Hemolymph of triatomines presents fungistatic activity against Cryptococcus neoformans and improves macrophage function through MCP-I/TNF-α increase.

Background: Triatomines are blood-feeding arthropods belonging to the subfamily Triatominae (Hemiptera; Reduviidae), capable of producing immunomodulatory and water-soluble molecules in their hemolymph, such as antimicrobial peptides (AMPs). In this work, we evaluated the antifungal and immunomodulatory activity of the hemolymph of Meccus pallidipennis (MPH) and Rhodnius prolixus (RPH) against Cryptococcus neoformans. Methods: We assessed the activity of the hemolymph of both insects on fungal growth by a minimum inhibitory concentration (MIC) assay. Further, RAW 264.7 macrophages were cultivated with hemolymph and challenged with C. neoformans. Then, their phagocytic and killing activities were assessed. The cytokines MCP-1, IFN-γ, TNF-α, IL-10, IL-12, and IL-6 were measured in culture supernatants 4- and 48-hours post-infection. Results: Both hemolymph samples directly affected the growth rate of the fungus in a dose-dependent manner. Either MPH or RPH was capable of inhibiting fungal growth by at least 70%, using the lowest dilution (1:20). Treatment of RAW 264.7 macrophages with hemolymph of both insects was capable of increasing the production of MCP-I and TNF-α. In addition, when these cells were stimulated with hemolymph in the presence of C. neoformans, a 2- and a 4-fold increase in phagocytic rate was observed with MPH and RPH, respectively, when compared to untreated cells. For the macrophage killing activity, MPH decreased in approximately 30% the number of viable yeasts inside the cells compared to untreated control; however, treatment with RPH could not reduce the total number of viable yeasts. MPH was also capable of increasing MHC-II expression on macrophages. Regarding the cytokine production, MCP-I and TNF-α, were increased in the supernatant of macrophages treated with both hemolymphs, 4 and 48 hours after stimulation. Conclusion: These results suggested that hemolymph of triatomines may represent a source of molecules capable of presenting antifungal and immunomodulatory activity in macrophages during fungal infection.

Hemolymph of triatomines presents fungistatic activity against Cryptococcus neoformans and improves macrophage function through MCP-I/TNF-α increase

Triatomines are blood-feeding arthropods belonging to the subfamily Triatominae (Hemiptera; Reduviidae), capable of producing immunomodulatory and water-soluble molecules in their hemolymph, such as antimicrobial peptides (AMPs). In this work, we evaluated the antifungal and immunomodulatory activity of the hemolymph of Meccus pallidipennis (MPH) and Rhodnius prolixus (RPH) against Cryptococcus neoformans. Methods: We assessed the activity of the hemolymph of both insects on fungal growth by a minimum inhibitory concentration (MIC) assay. Further, RAW 264.7 macrophages were cultivated with hemolymph and challenged with C. neoformans. Then, their phagocytic and killing activities were assessed. The cytokines MCP-1, IFN-γ, TNF-α, IL-10, IL-12, and IL-6 were measured in culture supernatants 4- and 48-hours post-infection. Results: Both hemolymph samples directly affected the growth rate of the fungus in a dose-dependent manner. Either MPH or RPH was capable of inhibiting fungal growth by at least 70%, using the lowest dilution (1:20). Treatment of RAW 264.7 macrophages with hemolymph of both insects was capable of increasing the production of MCP-I and TNF-α. In addition, when these cells were stimulated with hemolymph in the presence of C. neoformans, a 2- and a 4-fold increase in phagocytic rate was observed with MPH and RPH, respectively, when compared to untreated cells. For the macrophage killing activity, MPH decreased in approximately 30% the number of viable yeasts inside the cells compared to untreated control; however, treatment with RPH could not reduce the total number of viable yeasts. MPH was also capable of increasing MHC-II expression on macrophages. Regarding the cytokine production, MCP-I and TNF-α, were increased in the supernatant of macrophages treated with both hemolymphs, 4 and 48 hours after stimulation. Conclusion: These results suggested that hemolymph of triatomines may represent a source of molecules capable of presenting antifungal and immunomodulatory activity in macrophages during fungal infection.(AU)

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.

Bacteria cultured from the gut of Meccus pallidipennis (Hemiptera: Reduviidae), a triatomine species endemic to Mexico.

The study of intestinal microbiota in vector insects like triatomines is paramount in parasitology because many parasitic species inhabit the vector's gut. Although knowledge on the gut microbiota in various vectors of the parasitic flagellate Trypanosoma cruzi has grown, research efforts have focused on South American triatomines. This study reports the isolation of bacterial microbiota in the anterior and posterior gut of Meccus pallidipennis (a triatomine species endemic to Mexico) by culture, as well as its identification by phenotypic and biochemical tests and its quantification by counting colony-forming units. The study was performed on fifth-instar nymph and adult specimens of M. pallidipennis, either laboratory-bred or collected in the field and either infected or not with T. cruzi. Overall, 17 bacterial species were identified, with the genera Bacillus and Staphylococcus being the most prevalent regardless of the origin of the insects. No differences were observed in the number of bacterial species in the gut of laboratory-bred and field-collected insects, neither with respect to life stage or infection status. In general, the Shannon-Weaver diversity index was higher in non-infected insects than in infected ones. Further studies using non-culture methods are required to determine whether bacterial species diversity is modified by laboratory breeding.

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.

The cost of being a killer's accomplice: Trypanosoma cruzi impairs the fitness of kissing bugs.

Relatively little is known about the fitness effects and life history trade-offs in medically important parasites and their insect vectors. One such case is the triatomine bugs and the parasite Trypanosoma cruzi, the key actors in Chagas disease. Previous studies have revealed some costs but have not simultaneously examined traits related to development, reproduction, and survival or their possible trade-offs. In addition, these studies have not compared the effects of genetically different T. cruzi strains that differ in their weakening effects in their vertebrate hosts. We compared the body size of the bugs after infection, the number of eggs laid, hatching/non-hatching rate, hatching success, survival, and the resulting number of parasites in Meccus (Triatoma) pallidipennis bugs that were experimentally infected with two strains of T. cruzi (Chilpancingo [CH], the most debilitating in vertebrates; and Morelos [MO], the least debilitating) (both belonging to TcI group). Our results showed that infection affects size (MO < CH; MO and CH = control), number of eggs laid (MO and CH < control) hatching/non-hatching rate (MO < control < CH), hatching success (control < MO, CH = control = MO), and survival (Chilpancingo < Morelos < control). In addition, the CH strain produced more parasites than the MO strain. These results suggest that (a) infection costs depend on the parasite's origin, (b) the more debilitating effects of the CH strain are due to its increased proliferation in the host, and (c) differences in pathogenicity among T. cruzi strains can be maintained through their different effects on hosts' life history traits. Probably, the vectorial capacity mediated by a more aggressive strain could be reduced due to its costs on the triatomine, leading to a lower risk of vertebrate and invertebrate infection in natural populations.

Activity of the prophenoloxidase system and survival of triatomines infected with different Trypanosoma cruzi strains under different temperatures: understanding Chagas disease in the face of climate change.

BACKGROUND: Little is known about how human disease vectors will modify their life history patterns and survival capacity as a result of climate change. One case is that of Chagas disease, which has triatomine bugs and Trypanosoma cruzi as vectors and parasite, respectively. This work aimed to determine: (i) the activity of the prophenoloxidase system (prophenoloxidase and phenoloxidase activity, two indicators of immune ability) in three intestine regions (anterior midgut, posterior midgutand rectum) of the triatomine bug Meccus pallidipennis under three temperature conditions (20 °C, 30 °C and 34 °C) against two T. cruzi strains [ITRI/MX/14/CHIL (Chilpancingo) and ITRI/MX/12/MOR (Morelos)], and (ii) whether vector survival varies under these three temperatures after infection by these T. cruzi strains. RESULTS: Our results indicate that prophenoloxidase activity was lower at higher temperatures, that the level of prophenoloxidase activity elicited by each strain was different (higher in Chilpancingo than in Morelos strains), and that prophenoloxidase activity was more intense in the anterior midgut than in the posterior midgut or rectum. Survival rates were lower in insects maintained at higher temperatures and infected by Chilpancingo strains. CONCLUSIONS: These results indicate that climate change could lead to lower prophenoloxidase activity and survival rates in triatomines when infected with different T. cruzi strains, which could reduce the vector capacity of M. pallidipennis.

Biology of Meccus pallidipennis (Hemiptera: Reduviidae) to Other Conditions Than That Encountered in Their Native Habitat.

BACKGROUND: Meccus pallidipennis (Hemiptera: Reduviidae) is only found in Mexico and is one of the most important vectors for Trypanosoma cruzi transmission there. Because data concerning the ability of this bug to adapt to different environments are scarce, we aimed to elucidate its biology, behavior and ability to acclimatize to different environmental conditions. METHODS: From the eclosion of 90 1st instar nymphs, development was followed until the adult phase. Adults were fed after 30 days of fasting, and the average amount of blood ingested, the time between the beginning of the blood meal and the production of feces, and the frequency of stools/insect were recorded during their meals. After taking a blood meal, couples were isolated and monitored for 21 days, during which eggs were collected weekly. RESULTS: The development of M. pallidipennis took 171.74±7.03 days to complete its life cycle, and females ingested larger amounts of blood than males. Oviposition was constant and did not demonstrate a significant decrease during this study. CONCLUSION: Meccus pallidipennis was able to acclimatize to fluctuating laboratorial conditions other than those naturally found in Mexico.

Molecular Characterization of Trypanosoma cruzi in Infected Meccus pallidipennis in the Southern Region of the State of Mexico, Mexico.

PCR amplification and sequencing of Trypanosoma cruzi (T. cruzi) spliced-leader intergenic region of the mini-exon gene intergenic region (SL-IR) fragment was performed on intestinal tissue and fecal content DNA extracted from 19 Meccus pallidipennis (M. pallidipennis) specimens collected in the southern region of the State of Mexico. DNA sequence analysis from 49 bp T. cruzi SL-IR showed that all 19 samples corresponded to haplotype TcIa, and all of them were identical to GenBank sequence JQ028863. When extending the analysis to the whole 256 bp amplified sequence of the SL-IR, we found six sequences with a C insertion at position 10, one of which also presented a mutation (T/C) at position 54. One more sequence had an insertion (T) at position 223. Our findings suggest that two dominating TcIa clones are present in M. pallidipennis in the southern region of the State of Mexico. Interestingly, the SL-IR region of the dominating genotype was 100% identical to a circulating clone from Costa Rica present in humans, dogs, Triatoma dimidiata, and Panstrongylus rufotuberculatus. Future regional studies should explore the presence of this haplotype in humans and domestic animals.

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.

Evaluación de un Bioinsecticida Formulado con Metarhizium anisopliae y Beauveria bassiana (balsamo) Vuillemin para el control biológico de Meccus pallidipennis vector de la enfermedad de Chagas / Evaluation of a Bioinsecticide Formulated with Metarhizium anisopliae and Beauveria bassiana (balsam) Vuillemin for the biological control of Meccus pallidipennis vector of Chagas disease

La Tripanosomiasis americana, también conocida como enfermedad de Chagas, es un padecimiento potencialmente mortal cuyo agente etiológico es el parásito protozoo Trypanosoma cruzi (Chagas 1909 Kinestoplastida: Trypanosomatidae). A pesar de contar con una amplia evidencia clínica se desconocen las cifras de prevalencia en el país ya que para el sistema de salud público mexicano en este momento dicha enfermedad no constituye una prioridad. La existencia de al menos 33 especies de vectores y con elevados índices de infección natural redunda en la aparición de casos positivos que llegan a incluir niños, lo que demuestra una vía activa de transmisión y da fortalezas a criterios de endemicidad. La transmisión del parásito que produce la enfermedad se da a través de varias vías pero en el 33% de los casos se ha reconocido que es por vía vectorial, de allí que el éxito de las campañas de control de los insectos permitirá la disminución drástica del número de infectados. Los métodos de control químico han sido inefectivos debido a problemas operacionales en las campañas y a la falta de integración entre estos, así como a la resistencia de los insectos a los productos.

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.