Studies in search of a suitable experimental insect model for xenodiagnosis of hosts with Chagas' disease: 3 - On the interaction of vector species and parasite strain in the reaction of bugs to infection by Trypanosoma cruzi

The reaction of nine vector species of Chagas' disease to infection by seven different Trypanosoma cruzi strains; Berenice, Y, FL, CL, S. Felipe, Colombiana and Gávea, are examined and compared. On the basis of the insects' ability to establish and maintain the infection, vector species could be divided into two distinct groups which differ in their reaction to an acute infection by T. cruzi. While the proportion of positive bugs was found to be low in Triatoma infestans and Triatoma dimidiata it was high, ranging from 96.9% to 100% in the group of wild (Rhodnius neglectus, Triatoma rubrovaria)and essentially sylvatic vectors in process of adaptation to human dwellings, maintained under control following successful insecticidal elimination of Triatoma infestans (Panstrongylus megistus, Triatoma sordida and Triatoma pseudomaculata). An intermediate position is held by Triatoma brasiliensis and Rhodnius prolixus. This latter has been found to interchange between domestic and sylvatic environments. The most important finding is the strikingly good reaction between each species of the sylvatic bugs and practically all T. cruzi strains herein studied, thus indicating that the factors responsible for the excellent reaction of P.megistus to infection by Y strain, as previously reported also come into operation in the reaction of the same vector species to acute infections by five of the remaining T.cruzi strains. Comparison or data reported by other investigators with those herein described form the basis of the discussion of Dipetalogaster maximus as regards its superiority as a xenodiagnostic agent.

Studies in search of a suitable experimental insect model for xenodiagnosis of hosts with Chagas' disease: 4 - The reflection of parasite stock in the responsiveness of different vector species to chronic infection with different Trypanosoma cruzi stocks

Previous studies (1982,1987) have emphasized the superiority of sylvatic vector species over domestic species as xenodiagnostic agents in testing hosts with acute or chronic infections by T. cruzi "Y" stock. The present study, which is unique in that it contains data on both infectivity rates produced by the same stock in 11 different vector species and also the reaction of the same vector species to seven different parasite stocks, establishes the general validity of linking efficiency of xenodiagnosis to the biotope of its agent. For example, infectivity rates produced by "São Felipe" stock varied from 82.5% to 98.3% in sylvatic vectors but decreased to 42.5% to 71.3% in domestic species. "Colombiana" stock produced in the same sylvatic vectors infectivity rates ranging from 12.5% to 45%. These shrank to 5%-22.5% in domestic bugs. The functional role of the biotope in the vector-parasite interaction has not been eluddated. But since this phenomenon has been observed to be stable and easy to reproduce, it leads us to believe that the results obtained are valid. Data presented also provide increasing evidence that the infectivity rates exhibited by bugs from xenodiagnosis in chronic hosts, are parasite stock specific. For example, infectivity rates produced by "Berenice", "Y", "FL" and "CL" varied in R. neglectus from 26.3% to 75%; in P. megistus from 56.3% to 83.8%; in T. sordida from 28.8% to 58.8% in T. pseudomaculata from 41.3% to 66.3% and in T. rubrovaria from 48.8% to 85%. Data from xenodiagnosis in the same hosts, carrying acute infections by the same parasite stocks, gave the five sylvatic vectors a positive rating of approximately 100%, thus suggesting that the heavy loads of parasites circulating in the acute hosts obscured the characteristic interspecific differences for the parasite stock. Nonetheless these latter were revealed in the same hosts with chronic infections stimulated by very low numbers of the same parasite stocks. Certain observations here described lead us to speculate as to the possibility of further results from other parasite stocks, allowing the association of the infectivity rates produced in bugs by different parasite stocks with the isoenzymic patterns revealed by these stocks.

Resistance of mice immunized with killed culture trypomastigotes against infection by insect-derived trypomastigotes of Trypanosoma cruzi

Mice immunized with heat or merthiolate-killed culture trypomastigotes of the non-virulent G strain were resistant to the challenge by insect-derived trypomastigotes of the CL strain of Trypanosoma cruzi. No parasitemia was detected, by direct microscopic examination of blood samples, in 90% of immunized mice while all control animals developed a high parasitemia. Trypsinization before heat-inactivation, or fixation with paraformaldehyde, apparently reduced the immunogenicity of the G strain trypomastigotes. Mice immunized with trypomastigotes treated by either of these procedures were not protected against infection by virulent T. cruzi. Analysis of the 13I-labeled surface proteins of G strain trypomastigotes inactivated by the various methods suggests that these components are involved in eliciting protective immunity against T. cruzi infection.

Susceptibility of Aedes aegypti (L) to the insect growth regulators diflubenzuron and methoprene in Uberlândia, State of Minas Gerais

Aedes aegypti (L) (Diptera: Culicidae) was reared in several concentrations of diflubenzuron and methoprene under laboratory conditions in Uberlândia, State of Minas Gerais, southeastern Brazil. Characteristics such as LC50 and LC95, the susceptibility of immature stages of different ages to these insect growth regulators and their residual effects were studied. The LC50 and LC95 of diflubenzuron and methoprene were 5.19 and 12.24 ppb; 19.95 and 72.08 ppb, respectively. While diflubenzuron caused great mortality in all larval instars, methoprene was more effective when the mosquito was exposed from the start of the fourth larval instar onwards. Commercial concentrations of these two insect growth regulators close to LC95 presented greater residual activity than did their respective technical formulations. The parameters were compared with those obtained elsewhere. The characteristics investigated here indicate that these insect growth regulators are effective alternatives for controlling the dengue vector in the Uberlândia region.

Fauna de coleópteros aquáticos (insect: coleoptera) na Amazônia central, Brasil

Foram coletados 888 exemplares de Coleoptera aquáticos nos municípios de Manaus, Manacapuru e Presidente Figueiredo (AM), de fevereiro de 2000 a agosto de 2002, distribuídos em 12 famílias, 50 gêneros e 88 espécies ou morfoespécies. Novas ocorrências para o Brasil, incluem as seguintes espécies de Dytiscidae: Hydrodessus robinae, H. surinamensis, Hypodessus frustrator, Neobidessus confusus, N. spangleri e N. woodruffi. Os gêneros Agaporomorphus (Dytiscidae) e Pronoterus (Noteridae) são registrados pela primeira vez para o estado do Amazonas, assim como as espécies: P. punctipennis e Suphisellus nigrinus (Noteridae); Agaporomorphus grandisinuatus, Bidessonotus tibialis, Derovatellus lentus,Desmopachria nitida, Hydaticus xanthomelas, Laccophilus tarsalis, Liodessus affinis e Megadytes laevigatus (Dytiscidae). A família Dytiscidae foi a que apresentou maior riqueza, com 34 espécies, seguida de Hydrophilidae, com 20 e Noteridae, com 12 espécies. Os gêneros com maior número de espécies foram Gyretes (Gyrinidae) e Suphisellus (Noteridae) com 6 espécies, Copelatus (Dytiscidae) e Tropisternus (Hydrophilidae), com 5 espécies.

Studies in search of a suitable experimental insect model for xenodiagnosis of hosts with Chagas' disease. 1 - Comparative xenodiagnosis with nine triatomine species of animals with acute infections by Trypanosoma cruzi

In search of a suitable vector species for xenodiagnosis of humans and animals with chronic Chagas' disease we first investigated the reactions of different vector species to acute infection with Trypanosoma cruzi. Vector species utilized in this study were: Triatoma infestans, Rhodnius prolixus and Triatoma dimidiata, all well adapted to human habitats; Triatoma rubrovaria and Rhodnius neglectus both considered totally wild species; Panstrongylus megistus, Triatoma sordida, Triatoma pseudomaculata and Triatoma brasiliensis, all essentially sylvatic but some with domiciliary tendencies and others restricted to peridomestic biotopes with incipient colonization of human houses after successful eradication of T. infestans. Results summarized in Table IV suggest the following order of infectivity among the 9 studied vector species: P. megistus with 97.8% of infected bugs, T. rubrovaria with 95% of positive bugs a close second followed by T. Pseudomaculata with 94.3% and R. neglectus with 93.8% of infected bugs, almost identical thirds. R. prolixus, T. infestans and T. dimidiata exhibited low infection rates of 53.1%, 51.6% and 38.2% respectively, coupled with sharp decreases occuring with aging of infection (Fig. 1). The situation was intermediate in T. brasiliensis and T. sordida infection rates being 76.9% and 80% respectively. Results also point to the existence of a close correlation between prevalence and intensity of infection in that, species with high infection rates ranging from 93.8% to 97.8% exhibited relatively large proportions of insects (27.3% - 33.5%) harbouring very dense populations of T. cruzi. In species with low infection rates ranging from 38.2% to 53.1% the proportion of bugs demonstrating comparable parasite densities was at most 6%. No differences attributable to blood-meal size or to greater susceptibility of indigenous vector species to parasites of their own geographical area, as suggested in earlier...

Efficacy of ivermectin against the bloodsucking insect, Rhodnius prolixus (Hemiptera, Triatominae)

Ivermectin (0.2 mg/kg body weight) caused a high mortality in nymphs and adults of Rhodnius prolixus following a single meal in mice sub-cutaneously injected with the drug. This effect was more evident in nymphs of 1st-and 2nd-instar than in older nymphs and adults. Third-instar nymphs presented a high mortality when fed on mice treated with ivermectin 24 and 48 hours previously, while mortality was significantly reduced in nymphs fed on mice treated 72 hours before. Surviving 3rd-instar nymphs did not molt. When adult females were fed once on mice treated for 24 hours with ivermectin there was a considerable reduction in egg production. This inhibition was not reversed by a second feeding on normal mice. We concluded that sub-lethal doses of ivermectin caused toxic effects interfering in the neuro-endocrine control of development and reproduction of this bloodsucking insect.

Studies in search of a suitable experimental insect model for xenodiagnosis of hosts with Chagas' disease: 2 - Attempts to upgrade the reliability and the efficacy of xenodiagnosis in chronic Chagas' disease

In order to upgrade the reliability of xenodiagnosis, attention has been directed towards population dynamics of the parasite, with particular interest for the following factors: 1. Parasite density which by itself is not a research objective, but by giving an accurate portrayal of parasite development and multiplication, has been incorporated in screening of bugs for xenodiagnosis. 2. On the assumption that food availability might increase parasite density, bugs from xenodiagnosis have been refed at biweekly intervals on chicken blood. 3. Infectivity rates and positives harbouring large parasite yields were based on gut infections, in which the parasite population comprised of all developmental forms was more abundant and easier to detect than in fecal infections, thus minimizing the probability of recording false negatives. 4. Since parasite density, low in the first 15 days of infection, increases rapidly in the following 30 days, the interval of 45 days has been adopted for routine examination of bugs from xenodiagnosis. By following the enumerated measures, all aiming to reduce false negative cases, we are getting closer to a reliable xenodiagnostic procedure. Upgrading the efficacy of xenodiagnosis is also dependent on the xenodiagnostic agent. Of 9 investigated vector species, Panstrongylus megistus deserves top priority as a xenodiagnostic agent. Its extraordinary capability to support fast development and vigorous multiplication of the few parasites, ingested from the host with chronic Chagas' disease, has been revealed by the strikingly close infectivity rates of 91.2% vs. 96.4% among bugs engorged from the same host in the chronic and acute phase of the disease respectively (Table V), the latter comporting an estimated number of 12.3 x 10[raised to the power of 3] parasites in the circulation at the time of xenodiagnosis, as reported previously by the authors (1982).

The study of insect blood-feeding behaviour: 1. Feeding equipment, physical and endogenous factors, dose effect analysis, and diet destination

Experimental techniques that we have found useful during our studies of insect blood-feeding behaviour are reviewed. Some of the principal findings resulting from these techniques are discussed. Where directly applicable, the work of others is included, but no complete review of the subject has been attempted.

The study of insect blood-feeding behaviour: 2. Recording techniques and the use of flow charts

This paper continues a discussion of approaches and methodologies we have used in our studies of feeding in haematophagous insects. Described are techniques for directly monitoring behaviour: electrical recording of feeding behaviour via resistance changes in the food canal, optical methods for monitoring mouthpart activity, and a computer technique for behavioural event recording. Also described is the use of "flow charts" or "decision diagrams" to model interrelated sequences of behaviours.

Evolution of holometabola insect digestive systems: physiological and biochemical aspects

This review takes into account primarily the work done in our laboratory with insects from the major Holometabola orders. Only the most significant data for each insect will be presented and a proposal on the evolution of Holometabola insect digstive systems will be advanced.

The potassium impermeable apical membrane of insect epithelia: a target for development of safe pesticides

Columnar cell apical membranes (CCAM) in series with goblet cell apical membranes (GCAM) form an electroosmotic barrier separating the midgut lumen from epithelial cell cytoplasm. A unique K+ ATPase in GCAM generates three gradients across this barrier. A greater than 180 mV electrical gradient (lumen positive) drives amino acid uptake through voltage-dependent K+ symports. A greater than 1000-fold [H+] gradient (lumen alkaline) and a greater than 10-fold [K+] gradient (lumen concentrated) are adaptations to the high tannin and high K+ content, respectively, in dietary plant material. Agents which act on the apical membrane and disrupt the PD, H+, or K+ gradients are potential insecticides. Insect sensory epithelia and mammalian stria vascularis maintain similar PD and K+ gradients but would not be exposed to ingested anti-apical membrane insecticides. Following the demonstration by Sacchi et al. that Bacillus thuringiensis delta-endotoxin (Bt) induces specifically a K+ conductance increase in CCAM vesicles, we find that the K+ channel blocking agent, Ba2+, completely reverses Bt inhibition of the K+-carried short circuit current in the isolated midgut of Manduca sexta. Progress in characterizing the apical membrane includes finding that fluorosulfonylbenzoyladenosine binds specifically to certain GCAM polypeptides and that CCAM vesicles can be mass produced by Ca2+ or Mg2+ precipitation from Manduca sexta midgut.

The Azadirachtins: potent insect growth inhibitors

In the course of their coevolution with insects, plants have learnt to protect themselves by chemical means. Semiochemical act as antifeedants or deterrents, others by disrupting growth and development. By use of the Epilachna varivestis bioassay we isolated from Azadirachta indica seed a group of triterpenoids which interfee with larval growth and development in ppm range. Main components are the azadirachtins A and B with identical biological activity. Various other azadirachtins were obtained, either as minor seed components or by chemical modification of the naturally occuring compounds. Structure vs. activity relation studies enabled us to postulate a basic structural element that should still be biologically active and with much simpler chemical structure than natural compounds. What underlies the biological activity of these insect growth inhibitors? Their interference with the hormonal regulation of development and reproduction has been studied in Locusta migratoria and Rhodnius prolixus. In addition, tritiated dihydroazadirachtin A was used. With this approach, a precise correlation between administered dose, resulting effects, and retention of the compound was established. The azadirachtins either interrupt, delay, or deviate whole developmental programs. Results from these studies provide another chemical probe for studies in insect endocrinology and physiology.

Infection of a mammal by monogenetic insect trypanosomatids (Kinetoplastida, trypanosomatidae)

Monogenetic insect trypanosomatids of the genera Crithidia, Leptomonas and Herpetomonas, multiplied as in axenic cultures, for many months, in the lumen of the scent glands of the opossum Didelphis marsupialis. Specific antibodies were detected in the serum of the animals but there was no evidence of invasion of their tissues by the parasites.