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.

Coinfection by Trypanosoma cruzi and a fungal pathogen increases survival of Chagasic bugs: advice against a fungal control strategy.

Triatomine bugs carry the parasitic protozoa Trypanosoma cruzi, the causal agent of Chagas disease. It is known that both the parasite and entomopathogenic fungi can decrease bug survival, but the combined effect of both pathogens is not known, which is relevant for biological control purposes. Herein, the survival of the triatomine Meccus pallidipennis (Stal, 1872) was compared when it was coinfected with the fungus Metarhizium anisopliae (Metschnikoff) and T. cruzi, and when both pathogens acted separately. The immune response of the insect was also studied, using phenoloxidase activity in the bug gut and hemolymph, to understand our survival results. Contrary to expectations, triatomine survival was higher in multiple than in single challenges, even though the immune response was lower in cases of multiple infection. We postulate that T. cruzi exerts a protective effect and/or that the insect reduced the resources allocated to defend itself against both pathogens. Based on the present results, the use of M. anisopliae as a control agent should be re-considered.

Comparative genomics analysis of triatomines reveals common first line and inducible immunity-related genes and the absence of Imd canonical components among hemimetabolous arthropods.

BACKGROUND: Insects operate complex humoral and cellular immune strategies to fend against invading microorganisms. The majority of these have been characterized in Drosophila and other dipterans. Information on hemipterans, including Triatominae vectors of Chagas disease remains incomplete and fractionated. RESULTS: We identified putative immune-related homologs of three Triatominae vectors of Chagas disease, Triatoma pallidipennis, T. dimidiata and T. infestans (TTTs), using comparative transcriptomics based on established immune response gene references, in conjunction with the predicted proteomes of Rhodnius prolixus, Cimex lecticularis and Acyrthosiphon pisum hemimetabolous. We present a compressive description of the humoral and cellular innate immune components of these TTTs and extend the immune information of other related hemipterans. Key homologs of the constitutive and induced immunity genes were identified in all the studied hemipterans. CONCLUSIONS: Our results in the TTTs extend previous observations in other hemipterans lacking several components of the Imd signaling pathway. Comparison with other hexapods, using published data, revealed that the absence of various Imd canonical components is common in several hemimetabolous species.

Effect of the saliva from different triatomine species on the biology and immunity of TLR-4 ligand and Trypanosoma cruzi-stimulated dendritic cells.

BACKGROUND: Triatomines are blood-sucking vectors of Trypanosoma cruzi, the causative agent of Chagas disease. During feeding, triatomines surpass the skin host response through biomolecules present in their saliva. Dendritic cells (DCs) play a crucial role in the induction of the protection to aggressive agents, including blood-sucking arthropods. Here, we evaluated if salivary components of triatomines from different genera evade the host immunity by modulating the biology and the function of LPS- or T. cruzi-stimulated DCs. METHODS: Saliva of Panstrongylus lignarius, Meccus pallidipennis, Triatoma lecticularia and Rhodnius prolixus were obtained by dissection of salivary glands and the DCs were obtained from the differentiation of mouse bone marrow precursors. RESULTS: The differentiation of DCs was inhibited by saliva of all species tested. Saliva differentially inhibited the expression of MHC-II, CD40, CD80 and CD86 in LPS-matured DCs. Except for the saliva of R. prolixus, which induced IL-6 cytokine production, TNF-α, IL-12 and IL-6 were inhibited by the saliva of the other three tested species and IL-10 was increased in all of them. Saliva per se, also induced the production of IL-12, IL-6 and IL-10. Only the saliva of R. prolixus induced DCs apoptosis. The presence of PGE2 was not detected in the saliva of the four triatomines studied. Finally, T. cruzi invasion on DCs is enhanced by the presence of the triatomine saliva. CONCLUSIONS: These results demonstrate that saliva from different triatomine species exhibit immunomodulatory effects on LPS and T. cruzi-stimulated DCs. These effects could be related to hematophagy and transmission of T. cruzi during feeding.

Survival and immune response of the Chagas vector Meccus pallidipennis (Hemiptera: Reduviidae) against two entomopathogenic fungi, Metarhizium anisopliae and Isaria fumosorosea.

BACKGROUND: Chagas disease is a key health problem in Latin America and is caused and transmitted by Trypanosoma cruzi and triatomine bugs, respectively. Control of triatomines has largely relied on the use pyrethroids, which has proved to be ineffective in the long term. Alternatively, the use of entomopathogenic fungi has been implemented to control triatomine bugs. These fungi are highly efficient as they induce a reduction in immune response on insects. Meccus pallidipennis is the main triatomine vector of Chagas disease in Mexico. In this work we investigated the effects of two entomopathogenic fungi, Metarhizium anisopliae and Isaria fumosorosea, on M. pallidipennis nymphs in terms of insect survival and immune response. METHODS: We had an infected and a control group for each fungal species and assessed: a) insect survival during 30 days; and, b) phenoloxidase (PO) and prophenoloxidase (proPO; two key traits in insect immune response) at 24, 48, 96 and 144 h. For survival we used Kaplan-Meier survival analysis while for immune response we used factorial, repeated-measures ANOVA for each fungal species. RESULTS: Animals treated with M. anisopliae died sooner than animals treated with I. fumosorosea. Infected animals showed lower PO and proPO values than sham individuals, with a clear decrease in these parameters at 24 h with no further changes after this time. CONCLUSIONS: Our study widens the possibility of entomopathogenic fungi being used for triatomine control. The negative effect on PO and proPO seems mediated by a down-regulation of the triatomine immune response.

Immune defence mechanisms of triatomines against bacteria, viruses, fungi and parasites.

Triatomines are vectors that transmit the protozoan haemoflagellate Trypanosoma cruzi, the causative agent of Chagas disease. The aim of the current review is to provide a synthesis of the immune mechanisms of triatomines against bacteria, viruses, fungi and parasites to provide clues for areas of further research including biological control. Regarding bacteria, the triatomine immune response includes antimicrobial peptides (AMPs) such as defensins, lysozymes, attacins and cecropins, whose sites of synthesis are principally the fat body and haemocytes. These peptides are used against pathogenic bacteria (especially during ecdysis and feeding), and also attack symbiotic bacteria. In relation to viruses, Triatoma virus is the only one known to attack and kill triatomines. Although the immune response to this virus is unknown, we hypothesize that haemocytes, phenoloxidase (PO) and nitric oxide (NO) could be activated. Different fungal species have been described in a few triatomines and some immune components against these pathogens are PO and proPO. In relation to parasites, triatomines respond with AMPs, including PO, NO and lectin. In the case of T. cruzi this may be effective, but Trypanosoma rangeli seems to evade and suppress PO response. Although it is clear that three parasite-killing processes are used by triatomines - phagocytosis, nodule formation and encapsulation - the precise immune mechanisms of triatomines against invading agents, including trypanosomes, are as yet unknown. The signalling processes used in triatomine immune response are IMD, Toll and Jak-STAT. Based on the information compiled, we propose some lines of research that include strategic approaches of biological control.

Eficacia in vitro de tres endectocidas frente a Triatoma infestans / In vitro efficacy of three endectocides against Triatoma infestans

Introducción: distintos estudios han demostrado el papel preponderante que el peridomicilio cumple en la reinfestación de las viviendas por Triatoma infestans (vinchucas). Con el objetivo de eliminar focos residuales de T. infestans que habitan alrededor de los hogares se han desarrollado distintas estrategias. La administración de diferentes compuestos que tengan actividad contra T. infestans a los animales que habitan zonas cercanas a los domicilios y sirvan como fuente de alimentación a estos insectos, podría ser una buena manera de disminuir el riesgo de reinfestación domiciliaria. Objetivo: evaluar la eficacia in vitro de tres agentes antiparasitarios, doramectina (DRM), ivermectina (IVM) y eprinomectina (EPR) frente a ninfas de quinto estadio de Triatoma infestans. Métodos: se diseñaron alimentadores artificiales en donde se colocó sangre heparinizada y fortificada con distintas concentraciones de los tres endectocidas (100-0,4 ng/mL). Se utilizaron 600 ninfas de quinto estadio de T. infestans durante el experimento. Un grupo de vinchucas fue alimentada con sangre sin tratar (control). Luego de realizada la alimentación se observó el estado de los insectos cada 24 hs. durante el transcurso de una semana. Resultados: los tres endectocidas demostraron actividad frente a ninfas de quinto estadio de T. infestans. Comparando la actividad de las tres moléculas, DRM fue la que exhibió una mayor potencia contra los insectos, inclusive mantuvo su actividad frente T. infestans a 0,4 ng/mL (menor concentración evaluada). En el caso de IVM y EPR comenzaron a perder eficacia a concentraciones por debajo de los 6,25 y 3,15 ng/mL respectivamente, siendo totalmente inactivas a 0,4 ng/mL. Conclusiones: en base a estos resultados podemos aseverar que bajo nuestras condiciones experimentales, tanto IVM, EPR como DRM poseen una alta eficacia in vitro contra T. infestans, siendo la última la más efectiva de las tres evaluadas(AU)

Introduction: various studies have demonstrated the role that areas around the houses play in domiciliary re-infestation by Triatomainfestans (kissing bugs). With the aim of removing residual foci of T. infestans that inhabit in neighboring areas of houses, different strategies have been developed. The administration of different anti-T. infestans compounds to animals living in areas around the houses might be a good way to reduce the risk of domiciliary re-infestation. Objective: to evaluate the in vitro efficacy of three antiparasitic agents, doramectin (DRM), ivermectin (IVM) and eprinomectin (EPR) against fifth instar nymphs of Triatomainfestans. Methods: artificial feeders were designed , which contained heparinized and fortified blood with various concentrations of the three endectocides (100-0.4 ng/mL). We used 600 fifth instar nymphs of T. infestans during the experiment. A group of insects were fed with untreated blood (control). After feeding they were under observation to check their condition every 24 hours for a week. Results: the three molecules showed activity against T. infestans. In comparing the activity of the three molecules, DRM exhibited greater potency against insects, it even kept its activity against T. infestans at 0.4 ng/mL (lowest concentration tested). In the case of EPR and IVM, their efficacy began to lower at concentrations below 6.25 and 3.15 ng/mL respectively, being totally inactive at 0.4 ng/mL concentration. Conclusions: Based on these results, we can assert that under our experimental conditions, IVM, EPR and DRM show in vitro high efficacy against T. infestans, being the latter more effective than the other two molecules(AU)

Immune homeostasis to microorganisms in the guts of triatomines (Reduviidae)--a review.

Bacteria, fungi and parasites are in constant contact with the insect gut environment and can influence different aspects of the host gut physiology. Usually, some of these microorganisms develop and survive in the digestive tract. Therefore, the gut environment must be able to tolerate certain populations of these organisms for the establishment of interactions between non-pathogenic bacteria, parasites and the gut. This review provides a brief overview of the biological and molecular mechanisms that microorganisms use to interact with the gut epithelia in mosquitoes and speculates on their significances for the development of bacteria and Trypanosoma cruzi in the guts of triatomines.

Immune homeostasis to microorganisms in the guts of triatomines (Reduviidae): a review

Bacteria, fungi and parasites are in constant contact with the insect gut environment and can influence different aspects of the host gut physiology. Usually, some of these microorganisms develop and survive in the digestive tract. Therefore, the gut environment must be able to tolerate certain populations of these organisms for the establishment of interactions between non-pathogenic bacteria, parasites and the gut. This review provides a brief overview of the biological and molecular mechanisms that microorganisms use to interact with the gut epithelia in mosquitoes and speculates on their significances for the development of bacteria and Trypanosoma cruzi in the guts of triatomines.

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.

Susceptibility of different triatomine species to Trypanosoma rangeli experimental infection.

Trypanosoma rangeli is a kinetoplastid protozoan parasite that has been found in the majority of Latin American countries, overlapping its distribution area with that of Trypanosoma cruzi, the causative agent of Chagas disease. This parasite shares the same reservoirs and vectors as T. cruzi. Triatomines from genus Rhodnius are considered the most susceptible hosts to infection. In this work, we report the susceptibility of different triatomine species (Rhodnius neglectus, Panstrongylus megistus, Triatoma infestans, T. sordida, T. braziliensis, and T. vitticeps) to experimental infection by T. rangeli isolated from Didelphis albiventris in a highly endemic region for Chagas disease. An intense parasitism was evidenced in feces (56% to 81%) of the majority of the species studied on the 10th day after infection, decreasing during the period of the experiment (30 days). T. vitticeps did not present parasites in feces at any time. All triatomine species presented parasites in the hemolymph. In T. vitticeps and P. megistus, this parasitism was scarce (6.3% and 6.6%, respectively). In the other species, the parasitism was variable (62.5% to 100%). Triatomine mortality varied between 3% to 40%, increasing during the infection in all species studied. The lowest mortality was observed for T. infestans. Also, we showed that only trypomastigotes forms from salivary glands, and hemolymph were infective for mice. We conclude that all triatomine species used were susceptible to infection by T. rangeli at different levels. There was no direct correlation between intensity of parasitism and mortality.

Diminuiçäo da sobrevivência do Dipetalogaster maximus pela infecçäo da cepa Y do Trypanosoma cruzi em condiçöes de jejum absoluto / Aspects of biological interaction between Dipetalogaster maximus (Hemiptera, Reduviidae) and the Trypanosoma cruzi strain Y (Kinetoplastida, Trypanosomatidae)

Estudaram-se aspectos da relaçäo interespecífica entre o Dipetalogaster maximus e a cepa Y Trypanosoma cruzi, através do ciclo evolutivo e da resistência ao jejum. Os triatomíneos foram criados e separados em grupos de 60 insetos para cada experimento. Num grupo, ninfas de 1§ estádio de D.maximus foram infectadas no 10§ dia após a eclosäo, com a cepa Y de T.cruzi, com a finalidade de esclarecer a relaçäo interespecífica existente entre o inseto hospedeiro e o protozoário parasito. Nä houve diferença significativa entre a duraçäo média do ciclo evolutivo de triatomíneos näo infectados (201,1 e 202,6 dias) e infectados pelo T.cruzi (204,8 e 204,2 dias)...

Comportamento da parasitemia pelo Trypanosoma cruzi em chagásicos crônicos durante 13 anos / Aspects of parasitemia by Trypanosoma cruzi in chronic chagasic patients during 13 years

Foi estudada a parasitemia de 202 chagásicos crônicos por um período médio de 13 anos, através de repetidos xenodiagnósticos convencionais. Os pacientes tinham média de idade de 41,4 anos, residiam em área endêmica, sendo 124 do sexo feminino e 78 do sexo masculino. Foi observado que o nível de parasitemia aumentou em 14 indivíduos, diminuiu em 42 e permaneceu inalterado em 146. Porém, no geral, a parasitemia declinou. Os percentuais de chagásicos xenopositivos que foram 37,6 por cento, 48,5 por cento, 51 por cento no primeiro, segundo e terceiro xenodiagnósticos, respectivamente, em 1976/78, passaram a ser 30,2 por cento em 1988/91 (p = 0,00003). Os percentuais de pools positivos que foram 15,2 por cento, 20,9 por cento, 20,8 por cento no primeiro, segundo e terceiro xenodiagnósticos, respectivamente, em 1976/78, passaram a ser 10,4 por cento em 1988/91, (p = 0. 00000001). Houve 62 pacientes que tiveram todos os xenodiagnósticos negativos e 23 que apresentaram todos os exames positivos. Os percentuais de chagásicos com alta, média e baixa parasitemias que, em 1976/78, foram, respectivamente, 9,4 por cento; 20,8 por cento e 69,8 por cento passaram a ser, em 1988/91, respectivamente, 4,4 por cento, 12,9 por cento e 82,7 por cento

Human immune response to triatomine embryo extract

Dipetalogaster maximus embryo extracts were used to stimulate peripheral blood mononuclear cells (PBMC) and in ELISA with sera either from Trypanosoma cruzi infected or non-infected individuals. The results showed that there was significant proliferative response and high antibody, titers in sera of chagasic patients.

Human immune response to triatomine embryo extract.

Dipetalogaster maximus embryo extracts were used to stimulate peripheral blood mononuclear cells (PBMC) and in ELISA with sera either from Trypanosoma cruzi infected or non-infected individuals. The results showed that there was significant proliferative response and high antibody, titers in sera of chagasic patients.

Trypanosoma cruzi strain-specific monoclonal antibodies: identification of Colombian strain flagellates in the insect vector.

Spleen cells from mice immunized with insect-derived Trypanosoma cruzi metacyclic trypomastigotes were used to obtain Colombian strain-specific monoclonal antibodies. At least 4 different strain-specific antigens were recognized by the monoclonal antibodies on epimastigotes or metacyclic trypomastigotes. There was no reactivity with other stages of Colombian strain T. cruzi, nor with any stage of 15 other T. cruzi strains or isolates, nor with 22 other Trypanosomatidae. One of the monoclonal antibodies was used to identify, by indirect immunofluorescence, Colombian strain flagellates in cryostat sections or glass-slide smears of the insect vector's intestine.

Observaciones sobre el xenodiagnóstico en la infección experimental por Trypanosoma cruzi / Observations on the xenodiagnostic in the experimental infection by trypanosoma cruzi

En la tentativa de mejorar la sensibilidad del xenodiagnóstico, y para realizar algunas observaciones sobre su técnica, fueron realizados experimentos en 2 etapas. En la primera etapa ratas "Wistar", al final de la fase patente de la infección, fueron sometidas a la picada de ninfas de Triatoma brasiliensis santinacensis y la parasitemia medida cada hora durante las primeras 24 horas y luego 2 veces por día. En la segunda etapa se practicaron xenodiagnósticos únicos y seriados a ratas con 4 meses de infección por T. cruzi y los triatominos se examinaron a los 30 y 45 días por compresión y a los 60 días por disección del tubo digestivo. No se detectó ningún efecto de la picada de los triatominos en la parasitemia de las ratas, no siendo posible, por lo tanto, mejorar el rendimiento del xenodiagnóstico. El examen por compresión abdominal parece inducir mortalidad de los triatominos. La totalidad de los resultados positivos se obtuvieron examinando los triatominos. La totalidad de los resultados positivos se obtuvieron examinando los triatominos entre 30 y 45 días después de aplicado el examen. No hubo diferencias en los resultados obtenidos con xenodiagnósticos únicos y seriados. No hubo correlación entre la cantidad de sangre ingerida y el porcentaje de triatominos positivos; tampoco hubo correlación entre mudas y positividad. El examen por disección fue significativamente superior al examen por compresion

Species-specific allergens from the salivary glands of Triatominae (Heteroptera:Reduviidae).

We investigated allergenic cross-reactivity among species of the blood-feeding insects of the subfamily Triatominae. By skin testing, patients allergic to either Triatoma protracta or T. rubida gave positive responses only to the respective salivary antigen. RAST-inhibition experiments demonstrated that binding of IgE antibodies to T. protracta antigen was not inhibited by salivary extracts from T. rubida, T. cavernicola, T. rubrofasciata, or Rhodnius prolixus. The same level of species specificity was found for IgE antibodies to T. rubida. By direct RAST, no T. rubida positive serum bound T. protracta antigen, and 29 of 30 T. protracta positive sera failed to bind T. rubida. One serum from a T. protracta-allergic patient contained IgE antibodies to both T. protracta and T. rubida. RAST-inhibition experiments demonstrated that these antibodies did not cross-react and that this person had separate species-specific antibodies to T. protracta and T. rubida antigens. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of salivary extracts demonstrated that the lower molecular weight bands that contain the antigens responsible for human allergic reactions differed in number and size in all species tested. These studies demonstrate species specificity for the allergic response to Triatoma and stress the importance of accurate insect identification and the need for species-specific antigens for diagnosis and immunotherapy.

Identification and partial purification of species-specific allergens from Triatoma protracta (Heteroptera:Reduviidae).

This article describes the immunochemical characterization of allergens from Triatoma protracta, a hematophagous insect that causes IgE-mediated anaphylactic reactions when it bites sensitized allergic persons. Comparison of the allergenic potency of T. protracta salivary gland extract, thoracic and abdominal hemolymph, and a whole body extract by RAST inhibition demonstrated that salivary glands were the main source of T. protracta allergens. Concentrated salivary gland extracts were purified by gel filtration and isoelectric focusing. Fractions were tested for allergenic activity by RAST inhibition and for protein purity by polyacrylamide gel electrophoresis and immunoelectrophoresis. Two protein peaks were obtained on gel filtration. The high-molecular-weight peak contained a 70,000 MW protein/glycoprotein that had little allergenic activity. The low-molecular-weight peak comprised six proteins, molecular weight 17,000 to 25,000, and T. protracta allergen(s) eluted in parallel with this peak. These proteins were resolved by isoelectric focusing, and two fractions, pI 6.7 to 7.3 and pI 8.2, contained most of the allergenic activity. By RAST, 25/28 sera from T. protracta-allergic patients contained IgE antibody to these fractions, suggesting that they were major allergens. Each fraction demonstrated a single precipitin arc on immunoelectrophoresis and two bands, molecular weight 18,000 to 20,000, on gel electrophoresis. Cross-inhibition radioimmunoassays demonstrated that each fraction completely inhibited binding of the other fraction to IgE antibody, suggesting that they contained different isoelectric forms of the same allergen.