Eloi S. Garcia

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.

Origin, distribution, properties and functions of the major Rhodnius prolixus midgut hydrolases

Soluble β-acetylglucosaminidase, α-galactosidase, α-glucosidase and α-mannosidase predominate in anterior midgut lumen; soluble aminopeptidase, membrane-bound α-glucosidase and membrane-bound α-mannosidase, in posterior midgut tissue; and proteinases, sb-glucosidase and β-mannosidase, in posterior midgut lumen. Ultracentrifugation data suggest that there is only one major molecular species of the following soluble enzymes: aminopeptidase (Mr 61,000), with a broad specificity toward aminoacyl-β-naphthylamides; β-glucosidase (Mr 64,000), active on several β-glycosides; and acid phosphatase (Mr 82,000), which hydrolyzes many substrates and is competitively inhibited by tartrate (Ki 2 μM), molybdate (Ki 9 nM) and fluoride (three ligands per active site). There are major (85% of total) and minor (15%) thiol-activated hemoglobin hydrolase resolved by ultracentrifugation. The major activity (Mr 40,000) is cathepsin B-like hydrolyzing α-N-benzoyl-dl-arginine-p-nitroanilide, whereas the minor activity (Mr 88,000) is cathepsin d-like, being more active at pH 3.5 and being inhibited by pepstatin. The symbiont, Nocardia rhodnii, possesses an amylase which is found in Rhodnius prolixus midgut and an aminopeptidase and a BAPA hydrolase different from those of R. prolixus. The results suggest that digestion starts in the lumen of posterior midgut and ends with the action of enzymes trapped between extracellular membranes or on the surface of midgut cells. The use of cathepsin-like proteases is thought to be derived character evolved from a putative seed-sucker ancestor.

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.

Digestive enzymes trapped between and associated with the double plasma membranes of Rhodnius prolixus posterior midgut cells

Subcellular fractions of the cells of the posterior midgut of Rhodnius prolixus nymphs were obtained by conventional homogenization, under isotonic or hypotonic conditions, followed by differential centrifugation. Alkaline phosphatase and membrane-bound α-mannosidase are more abundant in fractions in which vesicles displaying brush-borders predominate. α-Glucosidase is associated with large membranous structures, although its subcellular distribution is different from that of alkaline phosphatase and α-mannosidase. α-Mannosidase-carrying membranes were resolved from α-glucosidase-carrying membranes in sucrose gradients, supporting the hypothesis that these enzymes are part of respectively, protein-rich inner and protein-poor outer microvillar membranes. To account for the soluble enzyme activities that sediment with vesicles displaying brush borders, major amounts of aminopeptidases are assumed to be trapped in the space between outer and inner microvillar membranes, from where they are set free by homogenization and (or) freezing and thawing. There are at least three different aminopeptidases, based on their activities toward several substrates and on sedimentation data. The results favor the view that oligomers derived from partial digestion of polymeric food are hydrolyzed down to monomers by enzymes trapped between microvillar membranes or on the surface of midgut cells. The use of microvillar membranes as a peritrophic membrane by R. prolixus is thought to be a derived character evolved from a putative phloem feeder Hemiptera ancestor.

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.

Azadirachtin depresses the release of prothoracicotropic hormone in Rhodnius prolixus larvae: evidence from head transplantations.

Azadirachtin A (1.0 μg/ml) if fed to last-instar larvae of Rhodnius prolixus through a blood meal, affects PTTH production as shown by a reduction or absence of stimulation of ecdysteroid titres in decapitation experiments. The host larvae, when decapitated 5 days after feeding, showed steadily declining haemolymph ecdysteroid titres whereas untreated larvae at this stage maintained their moulting hormone titre. Head transplantations from untreated donors 4–5 days after feeding onto headless larvae sustained hormone production for about 18 h. Heads from azadirachtin-treated donors were unable to sustain a constant ecdysteroid level which declined immediately after transplantation. In converse experiments, heads transplantation from untreated donors 5 days feeding stimulated the production of ecdysteroids in azadirachtin-treated recipients (40 days after azadirachtin A treatment) for about 18 h. It is suggested that the synthesis and release of PTTH was deficient in the azadirachtin-treated animals. The significance of these results is discussed in relation to a general mode of azadirachtin action in insects.

Aspects of classification of Hemiptera hemocytes from six triatomine species

The objective of this work was to characterize, and compare different morphological types of hemocytes of Rhodnius prolixus, Rhodnius robustus, Rhodnius neglectus, Triatoma infestans, Panstrongylus megistus, and Dipetalogaster maximus. This information provides the basis for studying the cellular immune systems of these insects. Seven morphological hemocyte types were identified by phase-contrast microscopy: prohemocytes, plasmatocytes, granular cells, cystocytes, oenocytoids, adipohemocytes and giant cells. All seven types of hemocytes are not present in every species. For example, adipohemocytes and oenocytoids were not observed in P. megistus and P. infestans, and giant cells were rarely found in any of the species studied. The hemocytes of Rhodnius and Dipetalogaster are more similar to each other than those from Triatoma and Panstronglus which in turn closely resemble each other. Emphasis is placed on methodological problems arising in this work which are discussed in detail.