Blood-sucking Bug Research Articles

The saliva proteome of the blood-feeding insect Triatoma infestans is rich in platelet-aggregation inhibitors

The saliva of the bloodsucking bug Triatoma infestans vector of Chagas disease contains an anti-hemostatic molecular cocktail that prevents coagulation, vasoconstriction and platelet aggregation in a vertebrate prey. In order to characterize T. infestans saliva proteome, we separated the secreted saliva by two-dimensional gel electrophoresis (2-DE). More than 200 salivary proteins were detected on the 2-DE map, mainly in the alkaline region. By nanoLC–MS/MS analysis using a LTQ–Orbitrap equipment followed by a combination of conventional and sequence-similarity searches, we identified 58 main protein spots. Most of such proteins possess potential blood-feeding associated functions, particularly anti-platelet aggregation proteins belonging to lipocalin and apyrase families. The saliva protein composition indicates a highly specific molecular mechanism of early response to platelet aggregation. This first proteome analysis of the T. infestans secreted saliva provides a basis for a better understanding of this fluid protein composition highly directed to counterpart hemostasis of the prey, thus promoting the bug's blood-feeding.

Allosteric regulation of 6-phosphofructo-1-kinase activity of fat body and flight muscle from the bloodsucking bug Rhodnius prolixus

6-Phosphofructo-1-kinase (phosphofructokinase; PFK) activity from Rhodnius prolixus, a haematophagous insect which is usually a poor flyer, was measured and compared in two metabolically active tissues - flight muscle and fat body. The activity of this important regulatory glycolytic enzyme was much more pronounced in muscle (15.1 +/- 1.4 U/mg) than in fat body extracts (3.6+/-0.4 U/mg), although the latter presented higher levels of enzyme per protein content, as measured by western-blotting. Muscle extracts are more responsible than fat body to ATP and fructose 6-phosphate, both substrates of PFK. Allosteric regulation exerted by different effectors such as ADP, AMP and fructose 2,6-phosphate presented a singular pattern for each tissue. Optimal pH (8.0-8.5) and sensitivity to pH variation was very similar, and citrate was unable to inhibit PFK activity in both extracts. Our results suggest the existence of a particular PFK activity for each tissue, with regulatory patterns that are consistent with their physiological roles.

Growth behaviour of two Trypanosoma cruzi strains in single and mixed infections: In vitro and in the intestinal tract of the blood-sucking bug, Triatoma brasiliensis

Competition and cooperation are well-recognized biological phenomena, even among parasites. Co-infection of parasites in a single host leads to several outcomes, one being competition for a limited resource. Here, the behaviour of mixed infection was evaluated using two isolates of Trypanosoma cruzi, previously typed as belonging to genotypes TcI and TcII. The growth in vitro and in the different compartments of the gut of Triatoma brasiliensis was studied. In vitro growth showed that MDID/BR/1999/M1 (TcI) has a doubling time of 19.5 h and MIDID/BR/1999/JCPD4 (TcII) of 9.6 h, while the mixed infection group presented a doubling time of 13.9 h. In vivo, three groups of infection were done: M1/TcI, JCPD4/TcII and mixed infection (50% of each strain), respectively. All comparisons among the groups were done using the Kruskal–Wallis non-parametric test. The data showed that the in vitro culture of mixed populations has a similar pattern to the growth of M1/TcI, apparently suggesting a positively selection for M1/TcI strain, in axenic culture. In the gut of the insects, M1/TcI isolate and mixed infections colonized predominantly the rectal wall and rectal lumen, in contrast to the JCPD4/TcII isolate, which was found mainly colonizing the small intestine. According to the isolates investigated, it could be concluded that the doubling time was not determinant factor for the final composition of a co-infection. Moreover, mixed infections resulted in a homogenous distribution of the parasites, comparing to the isolates studied separately. Apparently, in the gut of the bugs, the simultaneous presence of JCPD4/TcII isolate resulted in an improvement of the number of parasites from M1/TcI.

An update on Chagas disease (human American trypanosomiasis)

Human American trypanosomiasis or Chagas disease -- named after Carlos Chagas who first described it in 1909 -- exists only on the American continent. It is caused by a parasite, Trypanosoma cruzi, that is transmitted to humans by blood-sucking triatomine bugs, by blood transfusion, and transplacentally. Chagas disease has two, successive phases: acute and chronic. The acute phase lasts 6-8 weeks. After several years of starting the chronic phase, 20%-35% of infected individuals (the percentage varying with geographical area) develop irreversible lesions of the autonomous nervous system in the heart, the oesophagus, the colon and/or the peripheral nervous system. Data on the prevalence and distribution of Chagas disease markedly improved in quality during the 1980s, as a result of demographically representative, cross-sectional studies carried out in countries where no accurate information on these parameters was available. Experts had previously met in Brasilia, in 1979, and devised standard protocols for carrying out country-wide studies not only on the prevalence of human infection with T. cruzi but also on house infestation with the triatomine vectors. Thanks to a co-ordinated programme in the southernmost countries of South America (i.e.the 'Southern Cone'), transmission of T. cruzi by the vectors or blood transfusion has been successfully interrupted in Uruguay (from 1997), Chile (from 1999) and Brazil (from 2005), and the global incidence of new human infection with T. cruzi has decreased by 67%. Similar multi-country control initiatives have been launched in the Andean countries and in Central America, with the goal of interrupting all transmission of T. cruzi to humans by 2010 -- a goal set, in 1998, as a resolution of the World Health Assembly. Recent advances in basic research on T. cruzi include the genetic characterization of populations of the parasite and the sequencing of its genome.

Competitive displacement in Triatominae: the Triatoma infestans success

Brazil has just been certificated by Pan American Health Organization as 'free of Chagas disease transmission due to Triatoma infestans'. During the early 1980s, this species of blood-sucking bug alone was considered responsible for approximately 80% of Chagas disease transmission. But it was not always so. The species originally abundant in houses of central and eastern Brazil was Panstrongylus megistus, which seems to have been progressively displaced from houses by T. infestans during the past century. Indeed, T. infestans seems able to displace other Triatominae in artificial environments. Recent studies suggest that it might simply be because T. infestans feeds more efficiently than its Triatominae competitors.

Chemical Communication in Chagas Disease Vectors. Source, Identity, and Potential Function of Volatiles Released by the Metasternal and Brindley's Glands of Triatoma infestans Adults

Compounds from the metasternal and Brindley's glands of the blood-sucking bug, Triatoma infestans, were identified by solid phase microextraction (SPME) and gas chromatography-mass spectrometry. Volatile compounds released by adult bugs during copulation or after mechanical disturbance were also characterized. Six compounds were identified and found consistently in all samples from metasternal glands. The most abundant were 3-pentanone, 2-methylbutanol, 3-pentanol, and an unidentified compound. The metasternal gland blends did not differ qualitatively between sexes. Compounds found in Brindley's glands were short chain acids, alcohols, esters, and a ketone with no qualitative differences between sexes. Isobutyric acid was the main component of this blend, and two new confirmed compounds were described as products of these glands: 2-butanone and 2-methylbutyric acid. 3-Pentanone was collected from the headspace over 33% of the copulating pairs of T. infestans. Volatiles found in the headspace of disturbed T. infestans adults included short-chain fatty acids, alcohols, esters, and ketones, with no qualitative differences between sexes. Both types of glands apparently discharge their contents after disturbance. However, most of the volatiles released by bugs after disturbance came from Brindley's glands. The locomotor activity of fourth instars increased significantly after stimulation with the odors emitted by disturbed adults, as compared with larvae stimulated by the odor of undisturbed adults or by clean air. We also studied the directional behavioral response of fifth instars to the disturbance scent in a locomotion compensator. Larvae exposed to volatiles released by disturbed adults walked away from the direction of the odor. The results suggest that this blend or part of it functions as an alarm pheromone for T. infestans. We suggest that the metasternal glands of this species are involved both in the sexual and the alarm contexts, and that the Brindley's glands probably have both alarm and defensive roles.

Non-invasive blood sampling from primates using laboratory-bred blood-sucking bugs (Dipetalogaster maximus; Reduviidae, Heteroptera)

Primates are easily stressed by the conventional veterinary blood sampling routine and consequently, measured blood parameters may be biased. In this study, we tested blood-sucking bugs (Dipetalogaster maximus) on one lemur and two ape species (Microcebus murinus, Pongo abelii, Pan paniscus) as an alternative, non-invasive technique for bleeding primates. Within time periods of between 6 and 62 min we obtained blood volumes of 0.01-2.4 ml in 11 out of 12 trials from all three species. Therefore, we conclude that these bugs represent a new, gentle and effective tool for bleeding captive primates without stress.

A non-invasive technique to bleed incubating birds without trapping: a blood-sucking bug in a hollow egg

We describe a non-invasive technique to obtain blood samples from incubating birds without trapping and handling. A larval instar of the blood-sucking bug Dipetalogaster maximus (Heteroptera) was put in a hollowed artificial egg which was placed in a common tern (Sterna hirundo) nest. A gauze-covered hole in the egg allowed the bug to draw blood from the brood patch of breeding adults. We successfully collected 68 blood samples of sufficient amount (median=187 μl). The daily success rate was highest during the early breeding season and averaged 34% for all trials. We could not detect any visible response by the incubating bird to the sting of the bug. This technique allows for non-invasive blood collection from bird species of various sizes without disturbance.

Blood-sucking bugs as a gentle method for blood-collection in water budget studies using doubly labelled water

During doubly-labelled water (DLW) experiments, blood collection by venous puncture may traumatize animals and consequently affect the animals' behaviour and energy budget. Recent studies have shown that blood-sucking bugs (Triatominae; Heteroptera) can be used instead of conventional needles to obtain blood from animals. In this paper, we validate the bug method in captive nectar-feeding bats, Glossophaga soricina, for water budget analysis by comparing the daily water flux estimated with the DLW method with values measured by an energy balance method. As the mean daily water flux of the DLW method was not significantly deviating from the expected value, blood-sucking bugs may substitute more invasive methods of blood collection in DLW experiments. Based on the DLW estimates, daily energy and water intake rates were calculated and compared to values measured with the energy balance method. The DLW method and the energy balance method yielded on average similar results regarding the daily energy intake (DLW method: 48.8+/-14.2 kJ d(-1) versus energy balance method: 48.1+/-9.9 kJ d(-1)) and daily water intake (DLW method: 13.7+/-2.4 mL d(-1) versus energy balance method: 14.7+/-3.0 mL d(-1)). Based on the calculated water and sugar intake per day, we estimated the sugar concentration of ingested nectar to equal on average 16.2+/-2.4% (mass/mass), which fell close to the measured sugar concentration of 17% (mass/mass) bats fed on during the experiment. We conclude that it is possible to extrapolate mean daily energy and water intake for animal groups, populations and species based on DLW estimates, but due to the large variance of results (low accuracy), it seems inadequate to calculate values for single individuals.

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 lambda>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 microW/cm(2)) 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.

Nitrophorin synthesis is modulated by protein kinase CK2

Rhodnius prolixus is a blood-sucking bug whose saliva contains a family of nitric oxide-carrying proteins named nitrophorins (NPs). Saliva is injected into the host bloodstream during insect feeding. Nitric oxide is then released from NPs and will act on vascular smooth muscle, promoting vasodilation. Epithelial cells of salivary glands then undergo a massive synthesis of antihemostatics including NPs which produces saliva for the next blood meal. Here, we demonstrate the transient activation of a protein kinase in the salivary glands of R. prolixus after a blood meal. Biochemical, immunological, and pharmacological assays were used to identify this enzyme as protein kinase CK2. CK2 is activated after a blood meal and decreases to basal levels when salivary gland refilling is resumed. Inhibition of CK2 blocked [ 35S]methionine incorporation into newly synthesized salivary gland proteins in cultured tissue. Dissected salivary glands were then incubated with the heme fluorescent analog palladium (II) mesoporphyrin IX (Pd-MP) in the presence of a selective cell-permeable CK2 inhibitor, TBB (4,5,6,7-tetrabromobenzotriazole). NP synthesis was quantified based on fluorescence of the Pd-MP group bound to the NP heme pocket. TBB dramatically blocked NP synthesis. Altogether, these data are the first demonstration to show that antihemostatic synthesis in a blood-sucking arthropods is under protein phosphorylation control.

Trypanosomiasis: African and American

Abstract Human African trypanosomiasis has increased in many endemic areas in recent years. It is caused by Trypanosoma brucei gambiense or T. b. rhodesiense, and is transmitted by the tsetse fly. Diagnosis is difficult, particularly in the West African type, in which organisms are scanty. Determination of the stage of infection (early haemolymphatic stage or late meningoencephalitic stage) is also problematic, and the few drugs available are associated with significant toxicity. Advances in understanding of the cellular and molecular biology of the parasites causing African trypanosomiasis now must be matched with improvements in diagnosis and treatment of this condition, which has a current prevalence of about 300,000 cases. Chagas disease is caused by infection with T. cruzi carried by blood-sucking triatomine bugs. The infection is often latent for several years, before progressing in 15–30% of individuals to disabling organ involvement and ‘mega'-syndromes. This South American infection is difficult to diagnose and there is no effective treatment for established infection.

Rhodococcus triatomae sp. nov., isolated from a blood-sucking bug

Two bacterial isolates, strains IMMIB RIV-085(T) and IMMIB RIV-095, isolated from a blood-sucking bug of the genus Triatoma, were characterized by phenotypic and molecular taxonomic methods. Chemotaxonomic investigations revealed the presence of cell-wall chemotype IV and mycolic acids consistent with the genus Rhodococcus. Comparative 16S rRNA gene sequencing showed that the two isolates are genealogically highly related (100% sequence similarity) and constitute a new subline within the genus Rhodococcus, with Rhodococcus corynebacteroides and Rhodococcus rhodnii as their nearest phylogenetic neighbours (98.4 and 98.3% sequence similarity, respectively). However, DNA-DNA hybridization experiments demonstrated unambiguously that the isolates are genealogically distinct from R. corynebacteroides and R. rhodnii (32 and 43% relatedness, respectively). The isolates could be distinguished from their phylogenetic relatives and other members of the genus Rhodococcus by means of biochemical tests. On the basis of both phenotypic and phylogenetic evidence, it is proposed that these isolates be classified as a novel species, Rhodococcus triatomae sp. nov. The type strain is strain IMMIB RIV-085(T) (=CCUG 50202(T)=DSM 44892(T)).

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 °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.

Cathepsin D-mediated yolk protein degradation is blocked by acid phosphatase inhibitors

Vitellin (VT) is a lipoglycophosphoprotein stored inside the eggs of every oviparous organism during oogenesis. In the blood-sucking bug Rhodnius prolixus, VT is deposited inside growing oocytes together with two acid hydrolases: acid phosphatase (AP) and cathepsin D (CD). Egg fertilization triggers AP activity and VT proteolysis in vivo [Insect Biochem. Mol. Biol. 2002 (32) 847]. Here, we show that CD is the main protease targeting VT proteolysis during egg development. CD activity in total egg homogenates is blocked by the classical aspartyl protease inhibitor, pepstatin A. Surprisingly, AP inhibitors such as NaF, Na +/K + tartrate, and inorganic phosphate also block VT proteolysis, whereas this effect is not observed when tyrosine phosphatase inhibitors such as vanadate and phenylarsine oxide or an inhibitor of alkaline phosphatases such as levamisole are used in a VT proteolysis assay. NaF concentrations that block isolated AP activity do not affect the activity of partially purified CD. Therefore, a specific repressor of VT proteolysis must be dephosphorylated by AP in vivo. In conclusion, these results demonstrate for the first time that acid hydrolases act cooperatively to promote yolk degradation during egg development in arthropods.

The role of salivary lipocalins in blood feeding by Rhodnius prolixus

In order to overcome host mechanisms that prevent blood loss, the blood-sucking bug Rhodnius prolixus has evolved a complex salivary secretion containing dozens of different proteins. A number of these have been characterized and found to have roles in inhibiting various hemostatic or inflammatory systems. Interestingly, many of these biologically active salivary proteins belong to the lipocalin protein family. A proliferation of lipocalin genes has occurred via gene duplication and subsequent divergence. Functional genomic, proteomic, and functional studies have been performed to probe the role of salivary lipocalins in blood feeding. In the course of these investigations, anticoagulant, antiplatelet, antiinflammatory, and vasodilatory molecules have been described.

Edysteroid receptor (EcR) shows marked differences in temporal patterns between tissues during larval-adult development in Rhodnius prolixus: correlations with haemolymph ecdysteroid titres

The presence of ecdysteroid receptor (EcR) in various tissues was studied throughout larval-adult development of the blood-sucking bug, Rhodnius prolixus, using an antibody to EcR that recognizes all isoforms. On Western blots, the antibody recognizes three peptides of approximate molecular masses of 70, 68 and 64 kDa, from epidermis and fat body of developing larvae, which contain high levels of haemolymph ecdysteroids. These peptides are absent from both unfed larvae and adults, which are devoid of ecdysteroids. In vitro treatment of epidermis and fat body from unfed larvae with 20E induces the appearance of all three EcR immunoreactive peptides. The stage-specific appearance and 20E inducibility of the peptides implies that they represent the native EcR(s) of Rhodnius. Confocal fluorescence analysis using this antibody revealed a great diversity of temporal profiles of EcR in various tissues during development. Developmental profiles of EcR were examined in abdominal epidermis, fat body, spermatocytes, brain (including the medial neurosecretory cells), prothoracic glands (PGs), rectal epithelium and Malpighian tubules. EcR fluorescence was confined to the nuclei in close association with chromatin. EcR was absent from tissues of unfed larvae or adults, supporting the results from Western blots. Different tissues develop EcR at different developmental times and in the presence of radically different concentrations of haemolymph ecdysteroids, retain EcR for different lengths of time and lose EcR at different concentrations of ecdysteroids. These results suggest that each tissue possesses a distinctive response mechanism to ecdysteroids. An exception to this, are the PGs, which exhibited no EcR fluorescence at any time during development.

Activity and sequence characterization of two cysteine proteases in the digestive tract of the reduviid bug Triatoma infestans

Cathepsin B- and cathepsin L-like activities were identified in gut extracts of the blood-sucking bug Triatoma infestans using specific substrates and inhibitors. Activities decreased during the first 2 days after feeding but increased to a maximum value at 5 and 10 days post feeding. The deduced 332 and 328 amino acid sequences showed high levels of identity (50-60%) to other insect cathepsin B- and L-like proteases, respectively. The three amino acid residues of the catalytic domain, CHN, and the GCNGG motif were conserved in both cathepsins, but the occluding loop, characterizing B-like cathepsins, was present only in one. ERFNIN and GNFD motifs occurred in the other sequence, defining it as cathepsin L-like. The cathepsin B-like gene was expressed at low, constitutive levels in unfed and fed T. infestans.

Orientation Behaviour of the Blood-sucking Bug Triatoma infestans to Short-chain Fatty Acids: Synergistic Effect of L-Lactic Acid and Carbon Dioxide

The role of short-chain fatty acids in the host-seeking behaviour of Triatoma infestans larvae was investigated using a locomotion compensator. Several short-chain fatty acids were tested alone over a wide range of doses, or in combination with L-lactic acid (L-LA; 100 microg). Bugs showed no attractive response to single carboxylic acids, but when L-LA was added to airstreams carrying specific intensities of either propionic (C3; 100 microg), butyric (C4; 1 microg) or valeric acid (C5; 1 microg), these mixtures elicited an attractive response, evincing a synergistic effect. No orientation response was observed when caproic acid (C6) was offered with L-LA at the doses tested. Two blends were created: (1) C3, C4 and C5 combined at the effective doses when added with L-LA [C3C4C5 (1)], and (2) C3, C4 and C5 combined at a third of those intensities [C3C4C5 (2)]. Both blends were tested alone, with L-LA (100 microg), with a sub-threshold concentration of CO(2) (300 p.p.m. above the ambient level), and combined with both compounds together. Oriented responses of bugs were only observed with the blend (2) added with L-LA and with the combination of this lure with CO(2). This last combination evoked a behavioural response similar in intensity to that induced by a live mouse.

Purification, crystallization and preliminary X-ray analysis of Triatoma virus (TrV) from Triatoma infestans.

Triatoma virus (TrV) is a viral pathogen of the blood-sucking reduviid bug Triatoma infestans, the most important vector of American human trypanosomiasis (Chagas' disease). TrV has been putatively classified as a member of the Cripavirus genus (type cricket paralysis virus) in the Dicistroviridae family. This work describes the purification of TrV particles from infected T. infestans and their crystallization and preliminary crystallographic analyses. Two different crystal forms, rhombohedral and orthorhombic, were obtained at room temperature by the hanging-drop vapour-diffusion technique using polyethylene glycol and polyethylene glycol monomethylether as precipitants. The rhombohedral crystals have unit-cell parameters a = b = 306.6, c = 788.4 A (hexagonal setting), diffract to 3.2 A resolution and contain one-third of the viral particle per asymmetric unit. The orthorhombic crystals have cell parameters a = 336, b = 351, c = 332 A, diffract to about 2.5 A resolution, and contain one-half of a virus particle in the asymmetric unit. A complete diffraction data set has been collected to 3.2 A resolution, using synchrotron radiation, from a single rhombohedral crystal under cryogenic conditions.