Determination of insecticides' lethal concentrations and metabolic enzyme levels in Triatoma dimidiata.

OBJECTIVE: The feasibility of the use of WHO impregnated paper and biochemical assays to determine lethal concentrations (LC50 and LC99) and insecticide metabolic enzyme levels of Triatoma dimidiata. MATERIALS AND METHODS: LC50 and LC99 were calculated with WHO papers impregnated at different concentrations of malathion, propoxur and deltamethrin; the percentage of insensitive acetylcholinesterase (iAChE); and the levels of esterases, glutathione S-transferases, and monooxygenases in laboratory nymphs of the first stage (5 to 7 days), were undertaken using the WHO biochemical assays. RESULTS: Respectively the LC50 and LC99 µg/cm2 obtained for malathion were 43.83 and 114.38, propoxur 4.71 and 19.29, and deltamethrin 5.80 and 40.46. A 30% of the population had an iAChE, and only a few individuals had high P450 and ß-eterase levels. CONCLUSIONS: Impregnated papers and biochemical tests developed by WHO for other insects, proved to be feasible methods in monitoring insecticide resistance and metabolic enzymes involved in T. dimidiata.

OBJETIVO: La factibilidad de usar los papeles impregnados y ensayos bioquímicos según la OMS para determinar concentraciones letales (CL50 y CL99) y niveles enzimáticos en la resistencia a insecticidas en Triatoma dimidiata. MATERIAL Y MÉTODOS: Se calcularon la CL50 y CL99 con papeles impregnados según la OMS a diferentes concentraciones de malatión, propoxur y deltametrina; el porcentaje de acetilcolinesterasa insensible (iAChE); y los niveles de esterasas, glutatión S-transferasas, y monooxigenasas en ninfas de laboratorio del estadio I (5-7 días) se determinaron usando los ensayos bioquímicos según la OMS. RESULTADOS: Se obtuvieron las CL50 y CL99 µg / cm2 respectivamente para malatión 43.83 y 114.38, propoxur 4.71 y 19.29, y deltametrina 5.80 y 40.46. Un 30% de las chinches tuvo iAChE, y sólo pocos individuos tuvieron niveles superiores de P450 y ß-eterasas. CONCLUSIONES: Los papeles impregnados y ensayos bioquímicos que describe la OMS para otros insectos demostraron ser métodos factibles para monitorear la resistencia a insecticidas y las enzimas metabólicas involucradas en T. dimidiata.

Effect of the urease-derived peptide Jaburetox on the central nervous system of Triatoma infestans (Insecta: Heteroptera).

BACKGROUND: Triatoma infestans is the main vector of Chagas'disease in Southern Cone countries. In triatomines, symptoms suggesting neurotoxicity were observed after treatment with Jaburetox (Jbtx), the entomotoxic peptide obtained from jackbean urease. Here, we study its effect in the central nervous system (CNS) of this species. METHODS: Immunohistochemistry, Western blots, immunoprecipitation, two-dimensional electrophoresis, tandem mass spectrometry and enzymatic assays were performed. RESULTS: Anti-Jbtx antibody labeled somata of the antennal lobe only in Jbtx-treated insects. Western blot assays of nervous tissue using the same antibody reacted with a 61kDa protein band only in peptide-injected insects. Combination of immunoprecipitation, two-dimensional electrophoresis and tandem mass spectrometry identified UDP-N-acetylglucosamine pyrophosphorylase (UDP-GlcNAcP) as a molecular target for Jbtx. The activity of UDP-GlcNAcP increased significantly in the CNS of Jbtx-treated insects. The effect of Jbtx on the activity of nitric oxide synthase (NOS) and NO production was investigated as NO is a recognized messenger molecule in the CNS of T. infestans. NOS activity and NO levels decreased significantly in CNS homogenates of Jbtx-treated insects. CONCLUSIONS: UDP-GlcNAcP is a molecular target of Jbtx. Jbtx impaired the activity of T. infestans nitrergic system, which may be related with early behavioral effects. GENERAL SIGNIFICANCE: We report that the CNS of Triatoma infestans is a target for the entomotoxic peptide and propose that a specific area of the brain is involved. Besides potentially providing tools for control strategies of Chagas' disease vectors our data may be relevant in various fields of research as insect physiology, neurobiology and protein function.

Salivary antigen-5/CAP family members are Cu2+-dependent antioxidant enzymes that scavenge O2₋. and inhibit collagen-induced platelet aggregation and neutrophil oxidative burst.

The function of the antigen-5/CAP family of proteins found in the salivary gland of bloodsucking animals has remained elusive for decades. Antigen-5 members from the hematophagous insects Dipetalogaster maxima (DMAV) and Triatoma infestans (TIAV) were expressed and discovered to attenuate platelet aggregation, ATP secretion, and thromboxane A2 generation by low doses of collagen (<1 µg/ml) but no other agonists. DMAV did not interact with collagen, glycoprotein VI, or integrin α2ß1. This inhibitory profile resembles the effects of antioxidants Cu,Zn-superoxide dismutase (Cu,Zn-SOD) in platelet function. Accordingly, DMAV was found to inhibit cytochrome c reduction by O2[Symbol: see text] generated by the xanthine/xanthine oxidase, implying that it exhibits antioxidant activity. Moreover, our results demonstrate that DMAV blunts the luminescence signal of O2[Symbol: see text] generated by phorbol 12-myristate 13-acetate-stimulated neutrophils. Mechanistically, inductively coupled plasma mass spectrometry and fluorescence spectroscopy revealed that DMAV, like Cu,Zn-SOD, interacts with Cu(2+), which provides redox potential for catalytic removal of O2[Symbol: see text]. Notably, surface plasmon resonance experiments (BIAcore) determined that DMAV binds sulfated glycosaminoglycans (e.g. heparin, KD ~100 nmol/liter), as reported for extracellular SOD. Finally, fractions of the salivary gland of D. maxima with native DMAV contain Cu(2+) and display metal-dependent antioxidant properties. Antigen-5/CAP emerges as novel family of Cu(2+)-dependent antioxidant enzymes that inhibit neutrophil oxidative burst and negatively modulate platelet aggregation by a unique salivary mechanism.

Toxicological and biochemical analysis of the susceptibility of sylvatic Triatoma infestans from the Andean Valley of Bolivia to organophosphate insecticide.

To increase our knowledge of the natural susceptibility of Triatoma infestans to an organophosphate insecticide, we performed toxicological and biochemical studies on three sylvatic populations from Bolivia and two populations from domestic dwellings from Bolivia and Argentina. Fifty-per-cent lethal doses (LD50) were determined based on the topical application of fenitrothion on first instar nymphs and mortality was assessed at 24 h. Both type of populations exhibited LD50ratios significantly higher than 1 with a range of the values (1.42-2.47); the maximum value were found in a sylvatic (-S) population, Veinte de Octubre-S. Samples were biochemically analysed using a glutathione S-transferase activity assay. The highest significant activity was obtained for Veinte de Octubre-S and the lowest activity was obtained for the reference population (102.69 and 54.23 pmol per minute per mg of protein respectively). Two out of the three sylvatic populations (Veinte de Octubre-S and Kirus Mayu-S) exhibited significantly higher glutathione S-transferase activity than that of the reference population. Based on this analysis of the natural susceptibility of this organism to organophosphate insecticides, continental and focal surveys of organophosphate susceptibility should be conducted to evaluate the evolution and distribution of this phenomenon.

Analysing deltamethrin susceptibility and pyrethroid esterase activity variations in sylvatic and domestic Triatoma infestans at the embryonic stage.

The aim of the present work was to study the deltamethrin susceptibility of eggs from Triatoma infestans populations and the contribution of pyrethroid esterases to deltamethrin degradation. Insects were collected from sylvatic areas, including Veinte de Octubre and Kirus-Mayu (Bolivia) and from domiciliary areas, including El Palmar (Bolivia) and La Pista (Argentina). Deltamethrin susceptibility was determined by dose-response bioassays. Serial dilutions of deltamethrin (0.0005-1 mg/mL) were topically applied to 12-day-old eggs. Samples from El Palmar had the highest lethal dose ratio (LDR) value (44.90) compared to the susceptible reference strain (NFS), whereas the Veinte de Octubre samples had the lowest value (0.50). Pyrethroid esterases were evaluated using 7-coumaryl permethrate (7-CP) on individually homogenised eggs from each population and from NFS. The El Palmar and La Pista samples contained 40.11 and 36.64 pmol/min/mg protein, respectively, and these values were statistically similar to NFS (34.92 pmol/min/mg protein) and different from Kirus-Mayu and Veinte de Octubre (27.49 and 22.69 pmol/min/mg protein, respectively). The toxicological data indicate that the domestic populations were resistant to deltamethrin, but no statistical contribution of 7-CP esterases was observed. The sylvatic populations had similar LDR values to NFS, but lower 7-CP esterase activities. Moreover, this is the first study of the pyrethroid esterases on T. infestans eggs employing a specific substrate (7-CP).

Cholinesterase activity during embryonic development in the blood-feeding bug Triatoma patagonica.

Triatoma patagonica Del Ponte (Hemiptera: Reduviidae), a vector of Chagas' disease, is widely distributed in Argentina and is found in sylvatic and peridomiciliary ecotopes, as well as occasionally in human dwellings after the chemical control of Triatoma infestans. Anti-cholinesteratic products can be applied in peridomiciliary areas and thus knowledge of cholinesterase activity during embryonic development in this species might contribute further information relevant to effective chemical control. Cholinesterase activity was characterized by reactions to eserine 10(-5) m, to increasing concentrations of substrate and to varying centrifugal speeds. Acetylcholinesterase activity was detected on day 4 and was significant from day 5. A reduction in cholinesterase activity towards acetylthiocholine (ATC) was observed on days 9 and 10 of development. Cholinesterase activity towards ATC and butyrylthiocholine (BTC) in homogenates of eggs was inhibited by eserine 10(-5) m. The shape of the curve indicating levels of inhibition at different concentrations of ATC was typical of acetylcholinesterase. Activity towards BTC did not appear to be inhibited by excess substrate, which parallels the behaviour of butyrylcholinesterases. Cholinesterase activity towards ATC was reduced in supernatant centrifuged at 15 000 g compared with supernatant centrifuged at 1100 g. The cholinesterase system that hydrolyzes mainly ATC seems to belong to the nervous system, as indicated by its behaviour towards the substrates assayed, its greater insolubility and the fact that it evolves parallel to the development of the nervous system. Knowledge of biochemical changes associated with the development and maturation of the nervous system during embryonic development would contribute to the better understanding of anti-cholinesteratic compounds with ovicidal action that might be used in control campaigns against vectors of Chagas' disease.

Insecticide Resistance Mechanisms in Triatoma infestans (Reduviidae: Triatominae): The Putative Role of Enhanced Detoxification and Knockdown Resistance (kdr) Allele in a Resistant Hotspot From the Argentine Chaco.

Chagas disease affects around 6 million people in the world, and in Latin America, it is mainly transmitted by the kissing bug. Chemical control of the vector with pyrethroid insecticides has been the most frequently used tool to reduce the disease incidence. Failures of field control have been detected in areas of the Argentinian Gran Chaco that correlate with high levels of insecticide resistance. Here, we provide evidence of the mechanisms involved in the resistance to insecticides of field populations of T. infestans from General Güemes Department (Chaco Province, Argentina). The biochemical analysis suggests the increase in the activity of the degradative enzymes P450 oxidases and esterases as a minor contributive mechanism in low-resistance populations. The molecular study revealed high frequencies of the kdr L925I mutation at the voltage-gated sodium channel as responsible for the high resistance ratios detected. This knowledge contributes to the generation of comprehensive vector control strategies that reduce the incidence of the disease.

Novel Kdr mutations (K964R and A943V) in pyrethroid-resistant populations of Triatoma mazzottii and Triatoma longipennis from Mexico and detoxifying enzymes.

Although having five different ways of transmission the vector-borne is the principal way of transmission of Chagas disease, which involves insects of the subfamily Triatominae (Hemiptera: Reduviidae). Nineteen of the 31 species that occur in Mexico are associated with humans, and all are capable of transmitting the disease. Pyrethroids are the insecticides recommended for the control of these vectors in Mexico. We determined the susceptibility to the pyrethroids deltamethrin and permethrin of peridomestic populations of Triatoma mazzottii Usinger and two populations of Triatoma longipennis Usinger in comparison with a reference strain for each species. Bioassays were performed for the determination of the LD50 for both field populations and reference strains. A maximum of 27 fold resistance to deltamethrin was observed in T. mazzottii, meanwhile, for permethrin, T. longipennis from Jalisco show the highest value of 3.19 fold. There was significantly increased activity of esterases in field populations in comparison with their corresponding reference strain. The results of the search of kdr mutations related to the resistance to deltamethrin and permethrin in the evaluated species show the presence of mutations in the field populations, as is the case with individuals of T. mazzottii, for which the mutation was found A943V, and for the two populations of T. longipennis included in this study, we report the presence of the kdr mutation K964R. Evaluation of the various mechanisms involved in resistance to pyrethroids in triatomines from Mexico could guide us to the real justification for insecticide resistance monitoring.

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

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

Relative contribution of monooxygenase and esterase to pyrethroid resistance in Triatoma infestans (Hemiptera: Reduviidae) from Argentina and Bolivia.

Recently, high resistance to pyrethroid insecticides has been associated with ineffective field treatments against Triatoma infestans (Klug) (Hemiptera: Reduviidae) in northern Argentina. Samples were collected from two areas in Argentina (Salta and La Rioja) and one are in Bolivia (Yacuiba), and they were subjected to toxicological and biochemical assays. All populations were resistant to deltamethrin, but they showed different profiles to nonpyrethroid insecticides. The Salta population showed high resistance ratios (RRs) to deltamethrin and only slight differences in the susceptibility to fenitrothion and fipronil compared with the reference strain. Otherwise, the La Rioja population showed a lower RR to deltamethrin and no resistance to fenitrothion or fipronil. Finally, the Yacuiba population had high a RR to deltamethrin, but it was susceptibility to fenitrothion and fipronil. In several cases, deltamethrin-resistant populations had higher susceptibility to bendiocarb than the reference strain. Measured activity of P450 monooxygenase in individual insects (based on ethoxycoumarine-O-deethylase), tended to be higher in the deltamethrin-resistant populations, but the differences were not statistically significant. Activity of specific esterases determined by the hydrolysis of 7-coumaryl permethrate demonstrated an increase in the percentage of insects with higher esterase activity in the Salta and La Rioja populations. Unexpectedly, the Yacuiba population showed lower pyrethroid esterase activity than the reference strain. The different pyrethroid resistance patterns found in T. infestans from three geographical regions within Argentina and in Bolivia suggests that enzyme-based pyrethroid resistance in this species has multiple origins. Nevertheless, because nerve insensitivity (related to the presence of the kdr gene) is also an important mechanism related to pyrethroid resistance, further studies on the kdr gene should be carried to clarify the relative contribution of each pyrethroid-associated mechanism in deltamethrin-resistant populations of T.

Evolutionary relationships based on genetic and phenetic characters between Triatoma maculata, Triatoma pseudomaculata and morphologically related species (Reduviidae: Triatominae).

The maculata group currently comprises two species of Triatominae, Triatoma maculata and Triatoma pseudomaculata, which share morphologic and chromatic characteristics. In order to clarify the systematic status of these two vectors of Trypanosoma cruzi and to infer their evolutionary relationships, we performed an enzymatic, morphometric and cytogenetic comparison of them, also taking into account two sister species not included in the group (T. arthurneivai and T. wygodzinskyi). According to our results, T. maculata and T. pseudomaculata belong to distinct evolutionary lineages. Similarly, T. arthurneivai topotypes from Minas Gerais form an independent isolated group by morphometrics. Our results also support the specific status of the Triatoma population from São Paulo State (formerly referred to T. arthurneivai), and suggest the possibility that it is T. wygodzinskyi. Finally, we suggest that only the arboricolous T. pseudomaculata from northeast Brazil and the rupicolous sister species originated from São Paulo State should be classified together in the same group.

Salivary apyrases of Triatoma infestans are assembled into homo-oligomers.

Apyrase activity is present in the saliva of haematophagous arthropods. It is related to blood-feeding because of the apyrase ability to hydrolyse ADP, a key component of platelet aggregation. Five apyrases with apparent molecular masses of 88, 82, 79, 68 and 67 kDa were identified in the saliva of the vector of Chagas disease, Triatoma infestans. The large size observed during purification of these enzymes suggested oligomerization. In the present study, we confirmed, using gel-filtration and analytical ultracentrifugation, the presence of apyrase oligomers with molecular masses of 200 kDa in the saliva. Electrophoretic analyses showed that disulphide bonds were involved in homo-oligomerization. In addition, heterogeneity in disulphide bonds and in pI was detected, with the pI ranging from 4.9 to 5.4. The present study gives the first insights into the quaternary structure of soluble apyrases.

Toxicological, Enzymatic, and Molecular Assessment of the Insecticide Susceptibility Profile of Triatoma infestans (Hemiptera: Reduviidae, Triatominae) Populations From Rural Communities of Santa Cruz, Bolivia.

A wide range of insecticide resistance profiles has been reported across Bolivian domestic and sylvatic populations of Triatoma infestans (Klug, 1834) (Hemiptera, Reduviidae), including some with levels proven to be a threat for vector control. In this work, the insecticide profile of domestic T. infestans was studied with standardized toxicological bioassays, in an area that has not undergone consistent vector control. F1 first-instar nymphs hatched in laboratory from bugs captured in three communities from the Santa Cruz Department were evaluated with different insecticides. Moreover, the enzymatic activity of esterases and cytochrome P450 monooxygenases was measured in individual insects to evaluate the possible mechanism of metabolic resistance to pyrethroids. In addition, the DNA sequence of sodium channel gene (kdr) was screened for two point mutations associated with pyrethroid resistance previously reported in T. infestans.All populations showed reduced susceptibility to deltamethrin and α-cypermethrin, albeit the RR50 values varied significantly among them. Increased P450 monooxygenases and permethrate esterases suggest the contribution, as detoxifying mechanisms, to the observed resistance to deltamethrin in all studied populations. No individuals presented either mutation associated to resistance in the kdr gene. The level of susceptibility to α-cypermethrin, the insecticide used by the local vector control program, falls within an acceptable range to continue its use in these populations. However, the observed RR50 values evidence the possibility of selection for resistance to pyrethroids, especially to deltamethrin. Consequently, the use of pyrethroid insecticides should be closely monitored in these communities, which should be kept under entomological surveillance and sustained interventions.

Molecular taxonomic study of Chagas disease vectors from the phyllosoma, lecticularia, and rubrofasciata complexes.

The Triatiominae (Hemiptera: Reduviidae) are hematophagous hemipters of importance because they transmit Trypanosoma cruzi, the causal agent of Chagas disease. The aim of this study was to define the possible relationships between species of the Phyllosoma complex (Triatoma mazzottii, Triatoma pallidipennis, and Triatoma longipennis) and species of other complexes present in Mexico that have not been previously analyzed (Triatoma lecticularia and Triatoma rubida). In addition, it was determined the inclusion of Triatoma bassolsae in the Phyllosoma complex by using 10 isoenzymatic systems (corresponding to the 14 loci). Results of isoenzymatic study show that between the species of the Phyllosoma complex including Triatoma bassolsae, the polymorphism of the analyzed enzymes ranges from 14% to 50% (P < or = 0.95) and the species from external complexes showed polymorphism values of 43% (Triatoma lecticularia), 43% (Triatoma rubida), and 36% (Triatoma infestans). The genetic tree shows a clear difference between species of the Phyllosoma complex and the other complexes.

Determination of insecticides' lethal concentrations and metabolic enzyme levels in Triatoma dimidiata / Determinación de concentraciones letales y niveles de enzimas metabólicas de insecticidas en Triatoma dimidiata

Abstract: Objective: The feasibility of the use of WHO impregnated paper and biochemical assays to determine lethal concentrations (LC50 and LC99) and insecticide metabolic enzyme levels of Triatoma dimidiata. Materials and methods: LC50 and LC99 were calculated with WHO papers impregnated at different concentrations of malathion, propoxur and deltamethrin; the percentage of insensitive acetylcholinesterase (iAChE); and the levels of esterases, glutathione S-transferases, and monooxygenases in laboratory nymphs of the first stage (5 to 7 days), were undertaken using the WHO biochemical assays. Results: Respectively the LC50 and LC99 µg/cm2 obtained for malathion were 43.83 and 114.38, propoxur 4.71 and 19.29, and deltamethrin 5.80 and 40.46. A 30% of the population had an iAChE, and only a few individuals had high P450 and β-eterase levels. Conclusion: Impregnated papers and biochemical tests developed by WHO for other insects, proved to be feasible methods in monitoring insecticide resistance and metabolic enzymes involved in T. dimidiata.

Resumen: Objetivo: La factibilidad de usar los papeles impregnados y ensayos bioquímicos según la OMS para determinar concentraciones letales (CL50 y CL99) y niveles enzimáticos en la resistencia a insecticidas en Triatoma dimidiata. Material y métodos: Se calcularon la CL50 y CL99 con papeles impregnados según la OMS a diferentes concentraciones de malatión, propoxur y deltametrina; el porcentaje de acetilcolinesterasa insensible (iAChE); y los niveles de esterasas, glutatión S-transferasas, y monooxigenasas en ninfas de laboratorio del estadio I (5-7 días) se determinaron usando los ensayos bioquímicos según la OMS. Resultados: Se obtuvieron las CL50 y CL99 µg / cm2 respectivamente para malatión 43.83 y 114.38, propoxur 4.71 y 19.29, y deltametrina 5.80 y 40.46. Un 30% de las chinches tuvo iAChE, y sólo pocos individuos tuvieron niveles superiores de P450 y β-eterasas. Conclusión: Los papeles impregnados y ensayos bioquímicos que describe la OMS para otros insectos demostraron ser métodos factibles para monitorear la resistencia a insecticidas y las enzimas metabólicas involucradas en T. dimidiata.

Isoenzyme variability of five principal triatomine vector species of Chagas disease in Mexico.

Triatoma barberi, T. dimidiata, T. longipennis, T. pallidipennis and T. picturata, all key Chagas disease vectors in Mexico, were analysed by multilocus enzyme electrophoresis (MLEE) at 17 putative loci. The majority of insect specimens studied were collected from domestic and peridomestic structures from multiple geographic locations while others were collected from sylvatic areas. T. barberi was the least polymorphic species (P(0.95)=0.18), with polymorphism rates of the other species ranging from 0.29 to 0.50. T. barberi, a member of the protracta complex, clustered apart from the other studied species by Nei's genetic distance with >1.36, and at least eight loci were found to be diagnostic for this species. T. dimidiata was more related to T. longipennis, T. pallidipennis and T. picturata (phyllosoma complex) than to T. barberi, with a genetic distance averaging 0.36 with the phyllosoma complex species. In contrast, the genetic distances between the three phyllosoma complex species were not significantly different from zero, and there were no species-specific loci differentiating among them. The results strongly support the grouping of these three species in one complex, separate from the two other species studied.

Triapsin, an unusual activatable serine protease from the saliva of the hematophagous vector of Chagas' disease Triatoma infestans (Hemiptera: Reduviidae).

Salivary anticoagulant activities are widely distributed among hematophagous arthropods. Most of them are inhibitors of the serine proteases of the coagulation cascade. Here we show that the saliva of the exclusively hematophagous insect Triatoma infestans, an important vector in the transmission of Chagas' disease, contains an uncommon trypsin-like activity, triapsin. This novel enzyme was purified and characterized. It is a serine protease that is stored as a zymogen in the luminal content of the salivary glands D2. Triapsin is activated by trypsin treatment, or when the saliva is ejected during the insect bite. The enzyme was purified 300-fold from the released saliva by anion exchange chromatography in a HiTrap Q column, followed by chromatography in Phenyl-Superose, and Superdex HR75. The purified triapsin shows an apparent molecular mass of around 40 kDa in non-reduced SDS gels and in sieving chromatography, and 33 kDa in reduced SDS-gels. Its activity is lost after incubation with dithiothreitol indicating that cysteine bridges are essential for activity. Triapsin cleaves gelatin and synthetic substrates showing preference for arginine at P1 residues. The best p-nitroanilide substrate is isoleucyl-prolyl-arginine. It does not cleave bradykinin, angiotensin and other lysine containing substrates. The triapsin amidolytic activity against chromogenic substrates is similar to plasminogen activators, such as urokinase and tissue plasminogen activator. However, it does not activate plasminogen. The fact that triapsin is released at the bite in its active form suggests that it has a role in blood feeding.

Kinetics of expression of the salivary apyrases in Triatoma infestans.

Apyrases are nucleoside triphosphate-diphosphohydrolases that remove Pi from ATP and ADP. The blood feeding reduviid Triatoma infestans, which transmits the Trypanosoma cruzi agent of Chagas disease to animals and man, presents in its salivary glands five apyrases with molecular masses of 88, 82, 79, 68 and 67 kDa. These triatomine apyrases have been associated with the prevention of ADP induced platelet aggregation in the host. Here we provide biochemical data showing that these apyrases are stored in the lumen of the salivary gland D1 pairs, and that about one half of the pool of the enzyme is consumed during feeding. After the feeding recovery of apyrases to maximal activity level takes days, thus suggesting de novo protein synthesis. This hypothesis is supported by quantitative RT-PCR analysis which shows an upregulation of the 79 kDa apyrase mRNA level after feeding.

Genetic variability and geographic differentiation among three species of Triatomine bugs (Hemiptera-Reduviidae).

Three species of triatomine bugs, Triatoma sordida, T. guasayana, and T. patagonica, were examined by cytogenetic (C-banded karyotypes and male meiotic process) and isoenzymatic studies. These three species, with different importance as Chagas' disease vectors, were found to be closely related according to their known ethologic, ecologic, and morphologic traits. Although they have the same diploid chromosome number (2n = 22 constituted by 20 autosomes and an XY male/XX female sex mechanism), each species has a distinct chromosomal behavior during male meiosis and a specific amount and localization of C-heterochromatic blocks. Moreover, these chromosome characteristics allowed us to differentiate two T. sordida populations. Isoenzymatic data confirmed the taxonomic status of the three species and together with our cytogenetic results questioned the species homogeneity of T. sordida.

Genetic variability of Triatoma brasiliensis (Hemiptera: Reduviidae) populations.

Isoenzymes, random amplified polymorphic DNA (RAPD), and morphometry were used to compare genetic variability within and between three populations of Triatoma brasiliensis Neiva, 1911 from northeast Brazil. The isoenzyme profiles were identical for the three populations, whereas the complex RAPD patterns allowed clear discrimination between the three. Morphometric analysis, using characters of the head capsule, also showed discrimination between the three populations but only in comparisons of males, not females. The results show considerable genetic heterogeneity in T. brasiliensis, with an indication of geographic structuring possibly resulting from a recent series of domestication events.