Patrícia Azambuja

Insect natural products and processes: New treatments for human disease

In this overview, some of the more significant recent developments in bioengineering natural products from insects with use or potential use in modern medicine are described, as well as in utilisation of insects as models for studying essential mammalian processes such as immune responses to pathogens. To date, insects have been relatively neglected as sources of modern drugs although they have provided valuable natural products, including honey and silk, for at least 4-7000 years, and have featured in folklore medicine for thousands of years. Particular examples of Insect Folk Medicines will briefly be described which have subsequently led through the application of molecular and bioengineering techniques to the development of bioactive compounds with great potential as pharmaceuticals in modern medicine. Insect products reviewed have been derived from honey, venom, silk, cantharidin, whole insect extracts, maggots, and blood-sucking arthropods. Drug activities detected include powerful antimicrobials against antibiotic-resistant bacteria and HIV, as well as anti-cancer, anti-angiogenesis and anti-coagulant factors and wound healing agents. Finally, the many problems in developing these insect products as human therapeutic drugs are considered and the possible solutions emerging to these problems are described.

Immune responses in Rhodnius prolixus: influence of nutrition and ecdysone.

Starved larvae of Rhodnius prolixus, when challenged with Enterobacter cloacae B12, had their mortality related to their period of starvation. R. prolixus larvae fed on plasma alone, compared with insects fed on whole blood, had their immune reactivity affected as shown by: (i) a significant reduction in the ability to produce cecropin-like and lysozyme activities in the haemolymph when inoculated with E. cloacae; (ii) a reduction in numbers of haemocytes and nodule formation following challenge with bacteria; (iii) a decreased ability of plasma-fed insects in destroying their infection caused by inoculation of E. cloacae cells; and (iv) alpha-ecdysone therapy counteracted the immune depression in Rhodnius larvae fed on plasma alone. However, unlike other immune reactions, this set of experiments failed to demonstrate any interference of the plasma feeding on the prophenoloxidase-activating system, since melanin production was not reduced when the system was stimulated by the presence of bacteria in the haemolymph. The significance of these data is discussed in relation to the effect of diet components and the moulting hormone on the immune reactivity in insects.

Interactions between intestinal compounds of triatomines and Trypanosoma cruzi

Triatomine bugs are vectors of Trypanosoma cruzi, the etiologic agent of Chagas disease, a devastating disease that disables and leads to the death of many people in Latin America. In this review, factors from the insect vector are described, including digestive enzymes, hemolysins, agglutinins, microbiota and especially antimicrobial factors, which are potentially involved in regulating the development of T. cruzi in the gut. Differential regulation of parasite populations shows that some triatomine defense reactions discriminate not only between molecular signals specific for trypanosome infections but also between different strains of T. cruzi.

Towards an understanding of the interactions of Trypanosoma cruzi and Trypanosoma rangeli within the reduviid insect host Rhodnius prolixus

This review outlines aspects on the developmental stages of Trypanosoma cruzi and Trypanosoma rangeli in the invertebrate host, Rhodnius prolixus. Special attention is given to the interactions of these parasites with gut and hemolymph molecules and the effects of the organization of midgut epithelial cells on the parasite development. The vector insects permissiveness to T. cruzi, which develops in the vector gut, largely depends on the host nutritional state, the parasite strain and the molecular interactions with trypanolytic compounds, lectins and resident bacteria in the gut. T. rangeli invades the hemocoel and once in the hemolymph, can be recognized and activates the defense system of its insect vector, i.e., the prophenoloxidase system, phagocytosis, hemocyte microaggregation, superoxide and nitric oxide activity and the eicosanoid biosynthesis pathway. Taken together, these findings not only provide a better understanding of the interactions parasite-insect vector, but also offer new insights into basic physiological processes involved in the parasites transmission.

Immune-depression in Rhodnius prolixus induced by the growth inhibitor, azadirachtin

Abstract Azadirachtin (1.0 μg/ml) if fed to last-instar larvae of Rhodnius prolixus through a blood meal, affects the immune reactivity as shown by (i) a significant reduction in numbers of haemocytes and nodule formation following challenge with Enterobacter cloacae B12 (ii) a reduction in ability to produce antibacterial and lysozyme activities in the haemolymph when inoculated with bacteria, (iii) a decreased ability of azadirachtin-treated insects to destroy the primary infection caused by inoculation of E. cloacae cells. However, the present experiments, unlike other immune reactions, fail to demonstrate any interference of azadirachtin with the prophenoloxidase-activating system since the melanin production was not reduced when this system was stimulated by trypsin or by the presence of bacteria in the haemolymph. It is suggested that the immune response is deficient in the azadirachtin-treated insects. The significance of these results is discussed in relation to the general mode of azadirachtin action in insects.

Haemolytic factor from the crop of Rhodnius prolixus: Evidence and partial characterization

Abstract A haemolytic factor, which lysed sheep red cells in an isotonic buffer, was found in the crop of all larval stages and adult Rhodnius prolixus. Little or no haemolytic factor occurred in unfed insects but haemolytic activity increased for 2–4 days after feeding. From the 4th day on, the activity declined gradually. Fifth-instar larvae fed on whole blood, erythrocytes and haemoglobin produced large quantities of haemolytic factor, while those fed on plasma and erythrocyte stroma did not. The haemolytic factor was purified approximately 1200-fold by a two-step procedure: (1) Bio-Gel P-6 Gel-Filtration and (2) SP-Sephadex chromatography. Purified haemolytic factor was heatstable (100°C, 10 min), dialysable, inactivated by trypsin treatment, and could be recovered in the supernatant after addition of ethanol. It was concluded that the haemolytic factor is a peptide displaying a basic character.

Care and maintenance of triatomine colonies

The family Reduviidae, subfamily Triatominae, indudes more than 110 species, several of which are vectors of Trypanosoma cruzi, the causative agent of Chagas’ disease or American trypanosomiasis. Triatomines are common in the Americas, from the southern USA throughout Latin America, south to Patagonia. Chagas’ disease is endemic in 17 countries within this region. Some triatomines are sylvatic and are found in the safety of burrows and nests of wild vertebrates (didelphis, rodents and birds), rocks (especially associated with small rodents), fallen timber, hollow trees, roots, palms and bromeliads. Many species are found in peridomestic locations and in domestic animal houses, whilst others are domestic, occurring inside poor human habitation even when minimum conditions of shelter and food are offered. Domiciliary triatomines are quite opportunistic in their host selection and feed well on humans. They are poor flyers, thus the dispersion of triatomine species (or their eggs) is related to the transportation of man, his furniture, goods or migratory animals.

Cultivation-independent methods reveal differences among bacterial gut microbiota in triatomine vectors of Chagas disease

Background Chagas disease is a trypanosomiasis whose agent is the protozoan parasite Trypanosoma cruzi, which is transmitted to humans by hematophagous bugs known as triatomines. Even though insecticide treatments allow effective control of these bugs in most Latin American countries where Chagas disease is endemic, the disease still affects a large proportion of the population of South America. The features of the disease in humans have been extensively studied, and the genome of the parasite has been sequenced, but no effective drug is yet available to treat Chagas disease. The digestive tract of the insect vectors in which T. cruzi develops has been much less well investigated than blood from its human hosts and constitutes a dynamic environment with very different conditions. Thus, we investigated the composition of the predominant bacterial species of the microbiota in insect vectors from Rhodnius, Triatoma, Panstrongylus and Dipetalogaster genera. Methodology/Principal Findings Microbiota of triatomine guts were investigated using cultivation-independent methods, i.e., phylogenetic analysis of 16s rDNA using denaturing gradient gel electrophoresis (DGGE) and cloned-based sequencing. The Chao index showed that the diversity of bacterial species in triatomine guts is low, comprising fewer than 20 predominant species, and that these species vary between insect species. The analyses showed that Serratia predominates in Rhodnius, Arsenophonus predominates in Triatoma and Panstrongylus, while Candidatus Rohrkolberia predominates in Dipetalogaster. Conclusions/Significance The microbiota of triatomine guts represents one of the factors that may interfere with T. cruzi transmission and virulence in humans. The knowledge of its composition according to insect species is important for designing measures of biological control for T. cruzi. We found that the predominant species of the bacterial microbiota in triatomines form a group of low complexity whose structure differs according to the vector genus.

Suppression of the prophenoloxidase system in Rhodnius prolixus orally infected with Trypanosoma rangeli

Investigations were carried out to compare aspects of the prophenoloxidase (proPO)-activating pathway in Rhodnius prolixus hemolymph in response to oral infection and inoculation of the insects with two developmental forms of Trypanosoma rangeli epimastigotes strain H14. In vivo experiments demonstrated that in control insects fed on uninfected blood, inoculation challenge with short epimastigotes resulted in high phenoloxidase (PO) activity. In contrast, previous feeding on blood containing either short or long epimastigotes was able to suppress the proPO activation induced by thoracic inoculation of the short forms. In vitro assays in the presence of short epimastigotes demonstrated that control hemolymph or hemolymph provided by insects previously fed on blood containing epimastigotes incubated with fat body homogenates from control insects significantly increased the PO activity. However, fat body homogenates from insects previously fed on blood containing epimastigotes, incubated with hemolymph taken from insects fed on control blood or blood infected with epimastigotes, drastically reduced the proPO activation. The proteolytic activity in the fat body homogenates of control insects was significantly higher than in those obtained from fat body extracts of insects previously fed on blood containing epimastigotes. These findings indicate that the reduction of the proteolytic activities in the fat body from insects fed on infected blood no longer allows a significant response of the proPO system against parasite challenge. It also provides a better understanding of T. rangeli infection in the vector and offer novel insights into basic immune processes in their invertebrate hosts.

Infection of triatomines with Trypanosoma cruzi

The scientist who discovered American trypanosomiasis (Chagas’ disease), Carlos Chagas, not only wisely recognized the disease as a clinical entity, but also identified its causative agent (Trypanosoma cruzi) and its haematophagous (triatomine) insect vector (Chagas, 1909). Despite the fact that the reduviid vectors of the parasite have been well identified for more than eight decades, the understanding of the invertebrate cycle of the parasite remains remarkably limited. In T. cruzi, for instance, the trypomastigote stage does not multiply. It is the result of transformation of the parasite population that has been dividing as epimastigotes or amastigotes. Furthermore, the infective forms for both vertebrate and invertebrate hosts are meta-cyclic and bloodstream trypomastigotes, respectively.