponding isomers of 1 and 2. Relative configurations of the natural products and enantiomeric compositions of naturally occurring 1 and 2 were determined by comparison of their mass spectra and gas chromatographic retention times (co-injection) with those of authentic reference samples.