Identification and characterization of a collagen-induced platelet aggregation inhibitor, triplatin, from salivary glands of the assassin bug, Triatoma infestans

To facilitate feeding, certain hematophagous invertebrates possess inhibitors of collagen-induced platelet aggregation in their saliva. However, their mechanisms of action have not been fully elucidated. Here, we describe two major salivary proteins, triplatin-1 and -2, from the assassin bug, Triatoma infestans, which inhibited platelet aggregation induced by collagen but not by other agents including ADP, arachidonic acid, U46619 and thrombin. Furthermore, these triplatins also inhibited platelet aggregation induced by collagen-related peptide, a specific agonist of the major collagen-signaling receptor glycoprotein (GP)VI. Moreover, triplatin-1 inhibited Fc receptor gamma-chain phosphorylation induced by collagen, which is the first step of GPVI-mediated signaling. These results strongly suggest that triplatins target GPVI and inhibit signal transduction necessary for platelet activation by collagen. This is the first report on the mechanism of action of collagen-induced platelet aggregation inhibitors from hematophagus invertebrates.     

Change with age of feulgen-DNA values in the blood-sucking insect,Triatoma infestans klug

Summary Feulgen-DNA values were evaluated cytophotometrically in the Malpighian tubes of T. infestans at the nymphal, adult and ageing life periods. In all cases the total nuclear Feulgen-DNA contents were found to be distributed within the 32C and 64C classes. However, part of the Feulgen-DNA values is shifted to lower intervals in ageing insects, as compared with nymphal and adult individuals, promoted by decrease in Feulgen-DNA values of the euchromatins. Changed response to Feulgen reaction due to some aging-induced alteration in DNP complexes, and/or breaks and loss of DNA in the euchromatins, as part of the senescence process, are ascribed to be responsible for such phenomenon.     

Identification and characterization of a new kallikrein-kinin system inhibitor from the salivary glands of the malaria vector mosquito Anopheles stephensi

A new kallikrein-kinin system inhibitor, designated anophensin, was identified in the salivary glands of the malaria vector mosquito, Anopheles stephensi. In vitro reconstitution experiments showed that anophensin inhibits activation of the kallikrein-kinin system by inhibiting the reciprocal activation of factor XII (FXII) and prekallikrein (PK), and subsequent release of bradykinin. Additionally, anophensin inhibits activation of the kallikrein-kinin system on cultured human umbilical vein endothelial cells (HUVECs). Direct binding assays show that this inhibitory effect is due to Zn(2+)-dependent specific binding of anophensin to both FXII and high molecular weight kininogen (HK). Furthermore, anophensin interacts with both the N-terminus of FXII and domain D5 of HK, which are the binding domains for biological activating surfaces. These results suggest that anophensin inhibits activation of the kallikrein-kinin system by interfering with the association of FXII and HK with biological activating surfaces, resulting in the inhibition of bradykinin release in a host animal during insect blood-feeding.     

Eye pigments of the blood-sucking insect, Triatoma infestans Klug (Hemiptera, Reduviidae).

The pigmentation of black (wild) and red (mutant) eyes of Triatoma infestans was studied spectrophotometrically and compared with red-eyed (wild) and white-eyed (mutant) forms of Drosophila melanogaster. The spectral absorption profiles of the black and red eye pigments of T. infestans were similar to each other and to that of the wild-type eyes of D. melanogaster. The similarity to the wild form of D. melanogaster indicated that both eye forms of T. infestans contained ommochromes of the xanthommatin type, a finding confirmed by ascending paper chromatography. Pteridines, melanins, and ommins were not detected as eye pigments in T. infestans. The eye color difference in T. infestans was assumed to be a function of the xanthommatin concentration, with a smaller content of ommochrome in red eyes, although this probably did not affect the insect's visual acuity. These data support other findings regarding the similarities between black- and red-eyed specimens of T. infestans for other characteristics.     

Fine structure of the salivary glands of Triatoma infestans (Hemiptera: Reduviidae)

The fine structure of the salivary glands of adult Triatoma infestans (Hemiptera: Reduviidae) bugs has been analyzed. Stereomicroscopy and scanning electron microscopy showed that each insect presents a pair of salivary glands, each pair containing three distinct units (main, supplementary, and accessory) with different sizes and colors. Transmission electron microscopy demonstrated that all gland units consist of a monolayer of epithelial cells surrounding a large central lumen. The gland units are enveloped by a thick basal lamina containing bundles of muscle cells. Microvilli are present at the apical plasma membrane domain of the gland cells, thus enlarging the available membrane area for saliva secretion towards the large gland lumen, although occasionally budding vesicles could be observed among the microvilli. Cytochemical analysis showed that the salivary gland cells of T. infestans present abundant endoplasmic reticulum profiles and several lipid droplets.     

Nervous system of Triatoma infestans

The anatomy of the adult nervous system of the haematophagous bug Triatoma infestans has been studied by means of dissections and histology. The central nervous system comprises three nervous masses: the brain + suboesophageal ganglion, the prothoracic ganglion, and the posterior fused ganglion (meso + metathoracic + abdominal ganglia). The form of the brain is determined by the tubular head and the highly developed muscles of the pharyngeal pump. The prothoracic. ganglion is located near the posternum, the posterior ganglionic mass near the mesosternum. A significative variation of the branching pattern of abdominal nerves is reported. The innervations of mouth parts, salivary glands, muscles, retrocerebral complex, spiracles, rectum, reproductive organs, alary muscles, and peripheral nerves are described. © 1994 Wiley-Liss, Inc.     

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.     

Identification and characterization of a novel factor XIIa inhibitor in the hematophagous insect, Triatoma infestans (Hemiptera: Reduviidae)

Recently, we have cloned several Kazal-type serine protease inhibitors from the midgut of the Triatoma infestans bug. A single gene composed of multi Kazal-type domains, in tandem, encodes these inhibitors. In this work, we describe the purification and characterization of recombinant infestins 3-4 and 4, which are potent factor XIIa inhibitors (KI=67 pM and 128 pM, respectively). We also identified the first native factor XIIa inhibitor from a hematophagous insect. The factor XIIa inhibitory activity of infestin 4 demonstrates extremely efficient anticoagulant activity, prolonging activated partial thromboplastin time by approximately 3 times. Our results suggest that infestins perform a very important role in the T. infestans midgut during meal acquisition and digestion by controlling blood coagulation by means of inhibiting thrombin and factor XIIa.     

The response of the blood-sucking bug Triatoma infestans to carbon dioxide and other host odours

Behavioural responses of Triatoma infestans larvae to carbon dioxide and other odours of vertebrate origin were investigated in a locomotion compensator. T. infestans oriented towards airstreams enriched with carbon dioxide exhibiting a threshold response between 300 and 400 p.p.m. above the ambient CO(2) background. The accuracy of the oriented response to carbon dioxide improved with stimulus intensity. Remarkably, insects did not show any change in their sensitivity threshold to carbon dioxide with the starvation time. The attractiveness to carbon dioxide depended on the time of the day, i.e. these nocturnal bugs only oriented towards carbon dioxide-loaded airstreams during the first hours of the scotophase. L-lactic acid did not evoke oriented responses when it was presented as a single stimulus in a wide range of intensities. However, a marked synergism was evident when L-lactic acid was combined with a sub-threshold concentration of carbon dioxide. Under this condition, the threshold response to carbon dioxide decreased to 75-150 p.p.m. above ambient CO(2) background. The isomer D-lactic acid evoked no response, either alone or in combination with carbon dioxide. When insects were stimulated with 1-octen-3-ol a significant positive orientation was found. This response was not modified by the addition of carbon dioxide.     

Repeated plasticization and recovery of cuticular stiffness in the blood-sucking bug Triatoma infestans in the feeding context

Triatominae bugs experience changes in the mechanical properties of their cuticle prior to feeding. This process-plasticization-allows a rapid stretching of the unsclerotized abdominal cuticle of triatominae larvae and it is evoked by sensory inputs related to feeding. We tested: (a) whether the cuticle recovers its original mechanical properties after plasticization, (b) whether repeated stimulation would be able to evoke recurrent plasticization along the same larval instar, (c) the temporal course of recovering cuticular stiffness. We injected Ringer solution into the body cavity of the bugs at constant pressure, using the injection rate (ml/min) as a measure of the cuticle extensibility. To trigger plasticization, individuals were allowed to feed on blood from an artificial feeder at 32+/-2 degrees C. After plasticization occurred, the abdominal cuticle gradually recovered its original mechanical properties. Bugs were capable of plasticizing for a second time when repeatedly stimulated. The effects of plasticization vanished between 1 and 2 h after stimulation. Although one full meal could suffice to accomplish moult in other Triatomine species, Triatoma infestans is able to feed repeatedly during a single larval instar. Accordingly to this, their cuticle recovers stiffness in some hours and becomes able to respond repeatedly to sensory inputs associated with feeding.     

Spectral sensitivity of the photonegative reaction of the blood-sucking bug Triatoma infestans (Heteroptera: Reduviidae)

We studied the spectral sensitivity of the visual system of the blood-sucking bug Triatoma infestans, one of the main vectors of Chagas Disease in South America. We quantified the photonegative reaction of this insect in a rectangular arena, half of which was kept dark and the other half illuminated with various intensities of different monochromatic lights (or broadband stimuli for λ>665 nm). As a behavioral parameter of the photonegative response, we measured the time each insect spent in the dark half of the arena. We found that low intensity levels (under 0.06 μW/cm²) of monochromatic lights of 397, 458, 499, and 555 nm evoked a statistically significant (i.e., different from that of control groups) photonegative reaction. Insects were less sensitive to monochromatic lights of 357 nm (UV) and 621 nm (dark orange), and to broadband stimuli in the red part of the spectrum (665–695 nm). These findings indicate that the visual system of T. infestans is sensitive to broader regions of the spectrum than those previously reported.     

An insight into the sialome of the blood-sucking bug Triatoma infestans, a vector of Chagas' disease

Triatoma infestans is a hemiptera, vector of Chagas' disease that feeds exclusively on vertebrate blood in all life stages. Hematophagous insects' salivary glands (SG) produce potent pharmacological compounds that counteract host hemostasis, including anticlotting, antiplatelet, and vasodilatory molecules. To obtain a further insight into the salivary biochemical and pharmacological complexity of this insect, a cDNA library from its SG was randomly sequenced. Also, salivary proteins were submitted to two-dimensional gel (2D-gel) electrophoresis followed by MS analysis. We present the analysis of a set of 1534 (SG) cDNA sequences, 645 of which coded for proteins of a putative secretory nature. Most salivary proteins described as lipocalins matched peptide sequences obtained from proteomic results.     

Mating behavior of the hematophagous bug Triatoma infestans: role of Brindley's and metasternal glands

We investigated if Brindley's and metasternal glands are involved in the sexual behavior of Triatoma infestans. In laboratory assays, we analyzed the effect of selective occlusion of Brindley's and metasternal glands of the female (separately and together) on the behavior of males. Control assays without occlusion of glands were also performed. We quantitatively tested if such glands affect mating occurrence, the copulatory attempts of males, and the aggregation of males around a mating couple. The number of mating attempts by males did not differ between treatments, demonstrating that likelihood of males mating did not depend on which gland is occluded in the female. In the absence of any occlusion, T. infestans mated and males aggregated. The proportion of copulations and aggregation behavior of males did not differ between treatments when female's Brindley's glands were occluded. However, when metasternal glands were occluded, the proportion of mating couples decreased and males did not aggregate. We demonstrated that the metasternal glands of the female are involved in the sexual behavior of T. infestans, while Brindley's glands seem to have no effect on mating behavior. Copulation and aggregation behavior of males likely result from the eventual release of volatiles from the female's metasternal glands.     

A short proregion of trialysin, a pore-forming protein of Triatoma infestans salivary glands, controls activity by folding the N-terminal lytic motif

Triatoma infestans (Hemiptera: Reduviidae) is a hematophagous insect that transmits the protozoan parasite Trypanosoma cruzi, the etiological agent of Chagas' disease. Its saliva contains trialysin, a protein that forms pores in membranes. Peptides based on the N-terminus of trialysin lyse cells and fold into alpha-helical amphipathic segments resembling antimicrobial peptides. Using a specific antiserum against trialysin, we show here that trialysin is synthesized as a precursor that is less active than the protein released after saliva secretion. A synthetic peptide flanked by a fluorophore and a quencher including the acidic proregion and the lytic N-terminus of the protein is also less active against cells and liposomes, increasing activity upon proteolysis. Activation changes the peptide conformation as observed by fluorescence increase and CD spectroscopy. This mechanism of activation could provide a way to impair the toxic effects of trialysin inside the salivary glands, thus restricting damaging lytic activity to the bite site.     

Distribution and characterization of Corazonin in the central nervous system of Triatoma infestans (Insecta: Heteroptera)

The distribution of corazonin in the central nervous system of the heteropteran insect Triatoma infestans was studied by immunohistochemistry. The presence of corazonin isoforms was investigated using MALDI-TOF mass spectrometry in samples containing the brain, the subesophageal ganglion, the corpora cardiaca-corpus allatum complex and the anterior part of the aorta. Several groups of immunopositive perikarya were detected in the brain, the subesophageal ganglion and the thoracic ganglia. Regarding the brain, three clusters were observed in the protocerebrum. One of these clusters was formed by somata located near the entrance of the ocellar nerves whose fibers supplied the aorta and the corpora cardiaca. The remaining groups of the protocerebrum were located in the lateral soma cortex and at the boundary of the protocerebrum with the optic lobe. The optic lobe housed immunoreactive somata in the medial soma layer of the lobula and at the level of the first optic chiasma. The neuropils of the deutocerebrum and the tritocerebrum were immunostained, but no immunoreactive perikarya were detected. In the subesophageal ganglion, immunostained somata were found in the soma layers of the mandibular and labial neuromeres, whereas in the mesothoracic ganglionic mass, they were observed in the mesothoracic, metathoracic and abdominal neuromeres. Immunostained neurites were also found in the esophageal wall. The distribution pattern of corazonin like immunoreactivity in the central nervous system of this species suggests that corazonin may act as a neurohormone. Mass spectrometric analysis revealed that [Arg⁷]-corazonin was the only isoform of the neuropeptide present in T. infestans tissue samples.     

Circadian rhythm of behavioural responsiveness to carbon dioxide in the blood-sucking bug Triatoma infestans (Heteroptera: Reduviidae)

The temporal modulation of the behavioural response to carbon dioxide and its chronobiological basis were investigated in larvae of Triatoma infestans. We analysed the orientation towards CO(2) of insects kept under three different illumination regimes: (1) 12 h light/12 h darkness cycles (L/D), (2) constant darkness (D/D) and (3) constant light (L/L). When maintained under L/D conditions, insects exhibited an oriented response towards airstreams added with 1500 ppm of CO(2) during the first hours of the scotophase only. Bugs maintained under D/D also showed a positive orientation response towards CO(2) during the first hours of the subjective night, while bugs kept under L/L did not show a rhythmic oriented behaviour. Thus, T. infestans displayed a daily rhythm of orientation towards CO(2) (i.e. a potential food source) only at the beginning of the scotophase. The persistence of the rhythm under constant darkness reveals the existence of an endogenous circadian control of this behaviour.     

The role of water vapour in the orientation behaviour of the blood-sucking bug Triatoma infestans (Hemiptera,Reduviidae)

The behavioural response to water vapour of the haematophagous bug Triatoma infestans was analysed. Dry or humid discrete sources at different temperatures were used as stimuli for insects walking on a locomotion compensator. Humidity significantly increased the tendency of these bugs to orientate towards thermal sources. Furthermore, humid sources at room temperature were attractive to T. infestans, but this effect was limited to short-range distances. On the other hand, dynamic sources, i.e. airstreams carrying different water vapour contents did not affect the spontaneous anemotactic behaviour of this species, neither in sign (positive) nor in intensity. The anemotactic behaviour was also not influenced by the physiological water balance state of the bugs. Results are discussed in relation to the cues released by living hosts of triatomine bugs and in relation to their responses to air-currents.