VECTORS

• ^{VT 001-010}

• ^{VT 011-020}

• ^{VT 021-030}

• ^{VT 031-040}

• ^{VT 041-050}

• ^{VT 051-062}

VT-1 – INSECT PATHOGENICITY OF A TRYPANOSOMATID ISOLATED FROM PIEZODORUS GUILDINI

Baccan,G.B¹.; Falleiros, A F².; Pires,P.D¹.; Cavazzana Jr.,M.¹; Jankevicius, J.V¹. & Itow Jankevicius, S.¹.

¹Laboratorio de Biologia de Tripanosomatideos, Departamento de Microbiologia, Centro de Ciencias Biologicas, Universidade Estadual de Londrina.

²Departamento de Anatomia, , Centro de Ciencias Biologicas, Universidade Estadual de Londrina.

A Trypanosomatid (563 TD) was isolated from digestive tract of phytophagous Hemipteran Piezodorus guildini and cultivated in GYPMI medium at 25°C. This trypanosomatid showed to be fatal to phytophagous hemipterans.

An experimental infection was conducted on trypanosomatid non infected (" clean") phytofagous hemiptera Veneza zonata (Hemiptera, Coreidae), raised from collected eggs and the nymphs feed on clean sherry tomatoes obtained from seeds at laboratory. The infection was done via haemocelle (10²culture cells) or on experimentally infected tomatoes.

Via haemocelle , the cells of haemolymph were apparently normal, most of them plasmatocytes, with no differences between 0 and 3 hours of infection. Starting with 7 hours, it was an increase of the population of prohaemocytes, with cells dividing and the appearance of bacteria. The enhancement of bacterial population was progressive at about 19 hours, been observed also a budding phenomenon of haemocytes. All the haemocelle-infected insects die about 24 hours after infection.

In the infection via tomatoes, the protozoa were observed at digestive tract about 6 days of infection and at haemolymph about 9 days after infection. It was not observed salivary glands infection and die occurred in all insects between 12 and 14 days.

Externally there was no signals of the infection and the strain 563 TD, belonging to genus Leptomonas, was highly pathogenic to Veneza zonata, important vector of trypanosomatids of plants, probably by lowering insect defenses against bacterial infection.

Financial support: CAPES, CNPq, CPG/UEL

VT-2 – SELECTION OF AN AEDES FLUVIATILIS POPULATION TREATED WITH BACILLUS THURINGIENSIS AND THE RELATIONSHIP WITH PLASMODIUM GALLINACEUM INFECTION

Cunha, C.L., Secundino, N.F.C. & Pimenta, P.F.P.

Laboratory of Medical Entomology, Centro de Pesquisas René Rachou – CPqRR Fundação Oswaldo Cruz – FIOCRUZ P.O. Box 1743. Av. Augusto de Lima 1715, Belo Horizonte – M.G, Brazil. e-mail: [e-mail]

The cullicidae family has crucial medical importance because of the transmission of several pathogens to man including that causing malaria. Aedes fluviatilis infected with Plasmodium gallinaceum has been used as a model to study the interaction between the malarial parasites and mosquito vectors. The use of Bacillus thuriengiensis (Bti) as a larvicide is receiving increasing attention because of the appearance of resistance to conventional insecticides as well environmental problems caused by use of the latter. However, recent studies have also suggested some resistance of larva mosquitoes to Bti. In order to check this possibility and to know how Bti could affect the malaria infection rate of the mosquito vector in treated areas, we tested the vectorial ability of a population of Ae. fluviatilis selected for resistance in laboratory conditions. We used bioassays in which LC₅₀ was calculated to select survival of mosquitoes during 9 generations. After the 3^rd generation they already have developed 6 fold resistance. We dissected the midguts from larvae and adults and processed them for SDS-PAGE to know the effects of Bti resistance in the mosquito. Preliminary results demonstrated characteristic protein bands present only in the guts of the Bti-treated mosquitoes. This finding raised the possibility of a different interaction process of the Bti-treated mosquitoes with pathogens. To test this hypothesis, we fed the two groups of mosquitoes directly on P. gallinaceum-infected chickens. Seven days after the infected bloodmeal, the midguts were dissected and the percentage of infected mosquitoes and the number of oocysts compared with the controls. The statistical analysis of three experiments showed no significant differences among the control and the Bti-treated groups (P>0.10) and we conclude that the effect of the biological inseticide in the mosquito vector does not alter the malaria infection rate.

Financial support: FIOCRUZ, WHO, CNPq and PRONEX.

VT-3 – RHODNIUS PROLIXUS PROTEASE INDUCED BY TRYPANOSOMA CRUZI INFECTION

Feder, D; Gomes, SAO; Garcia, ES; Azambuja, P.

Departamento de Bioquimica e Biologia Molecular – Fundação Oswaldo Cruz

Rhodnius prolixus is an insect that has been used as a model in studies of parasite-vector interactions by our laboratory. The interaction protozoan parasites with vectors may be modulated by a complex reactions. Several trypanosomes contain proteases that may be released into their invertebrate hosts. Previously published data demonstrated metalloproteases activities in the haemolymph of R. prolixus infected with Enterobacter cloacae (Feder et al., 1998) and T. rangeli (Feder et al., in press). Based in these facts, we decided to investigate proteases of R. prolixus infected with T. cruzi.

Using SDS-polyacrylamida gel electroforesis containing gelatin as substrate, analysis of zimograms performed on samples of different tissues of controls and insects inoculated or orally infected. We observed protease activities in the haemolymph and fat body in a bloodsuking insect, R. prolixus, infected with T. cruzi (Dm28c). These proteases demonstrated distinct patterns of activities: (i) proteases were detected in the haemolymph of insects which were fed on, or inoculated with T. cruzi (Dm28c), but they were not observed in fat body of insects; (ii) protease was also presented in the fat bodies derived from naive insects or controls inoculated with sterile phosphate-saline buffer (49 KDa), as previously described with other microorganisms but it was not detected in the haemolymph of these insects. These proteases from the insect with T. cruzi will be characterized. The significance of these proteases will be discussed in relation to the success of the establishment of infection of these parasites in vector, R. prolixus.

This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico, Fundação Oswaldo Cruz (Papes) and PADCT. PA,ESG,SAOG are CNPq, and DF FAPERJ/FIOCRUZ research fellows.

VT-4 – PROTEIN PHOSPHORYLATION DURING SALIVARY GLAND GROWTH IN THE BLOOD-SUCKING INSECT RHODNIUS PROLIXUS

Mesquita, R.D. and Silva-Neto, M.A.C.

Departamento de Bioquímica Médica, ICB, CCS, UFRJ, Rio de Janeiro, RJ, Brasil.

Rhodnius prolixus salivary glands contain a number of different anti hemostatic components such as antifactor VIII ; an antiplatelet activity; an apyrase activity; an antithromboxane activity and finally a vasodilator activity with the general properties of nitric oxide (NO). Although the mechanisms which regulate the production of those molecules are unknown. Protein phosphorylation is by far the most powerful mechanisms which control cellular activities. The presence of protein kinase activity in this system was studied using ³²P-ATP and dephosphorylated casein as substrate. When salivary glands from blood fed 4^st nymphs were assayed for protein kinase activity this assay revelead a ten fold increase in enzyme activity 07 days after feeding. This enzyme activity (1.5 pmol ³²P incp./min/gland pair ± 0.2) is kept activated on the fist half of the feeding cycle. After moulting this activity decreases more than two fold (0.5 pmol ³²P incp./min/gland pair ± 0.05). Endogenous substrates of this enzyme include a major phosphorylated 30 kDa protein as determined by SDS-PAGE followed by autoradioagraphy. This protein is phosphorylated in vitro only after moulting when the protein kinase activity is smaller. Salivary gland extracts from days 07 and 30 after blood feeding were then fractionated on a Superose 6HR gel filtration collumn. Fractions were assayed for protein kinase activity using casein as substrate. These experiments revealead a single peak of protein kinase activity in both days with a molecular weight around 130 kDa. The inhibition of enzyme activity after moulting is also verified using this material. We further analyzed apyrase activity during blood feeding cycle. This activity is three fold increased after moulting (30 nmol Pi released/min/gland pair ± 5.0 on the fist half of the feeding cycle to 90 nmol Pi released/min/gland pair ± 5.0). When apyrase activity was analyzed on salivary gland extracts from days 07 and 30 after fractionation on Superose 6 Hr the increase in apyrase content is also found after moulting. Together these data suggest some relationship between protein phosphorylation events and the level and/or apyrase content in Rhodnius salivary glands.

Supported by CNPq, FAPERJ, TWAS and IFS.

VT-5 – ULTRASTRUCTURAL AND CYTOCHEMICAL ANALYSIS OF HEMOCYTES FROM RHODNIUS PROLIXUS (HEMIPTERA:REDUVIIDAE) INFECTED WITH TRYPANOSOMA RANGELI (TEJERA, 1920).

Oliveira, M.A.^* & DeSouza, W.^*,**

*LBCT; CBB; Universidade Estadual do Norte Fluminense; Av. Alberto Lamego, 2000; Campos dos Goytacazes-RJ; CEP28015620. **Lab. Ultraestrutura Celular Hertha Meyer; Instituto de Biofísica Carlos Chagas Filho, CCS; UFRJ.

Trypanosoma rangeli is an hemoflagelate protozoan that infects domestic and wild mammals, including man. Its distribution comprises Central and South America. The invertebrate host is the triatomine R. prolixus, the same vector of T. cruzi, the causative agent of Chagas' disease. It is not uncommon to find R. prolixus infected with both parasites. The life cycle of T. rangeli in invertebrate host begins in the intestine, where the parasites multiply and pass across the epithelial cell by an intracellular route, arriving into the hemocel. In this phase, the parasites are able to multiply freely in the hemolymph or invade hemocytes, where they apparently are able to multiply. The aim of this study is to determine which hemocytes are involved in cellular immune response against T. rangeli and verify if the parasite is able to multiply inside hemocytes. The insects were infected by an intra-celomic inoculation of 10⁴ parasites and after 24 hours the hemolymph was collected daily. For scanning electron microscopy, the hemocytes were adhered to coverslides with 6 mm diameter and fixed in 2,5% of glutaraldehyde, 4% of paraformaldehyde diluted in 0,1M cacodylate buffer, pH 7,2. After 2 hours cells were washed 3 times with the same buffer, post-fixed in 1% of osmium tetroxide and 0,8% of potassium ferricyanide for 1 hour, washed, dehydrated, critical point dried and covered with a 20nm layer of gold and observed in a scanning electron microscope. For transmission electron microscopy, the hemocytes were fixed as described above, dehydrated and embedded in Epon. 60-80nm thick sections were stained in uranyl acetate and lead citrate and observed in the transmission electron microscope. In addition we utilized two cytochemical techniques to analyze alterations of the hemocytes. Thiery technique and acid phosphatase detection were performed as described in the current literature. The results showed that parasites interact first with granular cells and plasmatocytes, and just the last type of hemocytes present parasites in its interior, confined inside a vacuole. In the interior of the plasmatocytes the parasite could present a normal or a degenerated aspect. It was common to find plasmatocytes presenting more than one parasite in the cytoplasm, always in different vacuoles. Thiery technique showed that inside vacuoles there is a significant amount of polysaccharides when the parasites was well preserved. The acid phosphatase detection showed that when the parasites are intact the vacuoles do not show a positive reaction for acid phosphatase. Until now it was not possible to observe parasites inside a vacuole presenting a positive reaction. We can suggest that just some parasites are able to multiply inside plasmatocytes, whereas others are killed. The data obtained by Thiery technique and acid phosphatase detection suggest that parasites inside hemocytes are able to modify or escape from microbicidal mechanisms.

Supported by PRONEX, CAPES, CNPq, FENORTE and FAPERJ

VT-6 – INTERACTION OF RHODNIUS PROLIXUS LIPOPHORIN WITH ITS BINDING SITE AT THE FAT BODY

E.G. Pontes, L.A.M. Grillo and K.C. Gondim.

Departamento de Bioquímica Médica, ICB, CCS, UFRJ, Rio de Janeiro, Brasil.

Lipophorin is the major hemolymphatic lipoprotein in insects, and it is known to be involved in the transport of lipids between different organs in the hemolymph of Rhodnius prolixus. In this insect lipophorin transfers phospholipids to the oocytes, midgut and fat body and can be reloaded with more lipids and reutilized. For delivering or taking up lipids, lipophorin interacts with tissues by means of specific binding sites at cell surface, and we are studying the lipophorin receptor on Rhodnus prolixus fat body.

Fat body membrane preparation was obtained from adult females four or five days after blood meal. Purified lipophorin was radiolabelled in protein moiety (¹²⁵I-LP) and incubated with membrane preparation in the presence of bovine serum albumin and calcium, for the receptor characterization.

In the presence of increasing concentrations of membrane protein, corresponding increases in lipophorin (¹²⁵I-LP) binding were observed. The specific binding of lipophorin to the membrane preparation was a saturable process, with a K_d of 2.1 ± 0.4x10^-7 M. Lipophorin binding did not depend on calcium and was strongly inhibited by an increased in the ionic strength. Suramin, a polisulfated polycyclic hydrocarbon know to inhibit the binding of lipoprotein to their receptor , also interfered with lipophorin binding to the fat body receptor, and binding was completely abolished at a concentration of 2 mM suramin but at concentration of 0.05 and 0.1 mM it seemed to increase the binding activity. Lipophorin from Manduca Sexta was also recognized by R. prolixus fat body receptor, as it competed with Rhodnius ¹²⁵I-LP for the binding to the membrane preparation. The study of this receptor properties is important for the understanding of the regulation of lipid transport in hematophagous insects.

Supported by CNPq, FINEP, Pronex, PADCT.

VT-7 – PRELIMINARY CHARACTERISATION OF THE SYLVATIC ECOTOPE OF TRYPANOSOMA CRUZI TRANSMISSION IN THE FEDERAL DISTRICT OF BRAZIL

Gurgel, R.G.*; Miles, M.A.**; Yeo, M.** & Cuba Cuba, C.A.*

Laboratório de Parasitologia, Departamento de Patologia, Universidade de Brasília*, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, UK**

Silvatic Trypanosoma cruzi transmission has not been proven in the Federal District of Brazil, nor is domestic transmission of T. cruzi documented. Silvatic triatomine bug species and mammals previously reported to be reservoir hosts of T. cruzi are abundant in the Biome Cerrado of Central Brazil (particularly in gallery forests). The WHO recommends further research on the distribution of non-domiciliated triatomine populations and reservoirs in enzootic foci. In preliminary work in gallery forest located in Núcleo Rural Rio Preto, Planaltina/DF the following methods were used: a) trapping and examination of marsupials and rodents by xenodiagnosis (with 10 nymphs of Rhodnius neglectus) and by thick films; b) tracking of trapped mammals to their refuges by means of a spool and line tracking device (Miles, M.A.;Souza, A.A. & Povoa, M.M.1981.J.Zool.,195:331-347) followed by microhabitat dissection; and c) examination of the crows of palm trees for the presence of triatomine bugs. Sixteen animals were trapped and examined (12 marsupials and 4 rodents). Didelphis albiventris was found naturally infected with T. cruzi principal zymodeme I, which is know to be associated with Didelphis and Rhodnius. No bugs have yet been retrieved from located nests of D. albiventris but 14% of "Buriti" palms (Mauritia flexuosa) within 250 m of human dwellings were found to be colonised by R. neglectus and Psammolestes coreodes. So far no triatomine bugs have been found infected with T. cruzi or T. rangeli. The source of infection to Didelphis albiventris and the extent of the enzootic focus, and the possible future risk to local inhabitants remains to be evaluated fully.

Partiallly sponsored by CNPq, FAP/DF-LSHTM-England

VT-8 – TRYPANOSOMA CRUZI DEVELOPMENT IN RHODNIUS PROLIXUS: INFLUENCE OF ECDYSONE AND DIFFERENT DIETS

Cortez, MGR(1); Gonzalez MS(1,2); Mello, CB(1,2); Garcia ES(1); Azambuja, P(1).

Departamento de Bioquimica e Biologia Molecular – Fundação Oswaldo Cruz and (1) Instituto de Biologia – Universidade Federal Fluminense (2).

Recently our laboratory demonstrated that (1) decapitation of Rhodnius prolixus drastically blocked the development of Trypanosoma cruzi (Dm28c) and (2) head transplantation or therapy with ecdysone reversed the T. cruzi infectivity in decapitated insects. In the present study we explore the effect of decapitation and ecdysone therapy in insects infected with T. cruzi fed on whole blood and plasma alone and quantified epimastigote and metacyclic trypomastigote forms in the stomach, intestine and rectum of the insect vector. In all experiments the insects were decapitated just after feeding and the infection was done by adding 1.6 X 10⁴/ml of diet and 6.1 X 10⁵/ml of T. cruzi forms of trypomatigotes and epimastigotes, respectively. At different intervals after the insects fed on infected whole blood or plasma alone, the stomach, intestine and rectum were removed and gently homogenized in 1 ml phosphate-buffered saline (pH 7.2). Different T. cruzi forms and the number of parasites were quantified in a Neubauer hemocytometer. We demonstrated that insect infected with T. cruzi and fed on whole blood if decapitated the total number of parasites drastically decreased until 15 after feeding in the entire gut. However, decapitated insect treated with ecdysone maintained the infection as high as the infection in control insects. The epimastigote infection also was reversed by the treatment of decapitated insects with ecdysone. However, the number of trypomastigotes significantly diminished in decapitated insects and in insects decapitated receiving ecdysone therapy. The infections in the stomach were similar in the different groups of insects but the intestinal compartment seems to be the responsible for the decrease of infection in the decapitated insects since the epimastigote infection was reversed by ecdysone treatment but the trypomastigote infection not. The parasite infection in the rectum followed the same pattern of the intestine. We also demonstrated that the T. cruzi infection in insects fed on plasma alone presented no significant different if the insects were treated with ecdysone. It seems that the presence of hemoglobin in the food and the hormone ecdysone may act interfering in the establishment of the T. cruzi infection in the intestine of R. prolixus.

This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico, Fundação Oswaldo Cruz (Papes) and PADCT. PA,ESG, CBM, are CNPq and MGRC is CAPES research fellows.

VT-9 – RADIATION EFFECT (CS 137) IN RHODNIUS PROLIXUS/TRYPANOSOMA RANGELI INTERACTION

Gomes, SAO^a; Nogueira, NFS^b; Oliveira, MA^b; Graciano, GL^b; De Souza, W^bc; Garcia, ES^a; Azambuja,P^a.

a Departamento de Bioqumica e Biologia Molecular - Fundação Oswaldo Cruz.

b Laboratório de Biologia Celular e Tecidual, Centro de Biociências e Biotecnologia -Universidade Estadual do Norte Fluminense.

c Lab. de Ultraestrutura Hertha Meyer, IBFCCF, CCS, UFRJ.

Trypanosoma rangeli Tejera, 1920, after being ingested as tripomastigotes by its vector Rhodnius prolixus, multiplies as epimastigotes in the midgut and invade the hemocoel. The epimastigotes survive in the hemolymph and/or inside the hemocytes, migrate to complete their development in the salivary glands. The literature is very poor about the effects of chemical and physical treatments on the interaction vector - parasite. In this study we investigated the radiation effect in the development of T. rangeli (H14 and Choachi strains) in R. prolixus. We applied different radiation doses ranged 400 to 4000 rads per insect. Our results demonstrated that doses higher than 1200 rads increased drastically the mortality. Insects treated with 400 and 1200 rads had the parasite colonization in the hemocoel antecipated for 7 days non radiated, and presented long and short epimastigotes of T. rangeli at high concentration in the hemolymph if compared with control infected insects. Since total counting of hemocyte and nodule formation were the same for all groups treated or not, aparently, the radiation did not affect the cellular immune response. Preliminary results demonstrated ultrastructural alterations in the midgut epithelial cells, mainly in cytoplasmatic organelles. Some cells presented a general disorganization while others presented just a morphological alteration of organelles. The ultraestructural alteration in the gut epithelium was directly correlated to the doses of radiation used. The significance of the modified cell's gut in radiated insects is discussed in relation to T. rangeli invasion in the hemolymph.

This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico, Fundação Oswaldo Cruz (Papes), PADCT and FENORTE, CAPES.

PA,ESG,SAOG are CNPq, research fellows.

NFSN, GLG and WDeS are FENORTE and MAO is CAPES research fellows.

VT-10 – BIOCHEMICAL ASPECTS OF HAEM POLYMERIZATION IN RHODNIUS PROLIXUS MIDGUT

Oliveira, M. F., Silva, J. R., Dansa-Petretski, M. & Oliveira, P. L.

Departamento de Bioquímica Médica, Bloco D-SS, CCS, UFRJ, Rio de Janeiro, Brazil

Free haem (Hm) is a powerful generator of reactive oxygen species that can damage many biomolecules. Moreover, Hm can associates with phospholipid bilayers and interferes with their physical integrity, leading to membrane disruption. Many organisms developed efficient defences against the deleterious effects of free Hm. A special situation is found in Plasmodium parasites that during its life-cycle digest up to 80% of host cell haemoglobin producing large amounts of its prosthetic group Hm. Part of the freed Hm is sequestered into a dark-brown crystalline polymer called malaria pigment or haemozoin (Hz). In a previous work we identified Hz, as a product of haemoglobin digestion, in the midgut of the blood-sucking bug Rhodnius prolixus. Here, we investigated the process of Hm polymerization in R. prolixus.

Firstly, the particulate fraction from midgut luminal content of plasma-fed insects was able to promote Hm polymerization in vitro. This fraction contains extracellular phospholipid bilayers known as perimicrovillar membranes (PMM) which ensheat the microvillar membranes of the epithelial midgut cells. Also, a great number of membrane patches and vesicles, derived from the PMM, are thrown for the lumen of the intestine. Previous incubation of this fraction at different temperatures showed that the Hm polymerase activity is heat-labile and is not associated to the Rhodnius actinomycete endosymbint Rhodococcus rhodnii. Moreover, this activity is strongly dependent of pH, temperature and time on Hz synthesis. The factor responsible for Hm polymerization is detergent soluble since the Hm polymerase activity increased more than three times when Hz synthesis promoted by midgut particulate fraction was carried out in the presence of n-octyl-b-D-glucopyranoside. Taken together these results suggest that Hm polymerase activity in R. prolixus midgut is promoted by an insect-derived factor and seems to be associated with PMM.

Financial Support: CNPq, Pronex, PADCT, and FINEP.

VT-11 – HAEM POLYMERIZATION INTO HAEMOZOIN IN RHODNIUS PROLIXUS REDUCES HAEM RELATED DELETERIOUS EFFECTS

Timm, B.L., Oliveira, M.F. & Oliveira, P.L.

Departamento de Bioquímica Médica, Bloco D-SS, CCS, UFRJ, Rio de Janeiro, Brazil

Blood-sucking insects use haemoglobin as the major food source and its digestion release large amounts of haem. Free haem is a powerful generator of reactive oxygen species that can damage a variety of biomolecules. Also, haem can associate with phospholipid bilayers, causing structural alterations on the membrane and leading to cell lysis. Several organisms developed mechanisms to avoid the deleterious effects of free haem. In Plasmodium parasites part of the free haem (Hm) is sequestered inside digestive vacuolum into an insoluble dark brown crystalline polymer named malaria pigment or haemozoin. In a previous work of our group haemozoin was isolated and identified in Rhodnius prolixus (rHz). Here we investigated the pro-oxidant activity of rHz by several methodological approaches.

Thiobarbituric acid reactive substances (TBARS) were produced in the presence of haem, but not with rHz using azolectin liposomes as substrate. This result is in accordance with our previous data when linolenic acid or 2-deoxyribose where used as substrates for TBARS assay. TBARS production occurred at both neutral and acid pH, indicating independence of the reduced oxidant activity of rHz on the pH. Incubation of 10 mM of rHz with a 0,5% suspension of rabbit red blood cells caused no damage to the cells, while the same concentration of Hm led to complete haemolysis. Oxygen consumption, measured by a Clark electrode, did not occurred significantly when azolectin liposomes were incubated with 10 mM of rHz. However the addition of 10 mM Hm caused an intense decrease of oxygen tension at the same experimental conditions. It is important to note that previous treatment of rHz with 0,1 N NaOH, a condition which depolymerize the pigment producing monomeric Hm, led to a similar behaviour as described to Hm in all the above mentioned protocols. These results point out to a reduced pro-oxidant activity of rHz, compared to the monomeric Hm. Our present results strength the hypothesis that haemozoin synthesis in the midgut of R. prolixus represents an efficient way to avoid haem toxicity.

Supported by CNPq, Pronex, PADCT and FINEP

VT-12 – ECTO-ATPASE ACTIVITIES ON MALPIGHIAN TUBULES OF RHODNIUS PROLIXUS

Ferraro, R.B.; Caruso-Neves, C.^*; Meyer-Fernandes, J.R.

Departamento de Bioquímica Médica, ICB, CCS, UFRJ, Rio de Janeiro, RJ, Brasil; ^* Instituto de Biofísica Carlos Chagas Filho, CCS, UFRJ, Rio de Janeiro, RJ, Brasil.

Chagas desease is an endemic desease caused by a protozoan disseminated in Brazil. The natural transmission occurs by the parasite excretion by the blood-feeding vector a reduviidae insect. Rhodnius prolixus is a hematophagous insect which ingests large volumes of blood during a single feeding session. The digestive and excretory system of this insect consist of anterior, medium and posterior midgut, the Malpighian tubules and the rectum (Phillips, JE 1981 Am. J. Physiol. 241: R241-R257). The Malpighian tubules are in number of four and structures that connect with the region between the medium and the posterior midgut. They are formed by two segments, superior and inferior, and are in permanent contact with the haemolymph. The inferior tubule was a blindend. The primary function of Malphighian tubules are the transepithelial transport, that results in secretion of ions and water excess(O'Donnell, MJ and Maddrell, SHP 1984 J Exp Biol 110:275-290). The Malpighian tubules can be compared to the vertebrate kidney.

Ecto-ATPases are transmembrane enzymes that catalyze the hydrolysis of extracellular ATP to ADP and inorganic phosphate. These ecto-enzymes are found in various tissues, as smooth and skeletal muscles, nervous tissue and organs as lung, liver and kidney in different organisms, including human. In this study we detected and partially characterized ecto-ATPase activities in R. prolixus Malpighian tubules.

Malpighian tubules were dissected from R. prolixus adult males, fasted for 5 weeks, and were collected in a cold isosmotic solution (100 mM NaCl, 8.6 mM KCl, 20mM Tris-Hepes (pH 7.0), 34 mM glucose, 2.9 mM alanine) with composition similar to the haemolymph (Maddrell, SHP 1969 J Exp Biol 51:71-97). In this saline it was possible to measure ecto-ATPase activities. The enzymatic activity was linear with time until at least one hour and with the number of tubules. The ecto-localization of this enzyme was comproved by its insensibility to common inhibitors, as vanadate, molibdate and fluoride. Moreover this activity was inhibited by suramine, an E-Type ATPase inhibitor and antagonist of P₂ receptor. The tubules incubated with 1mM EDTA showed Mg-independent ATPase activity (11.6 nmoles Pi x h^-1 x mg^-1). However, in the presence of 1mM MgCl₂ the ecto-ATPase activity was 8.2 nmoles Pi x h^-1 x mg^-1. These data are indicating that Malpighian tubules of Rhodnius prolixus probably possesses, at least, two Ecto-ATPase activities, a Mg-independent and a Mg-dependent activity.

Supported by CNPq, Faperj, CAPES and PRONEX

VT-13 – IDENTIFICATION AND CHARACTERIZATION OF AN ECTO-ATPASE PRESENT IN RHODNIUS PROLIXUS FAT BODY

Entringer,P.F. , Gondim,K.C. and Meyer-Fernandes,J.R.

Departamento de Bioquímica Médica, ICB, CCS, UFRJ, 21941-500,Rio de Janeiro - RJ

[e-mail]

Ecto-ATPases, are glicoproteic that hydrolyze extracellular nucleoside tri and/or diphosphates are located on the cell surface, and are present in a great variety of cell types. These ecto-enzymes are found in various tissues, as smooth and skeletal muscles, nervous tissue and organs as kidney, lung and liver in different organisms, including humans.

In this study we detected and partially characterized ecto-ATPase activities in Rhodnius prolixus fat body. This organ is involved with different aspects of the insect metabolism, as carbohydrate and lipid storage, synthesis of many proteins, and can be compared to the vertebrate liver.

Fat bodies dissected from Rhodnius prolixus adult females were incubated in the presence of [ g³²P ]-ATP, and ATPase activity was determined by measuring ATP hydrolysis, by liquid scintillation. Similarly to other ecto-ATPases, the Rhodnius fat body enzyme was stimulated by MgCl₂ , CaCl₂, MnCl₂, SrCl₂ and ZnCl₂ (5 mM). Two ecto-ATPase activities, a Mg dependent and a Mg independent, were identified and both activities incresed linearly with time until 30 minutes. The effect of pH was examined in both activities and in the pH range from 6.0 to 8.0 the ATPase activities were not affected.

In order to discard the contribution of other ATPase activities to the ATP hydrolysis and to better characterize this ecto-enzyme, various specifc inhibitors were tested. Both ATPase activities (Mg dependent and independent) were not inhibeted by vanadate (1mM), NaF (1mM), sodium phosphate (5mM), pNPP (5mM) and tartrat (1mM), indicating that ATP hydrolysis was not promoted by phosphatases . The lack of response to ammonium molybdate (1mM) indicated that a 5'nucleotidase did not contribute to the ATP hydrolysis. The absence of mitochondrial ATPase participation was shown by the use of sodium azide (1mM) and oligomycin (1mg/mL). Na⁺/K⁺ ATPase activity was not present as indicated by the use of ouabain (1mM). The Mg dependent activity was inhibited by ADP and 5'AMP, indicating the presence of an ATP diphosphohydrolase, whereas the MgCl₂ independent activity was not affected.

The external localization of ATPases here studied was confirmed by the utilization of 1mM 2',2'-diisothiocyanostylbene disulfonic acid (DIDS), that inhibited both activities.

Supported by: Faperj, CNPq, Finep, Pronex and PADCT.

VT-14 – RHODNIUS PROLIXUS' SALIVARY GLANDS PHOSPHOPROTEINS

Silva-Neto, M. A. C.

Departamento de Bioquímica Médica, ICB, CCS, UFRJ, Rio de Janeiro, RJ, Brazil.

Blood sucking arthropods salivary glands can harbor several different human parasites. Although the mechanisms which control salivary gland biology are unknown. Protein phosphorylation is the most powerful control mechanism for the regulation of cellular activities. In order to verifiy the presence of phosphoproteins under regulation during Rhodnius prolixus salivary gland growth 4^th instar nymphs were metabolic labelled with ³²Pi. Insects were then dissected in different days after blood feeding and salivary glands removed. When this material was analyzed through SDS-PAGE followed by autoradiography a single phosphorylated 30 kDa band was visualized. This phosphoprotein seems to be accumulated during salivary gland growth. Insects were fed with ³²Pi and dissected 30 days later. Salivary gland extracts were obtained and fractionated through cromatography on Superose 6 Hr gel filtration collumn. ³²P content of each fraction was analyzed through liquid scintillation counting. This experiment showed a major peak of ³²P associated to a the 20 kDa fraction of the column. Fracions were then pooled and applied on a Mono Q 5/5 Hr ion exchange column. Under these conditions two different peaks showed up, the first one is eluted in the void of the column and the second one is eluted around 0.2 M NaCl. Metabolic labelled salivary glands phosphoproteins were also analyzed through chromatography on chromatofocusing using a Mono P collumn. Extracts from 50 ³²Pi metabolic labelled salivary glands obtained in Tris-acetate 75 mM pH 9.3 were applied on the collumn equilibrated with the same buffer. A pH gradient with polybuffer acetate pH 6.0 was used to elute the proteins and revealed two different peaks of radioactivity. The first peak is eluted in the void of the collumn and the second one is eluted in pH 9.0. Both fractions are recognized by monoclonal antibodies against phosphoserine indicating the presence of this modified aminoacid in the molecule. Finally 30 day old salivary glands were in vitro cultured with ³²Pi. Under these conditions a major low molecular weight phosphoprotein can also be labelled in a time dependent manner. The results above point to the presence of a family of low molecular weight phosphoproteins in the salivary glands of the blood sucking insect Rhodnius prolixus. In the future the isolation of these molecules as well as the obtention of their NH₂-terminal sequences will provide some information about the role they play in salivary gland biology.

Supported by CNPq, FAPERJ, IFS and TWAS.

VT-15 – a-GLUCOSIDASE IS A BIOCHEMICAL MARKER OF PERIMICROVILLAR MEMBRANES IN THE KISSING BUG RHODNIUS PROLIXUS AND OTHER HEMIPTERANS

Silva J.R., Dansa-Petretski M. & Silva C.P.

Laboratório de Química e Função de Proteínas e Peptídeos, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense, Campos-RJ

The insects of the order Hemiptera are characterized by the absence of the peritrophic membrane, a chitinuous-proteic structure that envelopes the food bolus in the majority of insects. But the microvillar membranes of the intestinal cells are not in direct contact with the food bolus in hemipterans, because of the existence of the perimicrovillar membranes, which ensheathed the microvilli from their bases up to their tips and extending further to the gut lumen. These membranes, in contrast with the peritrophic membranes, are truly lipo-proteins containing membranes.

It has been suggested that a a-glucosidase is a biochemical marker of these structures in the cotton seed sucker bug Dysdercus peruvianus (Heteroptera: Pyrrhocoridae) (Silva et al., 1995). This is the only known example of a biochemical marker of these membranes. We were able to demonstrate that adults of the kissing bug R. prolixus has a major membrane bound a-glucosidase, which presents many properties similar to those found for the enzyme from D. peruvianus. By using in gel assays with a fluorogenic substrate after SDS-PAGE, we observed that the R. prolixus a-glucosidase showed an electrophoretic migration very similar to that of the D. peruvianus enzyme, indicating that both enzymes have a relative molecular weight of 60,000. The similarity between the a-glucosidase of R. prolixus and D. peruvianus was reinforced by the observation that a polyclonal antibody raised against the enzyme of the phytophagous bug recognized the kissing bug enzyme by using cryoultramicrotomy studies. Gold labeling was intensely detected in the perimicrovillar membranes of the intestinal cells of R. prolixus. The same antibody was also capable to recognize the a-glucosidase from a cicada and an aphid, suggesting that this enzyme is very widespread among the insects of the order Hemiptera. Our results point to the fact that the a-glucosidase and the perimicrovillar membranes are conserved among these insects, revealing them as potential targets to control of these economically important pests and disease vectors.

Reference: Silva et al. (1995) J. Insect Physiol. 41, 1093-1103.

Financial support: IFS, FAPERJ, CNPq, CAPES, FENORTE.

VT-16 – VITELLOGENIN PHOSPHORYLATION SITES AFTER ENDOCYTOSIS IN THE OOCYTES OF RHODNIUS PROLIXUS

Coelho, H.S.; Salerno,A. P.; Masuda,H. and Silva-Neto, M.A.C.

Departamento de Bioquímica Médica, ICB, CCS, UFRJ, Rio de Janeiro, RJ, Brazil.

Vitellogenin is a phospholipoglycoprotein which is stored in the oocytes during oogenesis. Inside the oocytes of nonmammalian vertebrates, vitellogenin is the target of specific proteases whose activity gives rise to a set of smaller yolk polypeptides: lipovitellins 1 (120 kDa) and 2 (30 kDa) and phosvitins (28-35 kDa). Phosvitin is the most phosphorylated protein found in nature: more than half of its residues are serines, and most of them are phosphorylated. The high degree of phosphorylation allows these proteins to bind cations. These ions will be further used during embryo growth. In invertebrates, vitellogenins contain less phosphate and fewer serines than in vertebrates and their proteolytic processing pathway after endocytosis is absent. Rhodnius vitellogenin (460 kDa) is also removed from insect hemolymph during oocyte growth giving rise to vitellin. We decided then to search for phosphoserine rich streches on vitellogenin which could be derived from some post endocytosis processing by oocyte protein kinases. These sites are phosphorylated for casein kinase II which recognizes specifically acidic phosphorylation sequences on its substrates. Rhodnius vitellogenin was purified through ultracentrifugation of hemolymph in a KBr gradient, followed by extensive dialysis against water. The material was then centrifuged and applied in a DEAE Toyo Pearl. Vitellogenin peak was then diluted and applied on a Mono Q 5/5 Hr in HPLC. This protocol allowed us to obtain highly purified vitellogenin free of other hemolymphatic proteins. A similar protocol was used for the purification of vitellin from chorionated oocytes. Both molecules were trypsin digested for 2 hours at pH 8.0 and this material reacted with monoclonal antibodies against phosphoserine. This treatment revealed a 80 kDa phosphorylated polypeptide which contains most of the phosphorylation sites of both molecules. This fragment is more phosphorylated on vitellin than in vitellogenin which indicates some kind of post endocytosis phosphorylation. In order to verify the presence of casein kinase II phosphorylation sites in both molecules this enzyme was also highly purified from Rhodnius oocytes. Phosphorylation reactions demonstrated that casein kinase II phosphorylate additional sites on vitellogenin which are absent or already phosphorylated on vitellin. We now intend to obtain internal sequences of the 80 kDa polypeptide in order to determine if those sequences share any similarity with phosphoserine clusters found on nonmammalian counterparts.

Supported by CNPq, IFS, TWAS and FAPERJ.

VT-17 – EFFECTS OF TWO PROTEINS (30 KDA AND 45 KDA), ISOLATED FROM CHORIONATED OOCYTES OF RHODNIUS PROLIXUS, ON CARDIAC CELLS AND FUNGI

*Bouts, D.M.D; *Porto, M.J.; *Paiva-Silva, G.O, ⁺Melo,A.C.A;*Silva-Neto, M.A.C; *Almeida, M.S; *Kurtenbach, E; ^#Masuda, M.O; ^#Nascimento, J.H.M. and *Masuda H.

*Departamento de Bioquímica Médica, ICB UFRJ, Rio de Janeiro, Brasil.

^# Instituto de Biofísica Carlos Chagas Filho, UFRJ, Rio de Janeiro, Brasil.

⁺Departamento de Patologia , CCB, UFPa, Pará, Brasil.

The egg of Rhodnius prolixus is composed of yolk, which contain a large amount of phosphorylated proteins, sugar and lipids. The yolk is surrounded by the cell membrane and it is protected against mechanical damage, desiccation and microorganisms by the eggshell. Here we describe the purification and partial characterization of two proteins from the eggshell which show a inhibition of Aspergillus niger growth. One of these proteins reversibly inhibits calcium channels of cardiac cells and the other apparently associate with cell membrane making it leaky. The proteins were extracted from eggshell in 8M urea and applied on SDS-PAGE . The 30 kDa and 45 kDa proteins were recognized, cut out from the gel and removed from using ammonium bicarbonate.

The purified proteins were used to test its effect on the the Aspergillus niger growth. The growth of Aspergillus niger was monitores at 25^oC during 96 h in 120 ml of culture medium (YNB) from Difco. Both proteins inhibited 100% of Aspergillus growth at the final concentration of 200 mg/ml.

Effects on calcium channel was evaluated in isolated cardiac cells using the whole cell configuration of patch clamp method.

Supported by CNPq,CAPES, PADCT and PRONEX

VT-18 – THE ROLE OF EICOSANOIDS IN THE ENDOCYTOSIS IN RHODNIUS PROLIXUS OOCYTES

Medeiros MN¹, Oliveira DMP¹, Carlos DCA¹, Paiva-Silva GO², Silva-Neto MAC², Masuda H² and Machado E.A¹

1-Programa de Parasitologia e Biologia Celular, IBCCF, CCS, UFRJ – Brasil

2-Departamento de Bioquímica, ICB, CCS, UFRJ - Brasil

Oogenesis plays a central role on insect reproduction. During its development a wide range of proteins are accumulated by insect oocytes. Several of these proteins are removed from hemolymph by specific receptors in oocyte's surface. A heme binding protein was isolated from the hemolymph and eggs of the blood sucking bug Rhodnius prolixus and characterized (Oliveira et al. 1995, JBC 270, pp:10897-10901). Purified RHBP labelled with ¹²⁵I in protein moiety was then used in this study in order to investigate its endocytosis by the ovaries in vivo and in vitro (as characterized by Machado et al., 1998, Arch. Insect Biochem. Physiol. 39, pp133-143). ¹²⁵IRHBP endocytosis was studied in vivo through injections of protein in insect hemolymph, and the radioactivity incorporated by ovaries was estimated by gama counting. In vitro, the ¹²⁵IRHBP incubated in the presence of ovaries was specifically removed from the media by selective receptors. These results show that Rhodnius oocytes have specific binding sites for RHBP at their surface. These approaches also permit to study endocytosis regulation in Rhodnius oocytes. In addition the participation of eicosanoids and second messengers in regulation of RHBP endocytosis was addressed. The rate of RHBP increased in the presence of indomethacin (a potent cyclooxigenase inhibitor), both in vitro (up to 50%) as in vivo (up to 20%). The serine/threonine protein phosphatase inhibitor, okadaic acid (1mM), decreased in vitro the RHBP uptake (up to 70%). Together these data show that RHBP endocytosis may be under protein phosphorylation control, and local mediators such as eicosanoids. Phosphoproteins involved in this process are now being identified in the ovaries by immunoblot with antiphosphoaminoacids antibodies.

Supported by CNPq and FAPERJ

VT-19 – MORPHOLOGICAL STUDIES OF RHODNIUS BRETHESI MATTA, 1919 (HEMIPTERA, REDUVIIDAE, TRIATOMINAE) BY SCANNING ELECTRON MICROSCOPE AND CONFOCAL

Ferro , Z. P. A., * Carvalho- Moreira , C. J., * Junqueira , A. C. V., * Spata , C.M., * * Silva, L. M. & Gonçalves , T. C. M.

Department of Entomology, * Department of Tropical Medicine, * * Department of Ultra-structure and Cellular Biology, IOC, Fiocruz, Av. Brazil 4365, 21045-900 Rio de Janeiro, RJ, Brazil.

The aim of this study is to analyze the ultra-structure of the eggs exochorion, and cuticular structures of nymphs and adults of R. brethesi with taxonomic diagnosis. The specimens were provided by the bug colony of the Tropical Medicine Department, Oswaldo Cruz Foundation. The methodology was the same as the one described by Ferro et. al., 1998 (Mem. Inst. Oswaldo. Cruz, RJ. Vol. 93, Suppl. II, Nov.). Micrographs were performed in scanning electron microscope and confocal. The eggs present a lateral flattening, characteristic of the genus Rhodnius.. The operculum exochorion presents hexagonal cells and some pentagonal , both of smooth aspect (Mascarenhas, 1982, Amazon Acta 12 (3): 661-664). The body of the egg presents exochorion with granular hexagonal cells in flat funnels and one follicular pit per cell. In the chorial rim were visualized spermatic groove, micropylae and aeropylae (Barata, 1981, Rev. Public Health, 15: 490-542), as well as a circular micro-structure, that is under analysis phase. In the antennas, sensillas denominated trichobothrias located in the second antennal segment or pedicel, were studied (Catalá, 1997, Atlas of Chagas' Desease Vectors in the Americas I: 74 - 83). A trichobothria in the subapical area of the pedicel in all nymphal instars and at least nine in the adult specimens that are distributed from the base up to the apex of the pedicel was visualized, different from what is shown in Lent & Wigodzinky, 1979 (Revision of the Triatominae (Hemiptera, Reduviidae) and their significance as vectors of Chagas' Disease. Bull. Am. Mus. Nat. Hist., 163 (3):146). The cuticular area, in the base of the trichobothria, presents regular grooves with thin, finger-shaped prolongation , different from the rest of the integument of the pedicel. The labrum is pear-shaped and it rests on the first segment of the rostrum that presents, laterally, a regular and symmetrical granulation. The integument of the labrum presents short and thick bristles, generally curved down. And, in the integument of the adult labrum, "star-like" structures were observed. The apex of the rostrum presents sensillas with different forms and sizes (Catalá, 1996, J. Morphology 228: 195 - 201). Stridulatory sulcus, where the apex of the rostrum rests, are formed by parallel bars. Two cuticular structures stand out in the apex of the tibiae: the ctenidium that is present in all nymphal instars and the spongy fossula present just in the adult specimens. So as to confirm some structures that are being analyzed, a greater number of R. brethesi will be observed.

VT-20 – THE INFLUENCE OF TEMPERATURE AND HUMIDITY ON THE LIFE CYCLE OF RHODNIUS NEGLECTUS LENT, 1954 IN LABORATORY (HEMIPTERA,REDUVIIDAE,TRIATOMINAE)

Rocha, D.S.; Galvão, C.; Jurberg, J; Cunha, V. & Carcavallo, R.U.

Laboratório Nacional e Internacional de Referência em Taxonomia de Triatomíneos, Departamento de Entomologia, Instituto Oswaldo Cruz, Av. Brasil 4365, Rio de Janeiro, RJ, Brasil, 21045-900, Fax: [phone], E-mail: [e-mail]

Global warming and the possible physical and ecological impacts are waking up researchers to several areas of interest. The impact that climatic change can have on human health has been a theme of debate among specialists of WHO (WHO, 1996 Climate change and human health, Geneva, 298 pp.). In the case of Chagas` disease, the probable alterations in the biological dynamics of the insect vectors would be: modifications in the geographical distribution of the species; alterations in the dispersion mechanism of flight; increase in the number of bloodmeals, increasing the probability of transmission of the Trypanosoma cruzi; reduction in the duration of the life cycle and a increase in population density, causing a significant impact on the epidemiology of Chagas` disease (Burgos et al. 1994, Entomol Vect 1: 69-78; Carcavallo et al. 1998, Entomol Vect 5: 137-138). Rhodnius neglectus Lent,1954 is distributed in States of Bahia, Goiás Mato Grosso, Minas Gerais and São Paulo (Carvalho & Verano 1956, Rev. Goiana Med. 2: 241;Barretto 1967, Rev. Soc. Bras. Med. Trop. 1:23 and Mello1980, Rev, Brasil Biol. 40: 323-326). In spite of being a sylvatic species, it is frequently found in dwellings, according to Corrêa (1968, Rev. Bras. Malariol. D. Trop. 20:39-81) and Barretto et al. (1968, Rev. Inst. Med, Trop. São Paulo 27:145-156) it demonstrates a tendency to domesticity. The objective of this work was to established the effect of different conditions of temperature and humidity on the development of R. neglectus. The insects used were from the colonies maintained at the Laboratório Nacional e Internacional de Referência em Taxonomia de Triatomíneos.150 eggs were collected from the colony, all oviposited on the same day. These eggs were divided into 3 groups and maintained under the following conditions: a) 33 ^oC and 70% of R. U., b) 33 ^oC and 40% of .R U. and c) 28 ^oC and 70% of R.U. The insects were fed weekly on mice, and observed daily, for evaluation of the following parameters: period of egg development to adult, period of development of each instar, number of bloodmeals accomplished by instars and the percentage of mortality. The development period (in days) of each instar increased to the adult phase in the three observed groups, the shortest period of egg development to adulthood was observed in group b (81 days) and the longest in group a (150 days), demonstrating a greater influence of low humidity, when combined with a high temperature. With regard to the number of accomplished bloodmeals, there were no significant differences observed between the studied groups (Mann-Whitney P <0,01). The largest percentage of mortality was observed in groups a and b, demonstrating that, unlike the development period, the highest temperatures had a greater influence on mortality.

Financial support: by CNPq, agreement FNS/Fiocruz 123/97 and ECLAT.

VT-21 – POPULATIONAL DYNAMICS OF RHODNIUS PALLESCENS BARBER, 1932 UNDER LABORATORY CONDITIONS (HEMIPTERA, REDUVIIDAE, TRIATOMINAE)

Borgerth, S.P.S.; Rocha, D.S.; Galvão, C.; Cunha, V.; Jurberg, J. & Carcavallo, R.U.

Laboratório Nacional e Internacional de Referência em Taxonomia de Triatomíneos, Departamento de Entomologia, Instituto Oswaldo Cruz, Av. Brasil 4365, 21045-900, Rio de Janeiro, RJ, Brasil.

Rhodnius pallescens is considered to be the main vector of Trypanosoma cruzi in rural areas of Panama and is the only vector known of T. rangeli in this country. This species can be found both peridomestically and in sylvatic habitats in Belize, Colombia and Costa Rica. The objective of present work is to study the populational dynamics of a colony of Rhodnius pallescens, for a period of 24 months. The following observations were made: the period of eggs incubation, the percentage of ecdysis and mortality, the period of development of the eggs to adults and the age pattern of the colony recorded every 30 days. The aim being to try to establish correlations between seasonality, density and phases of development. Insects were taken ramdomly from the colony, maintained in the insectary of the Laboratório Nacional e Internacional de Referência em Taxonomia de Triatomíneos. Specimens of 5 ^th instar nymphs to were coupled and placed in 3 glass container, maintained in ambient temperature, were in the observed period varied between 23.3 ^oC to 26.9 ^oC. Mice were offered for feeding every week for six hours. To this date only one couple has been. After 111 days of observation 136 eggs were obtained, of these 79 (58%) hatching. The percentage of mortality in first instar be considered dicrease that compare to the other species of the genus. The percentage of mortality in first instar is 11.1% . 19.1% is the second instar nymphs, 8.8% in third instar and only one specimen in fourth instar. The longevity of the male was 104 days. This preliminary results have demonstrated a low biotic potential. It is important to point out that these observations were make in the winter , and a different pattern of results maybe obtained by running the experiment in the summer month under conditions of increased temperature.

Financial support: CNPq, agreement FNS/Fiocruz 123/97 and ECLAT.

VT-22 – TRYPANOSOMA CRUZI TRANSMISSION CYCLE: SELECTION CONDITIONS IN THE SYLVATIC ENVIRONMENT

Pinho, A.P.; Pinto, R.C.V.; Ivo, A. & Jansen, A.M.

Lab.de Biologia de Tripanosomatídeos, Depto.de Protozoologia - IOC-FIOCRUZ.

Trypanosoma cruzi is transmitted by dozens Reduviidae species that may act as biological filters , influencing growth and/or metacyclogenesis rates. Also mammalian reservoirs can act as selective forces, maintaining or eliminating a given subpopulation of the parasite. Aiming to better comprehend the consequences of this interplay in the transmission cycle of T.cruzi in a given sylvatic environment we performed a study of the natural infection of marsupials (Didelphis marsupialis and Philander opossum ) in Teresópolis, Rio de Janeiro State. The characterization of the isolates through biological parameters(mortality for swiss mice) we observed that the isolates of T.cruzi could be separated in two groups one, mainly associated with P.opossum and the other with D.marsupialis. The isolates derived from Rhodnius prolixus , the only triatomine species collected in the area were biologically similar to the D. marsupialis isolates. As we knew from experimental infections that P.opossum is able to maintain D.marsupialis subpopulations of T.cruzi, we suggested that two distinct transmission cycles were occurring sympatrically between these two phylogenetically related species. To confirm this hypothesis we followed up the experimental infection of R. prolixus with, respectively, one isolate from P.opossum and one from D.marsupialis. . We also compared the pattern of the experimental infection of bugs derived from our field specimens of R. prolixus , with insects from a long time laboratory maintained R. prolixus colony . Two groups of 50 bugs from each insect colony were allowed to feed through an artificial membrane on defibrinated rabbit blood containing 1 x 10⁶ parasites/ml respectively, D. marsupialis and P. opossum strains ( G-645 and PO13). Non engorged insects were eliminated and the remnants dissected 20, 30 60 days after the infecting meal. The whole intestine was grounded in PBS buffer and countings in Neubauer chamber were performed. It was observed that, in R. prolixus from the laboratory colony, both T.cruzi isolates developed and differentiated well and in a similar pattern. But the course of experimental infection of R. prolixus from the field colony differed significantly depending if Philander or Didelphis strain was used: infections with G-645 strain resulted in ten fold higher number of flagellates (epimastigotes and metacyclic forms) 20 and 30 days after the infecting meal. On the contrary, PO13 strain yielded 3x more parasites 60 days after the infection. We concluded: a) in Teresópolis, both T. cruzi isolates are well adapted to the insect vector therefore, P.opossum and D. marsupialis are participating from two distinct transmission cycles confirming our previous hypothesis on the complexity of the transmission cycle of Trypanosoma cruzi in the sylvatic ecotope. b) Not only the vector species but also its maintenance conditions influences the development of T. cruzi.

Supported by PAPES/FIOCRUZ.

VT-23 – HIGH LEVELS OF MITOCHONDRIAL DNA SEQUENCE DIVERGENCE AMONG TRIATOMA BRASILIENSIS NEIVA, 1911 POPULATIONS. (HEMIPTERA, REDUVIIDAE, TRIATOMINAE)

Monteiro FA^1,3, Costa J² & Beard CB³.

¹Laboratório Nacional e Internacional de Referência em Taxonomia de Triatomíneos,

²Núcleo de Informatização, Coleção Entomológica, Departamento de Entomologia, Instituto Oswaldo Cruz, Rio de Janeiro, Brasil.

³Entomology Brach, Centers for Disease Control and Prevention, Atlanta, USA.

Triatoma brasiliensis is the most important Chagas disease vector in the semiarid areas of northeast Brazil. The high degree of chromatic and morphological variation presented by this species has led, in the past, to its subdivision in three subspecies (T. b. brasiliensis, T. b. macromelasoma and T. b. melanica). Lent and Wygodzinsky (1979), however, invalidated these subspecific ranks stating that natural intermediate forms between them could be found. Recently, Costa et al. (1997a, b, 1998) and Costa and Marchon-Silva (1998) not only confirmed the distinctness of the three populations (subspecies) of T. brasiliensis based on morphological, isoenzymatical, ecological and biological data but also revealed the existance of a fourth undescribed population. In this study we compared by mitochondrial DNA sequence analysis, specimens belonging to the three proposed subspecies and also specimens from the recently reported chromatic population (juazeiro population). A 510 base pair region of the mitochondrial cytochrome B gene was sequenced from two specimens of each population (except for the melanica population where 4 individuals were analyzed). The sequences were aligned by eye and analyzed according to the Neighbor-Joining method, yielding a tree that reflected the following relationship: (melanica (juazeiro (brasiliensis, macromelasoma))). All pairs of specimens of each population analyzed presented the same haplotype. However, the pairwise sequence comparisons among the four populations revealed a large degree of sequence divergence, ranging from 2.7 (for the brasiliensis-macromelasoma pair) to 11.2% (for the brasiliensis-melanica pair). To assess the taxonomic meaning of such measures, pairs of closely related species, namely T. infestans / T. platensis and T. sordida / T. garciabesi were also analyzed. The percentages of sequence divergence obtained were 7.7 and 7.5%, respectively. These results show that the levels of sequence divergence found between some populations of T. brasiliensis is even higher than those found between other closely related triatomine species, and therefore strengthen previous observations suggesting that T. brasiliensis may hold more than a single biological species.

Financial Support: CAPES and FNS

VT-24 – COMPARATIVE STUDY OF INTERNAL ANATOMY AND MORPHOMETRY OF MALE REPRODUCTIVE SYSTEM OF TWO POPULATIONS OF TRIATOMA BRASILIENSIS NEIVA, 1911 (HEMIPTERA, REDUVIIDAE, TRIATOMINAE) – PRELIMINARY RESULTS

Freitas, S.P.C., Santos-Mallet, J.R., Costa, J.M.^* & Gonçalves, T.C.M.

Núcleo de Morfologia, Anatomia Interna e Ultraestrutura de Artrópodos Vetores/ Laboratório de Transmissores de Leishmaniose e Oncocercose, ^*Núcleo de Informatização da Coleção Entomológica, Departamento de Entomologia, Instituto Oswaldo Cruz – FIOCRUZ, Rio de Janeiro – RJ.

Morphology of the genitalia has been largely used for taxonomic purposes in insects, however there are only few studies on the morphology of portions of the internal reproductive system of Reduviid bugs. In the present study we have performed a morphological analysis of the male reproductive system of Triatoma brasiliensis Neiva,1911 (Hemiptera, Reduviidae, Triatominae), including a morphometry of the testicular follicles by both light and scanning electron microscopy. The specimens were obtained from colonies held in laboratory for 4 years, initiated with specimens caught in Caicó/RN (brasiliensis population), Espinosa/MG (melânica population), Petrolina/PE (macromelasoma population) and Juazeiro/BA (juazeiro population),Brazil. According to Costa (1997), these four populations can be distinguished by their morphological, biological, isoenzymatical and ecological aspects. The insects were fed weekly on Swiss albino mice and kept in a BOD incubator at 29°C and 80%RU with a 12 hours photoperiod. The dissection was done in a salt solution (0,7% NaCl 0,3% KCl) at the 3rd day after the imaginal ecdysis. The covering membrane of the testicular follicles was removed in order to allow their distention. Each follicle was drawn at a camera lucida and subsequently measurements were made from the drawings using a curvimeter along both side. Only the highest values were considered. Preliminary inter-specific comparisons, to long, medium and short testicular follicles were made through the analysis of variance between brasiliensis and juazeiro populations. The comparison have shown a significant difference (P³ 0,001) to the long follicles in the specimens obtained until now. In the medium and short follicles there were no significant difference. The others populations will be analyzed in the future.

Support - FNS

VT-25 – INFLUENCE OF BLOOD SOURCE ON TRIATOMA BIOLOGY

Guarneri, A.A.¹, Pereira, M. H.², Diotaiuti, L.1

1Laboratório de Triatomíneos e Epidemiologia da Doença de Chagas, Centro de Pesquisas René Rachou, FIOCRUZ, Caixa Postal 1743, 30190-901, Belo Horizonte, MG.

²Departamento de Parasitologia, Instituto de Ciências Biológicas, UFMG, Av. Antônio Carlos, 6627, 31270-901, Belo Horizonte, MG.

The objective of this study was to determine the influence of the blood meal source on the life cycle and reproductive development of females of Triatoma infestans (Klug, 1834), Triatoma brasiliensis Neiva, 1911, Triatoma sordida (Stal, 1859) and Triatoma pseudomaculata Corrêa and Espínola, 1964. The life cycle, mortality and fecundity of females were evaluate in insects fed on mice or pigeons. In all triatomine species studied, the life cycle was shorter for the groups fed on mice than for those fed on pigeons, the range of differences being between 1.5 times (T. pseudomaculata and T. infestans) and 2.4 times (T. brasiliensis). The values of total life cycle obtained on pigeons and mice were, respectively, 282.0 ± 13.2 and 119.7 ± 3.2 to T. brasiliensis, 247.0 ± 11.8 and 161.9 ± 4.1 to T. infestans, 335.4 ± 12.4 and 232.6 ± 9.7 to T. pseudomaculata, and 252.7 ± 18.4 and 119.7 ± 2.2 to T. sordida. The mortality rate of nymphs during the life cycle tended to be greater in insects fed on pigeons than those fed on mice, the differences for T. brasiliensis being statistically significant. The fecundity of females was different when the two feeding sources were compared, with the exception of T. pseudomaculata. For T. brasiliensis and T. sordida the higher mean of eggs/female were observed in females fed on mice, while for T. infestans these values were higher when females were fed on pigeons. Females of T. sordida and T. pseudomaculata had a greater fecundity than T. infestans and T. brasiliensis independently of the blood meal source. The differences of fecundity observed, probably reflect differences in the availability of blood in the natural ecotopes of these species, meals being more frequent for T. infestans and T. brasiliensis, which live at high densities in association with rodents in highly stable ecotopes. Since T. sordida and T. pseudomaculata live in more unstable ecotopes with fewer sources of blood they form small sparse colonies and invest more energy in reproduction than maintenance.

Financial support: FAPEMIG, CNPq, CPqRR/FIOCRUZ.

VT-26 – MORPHOMETRIC AND MOLECULAR STUDIES OF INTRADOMICILIARY POPULATIONS OF TRIATOMA BRASILIENSIS NEIVA, 1911

Borges, EC¹, Romanha, AJ¹, Dujardin, JP² & Diotaiuti, L1

1Centro de Pesquisas René Rachou, Av. Augusto de Lima, 1715, Belo Horizonte (MG), Brazil ²Institut Français de Recherche Scientifique pour le Développement en Coopération (ORSTOM-IRD), La Paz, Bolívia.

The objective of this study was to determine the existence of genetic variability of populations of T. brasiliensis from intradomiciliary ecotopes, obtained in two States of the Brazilian Northeast (Ceará and Piauí). In Ceará, the insects were captured in two counties - Independência and Novo Oriente, and in Piauí, in the county of Simplício Mendes. The colonies of Independência and Simplício Mendes were founded starting from insects captured at several homes, and the one of Novo Oriente, of insects coming from just an intradomicílio. For the morphometric studies (Dujardin et al.,1998), were used heads of 15 males and 15 females of each population. For the molecular studies, using the RAPD (" Random Amplified Polymorphic DNA "), legs of 10 males and 10 females of each population were processed. The results of the morphometric studies indicate that the coming insects of Independência are larger, and that the males, analysed by they form, can be better differentiated than the females, suggesting the existence of a larger variability among them. The evolutionary tree built by the analysis of RAPD shows clear separation of the 3 populations, belonging the two of Ceará to a same group, and Simplício Mendes's population the other. Those results indicate the existence of great interpopulational heterogeneity in T. brasiliensis, that proceeds being investigated with insects from another states as well as from another ecotopes.

Financial Support: CPqRR/FIOCRUZ, FAPEMIG, ECLAT.

VT-27 – CHARACTERIZATION OF THE I UROTERGITE PROCESS IN TRIATOMA DELPONTEI, ROMANà & ABALOS,1947 AND TRIATOMA PLATENSIS NEIVA,1913 THROUGH ELECTRONIC MICROSCOPY AND OPTIC MICROSCOPY

Barata, J.M.S. ¹, Rosa, J.A. da², Urbinatti, P.R.¹, Ceretti-Jr, W. ¹, Marassá, A.M. ³ Gonçalves de Andrade, R.M⁴, ,

1 Faculdade de Saúde Pública/USP, São Paulo, SP; Brasil, 2 Depto. de Parasitologia/UNESP, Araraquara, SP; Brasil, 3 Instituto Adolfo Lutz, 4 Instituto Butantan, São Paulo, SP, Brasil.

The I Urotergite Process has been characterized to Triatominae species from different places in Brazil and South America. Aiming to extend this study to other species of importance in the Chagas Disease, we are describing this structure to Triatoma delpontei which occurs in territories of Argentina, Uruguai and Paraguai and Triatoma platensis which is distributed in territories of Argentina, south of Bolivia and Paraguai. Adult males of Triatoma delpontei Romanã & Abalos 1947 from Santiago del Estero (CTA-129) were studied. On dorsal view T.delpontei presents the I Urotergite on a higher level than the II Urotergite as in T. brasiliensis and T. rubrovaria (Barata et al;1998).The triangular plate has a trapezium shape as in T.infestans (Barata et al;1994); its apex is rhomboid as in T.sordida (Barata et al. 1996a,1996b) and it is lodged into a sunken surface which is in the circular area of the II Urotergite.. The I Urotergite presents a conspicuous transversal groove that is located at the bottom of this structure. Wrinkles are distinctly concentrated at the lateral and central parts of the I Urotergite; where plenty of long bristles can be seen. The circular area of the II Urotergite is smooth in almost its extension and presents in its central portion a sunken surface where the apex of the I urotergite is lodged. The circular area is limited by two prominent divergent, short, vertical grooves. The wrinkling of the remainder surface of the I Urotergite is scarcely visible and it is more evident on the sides. Bristles can't be seen at the Optic Microscope.Triatoma platensis Neiva, 1913 on dorsal view presents the I Urotergite on a higher level than the II Urotergite (Barata et al,1994,1996a ,1996 b,1998). The triangular plate has a triangular shape whose sides are narrow and slightly concave, not reaching the adjacent angle of the II Urotergite as in the other species that have been studied. The I urotergite apex presents a downward pronounced slope and according to the geographical region, morphological changes were observed, as it was in T.rubrovaria (Barata et al,1998).While the sample from Cordoba has a pointed apex, the one from La Pampa has a rhomboid one. The II Urotergite usually presents a circular concave area limited by a pair of concentric, long, vertical grooves. In all extension of the rectangular plate wrinkles are distributed and they are more evident near the circular area. In the sample from La Pampa, the rectangular plate is extremely wrinkled. Long bristles are distributed in the circular area and on the lateral fringes and are plentifully spread in some specimens.

VT-28 – CHARACTERIZATION OF THE II UROTERGITE PROCESS IN TRIATOMA PROTRACTA UHLER, 1894 AND TRIATOMA LECCTICULARIA STAL, 1859 THROUGH SCANNING ELECTRONIC MICROSCOPY AND OPTIC MICROSCOPY

Barata, J.M.S. ¹, Rosa, J.A. da², Urbinatti, P.R.¹, Ceretti-Jr, W. ¹, Marassá, A.M. ³ Gonçalves de Andrade, R.M⁴, ,

1 Faculdade de Saúde Pública/USP, São Paulo, SP; Brasil, 2 Depto. de Parasitologia/UNESP, Araraquara, SP; Brasil, 3 Instituto Adolfo Lutz, 4 Instituto Butantan, São Paulo, SP, Brasil.

The I Urotergite Process was firstly described to some Triatominae species that are involved in the transmission of Chagas Disease at the South American Continent in areas of Bolivia, Argentina, Paraguai, Uruguai and Brazil. At this short communication,

we are extending this characterization to two North American species that have been found naturally infected and can be associated to the enzootic cycle of this disease in Mexico middle south of the USA Adult males of Triatoma protracta Uhler,1894 from Monte Diablo CA (CTA-045) were studied. The triangular plate has an isosceles shape; it is on a higher level than the II Urotergite and display wrinkles and parallel, conspicuous grooves which take place in most of the structure. Its apex doesn't preserve an angular process as it was observed in the other species that have been studied. Conspicuous short bristles can be seen and they are distributed in the median portion of the I Urotergite. A different light coloured area distinguishes the apex border of the I Urotergite, including its apex. The rectangular plate presents a plane circular area well defined by a pair of divergent, vertical grooves. In the median portion of the circular area, not prominent parallel horizontal striations can be seen and in some specimens can also reach the adjacent portion of the II Urotergite. The II Urotergite is extremely wrinkled and striated, not showing bristles at the Optic Microscope. Adult males of Triatoma lecticularia from Oklahoma- USA (CTA-021) were studied. The triangular plate is on a higher level than the II Urotergite. At the middle of the plate there is a centre apical slope that is contiguous to the angular process and take the shape of a lozenge. This structure is distinctively seen in some specimens. Bristles are distributed at the central part of the plate and wrinkles are concentrated on the lateral fringes of the triangular plate. A wrinkly dark area surrounds the apex border of the I Urotergite including its apex. Parallel, vertical grooves are displayed at the base of the triangular plate. The II Urotergite presents a plane circular area well defined by a pair of divergent, vertical grooves. Wrinkles are distributed in all extension of the rectangular plate and the ones which are next to the border of the I Urotergite are more evident and darker than the others. Bristles could not be observed at the Optic Microscope.

VT-29 – MORPHOLOGICAL STUDY OF THE FORE WINGS OF TRIATOMA GUAZU LENT & WIGODZINSKY, 1979 (HEMIPTERA; REDUVIIDAE; TRIATOMINAE) SEEN BY OPTICAL AND SCANNING ELECTRON MICROSCOPY (S.E.M.)

Bulhões, D. M., Carcavallo, R., Barbosa, H. S* & Jurberg, J.

Laboratório Nacional e Internacional de Referência em Taxonomia de Triatomíneos (LNIRTT), Dept° de Entomologia, *Dept° de Ultraestrutura e Biologia Celular, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Av. Brasil, 4365, Rio de Janeiro, RJ – 21045-900 .Fax: [phone].

Triatoma guazu, described with reference to only one specimen (female holotype), collected in Paraguay (Lent & Wigodzinsky, 1979, Bull. Am. Mus. Nat. Hist. 163:123-520) it received the specific name guazu (big in guarany), for being one of genus Triatoma¢s largest representatives. In 1996, the male allotype, collected in Mato Grosso (Brazil), was described with particular attention to external genitalia (Lent et al., 1996, Mem. Inst. Oswaldo Cruz, 91 (3): 313-315). The objective of this work was to obtain additional morphological informations by the study of particular undescribed structures existing in the fore wings of T. guazu. Ten specimens (five males and five females) were provided by the Department of Entomology, National and International Laboratory of Reference in Taxonomy of Triatomines, Institute Oswaldo Cruz, were used. The fore wings were removed and submerged in 98% alcohol, mounted under coverslips using Canada of balsam for observation and drawings were made at X12 magnification using standard lucid camara Wild M5. For scanning electron microscope observation, the wings were dissected into pieces, individually fixed to a support, exhibiting dorsal and ventral surfaces, metalized with gold and photographed under various optical magnifications. Results indicated a corium with corrugated integument on dorsal surface of the wing, covered with spines, with setae and sensilla implanted among the spines. They were observed numerous setae surrounded by mechanoreceptors over the subcostal vein. In the middle of the membrane surface was evidenced a single campaniform sensillum. On the ventral surface, a ctenidium was observed, with a comblike structure made up of a row of flattened spines on the surface of the wing, as a part of a coaptor system, constituting an elementary component part of a morphological mechanism adapted for flight. This element is made up of thirty-three spines juxtaposed to each other, limited by a corrugated protuberance and a depression that allows the fitness of the hind wing. It is expected that the findings of these structures herein described will help further studies of comparative morphology and phyllogeny of the group.

Supported by CNPq. Convênio 123/97 FIOCRUZ/FNS e ECLAT (European Commission Latin America Triatominae Research Network) Contract ERB IC 18-CT96-0054.

VT-30 – DESCRIPTION OF THE EIGHTH AND NINETH UROSTERNITE OF MALE AND FEMALE FIFTH INSTAR NYMPHS OF TRIATOMA GUAZU LENT AND WYGODZINSKY, 1979 AND TRIATOMA RUBROFASCIATA (DE GEER, 1773) (HEMIPTERA, REDUVIIDAE)

Rosa, JA da, Barata, JMS*, Belda Neto, FM, Justino, HHG, Cilense, M**, Valente, SAS***

Faculdade de Ciências Farmacêuticas, Universidade Estadual Paulista, Araraquara, 14801-902, SP, Brasil.

*Faculdade de Saúde Pública, Universidade de São Paulo, São Paulo, 01246-900, SP, Brasil

** Instituto de Química, Universidade Estadual Paulista, Araraquara, 14801-970, SP, Brasil.

*** Instituto Evandro Chagas, Ananindeua, 67970-070, PA, Brasil.

Galliard (1935) defined the distinction of male and female fifth instar nymph of Triatominae. Rosa et alii suggested the possibility of Triatominae specific distinction in the fifth instar based on 8^th and 9^th urosternites characteres. Afterwards three Panstrongylus, one Rhodnius and seventeen Triatoma species were studied. Now are described the 8^th and 9^th urosternite of fifth instar female and male nymph of T. guazu and T. rubrofasciata, through scanning electron microscope Topcon SM300. The male nymph of fifth instar of T. guazu shows a longitudinal swift depression across the nineth ventral segment, one pore in the posterior limit of this segment and one slit in the adjacent central portion with the tenth segment. The nineth abdominal ventral segment of female nymph of the T. guazu are united to the eighth segment in the central portion , shows (1+1) lateral slits and one "V" form depression in the adjacent tenth segment. The male nymph of fifth instar of the T. rubrofasciata shows one strong longitudinal depression across the nineth abdominal ventral segment and one depth slit in the adjoining portion with the tenth segment. The female fifth instar nymph of T. rubrofasciata has (1+1) projections in the eighth segment that covered part of the nineth abdominal ventral segment. The nineth segment of T. rubrofasciata has (1+1) lateral slits and one depression in the central portion. Another characteristic of this segment is the short dimension. The morphological structures of the nineth ventral abdominal segment of female fifth instar nymph of T. guazu and T. rubrofasciata are different of the following species: T. arthurneivai, T. brasiliensis, T. circummaculata, T. costalimai, T. dimidiata, T. infestans, T. lecticularia, T. maculata, T. matogrossensis, T. platensis, T. pseudomaculata, T. protacta, T. rubrovaria, T. sordida, T. tibiamacualta, T. vitticeps and T. williami.

Aknowledgments: João Luiz Molina Gil and João Maurício Nobrega da Silva Filho (Serviço Especial de Saúde de Araraquara).

Supported by FAPESP, PADC/FCF-UNESP.

VT-31 – FLIGHT INITIATION IN TRIATOMA INFESTANS (KLUG,1834) AND T. MELANOSOMA MARTINEZ, OLMEDO & CARCAVALLO, 1987 (HEMIPTERA, REDUVIIDAE)

Galvão C., Rocha D.S., Carcavallo R.U. & Jurberg J.

Laboratório Nacional e Internacional de Referência em Taxonomia de Triatomíneos, Departamento de Entomologia, Instituto Oswaldo Cruz, Av. Brasil 4365, Rio de Janeiro, RJ, Brasil, 21045-900, Fax: [phone], E-mail: [e-mail].

Domestic populations of T. infestans can be readily eliminated by residual spraying of infested houses using modern pyrethroid insecticides, but the treated premises then remain at risk to reinfestation by bugs coming from untreated foci. Dispersal of the bugs is frequently by passive carriage, for example amongst the clothes and utensils of people travelling between different villages, but the bugs can also disperse as adults flying between houses. For T. infestans, the mean effective range of adult flight seems to be around 200 metres (Schofield & Matthews 1985, J. Trop. Med. Hyg. 88: 211-222), although flights in excess of 1 Km have been recorded in the field (Schweigmann et al. 1988, Med. Vet. Ent. 2:401-404). Species occupying silvatic habitats may be expected to retain a greater capacity for active flight, as indicated by comparison of the flight capacity of silvatic T. sordida (Schofield et al. 1991, Trans R. Soc. Trop. Med. Hyg. 85: 676-678) with that of domestic T. infestans (Schofield et al. 1992, Med. Vet. Ent. 6: 51-56) under similar field conditions. We offer a further comparison of flight initiation, comparing T. infestans with the very closely-related T. melanosoma. Fifth instar nymphs of T. infestans and T. melanosoma were randomly chosen from the colonies maintained at Instituto Oswaldo Cruz, Rio de Janeiro. After moulting to adults, the bugs were individually placed in Borrel flasks, starved for 7 days, and then allowed to feed for 4 hours on restrained pigeons. Those that fully engorged were kept isolated for a further 14 days and then marked individually with paint on the thorax following the system of MacCord et al. (1983, Mem. Inst. Oswaldo Cruz 78:473-476). These individually marked bugs were then placed into a flight apparatus following the design of Lehane & Schofield (1982, Bull. Ent. Res. 72:497-510). Bugs were placed in the flight apparatus in the evening, and removed the following morning, so that a daily record was maintained of flight until all bugs had died. In total, observations were made on 46 specimens of T. melanosoma and 50 of T. infestans, although the number of bugs present in each flight apparatus on each occasion varied from 1 to 18 because of their different emergence times. Most of the bugs initiated flight within the first 5 days of observation. During this time, 70% of male and 85% of female T. infestans initiated at least one flight, compared to 77% of male and 65% of female T. melanosoma. The proportion of flights then steadily declined with time in both species, although females continued to initiate flight more frequently than males. During the whole period of observation up to death of the final adult (183 days for T. infestans, 232 days for T. melanosoma) females of T. melanosoma flew on 57% of occasions and females of T. infestans flew on 46% of occasions, whereas males of the two species flew on 47% and 39% of occasions, respectively. Similarly, 96% of female T. melanosoma and 87% of female T. infestans initiated at least one flight during the observation period, compared to 90% of male T. melanosoma and 74% of male T. infestans. However, none of these differences is significant (Mann-Whitney test) either between species or between sexes. Our results confirm those of earlier observations on T. infestans (Lehane & Schofield, 1982 op.cit.) showing that flight initiation generally declines from a peak at about 14 days post-feeding, and is generally more marked in females than in males – though rarely significantly so.

Financial support: by CNPq, agreement FNS/Fiocruz 123/97 and ECLAT.

VT-32 – MORPHOMETRIC STUDIES OF LEGS OF 1^ST AND 2^ND INSTAR NYMPHS OF MEPRAIA SPINOLAI AND TRIATOMA INFESTANS (HEMIPTERA, REDUVIIDADE)

Naegele, MP, Martinez, I, Fernandes, MZT, Belda Neto, FM, Valente, SAS*, Barata, JMS**, Rosa, Ja da.

Faculdade de Ciências Farmacêuticas, Universidade Estadual Paulista, Araraquara, 14801- 902, SP, Brasil.

* Instituto Evandro Chagas, Ananindeua, 67970-070, PA, Brasil.

** Faculdade de Saúde Pública , Universidade de São Paulo, São Paulo, 01246-900, SP, Brasil.

Chagas's disease occurs in 17 countries of Latin America and the most important mechanism of Trypanosoma cruzi man transmission is Triatominae feaces, being T. infestans the main vector (Silveira; 1983; OMS, 1991). M. spinolai, wild species belonging to Triatominae subfamily is unique in Mepraia genus, and unique in Triatominae subfamily to have males macropterous, brachypterous, or micropterous and females micropterous. Another characteristic of this species is to have long and slender legs, mainly those of 3^rd pair (Lent and Wygodzinsky, 1979). In this study, the segments of the legs of the three pairs of 1^st and 2^nd instars nymphs of Mepraia spinolai and Triatoma infestans were measured by Nikon profile projector model 6C. Statistical analysis of the segments of the legs measured was done by INSTAT program, where we obtained average, standard deviation and standard error. Significant difference was not observed between right and left segments of the legs of 1^st and 2^nd instars nymphs of Mepraia spinolai and Triatoma infestans. Second instars nymphs of Triatoma infestans showed segments of legs significantly bigger than the 1^st instars nymphs. The segments of the legs of the 1^st instars nymphs of Mepraia spinolai presented the following measurement differences: tíbñ fêmñ tarñ troñ cox, while the 2^nd instars nymphs showed tíbñ fêmñ tarñ coxñ tro. The 2^nd and 3^rd pairs of legs of the 1^st and 2^nd instar nymphs of Triatoma infestans showed tíbñ fêmñ tarñ troñ cox, while in 1^st pair of legs the relation was fêmñ tíbñ tarñ troñ cox . Mepraia spinolai's femur and tibia are bigger than Triatoma infestans' ones in both instars.

Supported by CNPq

VT-33 – WEIGTH REDUCTION OF EACH INSTAR OF TRIATOMA JURBERGI CARCAVALLO, GALVÃO & LENT, 1998, (HEMIPTERA, REDUVIIDAE) PERMITTED ONLY ONE BLOODMEAL

Silva, Mba.; Rocha,D.s.; Galvão,C.;Cunha,V.; Carcavallo,R.u.; & Jurberg,J.

Laboratório Nacional e Internacional de Referência em Taxonomia de Triatomíneos, Departamento de Entomologia, Instituto Oswaldo Cruz, Av.Brasil 4365, Rio de Janeiro, RJ, Brasil, 21045-900.

Triatoma jurbergi, a closely-related species of Triatoma guazu Lent & Wygodzinsky,1979, was described recently by Carcavallo et al.(1998, Mem. Inst. Oswaldo Cruz, 93(4):459-464 based on specimens collected in Rondonópolis, Mato Grosso, Brazil. It is a sylvatic species, that has been found occasionally in the surroundings of rural houses, however, its role in the epidemiology of the Chagas' disease not will established yet. The objective of the present work is to assess losses of weight for each evaluated instar after a single bloodmead. Reared insects were obtainned from the Triatominae International Reference Laboratory at the Departament of Entomology of Instituto Oswaldo Cruz Rio de Janeiro, Brasil. Fifty eggs and 50 specimens of each instar were randomly removed from the rearing colony, individually enclosed Borrel flasks and maintained under enviromented controlled conditions. Insects were fed on pigeons (Columba livia) as protein food source. Soon after eclosion and/or moult, insects were-weighted and allowed to have only one bloodmeal.After each bloodmeal, insects were individually weighted again and weighting every seven days there after.Until next ecdyse for death. Results on body weight of insects submited to diffents bloodmeal and weight losses during each phase of development( table included).Indicated a longer life span period the second instar, in spite of the fact that these nynphs ingested, proportionnally, lesser amount of blood.

Financial support: CNPq; agreement 123/97-FNS/Fiocruz and ECLAT.

VT-34 – TRIATOMA INFESTANS SALIVA APYRASES ARE 480 KDA OLIGOMERS

Faudry E¹, Lozzi SP¹, Santana JM¹, Sousa MV², Ricart CAO², Rocha PS¹, Macedo TC¹ and Teixeira ARL¹. From ¹Laboratory for Multidisciplinary Research in Chagas' Disease, and from ²Centro Brasileiro de Serviços e Pesquisas em Proteínas, University of Brasília.

Triatoma infestans feeding ability, here understood as its capacity to overcome the host homeostatic response, can be related to various compounds secreted in its saliva. In a previous study, we identified and characterized apyrase activity in the saliva of T. infestans, the main vector of Chagas' disease in Brazil. The apyrase, a diphosphohydrolase that removes Pi from ATP and ADP, is a potent platelet anti-aggregator, which has been associated with fluidity of the blood essential for the insect feeding and thriving.

This activity was purified by gel filtration chromatography as a 480 kDa protein. The two dimensional electrophoresis analyses showed the presence of isoenzymes composed of peptide chains of 79, 82 and 88 kDa. The Peptide Mass Fingerprinting technique showed the 79 and 88 kDa polypeptides to be homologous to the Aedes aegypti apyrase. Cross-linking experiment with glutaraldehyde demonstrated those chains are assembled in a high molecular mass complex. The purified apyrase molecular complex was submitted to native PAGE and formed a single 460 kDa band under silver staining, which co-migrates with the in-gel apyrase activity. These findings show that this polypeptide oligomer encloses all apyrase activity in the saliva of T. infestans.

Supported by CNPq and FINEP - Financiadora de Estudos e Projetos, Brasil.

VT-35 – STUDY OF ENZYMES IN TESTES AND OVARIES FROM ADULT DIPETALOGASTER MAXIMUS AND TRIATOMA INFESTANS. CORRELATION WITH ULTRASTRUCTURE

Scaraffia, P. Y., Maldonado, C.,¹ Aoki, A.¹ & Gerez de Burgos, N. M.

Cátedra de Química Biológica and ¹Centro de Microscopía Electrónica, Facultad de Ciencias Médicas, Universidad Nacional de Córdoba. 5016 Córdoba, Argentina. E-mail: [e-mail]

D. maximus and T. infestans are vectors of Trypanosoma cruzi, the etiological agent of American trypanosomiasis. Knowledge of functional aspects of organs involved in reproduction of these species may be useful for the design of control methods. Activity of enzymes related to glycolysis: hexokinase (HK), fructose-6-phosphate kinase (F6PK); pentose pathway: glucose-6-phosphate dehydrogenase (G6PDH); amino acid catabolism: glutamate dehydrogenase (GlutDH) and involved in shuttle systems for the transfer of reducing equivalents to the mitochondria: aspartate aminotransferase (AAT), malate dehydrogenase (MDH) and glycerol-3-phosphate dehydrogenase (GPDH) were determined on extracts of adult testes and ovaries from D. maximus and T. infestans. The fine structure organization of the same organs were studied by electron microscopy. Testes: Activities of F6PK in D. maximus and T. infestans, either expressed in U/mg protein or U/g wet tissue, were higher than that of G6PDH. HK activity was lower than F6PK in D. maximus; in contrast, HK was higher than F6PK in T. infestans. The activity of F6PK was twice as high as GlutDH in both species. AAT and MDH showed higher activity than GPDH. Ovaries: F6PK showed similar activity to G6PDH. At variance with T. infestans, HK activity in D. maximus was lower than that for F6PK. GlutDH was higher than F6PK in both species. AAT and MDH activities were greater than that of GPDH. Levels of enzymes would be an index of the activity of the pathways in which those enzymes are integrated. In testes from both species most of the glucose would be utilized through the glycolytic pathway. Number of mitochondria and development of the rough endoplasmic reticulum in cell of the spermatogenic line indicate very active oxidative metabolism and protein synthesis. In ovaries, the existence of an active pentose pathway supply the NADPH required for fat and ecdysteroids synthesis. Catabolism of amino acid ending in alpha-ketoglutarate appears to be relatively more important in ovary than in testis. Fat and glycogen are stored in follicular cells of D. maximus; oocytes of both species contain numerous fat droplets. Abundant mitocondria are present in follicular cells and oocytes. Rough endoplasmic reticulum and free ribosomes are also conspicuous in these cells.The malate/aspartate H-transfer system seems to be relatively more important than the glycerophosphate shuttle in ovaries as well as in testes, at variance with our finding in muscles from the same insects (Comp. Biochem. Physiol 116A:173-179, 1997)

VT-36 – STUDY ON THE FEEDING BEHAVIOR OF TRIATOMA SORDIDA (STAL 1859) (HEMIPTERA – REDUVIIDAE) CAPTURED IN MATO GROSSO STATE BRAZIL USING THE PRECIPITIN TECHNIQUE AND DEGREE OF INFECTIVITY

Lorosa¹, E.S.; Bulhões ¹, D.M.; Cunha¹, V.; Andrade¹, R.E.; Figueredo², J.F. & Jurberg¹, J.

1-Laboratório Nacional e Internacional de Referencia em Taxonomia de Triatomíneos – Deptº de Entomologia – Instituto Oswaldo Cruz – Av.Brasil 4365, 21045-900, Rio de Janeiro, RJ., Brasil, tele-fax 0—21-290-9339.

E-mail: [e-mail]

2- Coordenadoria Regional de Mato Grosso – MT, Fundação Nacional de Saúde

The authors assessed using precipitin technique, food source of two hundred thirty four specimens of Triatoma sordida ( Stal,1859) captured in domiciliar and peri-domiciliar environment of Rondonópolis, Mato Grosso State, Brazil. The series of anti-serum used and respective titles ware as follows: Anti-human 1:15.000, anti-bird 1:10.000, anti-dog 1:15.000, anti-cat 1:12.000, anti-horse 1:16.000, anti-goat 1:14.000, anti-cow 1:15.000, anti-pig 1:10.000, anti-rodent 1:17.000, anti-opossum 1:15.000, anti-armadillo 1:15.000. Natural infection was assessed simultaneously using standard microscope examination.

Results indicated that one hundred twenty eight specimens reacted with following food source, respectively: goat (13.7%), opossum (marsupial) ( 13.7%), rodent (10.7%), bird ( 6.4%), dog (3%), pig (3%), human (2.6%), lizard (0.85%), frog (0.85%). One hundred and six specimens reacted with more them one food source as follows: rodent-opossum (7.3%), goat-bird (5.1%), goat-pig (2.6%), bird-frog (2.6%), bird-opossum (2.6%), bird-rodent (1.7%), frog-opossum (1.7%), bird-lizard (1.7%), opossum-pig ( 1.7%), goat-pig (1.3%), opossum-goat (1.3%), dog-opossum (1.3%), dog-frog (0.85%), lizard-bird (0.85%), dog-goat (0.85%), pig-lizard (0.85%), rodent-lizard (0.85%), pig-bird (0.85%), human-opossum (0.85%), pig-rodent (0.43%), frog-pig (0.43%), lizard-goat (0.43%), dog-bird (0.43%), rodent-lizard (0.43%), goat-rodent-opossum (1.7%), opossum-bird-rodent (0.85%), bird-opossum-goat (0.85%), rodent-bird-goat (0.85%), opossum-goat-dog (0.43%), goat-frog-bird (0.43%) rodent-bird-pig (0.43%), opossum-goat-rodent (0.43%), pig-bird-opossum (0.43%), rodent-opossum-goat-bird (0.85%) and bird-rodent-goat-lizard (0.43%).

None of the simples reacted with anti-cat, anti-horse, anti-cow neither anti-armadillo. Bigger incidence was observed in opossum and goat followed by rodent and bird. On the other hand natural infection for Trypanosoma cruzi like was detected in one hundred five (44.9%) specimens.

Trabalho realizado com auxílio do convênio N.123/97 Fiocruz/FNS;CNPq; ECLAT – European Communit Latin America Triatominae Research Network IC 18- CT- 960054

VT-37 – FEEDING STUDY OF SOME SPECIES OF TRIATOMINAE CAPTURED IN RIO GRANDE DO SUL STATE, BRAZIL, USING THE PRECIPITIN TECHNIQUE AND DEGREE OF THE INFECTIVITY

Lorosa⁽¹⁾, E.S.; Nunes⁽²⁾, I.M.; Vinhaes⁽³⁾, M.C.; Pereira⁽¹⁾, C.A.; Castro⁽¹⁾, P.R.P. & Andrade⁽¹⁾, R.E.

1- Laboratório Nacional e Internacional de Referência em Taxonomia de Triatomíneos – Deptº de Entomologia – Instituto Oswaldo Cruz- Av. Brasil, 4365, 21045-900, Rio de Janeiro, R.J, Brasil, tele-fax [phone]

e-mail: [e-mail]

2- Fundação Nacional de Saúde – Gerência de Chagas – Rio Grande do Sul - RS

3- Gerência Nacional do programa de controlo da Doença de Chagas – FNS – Brasília – D.F.

The objective of the work was to make a comparative feeding study of some species of Triatominae using the technique of precipitin, and natural infection was assessed using standard microscope examination.

The following species and number of individuals, respectively, were assessed: Triatoma infestans, 81 specimens; Panstrongylus megistus, 78 specimens; P.tupynambai, 7 specimens and Triatoma rubrovaria, 271 specimens, all captured in the state of Rio Grande do Sul, Brazil.

A Batery of anti-serum and respective titles used were as follows: anti-human 1:15.000, anti-bird 1:10.000, anti-dog 1:15.000, anti-cat 1:12.000, anti-horse 1:16.000, anti-goat 1:14.000, anti-cow 1;15.000, anti-pig 1:10.000, anti-rodent 1:17.000, anti-opossum 1:15.000, anti-armadillo 1:15.000 e anti-sheep 1:8.000.

Results indicated that 27 specimes of T.infestans fed on rodents 9.9%, birds 9.9%, humans 2.5% and horses 2.5% and 52 reacted with more them one food source birds-rodents 19.7%, birds-opussums 19.7%, rodents-opussums 12.3% and birds-rodents-opossums 3.7%. 61 specimes of the P.megistus, birds 19.2%, humans 15.4%, dogs 14.1% and horses 11.5%,. 17 specimes reacted with more them one food source, rodents-birds 5.12%, humans-birds 2.6%, birds-dogs 2.6%, dogs-rodents 2.6%, birds-dogs-horses 1.3% and humans-birds-rodents-opossums 1.3%. 3 specimes of the P. tupynambai fed on: dogs 28.5%, rodents 14.3%, 4 specimes reacted with more them one food source: humans-rodents 14.3%, birds-rodents 14.3%, humans-dogs 14.3%, birds-dogs-rodents 14.3%. 189 specimes of T.rubrovaria fed on rodents 19.5%, birds 16.2%, dogs 12.1% and humans 7.0%. 75 specimes reacted with more them one food source as follows: dogs-rodents 3.7%, humans-birds 3.7%, humans-rodents 1.8%, humans-birds-dogs 1.1% and rodents-humans-birds 0.36%.

Results of those naturally infected by Trypanosoma cruzi like indicated 33 (40%) individuals of Triatoma infestans positive, 32 (39.7%) of Triatoma megistus positive, 1 (14.3%) specimen of P.tupynambai positive and 52 (19.2%) specimens of T.rubrovaria positive.

Trabalho realizado com auxílio do convênio N.123/97 Fiocruz/FNS;CNPq; ECLAT – European Communit Latin America Triatominae Research Network IC 18- CT- 960054

VT-38 – MORPHOMETRIC STUDIES OF ANTENNAL SEGMENTS OF NYMPHS AND ADULTS OF PANSTRONGYLUS MEGISTUS AND RHODNIUS NEGLECTUS (HEMIPTERA, REDUVIIDAE)

Freitas, SCM, Fernandes, MZT, Martinez, I, Belda Neto, FM, Barata, JMS*, Rosa, JA da

Faculdade de Ciências Farmacêuticas, Universidade Estadual Paulista, Araraquara, 14801-902, SP, Brasil.

*Faculdade de Saúde Pública, Universidade de São Paulo, São Paulo, 01246-900, SP, Brasil

The Chagas disease affect a universe of 16 million of people in Latin America (WHO, 1991). Five million of people in Brazil are infected by Trypanosoma cruzi (Dias, 1987). These index show us the importance and necessity of the Triatominae studies. The relative lenght of the four antennal segments are used as taxonomic character (Lent & Wygodzinsky, 1979). Fifteen samples of P. megistus and R .neglectus were utilized for the mensuration of four right and left antennal segments of the first to the fourth instar nymphs and, male and female fifth instar nymphs and adults, through Nikon profile projector model 6C. The measurations were statisticaly evaluated by the INSTAT program through ANOVA test, t-student paired test and t-student non paired test. Signifcant and diferent remarks were observed betwen the four antennal segments of five instar nymphs and adults. The following results were obtained for the first and second instars nymphs of P. megistus: 4^th>3^rd>2^nd>1 ^st, for the third instar nymphs: 3^rd>4^th>2^nd>1 ^st, for fourth and fifth instars nymphs and adults : 2^nd>3^rd>4^th>1 ^st. The differences noted between antennal segments of R. neglectus were: in first and second instars nymphs: 3^rd>2^nd>4^th>1 ^st, in the third and fourth instar nymphs: 3^rd>2^nd>4^th>1 ^st and fifth instar nymphs and adults: 2^nd>3^rd>4^th>1 ^st. Significant diference was not observed between right and left segments , between male and female in both species. Based on the results of P.megistus we could note and compare the similarity with T. rubrovaria (Tres et alii, 1996, 1997). The research measurement data of antennal segments of R. neglectus are similar to T. rubrovaria and P. megistus in the fifth instar nymphs and adults, on the others instars standard measure are distincts.

Supported by CNPq.

VT-39 – CHARACTERIZATION OF THE II UROTERGITE PROCESS IN TRIATOMA PROTRACTA UHLER, 1894 AND TRIATOMA LECTICULARIA STAL, 1859 THROUGH SCANNING ELECTRONIC MICROSCOPY AND OPTIC MICROSCOPY

Barata, J.M.S. ¹, Rosa, J.A. da², Urbinatti, P.R.¹, Ceretti-Jr, W. ¹, Marassá, A.M. ³ Gonçalves de Andrade, R.M⁴, , 1 Faculdade de Saúde Pública/USP, São Paulo, SP; Brasil, 2 Depto. de Parasitologia/UNESP, Araraquara, SP; Brasil, 3 Instituto Adolfo Lutz, 4 Instituto Butantan, São Paulo, SP, Brasil.

The I Urotergite Process was firstly described to some Triatominae species that are involved in the transmission of Chagas Disease at the South American Continent in areas of Bolivia, Argentina, Paraguai, Uruguai and Brazil. At this short communication,

we are extending this characterization to two North American species that have been found naturally infected and can be associated to the enzootic cycle of this disease in Mexico middle south of the USA Adult males of Triatoma protracta Uhler,1894 from Monte Diablo CA (CTA-045) were studied. The triangular plate has an isosceles shape; it is on a higher level than the II Urotergite and display wrinkles and parallel, conspicuous grooves which take place in most of the structure. Its apex doesn't preserve an angular process as it was observed in the other species that have been studied. Conspicuous short bristles can be seen and they are distributed in the median portion of the I Urotergite. A different light coloured area distinguishes the apex border of the I Urotergite, including its apex. The rectangular plate presents a plane circular area well defined by a pair of divergent, vertical grooves. In the median portion of the circular area, not prominent parallel horizontal striations can be seen and in some specimens can also reach the adjacent portion of the II Urotergite. The II Urotergite is extremely wrinkled and striated, not showing bristles at the Optic Microscope. Adult males of Triatoma lecticularia from Oklahoma- USA (CTA-021) were studied. The triangular plate is on a higher level than the II Urotergite. At the middle of the plate there is a centre apical slope that is contiguous to the angular process and take the shape of a lozenge. This structure is distinctively seen in some specimens. Bristles are distributed at the central part of the plate and wrinkles are concentrated on the lateral fringes of the triangular plate. A wrinkly dark area surrounds the apex border of the I Urotergite including its apex. Parallel, vertical grooves are displayed at the base of the triangular plate. The II Urotergite presents a plane circular area well defined by a pair of divergent, vertical grooves. Wrinkles are distributed in all extension of the rectangular plate and the ones which are next to the border of the I Urotergite are more evident and darker than the others. Bristles could not be observed at the Optic Microscope.

VT-40 – MORPHOMETRICS OF THE GENUS PANSTRONGYLUS BERG, 1879 (HEMIPTERA, REDUVIIDAE, TRIATOMINAE)

Santos, C.M., Jurberg, J., Galvão, C., Rocha, D.S. & Galíndez-Girón, I.+

Laboratório Nacional e Internacional de Referência em Taxonomia de Triatomíneos, Departamento de Entomologia, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, RJ + Universidad de Los Andes, Núcleo "Rafael Rangel", Laboratorio de Biologia de Triatominae, Villa Universitaria, La Concepción, Trujillo, Venezuela.

The genus Panstrongylus Berg, 1879, is composed by 13 especies, all have a neotropical distribution; they are prevalent from the South of Mexico to the Patagonian region in Southern Argentina (Carcavallo et al., 1994, Entomol.Vect., 1: 113-120). All species of this genus have been described in detail through the study of external and internal morphology, showing significant differences between male genitalia (Lent & Jurberg, 1968, Rev. Brasil. Biol., 28: 499-520; Lent & Jurberg, 1975, Rev. Brasil. Biol., 35: 379-438; Lent & Wygodzinsky, 1979, Bull. Am. Mus. Nat. Hist. 163: 127-130). Some of them have importance as vectors' of Trypanosoma cruzi, the causative agent of Chagas' disease, such as P. megistus Burmeister, 1835 and P. geniculatus (Latreille, 1811) (Curto de Casas et al., 1996, Entomol. Vect. 3 (2): 43-58). Morphometry is suggested here as a new "tool" capable not only of solving taxonomic problems about Triatominae, but also to try to create a philogenetic approach. Recent works by Dujardin et al. (1999, Mem. Inst. Oswaldo Cruz 94: 565-569) suggest that changes in the sexual size dimorphism of Triatominae can be used as a new character to study the species of Triatominae in the transition from natural to artificial habitats, like domestic ecotopes. The present work uses all species of Panstrongylus, deposited in the "Herman Lent" collection of the Laboratório Nacional e Internacional de Referência em Taxonomia de Triatomíneos, Departamento de Entomologia, Instituto Oswaldo Cruz, FIOCRUZ, Brasil. The measurements were made using a stereoscopic microscope ("Wild M5-51844"), with micrometer eyepiece; 13 measurements were taken from the head, of each adult specimen, as described by Lent & Wygodzinsky (1979, Bull. Am. Mus. Nat. Hist. 163: 127-130) importants in the taxonomy of the Triatominae. To this date 56 specimens have been measured: 9 of P. chinai (Del Ponte, 1929); 4 of P. humeralis (Usinger, 1939); 4 of P. lignarius (Walker, 1873); 29 P. megistus and 10 of P. rufotuberculatus (Champion, 1899), from different places. The preliminary results corroborate the sexual dimorphism already known, but in P. megistus these differences were more obvious than in the other species because the females were significantly larger than males, from differents localities, in all the measured variables, suggesting a possible populational variability.

Financial Support: CNPq; Convênio n° 123/97 - FNS/FIOCRUZ; ECLAT

VT-41 – COLONIZATION OF LUTZOMYIA LONGIPALPIS: MORPHOLOGICAL STUDY AND SUSCEPTIBILITY TO INFECTION BY LEISHMANIA SPP.

¹Secundino, N.F.C., ²Braga, E.M. and ¹Pimenta, P.F.P.

¹Laboratory of Medical Entomology, Centro de Pesquisas Rene Rachou - CPqRR, Fundação Oswaldo Cruz- FIOCRUZ. Av. Augusto de Lima 1715, Belo Horizonte-MG and ²Department of Parasitology, Universidade Federal de Minas Gerais, Brazil.e-mail: [e-mail] and [e-mail]

A colony of the phlebotomine Lutzomyia longipalpis from the Lapinha cave (Minas Gerais state, Brazil) was established in a short time in our Laboratory. The intention was to obtain enough flies to develop several experiments as: (a) to study the aspects of each developmental stage of the sandfly; (b) to study the morphology of the midgut and (c) to investigate the susceptibly of the sandfly to the Leishmania infections. The colony started with 850 adult females from the wild life, caught during nighttime with CDC light traps. After the females came from the field, they were blood fed in anesthetized hamster for the next four days. The engorged females were separated and individualized for oviposition and identification. The eggs were counted, separated and placed in pots of plaster for creation. The larvae were daily fed on a special diet. The insects were all time maintained at 26° C in a B.O.D. incubator. The adult releases were completed in a period of about 30-35 days. After this, the colony was established and maintained until now, the 11^th generation. In following experiments we used the scanning electron microscopy to observe the eggs and larvae morphology. The external surfaces of the eggs are covered with an exochorion characterized by arrangements of a series of parallel, discontinuous, longitudinal ridges that converge at egg ends. There are not lateral connections between the ridges. The larvae stages were distinguished by the presence of one or two pairs of caudal filaments. The abdominal segments were recognized by the presence of stump projecting structures on the dorsal surface called prolegs. The lateroventral surface is covered with different brush-like hairs. In the pupae stage is easy to observe the abdominal segments with lot of spiculates, the wings and the legs. We also developed a histological study of the adult midgut, the region where the Leishmania parasites initiate the infection. Conventional staining of Historesin sections showed details of the organ, demonstrating differences between the thoracic and abdominal regions of the midgut. Another experiments were done in order to verify the infection of the Lu. longipalpis by Leishmania. We verified that insects from the colony are much more susceptible to infection than those from the wild life. In order to better understand this aspect we developed biochemistry studies to compare the proteins present in the midgut and in the saliva of these insects. We concluded that they are also different in the protein contents of these organs. Further studies are necessary to understand these aspects and possible relationship with the parasite-vector interaction.

Financial support: FIOCRUZ, WHO, CNPq and PRONEX.

VT-42 – MOLECULAR POLYMORPHISM WITHIN A PUTATIVE SONG GENE OF LUTZOMYIA LONGIPALPIS

Oliveira, S.G.*, Souza, N.A. ^$, Kyriacou, C.P.^# & Peixoto, A.A.*

* Departamento de Bioquímica e Biologia Molecular, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil

^$ Departamento de Entomologia, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil

^# Department of Genetics, University of Leicester, Leicester, U.K.

During courtship, males of Drosophila produce a "lovesong" by vibrating their wings, and this species-specific acoustic signal has been implicated in the reproductive isolation of closely related species. Any gene that influences the characteristics of this courtship song is therefore a candidate for influencing the speciation process. cacophony (cac) is a calcium-channel gene that determines particular features of the courtship song in Drosophila melanogaster. cac is particularly interesting from an evolutionary point of view because one of its mutant alleles produces a song that resembles those of other Drosophila species.

The sandfly Lutzomyia longipalpis (Psychodidae; Phlebotominae), the vector of visceral leishmaniasis in Latin America, is considered a complex of sibling species. Courtship songs have been recorded from L. longipalpis and, as in Drosophilids, it is possible that they play a role in the reproductive isolation of sandflies. As part of a molecular and behavioural analysis of courtship songs in neotropical sandflies, we recently isolated and sequenced from L. longipalpis, a DNA fragment homologous to cacophony. We are now analysing the molecular polymorphism within this putative sandfly song gene using PCR and DNA sequencing. A number of alleles were sequenced from two populations of L. longipalpis (Natal, RN and Lapinha, MG) and preliminary analysis suggests that cacophony will be a good molecular marker for speciation studies within this species complex.

Finantial Support: Wellcome Trust, Faperj, Fiocruz, CNPq.

VT-43 – TRANSIENT EXPRESSION OF DIFFERENT GENES IN CELL LINE AND MIDGUT OF LUTZOMYIA LONGIPALPIS

Fampa, P¹, Oliveira, SMP³, Carvalho, TU², Silva, NLC² & Saraiva, EMB1

1Laboratório de Imunobiologia das Leishmanioses, Dept^o Imunologia, Instituto Microbiologia, ²Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro & ³Dept^o Entomologia, Instituto Oswaldo Cruz, Rio de Janeiro, RJ, Brasil.

Genetic manipulation of insect vectors, in order to alter their ability to transmit parasites, may provide a new approach in the control of vector-borne diseases. Sandfly midgut plays an important role in the transmission of Leishmania parasites. The ability to express genes in sandfly would be a powerful approach to characterize midgut genes, and to reveal important vector determinants of pathogen transmission. In the present study, we report a method for the transfection of midguts from Lutzomyia longipalpis, the Leishmania chagasi vector. Midguts are important for parasite metacyclogenesis, and as such, the prime target for the expression of deleterious genes to the parasite. A lipofectin-based assay was used to transfect plasmids carrying the luciferase or b-galactosidase reporter genes under control of the Drosophila heat-shock protein 70 promoter. Indirect immunofluorescence was used to characterize the luciferase expression at different plasmid concentrations and times post-transfection. The guts viability in 24 h culture was tested by Leishmania promastigotes binding assays in vitro and by ³⁵S methionine incorporation. The number of parasites that remained bound to transfected guts and the protein synthesis were comparable to the controls.

Supported by: CNPq, Finep, FUJB, FAPERJ & PRONEX.

VT-44 – EFFECT OF A SECOND BLOOD-MEAL OF LUTZOMYIA MIGONEI (DIPTERA:PSYCHODIDAE) ON DEVELOPMENT OF LEISHMANIA BRAZILIENSIS

Nieves, E. & Pimenta, F. P. P.

Laboratory of Medical Entomology, Centro de Pesquisas Rene Rachou - CPqRR, Fundação Oswaldo Cruz- FIOCRUZ. Av. Augusto de Lima 1715, Belo Horizonte-MG, Brazil. e-mail: [e-mail] and [e-mail]

A few experimental studies have shown the effects of vector physiology on the life cycle of Leishmania parasites (Killick-Kendrick, R., 1979, Biology of Kinetoplastida, vol. 2, Academic Press, 395-460). In the present laboratory study we demonstrated that a second sand fly bloodmeal induces the multiplication and differentiation of Leishmania in early development. Leishmania promastigotes undergo sequential steps of proliferation and differentiation within their sand fly vectors to produce a final stage infective to the vertebrate host. Infection of laboratory-reared Lutzomyia migonei was carried out using artificial feeders containing a suspension of 2x10⁷Le. braziliensis amastigotes per ml of mouse blood. Approximately 300 female sand flies divided among 5 experimental groups were allowed to feed through chick-skin membranes stretched over the mouth of the feeders, which were placed over plastic rearing containers. Each sand fly was estimated to ingest approximately 2000 parasites. A second bloodmeal was offered on anesthetized, uninfected hamsters at 4 days after the infection. The re-fed females were counted and isolated in a separate cage from the unfed insects, the latter being used as a control. Both groups were provided with fresh sucrose solution and examined for Leishmania development. Females were dissected daily from 1-10 days after infection and their guts examined for presence of promastigotes under phase contrast microscopy, the numbers of parasites being counted in a hematocytometer. In addition Giemsa-stained smears were used to observe each of the different promastigote forms of Leishmania. The sand flies dissected 4 days after the infection (n=975) a total of 407 females had engorged or taken a partial bloodmeal within the 5 experimental groups, an average re-feeding rate of 55% ± 12.4 (range=37-75). The results showed that the second blood meal caused no deliterious effects on Leishmania-infected sand flies. An increase in parasite density, with a slight change in the early development was detected in the females that took the second blood-meal. Similar proportions of the different promastigote forms of the parasites were noted in both groups. An increase in the proportion of the metacyclic forms in the proboscis would increase the efficiency of transmission after the intake of the second blood-meal.

Financial Support: FIOCRUZ, CNPq, PRONEX and CONICIT (Venezuela).

VT-45 – COMPARATIVE MOLECULAR STUDIES OF GUTS FROM FED AND UNFED SAND FLIES

Ramalho Ortigão JM, Temporal P and Traub-Cseko YM

Oswaldo Cruz Institute - FIOCRUZ

Department of Biochemistry and Molecular Biology

P.O Box 962, 21045-900 Rio de Janeiro, RJ

One of the possible approaches for the control of insect-borne diseases is through the control of the vector. Besides the traditional methods involving insecticides, recent developments, mostly related to mosquitoes, aim at the generation of insects less fit to transmit diseases. A first step necessary in the development of these studies is the basic knowledge of molecules involved in feeding mechanisms, and eventually in the parasite/host interactions. We have initiated studies on the characterization of such molecules in sand flies.

Gut of sand flies unfed or fed on rabbit blood were compared molecularly through differential display reverse transcription (DDRT) PCR employing various combinations of anchored oligo dT plus arbitrary primers (APs). Specific bands were excised from the acrylamide gel, the DNA eluted, reamplified by PCR and cloned, either using the pCR 2.1 TOPO vector (TOPO TA cloning kit, Stratagene) or the pBluescript plasmid digested with Hind III. We have also applied RAPD-PCR (using OPG primers kindly donated by Dr. Edelberto Dias, CPqRR, Fiocruz) to amplify RNAs from unfed and fed guts as well as from salivary glands. With this latter approach we have also obtained a different pattern of bands which were separated on agarose gel, purified and cloned.

Upon sequencing of several clones we detected one that displayed a motif found to be homologous to an Anopheles gambiae gut-specific chitinase. When this clone was used as a probe in a Northern blot, containing whole insects RNA from blood-fed and sugar-fed Lutzomyia longipalpis, a signal was detected only against those that were fed on blood. Furthermore, this signal appeared only at approximately 30 hours after feeding with no signal detected at 16 hours after feeding, demonstrating the specificity of expression of this chitinase. Due to the likely fundamental physiological role a gut-specific chitinase may play in this vector, this and other clones obtained are being further characterized.

Acknowledgement: We thank Dr. Elizabeth Rangel and Dr. Sandra de Oliveira for the generous gift of insects. Supported by the Oswaldo Cruz Institute and CNPq.

VT-46 – RESEARCH ON PHLEBOTOMINAE (DÍPTERA: PSYCHODIDAE) IN GRUMARI, AN AREA OF ENVIRONMENTAL PROTECTION OF THE COUNTY OF RIO DE JANEIRO, RJ, BRAZIL

Carreira – Alves, J.R.; Cunha, S.P.; de Barros, L.C.V.; Gammaro, A.T.; Soares, A.V.*; & Soares, C. A.G.*

FNS/RJ-DSRJ – Laboratório de Entomologia, Av. Pedro II, CEP 20941-070, São Cristóvão, Rio de Janeiro, RJ, Brasil. *FNS/RJ-NERJ, Av. Brasil, 4036/109, Rio de Janeiro, RJ, E-mail: [e-mail]

Grumari is an area of environmental protection, located in the Jacarepagua lowlands, a place in the west zone of the county of Rio de Janeiro, between Recreio e Barra de Guaratiba, with the Atlantic Ocean in the south. This area belongs to XXIV Administrative Regional Office of Barra da Tijuca. In September 1998, there occurred an autochthon case of visceral leishmaniasis. With an aim to investigate and keep updated the Phlebotominae fauna, as well as promoting a more effective entomological observation, mainly concerning the evaluation of the watering of insecticidal products, 9 (nine) captures were done in 4 distinct areas. Three of took us 4 hours and the others, for which we used a CDC trap, took us 12 hours, from November 1998 till May 1999.

A total of 5,810 sandflies were captured and identified, such as: L. intermedia, L. migonei, L. firmatoi, L. schreiberi, Lutzomyia (Pintomyia) sp. and Brumptomyia cunhai. The most abundant species was the L. migonei with 54,81% of samples, which, due to its anthropophily, makes it possible to see it as vectors of this disease in the meridional region of the South-American Continent (Forattini, 1973). The L. intermedia was 44,5% out of the sandflies collected. This species is suspected to be the vector of LTA in the Southeast region (Rangel et al., 1984; 1986; Rangel, 1995). It should be pointed out that no samples of L. longipalpis were captured during this period, though the visceral leishmaniasis case mentioned above and 10 positive dogs in the region were recorded.

VT-47 – ACTION OF HUMAN SERA AND VENEZA ZONATA (HEMIPTERA, COREIDAE) HAEMOLYMPH ON CULTURE FORMS OF TRYPANOSOMATIDS

Baccan,G.B¹.;Bernardo,B.S¹ .;Girotto,E¹.;,Souza,W², Attias,M²;Jankevicius,J.V¹. & Itow Jankevicius,S.¹.

¹Laboratorio de Biologia de Tripanosomatideos, Departamento de Microbiologia, Centro de Ciencias Biologicas, Universidade Estadual de Londrina,

²Laboratorio de Ultraestrutura Celular Hertha Meyer, Instituto de Biofisica Carlos Chagas Filho, CCS, Bloco G, Subsolo, Cidade Universitaria, UFRJ, Rio de Janeiro, RJ

The interaction of Trypanosomatids with phytophagous hemipterans can define the localization of the flagellates in the insects (digestive tract and/or salivary glands) and classify the trypanosomatids as monoxenous (parasites of insects) or heteroxenous (involving parasites of insects and plants). The action of human sera and Veneza zonata haemolymph was analyzed on 3 groups of Trypanosomatids. The first group was composed by the trypanosomatids Phytomonas serpens, P.mcgheei and the strain 714 TD, isolated from digestive tract of V.zonata, all of them establishing infection in the salivary glands of the insect V. zonata, and transmitting infection to plants. The second group (Leishmania (L.) amazonensis) represents trypanosomatids that could not established an infection in V. zonata. The third group represents the strain 563 TD, a trypanosomatid isolated from the digestive tract of the phytophagous hemipteran Piezodorus guildini, pathogenic to the V. zonata.

The experiments with human sera used 30mL of undiluted normal sera mixed with 30mL of culture forms of trypanosomatids (10⁶ cells/mL) in Eppendorf tubes, incubated at 25°C and aliquots examined after periods of time, directly and stained with May-Grunwald-Giemsa. Almost immediately after the contact with human serum, the flagellates suffers strong agglutination. Groups 1 and 3 showed flagella-flagella, body-body and body-flagella interactions, whereas group 2 (Leishmania (L.) amazonensis) showed a faster flagellar agglutination.

With Veneza zonata haemolymph, collected with automatic pipette (30mL) from a leg of V. zonata ,was processed as with serum samples. The results of group 1 showed a recognition of protozoa by haemocytes in 5 minutes and at 35 minutes it was seen several nodules, without agglutination and in 1 hour, several culture forms presents round forms. L. (L.) amazonensis seemed to be more sensitive than isolates from group 1. The flagellates adhered to haemocytes at time 0, with much more intense agglutination and at time 10 min., it was observed large rosettes of flagellates and at time 30 minutes, including haemocytes. The group 3 showed no significant differences from group 1. The morphologic alteration is studied by electronic microscopy and fenoloxidase biochemistry.

Financial Support: CAPES, CNPq, CPG/UEL

VT-48 – CULICOIDES MIDGES (DIPTERA:CERATOPOGONIDAE) FROM LOCALITY OF CAMPOS DOS GOYTACAZES, RIO DE JANEIRO, BRAZIL

Felippe-Bauer,M.L., Silva C.S., Basto,F.N. *

Laboratório de Díptera, Depto. de Entomologia, Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil 4365, Manguinhos, Cx. Postal 926, 21045-900, Rio de Janeiro, Brasil.

The ceratopogonid midges of the genus Culicoides are common bloodsuckers, which have been involved in the transmission of human and animal diseases.They are known to serve as vector of protozoan and nematode parasites to birds and mammals, and virus to humans.

At present, 246 valid species were assignaled for the Neotropical Region. Only 66 have been cited south of the Amazonian Region. Very little work with systematical capture has been done in the Neotropical Region if compared with mosquitoes and other nematocerous diptera and more studies are necessary to best know the distribution of the Brazilian species.

We have had the opportunity to study material from light trap collection made by Fundação Nacional de Engenharia e Meio Ambiente (FEEMA) in 69 localities of the state of Rio de Janeiro, Brazil, during February 1989 to March 1990. First, we have studied the locality of Campos dos Goytacazes. After examination of 27 of the 50 total samples, we have obtained 75 specimens corresponding to the period of February to September 1989. The proportion of the species represented in these collections is as follows: C. limai group (47%), C. insignis (17,5%), C. pusillus group (17%), C. guyanensis (8%), C. guttatus group (4%), C. foxi (1,5%) and C. neoparaensis (1,5%).

At present, Culicoides of the limai group is more frequently found in this Region, considering this method of capture. Culicoides insignis and pusillus were also found in significant number. These last two species have been incriminated in the transmission of the bluetongue virus on cattle in the Central and South America.

* Programa de Vocação Científica da Fundação Oswaldo Cruz (PROVOC).

VT-49 – A HEME-BINDING ASPARTIC PROTEINASE FROM THE EGGS OF THE HARD TICK BOOPHILUS MICROPLUS

Sorgine, M.H.F., Paiva-Silva, G.O., Logullo, C., and Oliveira, P.L..

From Departamento de Bioquímica Médica, Instituto de Ciências Biomédicas, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro- RJ, Brazil, CEP 21910-590, Fax: [phone]. # E-mail: [e-mail]

The hard tick Boophilus microplus represents a major problem to cattle raising in tropical and subtropical areas of the globe, being the vector of many important cattle parasites such as Babesia bovis and Babesia bigemina. We have purified a new aspartic proteinase from the eggs this tick. This proteinase is able to bind heme with a 1:1 stoichiometry. The proteinase, herein named Thap (Tick Heme-binding Aspartic Proteinase), was classified as an aspartic proteinase based on its NH₂-terminal sequence, acidic optimum pH and inhibition by pepstatin.

THAP showed hydrolytic activity against hemoglobin. However, very low activity was observed against globin devoid of the heme prosthetic group. Hydrolysis of globin polypeptide chain by THAP increased as increasing amounts of heme were added to globin, with maximum activation at a heme:globin relation of 1:1. Further additions of heme to the reaction medium inhibited proteolysis, back to a level similar to that observed against globin alone. Addition of heme did not change THAP activity towards ribonuclease, a non-hemeprotein substrate. The major storage protein of tick eggs, vitellin is a hemeprotein. Hydrolysis of vitellin by THAP is also inhibited by addition of heme to the incubation media. In a ligand blot assay, THAP is able to bind to either hemoglobin or vitellin being this binding totally inhibited by the addition of free heme to the reaction media. Taken together, our results suggest that THAP uses heme bound to protein substrates as a docking site to increase specificity and regulate VT degradation accordingly to heme availability.

Supported by: PADCT, CNPq, FINEP, FAPERJ, CAPES and PRONEX

VT-50 – TEST OF DIFFERENT PROTOCOLS AIMING AT THE MORPHOLOGICAL CHARACTERIZATION OF INTERNAL STRUCTURES OF EMBRYOS FROM THE TICK BOOPHILUS MICROPILUS AND NEOTROPICAL ANOPHELES

Pedro Paulo de Abreu Manso (1, 2, 3); Luzia Fátima Gonçalvez Caputo (2); Leonardo de Araujo Abreu (4); Carlos Logullo (4, 5); Flávio Lara (5); Marcella Pereira da Cunha (3); Henrique Leonel Lenzi (2); José Bento Pereira Lima (3) and Denise Valle (2, 3)

(1) Depto. de Entomologia / FIOCRUZ-RJ; (2) Departamento de Patologia / FIOCRUZ-RJ, (3) USAMRU-B / Instituto de Biologia do Exército-RJ; (4) Depto. de Biologia Celular e Molecular-Instituto de Biologia / UFF-RJ; (5) Depto. de Biologia Médica / ICB / UFRJ-RJ

Our final aim is to assist in the development of new control strategies of the hematophagous arthropod vectors Boophilus micropilus and neotropical Anopheles. To accomplish this, knowledge of their biology, including the unraveling of morphological aspects of their embryonic development is a prerequisite. Here we describe attempts on the observation of embryonic internal structures at the optic microscopy level.

Previous attempts to analyze tick embryo morphology, by Transmission Electron Microscopy, have proved unsuccessful. Examination of resulting samples suggested lack of penetration of resin and fixative. We have then reasoned that one of the major problems to be overcome was the impermeability of tick eggs. The protocol classically used to dechorionate Drosophila eggs was tested over Boophilus micropilus ones. Additionally, different fixatives were employed (glutaraldehyde, paraformaldehyde, Millonig) and results will be presented. To our surprise, the conventional histological processing utilized has rendered B. micropilus eggs permeable to paraffin impregnation. Also, the internal embryo tick structures were well preserved.

Concerning mosquito eggs, dechorionation or, alternatively, permeabilization of eggshell layers is needed, since mosquito embryos are covered by a highly sclerotized chorion. We have tested different procedures leading to the withdrawal or weakening of An. albitarsis eggs: the dechorionation protocol classically used for Drosophila eggs, a sclerotization inhibitor, an egg clarification method and various combinations of these assays. After these treatments, Anopheles eggs were submitted to the conventional steps involved in paraffin impregnation. Comparison of all samples after sectioning and staining revealed preservation of internal structures in clarified embryos, treated or not with the sclerotization inhibitor.

We aim at utilizing the protocols here defined to characterize the morphogenesis of internal structures during the whole embryonic development of both ticks and Anopheles mosquitoes.

Supported by: UNDP / WORLD BANK / WHO, PAPES II Program (Fundação Oswaldo Cruz), USAMRU-B, FAPERJ

VT-51 – EFFECT OF DIFFERENT SCLEROTIZATION INHIBITORS OVER ANOPHELES ALBITARSIS EGG DARKENING AND HARDENING

Jesus Martins, Ademir Jr. (1, 2); Cardozo, Sabrina Gonçalves de Carvalho (1, 2); Pereira da Cunha, Marcella (2); Pereira Lima, José Bento (2); and Valle, Denise (1, 2)

(1) Departamento de Entomologia/FIOCRUZ-RJ, (2) USAMRU-B / Instituto de Biologia do Exército-RJ

The analysis of Anopheles embryo development is one of the requirements towards the construction of genetically modified malaria vectors refractory to Plasmodium.

In order to collaborate with the neotropical Anopheles embryo characterization, some drugs are being tested, over An. albitarsis synchronized eggs, since oviposition. These drugs are intended to keep the eggs bright and permeable, since they inhibit some steps on the sclerotization enzymatic cascade. In the present work the influence of these drugs on both egg darkening and egg permeabilization are presented. The effects of the sclerotized inhibitors used on viability are shown elsewhere (Cardozo et al., 1999).

For the eggs darkening inhibition assays, different concentrations of a Dopa decarboxylase (DDC) inhibitor were used in association with an anti-oxidant. Control eggs start darkening from oviposition on and, in about 50-60 minutes all of them are black. On the other hand, experimental eggs don't darken completely for at least 270 minutes. In some cases, embryos can be observed inside the egg through the stereomicroscope, until larvae hatching. Other substances are also intended to be tested, like a Phenol oxidase inhibitor.

Both darkening and hardening of mosquito eggshell are seen as consequences of the sclerotization process. We then reasoned that the same drugs that are able to inhibit egg darkening would also restrain egg hardening and consequently, render the chorion permeable. For permeability assays, control (laid over water) and experimental (laid over sclerotization inhibitors) An. abitarsis eggs were put in contact with ethylene glycol (known as a cryopreservant) at different moments after egg laying. The shrinkage of the eggs was recorded at each five minutes for a total period of 1 hour. The results of these assays point to a shrinkage delay in experimental eggs in comparison to the controls. Shrunken eggs never attain their normal volume again. Even eggs at oviposition (control and experimental) seem to shrunk when in contact with ethylene glycol. If these data are confirmed, the existence of another factors, besides sclerotization, influencing the egg permeability will have to be considered.

We aim to define permeabilization conditions sufficiently strong to enable the cryopreservation of Anopheles embryos.

Supported by: UNDP / WORLD BANK / WHO, PAPES II Program (FIOCRUZ), USAMRU-B.

VT-52 – THE SEARCH FOR ROSS CELLS IN THE ANOPHELES AQUASALIS MIDGUT: A POSSIBLE TARGET FOR PLASMODIUM INVASION

^1,2 Ferreira, M.A.P. & ¹Pimenta, P.F.P.

¹Laboratório de Entomologia Médica, Centro de Pesquisas René Rachou – CPqRR Fundação Oswaldo Cruz – FIOCRUZ P.O. Box 1743. Av. Augusto de Lima 1715, Belo Horizonte – M.G. Cep 30.190-002 and ²Laboratório de Histologia e Embriologia Comparada, Departamento de Histologia e Embriologia, Universidade Federal do Pará, Belém – PA Fax [phone] e-mail: [e-mail] and [e-mail]

Anopheles mosquitoes are the main vectors of malarial Plasmodium to the vertebrates. Anopheles aquasalis is a primary vector of human malaria in the coastal Brazil mainly in the North and Northeast regions. The parasite life cycle is initiated when the infected blood from the vertebrate is ingested by the female mosquito. The establishment of the infection occurs in the midgut during the bloodmeal digestion. The parasites need to interact and cross the epithelial cells of the midgut in order to produce oocystis. At the present this interaction process is not well understood. The main goal of this study is to recognize the morphology of the midgut epithelium and the Ross cells, the target for Plasmodium invasion in Aedes aegypti (Shahabuddin and Pimenta, 1998 PNAS-USA 95 3385-3389). This information will help further interaction studies. Mosquito midguts were dissected and divided in two parts: thoracic and abdominal regions, and processed routinely for transmission electron microscopy. The ultrastructural analysis of the thoracic midgut demonstrated a typical epithelium with the presence of a main cell population composed by columnar cells. The apical region of these cells are completely covered by microvilli, the cytoplasm is dense with a central nucleus and prominent nucleolus. In the basal region, the plasma membrane is folded in contact with a basal lamina. These muscle cells have variable shapes and are continuous with the epithelium basal lamina. The structural aspect of abdominal midgut is slightly different from the thoracic part. Besides the main epithelial cells, large cells projecting to the midgut lumen can be observed. The apical region of these cells is irregular with few microvilli and dense cytoplasm with no visible organelles. The aspect of this cell population is very similar to the Ross cells present in the Ae. aegypti midgut. This preliminary study is the basis for further analysis of the interaction process between the midgut and the malarial parasites, which could help provide essential information on how to block the infection in the mosquitoes.

Financial support: FIOCRUZ, CNPq, CAPES/PICDT/UFPA and PRONEX.

VT-53 – PARTIAL CHARACTERIZATION OF GLUTATHIONE S-TRANSFERASE AND ARGININE KINASE IN THE SALIVARY GLANDS OF THE MALARIA VECTOR, ANOPHELES DARLINGI

Duarte, E. C. & Marinotti, O.

Departamento de Parasitologia, ICB - USP, São Paulo – SP, Brazil.

Anopheles darlingi is the main vector of malaria in Brazil. Their salivary glands are directly involved in transmission of malaria parasites. The sporozoites, infective forms of Plasmodium sp, are located in the salivary glands before being inoculated in vertebrates hosts during mosquito feeding. We have investigated the expression of genes in the salivary glands of Anopheles darlingi. Two cDNAs were partially characterized. One of them, corresponds to a glutathione S-transferase (GST) cDNA, showing high similarity to a previously studied Anopheles gambiae GST. Glutathione S-tranferase is a family of enzymes catalyzing the conjugation of reduced glutathione (GSH) to a wide variety of eletrophilic compounds. In insects GSTs have been associated with insecticide resistance. The activity of GST in the salivary glands were detected by biochemical analysis utilizing like substrate the 1-chloro-2,4-dinitrobenzene (CDNB). The activity of GST in the salivary glands extract showed higher then in mosquito extract. The sequence of the second cDNA displays similarity to arginine kinase (AK) cDNAs. AK is a member of the phosphagen kinase, which reversibly catalyze the transfer of phosphate from phosphoguanidine to ADP, yielding ATP, in cells which have variable metabolic outputs such as muscle, neurons, transport epithelia, photoreceptores and spermatozoa. Antibodies against Drosophila melanogaster muscle AK were used to identify the protein in the salivary glands of Anopheles darlingi. The anti Drosophila melanogaster AK serum recognizes specifically a polypeptide with approximately 40 KDa in Anopheles darlingi salivary glands extract.

Supported by: FAPESP.

VT-54 – COMPARATIVE WESTERN BLOT ANALYSIS OF THE MIDGUTS OF MALARIA MOSQUITO VECTORS

¹Macedo, E. M., ²Shahabuddin, M., ²Fields, I., ²Wang, P., ³Lima, J.B.P., ⁴Braga, E.M. & ¹Pimenta, P.F.P.

¹Laboratório de Entomologia Médica, Centro de Pesquisas René Rachou – CPqRR Fundação Oswaldo Cruz – FIOCRUZ P.O. Box 1743. Av. Augusto de Lima 1715, Belo Horizonte – MG, ²Laboratory of Parasitic Diseases, NIAID-NIH, Bethesda, MD- USA and ³US Army Medical Research Center Unit, Instituto de Biologia do Exército, Rio de Janeiro, Brasil. Departamento of Parasitology, Universidade Federal de Minas Gerais, Belo Horizonte, MG., Brazil. ([e-mail])

The development of malarial Plasmodium in mosquitoes is related to several conditions involved in the interaction between the parasite and the vectors. The parasites are ingested with the bloodmeal and are initially located inside the mosquito midgut. Inside the midgut the parasites have to deal with temperature change, pH, peritrophic matrix and digestive enzymes until they can differentiate to ookinetes and become able to attach and cross the intestinal epithelium into the hemocel. The attachment of the parasites occur directly to the microvilli of the epithelial cells and the initial invasion throughout the Ross cells, as described by (Shahabuddin and Pimenta, 1998. PNAS-USA 95: 3385-3389). Preliminary studies of Altaf et. al. (Infection and Immunity 1994. 62: 316-318) have demonstrated that antibodies against the mosquito midgut disturb the invasion process and reduce the number of oocists. In this study we used monoclonal antibodies produced against the Aedes aegypti midgut, to see the expression of similar components in the midguts of: Anopheles gambiae, An. aquasalis and Ae. fluvl1iatilis; all of which are natural mosquito vectors of distinct species of Plasmodium. The initial binding of the antibodies to the midguts was seen using immunofluorescence assays. It was observed that the midguts of the all the studied mosquitoes bound the antibodies raised against Ae. aegypti. idgut extracts from the same mosquitoes were submitted to SDS-PAGE and Western blot analysis. Comparative observation demonstrated that Ae. fluviatilis has a midgut protein pattern distinct from Ae. aegypti but similar to An. gambiae and An. aquasalis. The identification of these proteins and elucidation of their role in the interaction of the mosquitoes with Plasmodium will be attempted in future studies.

Financial support: FIOCRUZ, WHO, CNPq and PRONEX.

VT-55 – BLOOD MEAL INDUCED DIGESTIVE ENZYMES IN THE MIDGUT OF BRAZILIAN MALARIA VECTORS

caroci, A. de S.*; Valle, D.°; Marinotti, O.* & Ribolla, P.E.M.*

*Departamento de Parasitologia, ICB II – USP, São Paulo – SP, Brazil

°Fundação Instituto Osvaldo Cruz, Manguinhos – RJ, Brazil

We have analyzed the presence of several digestive enzymes in the midgut of blood fed anopheline mosquitoes (Anopheles darlingi, Anopheles aquasalis and Anopheles albitarsis). The major digestive protease activity, trypsin, was found restricted to the posterior portion of the midgut. The trypsin activity increases rapidly after blood ingestion and attains a maximum between 18 and 24 hours after feeding. In the midgut of sugar fed mosquitoes, only about 7% of the maximum trypsin activity are found.

Amonipeptidase activity is also restricted to the posterior midgut, however presents a different induction profile after blood meal. The aminopeptidase activity starts to increase only at 3 hours after blood ingestion. The maximum activity of the aminopeptidase seems to be delayed compared with trypsin.

Different from the proteases, a-glucosidase activity is high in the midgut of sugar fed mosquitoes (about 50% of the maximum in blood insects). Just after blood ingestion, a-glucosidase activity in the anterior portion of the midgut decreases, indicating that the enzyme is carried to the posterior region with blood. The maximum level of a-glucosidase activity in blood fed mosquitoes is only 1.5 times higher than in sugar fed mosquitoes.

Our data indicate some differences on the pattern of digestive enzymes induction of Brazilian anophelines, when compared to the Africa and Asian mosquitoes (Anopheles gambiae and Anopheles stephensi). These differences could influence the capacity of Brazilian mosquitoes to transmit malaria, since the parasites have to survive and suffer modifications in the midgut lumen.

The study of gut specific genes coding for proteases and other enzymes appears as a good model not only because the use of a mosquito defective in proteases could lead to an interference with the parasite penetration through the peritrophic membrane, but also, and most important, because gut specific blood meal induced promoters are good candidates for driving the expression of molecules able to block the parasite invasion inside the insect.

Financial Support: FAPESP

VT-56 – EFFECTS ON THE VIABILITY OF ANOPHELES ALBITARSIS EGGS TREATED WITH SCLEROTIZATION INHIBITORS

Cardozo, Sabrina Gonçalves de Carvalho (1, 2); Jesus Martins, Ademir Jr. (1, 2), Pereira da Cunha, Marcella (2); Pereira Lima, José Bento (2); and Valle, Denise (1, 2)

(1) Departamento de Entomologia/FIOCRUZ-RJ, (2) USAMRU-B / Instituto de Biologia do Exército-RJ

The construction of genetically modified Anopheles mosquitoes, refractory to the Plasmodium, appears as an alternative to malaria control. To attain this objective, the injection of exogenous DNA into eggs and the cryopreservation of embryos from different mosquito mutant and transgenic lines are some of the requirements to be achieved. In both cases a chorion permeabilization method is necessary, since mosquito eggshell is extremely sclerotized.

The action of some sclerotizing inhibitors on eggshell layers hardening and darkening has been tested (Jesus-Martins et al, 1999). However, it is also necessary to determine the role of these drugs on mosquito development, since reduction of viability could impair attempts of using these sclerotizing inhibitors on both exogenous DNA injection and embryo cryopreservation.

The effect on egg viability of different sclerotization inhibitors was tested since oviposition. In some cases several concentrations were used. The combined effect of sclerotization inhibitors and an antioxidant was also tested. Samples were analysed daily to record larvae hatching. We verified that, in the conditions established for an adequate hardening and darkening inhibition, eggs hatch at least as efficiently as controls.

We intend to test if the whole mosquito development is also normal when sclerotizing inhibitors are used during embryogenesis. This includes the efficacy of attaining adult stage, of mating and egglaying.

Supported by: UNDP / WORLD BANK / WHO, PAPES II Program (FIOCRUZ), USAMRU-B.

VT-57 – COMPARATIVE BIOCHEMICAL STUDY OF THE AEDES FLUVIATILIS MIDGUTS INFECTED OR NOT WITH PLASMODIUM GALLINACEUM

¹Carvalho, J.C., ²Braga, E.M., ¹Macedo, M.E. and ¹Pimenta, P.F.P.

¹Laboratory of Medical Entomology, Centro de Pesquisas Rene Rachou - CPqRR, Fundação Oswaldo Cruz- FIOCRUZ. Av. Augusto de Lima 1715, Belo Horizonte-MG and ²Department of Parasitology, Universidade Federal de Minas Gerais, Brazil.e-mail: [e-mail] and [e-mail]

The main goal of this study was to detect possible differences during the process of the bloodmeal digestion between Aedes aegypti mosquitoes, infected or not with Plasmodium gallinaceum. The mosquito midgut contents were submitted to SDS-PAGE and Western blot analysis. In order to process the experiments, mosquitoes were fed on normal chicken or on P. gallinaceum infected chicken. Three groups were analyzed: (1) mosquitoes fed on normal chicken; (2) mosquitoes fed on P. gallinaceum infected chicken; and (3) 15 day P. gallinaceum infected mosquitoes fed on normal chicken. The midguts were dissected at different times after the bloodmeal (immediately, 3h, 6h, 12h, 24h, 48h and 72h) and macerated in 0.05% Tween/PBS in the presence of protease inhibitors. The midgut extracts were submitted to 10% SDS-PAGE and analyzed. There were no visualized bands in the midgut extracts collected after 48h suggesting the total digestion of the bloodmeal after this time. Several protein bands (209, 182, 120, 68, 57 e 40 kDa) were visualized only on the groups 2 and 3, originated from infected mosquitoes or from mosquitoes fed on infected bloodmeal. Therefore, midgut extracts (3h after the bloodmeal) from the three mosquito groups were processed for Western blot analysis using antibodies against P. gallinaceum raised in mouse or obtained from the serum of infected chicken. A specific 120 kDa protein band was recognized by the mouse antibody and two bands, 73 and 81 kDa, recognized by the infected chicken serum. There was no detection of any protein bands in the midgut extracts from the group 3, the normal mosquitoes fed on the normal blood. Further studies are necessary to characterize these differences and to determine which proteins are those specific expressed during the blood digestion of the infected mosquitoes.

Financial support: FIOCRUZ, WHO, CNPq and PRONEX.

VT-58 – PARTIAL PURIFICATION OF AEDES AEGYPTI GUT CHITINASE

¹Souza-Neto, J.A, ²Gomes, V.M. ¹Abreu, T.A, Pimenta, ³P.F.P. & ¹Lemos, F.J.A.

¹Laboratório de Biotecnologia, ²Laboratório de Fisiologia e Bioquímica de Microrganismos; Universidade Estadual do Norte Fluminense, CEP: 28015-620 – Campos, RJ. Centro de Pesquisas René Rachou – CPqRR; Fundação Oswaldo Cruz – FIOCRUZ, P.O. Box 1743, CEP 30000, Belo Horizonte, MG, Brazil.

The peritrophic matrix (PM) is a extracelular chitin-protein structure present in the midgut of the mosquitoes involving the food bolus. It is recognized as a potential physical barrier to parasites which develop and multiply within the midgut. The knowledge about which set of enzymes are involved in the PM synthesis and degradation is still unknown. The aim of this study is to purify and characterize the chitinases of the midgut of A. aegypti and correlate this enzyme with the formation and degradation of the PM. Measurement of chitinolytic activity in homogenates of A. aegypti midguts, dissected at different times after blood feeding, with the substrate 4-methyllubelliferyl-D-N,N',N''-triacetylchitotriose, showed that highest enzyme activity appeared between 40-48h after the blood meal. Approximately 300 midguts from 44-48h blood fed mosquitoes were homogeneized in 0.1M Tris-HCl, pH 8.0 and centrifuged at 14000g for 10 min. The supernatant was applied to DEAE-Sepharose and two chitinase activities were separated. The highest activity, the unbound chitinase, was dialysed against 50mM acetate buffer, pH 5.5. After dialysis the sample was submitted to affinity chromatography using chitin as a matrix. The enzyme was eluted with 0.1N HCl, partially concentrated and loaded into a SDS/PAGE eletrophoresis. The eletrophoretic profile showed seven protein bands. We also identified the chitinase activity using a SDS/PAGE. After the eletrophoresis, the gel containing 0.01% glycol chitin was incubated in 0.1M acetate buffer, pH 5.0 with 1% Triton X-100. After incubation, the gel was transferred in a fresh solution of 0.01% Calcofluor in 0.5M Tris-HCl (pH 8.9). The chitinase band activity was visualized as a dark band in a fluorescent background under an UV transilluminator. Further purification steps are in progress.

Supported by CNPq and FENORTE.

VT-59 – CALIBRATION OF AN AEDES AEGYPTI INSECTICIDE RESISTANCE DETECTION METHOD BASED ON THE USE OF IMPREGNATED BOTTLES (AS RECOMENDED BY CDC) IN ORDER TO COOPERATE WITH THE DENGUE CONTROL AT BRASIL

Pereira da Cunha, Marcella (1); Lima, José Bento Pereira (1,2)

(1) USAMRU-B / Instituto de Biologia do Exército-RJ; (2) Departamento de Entomologia / FIOCRUZ-RJ.

Development of insecticide resistance is nowadays a serious problem, mainly among vectors of emerging diseases. Until 1992 it has been reported that 56 Anopheles and 39 Culex species presented some insecticide resistance, which interferes with vector control programs.

The World Health Organization recommends the use of impregnated papers as a standard evaluation method for insecticide resistance of adult mosquitoes (WHO, 1981). However, some difficulties associated with this protocol are: 1) this test has to be made with adults directly caught in the field, which poses problems concerning the age and the number of mosquitoes; 2) it is recommended that the test takes place with fed mosquitoes, which is not always possible in the laboratories; 3) results have to be recorded during 24 hours, and 4) few places in the world produce the impregnated papers and, consequently, their availability to the majority of the laboratories is very limited.

The Centers for Disease Control (CDC, Atlanta), has developed an alternative methodology to detect resistance to insecticides. This methodology is based on the use of 250 ml bottles impregnated with diluted insecticides. Some advantages of these assays are: 1) F1 mosquitoes reared in the laboratory are used, overcoming problems such as specimen number an age; 2) mosquitoes are submitted to the test without a blood meal; 3) recording of results takes only 2 hours, maximum and, 4) any technician can be trained in the bottle impregnation methodology.

We set up this alternative method in our laboratory using the susceptible Aedes aegypti Rock strain (provided by CDC) as reference lineage. The insecticides tested were malathion, fenitrothion and temephos. In our hands, 100% of mortality are attained at 30 minutes for malathion at 400 ug/bottle, 60 minutes for fenitrothion at 800 ug/bottle and 75 minutes for temephos at 900 ug/bottle.

Since we have both a reference susceptible Aedes aegypti strain and a calibrated protocol to record insecticide resistance, we intend to test this methodology on wild mosquito populations in order to assist vector control programs.

Supported by: USAMRU-B.

VT-60 – PLANT EXTRACTS INDUCE STRUCTURAL MODIFICATIONS AND PROTEIN SYNTHESIS IN PERITROPHIC MATRIX OF AEDES AEGYPTI LARVAE

*Gusmão, D.S., *Lemos, V.P., +Mathias, L., +Vieira, I.J.C., +Braz-Filho, R. & *Lemos, F.J.A.

*Laboratório de Biotecnologia, Centro de Biociências e Biotecnologia. + Setor de Produtos Naturais e Fisicoquimica Orgânica, Centro de Ciências Tecnológicas e Agropecuárias, Universidade Estadual do Norte Fluminense, Av. Alberto Lamego, 2000, Campos dos Goytacazes, RJ, Brazil.

The gut of most of the insects contains a layer of acelular material containing chitin separating the food of the epithelial cells of the gut.This layer is called peritrophic matrix (PM). Our knowledge concerning this structure is still quite incomplete. However, there is increasealy interest for the study of this structure due to its probable function as a barrier against pathogens. The most obvious PM function it is the mechanical protection of the intestinal epithelium against the damages caused by the abrasion of food particles. Peritrophic matrix of some insects also can carry out a secondary function as a barrier against the chemical attack of potentially toxic agents. The aim of our work is to test different crude alcoholic extracts of Leguminosae plants for larvicidae activity against Aedes aegypti larvae. We also ascertained alterations in the structure and protein constitution of PM caused by the extracts. We tested fourteen extracts of microorganism resistant plants. The extract of Derris urucu was the most active of all extracted tested. It promoted the death of 100% of the larvae at 250 ug/ml. The other toxic plants were Dalbergia nigra, Mirocarpus fastiatus and Zollernia illicifolia. The larvae submitted to the extract of these three plant species excreted long feces, being constituted mostly by entire PMs indicating hese extracts probably contain a chitinase inhibidor. PMs, presents in the feces of larvas maintained in the presence of active extracts, were collected and its protein content was analyzed by SDS/PAGE. We observed that these extract seems to induce novel PM proteins. Second instar larvae maintained in the presence of 150ug/ml of the leaf extracts of Myrocarpus fastigatus, Dalbergia nigra and Zollernia illicifolia had their development retarded. By light microscopy, we observed that the structure of PM is strongly altered by these extracts. These extracts are been fractioned phytochemically to identify the active components responsible for the observed activity against A. aegypti larvae.

Supported by CNPq and FENORTE.

VT-61 – IDENTIFICATION OF HEME BINDING PROTEINS IN PERITROPHIC MATRIX OF AEDES AEGYPTI

¹Lemos, V.P, ²Petretski, M.D.A., ³Oliveira, P.L., ²Silva, J.R., ⁴Jacobs-Lorena, M. & ¹Lemos, F.J.A.

¹Laboratório de Biotecnologia and ²Laboratório de Química e Função de Proteínas e Peptídeos, CBB, UENF, Campos, RJ. ³Departamento de Bioquímica Médica, CCS, UFRJ, Rio de Janeiro, RJ, Brazil. ⁴Department of Genetics, School of Medicine, CWRU, Cleveland, USA.

An important event is observed in the adult females mosquitoes in response to the blood meal: the formation of an extracellular matrix containing chitin, separating ingested food from the gut epithelial cells. This layer is called peritrophic matrix (PM). Our goal is to investigate the PM physiological role as a chemical barrier to the potencially toxic heme released from ingested red cell hemoglobin. By spectrophotometry, we showed that the amount of heme stays practically constant in the gut during digestion. Histochemical studies revealed that around 12 hr after blood meal (abm) a discrete and uniform layer surrounding the blood bolus could be seen. At 14hr abm the PM was thicker and yellow and 24h abm the PM was completely formed with an intense yellow color. The ultrastructural observations confirmed the results described above showing electron-dense inclusions characteristic of heme deposition on the PM at 24hr abm. We could also observe the enormous net of microfibers of which PM is constituted and how complex it is at this time (24h). We also carried out `in vitro' heme binding studies. Isolated dissected PM can bind nearly 5% of the heme present in the midgut. Several heme-binding protein bands were detected in polyacrilamide gels stained with dimethoxybenzidine. By Coomassie staining method, the eletrophoresis profile showed the presence of two predominant A. aegypti PM heme-binding proteins. Additional studies are being carrying out in order to purify, to characterize and to understand the physiological antioxidant role of these proteins.

Supported by FENORTE and PRONEX.

VT-62 – ANALYSIS OF SALT STRESS PROTEIN RESPONSE IN PERITROPHIC MATRIX OF AEDES AEGYPTI LARVAE

Gusmão, D.S. & Lemos, F.J.A.

Laboratório de Biotecnologia, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense, Campos dos Goytacazes, R.J., CEP 28015-620, Brasil

The distribution of mosquitoes is largely based on the availability of suitable sites for larval growth. Because mosquitoes larvae are aquatic, water is essential. However, water sources vary considerably in composition. Mosquitos larvae have developed physiological mechanisms that permit them to survive in extreme conditions in terms of salt composition. Mosquitoes like other hematophagous insects synthesize a peritrophic matrix containing chitin, protein, glycoprotein and proteoglycan that separate the ingested food from the gut. Proteoglycans and glycoproteins could play na importante role in regulating the movement of water and ions across the PM. Depending of the mosquito life stages, the PM is formed from a secretion produced by midgut epithelial cells (PM I, in adults), or by specialized cells of the cardia, at the junction of the foregut and midgut (PM II, in larvae). It can be an important physical and chemical barrier in the insect gut. We decided to investigate a possible alteration of the protein composition of the Aedes aegypti PM after submission to salt stress. The PMs, present in fecal pellet, were collected and their protein content was analyzed by SDS-PAGE. We observed that PM proteins were induced in larvae maintained in concentrations of NaCl between 200 mM and 520 mM. Also, we observed the induction of novel PM proteins in larvae maintained in concentration of 260 mM of KCl and CaCl2. The larvae when maintained under stress conditions produce great amount of feces. The first step of purification of these proteins was accomplished through a affinity cromatography using chitin as a matrix. With further characterization and aminoacid sequecing of these proteins it would de possible to identify their physiological role.

Supported by FENORTE.

Publication Dates

• Publication in this collection
  25 Aug 2000
• Date of issue
  Nov 1999