Insights into a hotspot in the Brasiliensis subcomplex (Hemiptera, Triatominae) by analysis of D2 domain of the nuclear gene 28S.

The Brasiliensis subcomplex is a monophyletic group formed by the species Triatoma brasiliensis brasiliensis, T. b. macromelasoma, T. juazeirensis, T. melanica, and T. sherlocki. However, using cytogenetic data and experimental hybrid crosses, T. lenti and T. petrochiae were also grouped into this subcomplex. This study aims to analyze the properties of hotspot in the D2 domain of the nuclear gene 28S in all species of the Brasiliensis subcomplex as well as T. lenti and T. petrochiae. These species show two transversions at position 385 (G↔C and T↔G). We suggest that this mutation in haplotype 4 may be an initial molecular tool that supports the relationship of these species with the subcomplex. In addition to the transversion at haplotype 4, these species, aside from T. melanica, also possess a transversion at position 385 (G↔T) in haplotype 1. Thus, we describe the hotspot mutations of the D2 domain of the nuclear gene 28S for species in Brasiliensis subcomplex as follows: three transversions are present at position 385 of haplotypes 1 and 4, which are shared by members of the subcomplex as well as T. lenti and T. petrochiae. These transversions may be considered a synapomorphy between these species. However, we emphasize that new phylogenetic studies should be conducted to evaluate whether T. lenti and T. petrochiae are truly members of the Brasiliensis subcomplex.

Distribution of constitutive heterochromatin in Triatoma melanocephala (Hemiptera, Triatominae).

In principle, Triatoma melanocephala was included in the Brasiliensis subcomplex on the basis of morphological parameters and geographical layout, since there were no other relevant data available in the literature. On the basis of karyotype, it has been proposed to exclude T. melanocephala, as well as of T. vitticeps and T. tibiamaculata, from the subcomplex, which shows fragmentation of the X sex chromosomes, thereby approaching the species of North America. Therefore, the present study aimed to determine the pattern of constitutive heterochromatin of T. melanocephala to provide new data on the cytotaxonomy of this vector of Chagas disease and especially to try to relate this species to some group, complex or subcomplex of triatomine species, aiding in their classification. This species showed no constitutive heterochromatin in the autosomes or X sex chromosome, but only the Y sex chromosome. The number of chromosomes and heterochromatin pattern of T. melanocephala proved to be identical to that described for Panstrongylus lutzi. Thus, the present study demonstrated a tentative relationship between T. melanocephala and P. lutzi. However, we emphasize that other comparative studies should be conducted between these species, such as experimental crosses and molecular, enzymatic, morphological, and morphometric analyses to determine whether these species are actually evolutionarily related or if the number of chromosomes and the heterochromatin pattern emerged as homoplasies in T. melanocephala and P. lutzi.

Insight into the salivary transcriptome and proteome of Dipetalogaster maxima.

Dipetalogaster maxima is a blood-sucking Hemiptera that inhabits sylvatic areas in Mexico. It usually takes its blood meal from lizards, but following human population growth, it invaded suburban areas, feeding also on humans and domestic animals. Hematophagous insect salivary glands produce potent pharmacologic compounds that counteract host hemostasis, including anticlotting, antiplatelet, and vasodilatory molecules. To obtain further insight into the salivary biochemical and pharmacologic complexity of this insect, a cDNA library from its salivary glands was randomly sequenced. Salivary proteins were also submitted to one- and two-dimensional gel electrophoresis (1DE and 2DE) followed by mass spectrometry analysis. We present the analysis of a set of 2728 cDNA sequences, 1375 of which coded for proteins of a putative secretory nature. The saliva 2DE proteome displayed approximately 150 spots. The mass spectrometry analysis revealed mainly lipocalins, pallidipins, antigen 5-like proteins, and apyrases. The redundancy of sequence identification of saliva-secreted proteins suggests that proteins are present in multiple isoforms or derive from gene duplications.

Dipetalodipin, a novel multifunctional salivary lipocalin that inhibits platelet aggregation, vasoconstriction, and angiogenesis through unique binding specificity for TXA2, PGF2alpha, and 15(S)-HETE.

Dipetalodipin (DPTL) is an 18 kDa protein cloned from salivary glands of the triatomine Dipetalogaster maxima. DPTL belongs to the lipocalin superfamily and has strong sequence similarity to pallidipin, a salivary inhibitor of collagen-induced platelet aggregation. DPTL expressed in Escherichia coli was found to inhibit platelet aggregation by collagen, U-46619, or arachidonic acid without affecting aggregation induced by ADP, convulxin, PMA, and ristocetin. An assay based on incubation of DPTL with small molecules (e.g. prostanoids, leukotrienes, lipids, biogenic amines) followed by chromatography, mass spectrometry, and isothermal titration calorimetry showed that DPTL binds with high affinity to carbocyclic TXA(2), TXA(2) mimetic (U-46619), TXB(2), PGH(2) mimetic (U-51605), PGD(2,) PGJ(2), and PGF(2α). It also interacts with 15(S)-HETE, being the first lipocalin described to date to bind to a derivative of 15-lipoxygenase. Binding was not observed to other prostaglandins (e.g. PGE(1), PGE(2), 8-iso-PGF(2α), prostacyclin), leukotrienes (e.g. LTB(4), LTC(4), LTD(4), LTE(4)), HETEs (e.g. 5(S)-HETE, 12(S)-HETE, 20-HETE), lipids (e.g. arachidonic acid, PAF), and biogenic amines (e.g. ADP, serotonin, epinephrine, norepinephrine, histamine). Consistent with its binding specificity, DPTL prevents contraction of rat uterus stimulated by PGF(2α) and induces relaxation of aorta previously contracted with U-46619. Moreover, it inhibits angiogenesis mediated by 15(S)-HETE and did not enhance inhibition of collagen-induced platelet aggregation by SQ29548 (TXA(2) antagonist) and indomethacin. A 3-D model for DPTL and pallidipin is presented that indicates the presence of a conserved Arg(39) and Gln(135) in the binding pocket of both lipocalins. Results suggest that DPTL blocks platelet aggregation, vasoconstriction, and angiogenesis through binding to distinct eicosanoids involved in inflammation.

On the mechanisms involved in biological heme crystallization.

Blood-feeding organisms digest hemoglobin, releasing large quantities of heme inside their digestive tracts. Free heme is very toxic, and these organisms have evolved several mechanisms to protect against its deleterious effects. One of these adaptations is the crystallization of heme into the dark-brown pigment hemozoin (Hz). Here we review the process of Hz formation, focusing on organisms other than Plasmodium that have contributed to a better understanding of heme crystallization. Hemozoin has been found in several distinct classes of organisms including protozoa, helminths and insects and Hz formation is the predominant form of heme detoxification. The available evidence indicates that amphiphilic structures such as phospholipid membranes and lipid droplets accompanied by specific proteins play a major role in heme crystallization. Because this process is specific to a number of blood-feeding organisms and absent in their hosts, Hz formation is an attractive target for the development of novel drugs to control illnesses associated with these hematophagous organisms.

Juvenile hormone mediates lipid storage in the oocytes of Dipetalogaster maxima.

Triatomines are vectors of Chagas disease and important model organisms in insect physiology. "Kissing bugs" are obligatory hematophagous insects. A blood meal is required to successfully complete oogenesis, a process primarily controlled by juvenile hormone (JH). We used Dipetalogaster maxima as an experimental model to further understand the roles of JH in the regulation of vitellogenesis and oogenesis. A particular focus was set on the role of JH controlling lipid and protein recruitment by the oocytes. The hemolymph titer of JH III skipped bisepoxide increased after a blood meal. Following a blood meal there were increased levels of mRNAs in the fat body for the yolk protein precursors, vitellogenin (Vg) and lipophorin (Lp), as well as of their protein products in the hemolymph; mRNAs of the Vg and Lp receptors (VgR and LpR) were concomitantly up-regulated in the ovaries. Topical administration of JH induced the expression of Lp/LpR and Vg/VgR genes, and prompted the uptake of Lp and Vg in pre-vitellogenic females. Knockdown of the expression of LpR by RNA interference in fed females did not impair the Lp-mediated lipid transfer to oocytes, suggesting that the bulk of lipid acquisition by oocytes occurred by other pathways rather than by the endocytic Lp/LpR pathway. In conclusion, our results strongly suggest that JH signaling is critical for lipid storage in oocytes, by regulating Vg and Lp gene expression in the fat body as well as by modulating the expression of LpR and VgR genes in ovaries.

Trypanosoma cruzi could affect wild triatomine approaching behaviour to humans by altering vector nutritional status: A field test.

Hematophagous insects exhibit complex behaviour when searching for blood-meals, responding to several host stimuli. The hematophagous insect Mepraia spinolai is a wild vector of Trypanosoma cruzi, causative agent of Chagas disease in humans, in the semiarid-Mediterranean ecosystem of Chile. In this study, we evaluated the association between the approaching behaviour to a human host, with T. cruzi infection status and nutritional condition of M. spinolai. To this end, we captured 501 individuals in six consecutive 10 min-timespan, using a human as bait. Captured vectors were weighed, photographed and measured to calculate their nutritional status by means of a Standardized Body Mass Index. Trypanosoma cruzi infection was assessed in the intestinal content by using a real-time PCR assay. Ordinal logistic regressions were performed separately for infected and uninfected groups to evaluate if the nutritional status was associated with the approaching behaviour to a human host, recorded as the time-span of capture. Nutritional status of uninfected triatomines was higher than that from infected ones (p < 0.005). Among the infected, those with higher nutritional status approached first (p < 0.01); there was no effect of nutritional status in the uninfected group. Trypanosoma cruzi infection might affect the foraging behaviour of M. spinolai under natural conditions, probably deteriorating nutritional status and/or altering vector detection abilities.

Study of the nucleolar cycle and ribosomal RNA distribution during meiosis in triatomines (Triatominae, Heteroptera).

Aspects of nucleolar activity during spermatogenesis were assessed in three triatomine species (Panstrongylus megistus, Rhodnius pallescens and Triatoma infestans) using cytochemical and fluorescent staining techniques. Toluidine blue and a variant of critical electrolytic concentration (CEC) allowed the discrimination of rRNA providing structural details of the nucleolus and RNA distribution during meiotic cell division. Acridine orange fluorochrome staining permitted the differentiation of nucleic acids, and silver-ion impregnation made possible the observation of pre-nucleolar bodies (PNBs). Our results support the phenomenon known as "persistence of the nucleolar material", and the hypothesis of post-meiotic reactivation of rRNA genes. Nucleolar organizer regions (NORs) were observed in some metaphasic spermatogonial chromosomes in P. megistus and T. infestans. In P. megistus at diplotene-diakinesis, NORs were also detected in one of the sex chromosomes and in an autosome. Therefore, it may be inferred that, in triatomines, the nucleolus does not completely disappear, but persists in the form of small bodies that get together to form the next nucleolar cycle which, in the case of meiosis, will be completed if fertilization occurs and a new zygote is formed.

An alternative, less invasive blood sample collection technique for serologic studies utilizing triatomine bugs (Heteroptera; Insecta).

The collection of blood samples for serological studies is often stressful for the focus animal. Recently, the use of bloodsucking bugs, such as Dipetalogaster maximus or Triatoma infestans (Reduviidae; Triatominae; Heteroptera), has been suggested as a new and less invasive method for blood collection. To evaluate this technique, we collected paired blood samples from 20 domestic rabbits (Oryctolagus cuniculus) during a study of rabbit hemorrhagic disease virus (RHDV). For each rabbit, blood samples were collected by the conventional method (needle and syringe from the vena auricularis) and through feeding by D. maximus. Samples were tested for RHDV antibodies using standard test kits at three different dilutions. Antibody titers were identical for 56 paired samples and differed in only four cases. The simple matching indices were 1 for the 1:10 dilution and 0.9 for the 1:100 and 1:1000 dilutions. The major advantages of the new technique are 1) the possibility to obtain blood from animals where veins are inaccessible and 2) the fact that anesthesia of focus animals may not be necessary.

Salivary Thromboxane A2-Binding Proteins from Triatomine Vectors of Chagas Disease Inhibit Platelet-Mediated Neutrophil Extracellular Traps (NETs) Formation and Arterial Thrombosis.

BACKGROUND: The saliva of blood-feeding arthropods contains a notable diversity of molecules that target the hemostatic and immune systems of the host. Dipetalodipin and triplatin are triatomine salivary proteins that exhibit high affinity binding to prostanoids, such as TXA2, thus resulting in potent inhibitory effect on platelet aggregation in vitro. It was recently demonstrated that platelet-derived TXA2 mediates the formation of neutrophil extracellular traps (NETs), a newly recognized link between inflammation and thrombosis that promote thrombus growth and stability. METHODOLOGY/PRINCIPAL FINDINGS: This study evaluated the ability of dipetalodipin and triplatin to block NETs formation in vitro. We also investigated the in vivo antithrombotic activity of TXA2 binding proteins by employing two murine models of experimental thrombosis. Remarkably, we observed that both inhibitors abolished the platelet-mediated formation of NETs in vitro. Dipetalodipin and triplatin significantly increased carotid artery occlusion time in a FeCl3-induced injury model. Treatment with TXA2-binding proteins also protected mice from lethal pulmonary thromboembolism evoked by the intravenous injection of collagen and epinephrine. Effective antithrombotic doses of dipetalodipin and triplatin did not increase blood loss, which was estimated using the tail transection method. CONCLUSIONS/SIGNIFICANCE: Salivary TXA2-binding proteins, dipetalodipin and triplatin, are capable to prevent platelet-mediated NETs formation in vitro. This ability may contribute to the antithrombotic effects in vivo. Notably, both molecules inhibit arterial thrombosis without promoting excessive bleeding. Our results provide new insight into the antihemostatic effects of TXA2-binding proteins and may have important significance in elucidating the mechanisms of saliva to avoid host's hemostatic responses and innate immune system.

A subpopulation of dorsal unpaired median neurons in the blood-feeding insect Rhodnius prolixus displays serotonin-like immunoreactivity.

We describe, for the first time in insects, the presence of serotonin-like immunoreactive dorsal unpaired median (DUM) neurons. In unfed, untreated Rhodnius prolixus, the cell bodies of these DUM neurons displayed only faint serotonin-like immunofluorescence without any detectable fluorescence in the axons. There was, however, an extensive serotonin-like immunoreactive peripheral complex. We have enhanced the immunostaining of the cell bodies and axons of these DUM neurons by using 5,7-dihydroxytryptamine (5,7-DHT) and nerve transection, and have further defined their morphology with cobalt backfilling and Lucifer yellow injection. Injection of 5,7-DHT resulted in enhanced serotonin-like immunostaining, after 24 hours, of five DUM neurons in the mesothoracic ganglionic mass. Each DUM neuron possessed bifurcating serotonin-like immunoreactive axons projecting to the periphery via one of the five bilaterally paired abdominal nerves. Concomitant with the enhanced immunostaining in the cell body and axons of these DUM neurons was the depletion of a serotonin-like immunoreactive neurohaemal area lying on each of the abdominal nerves and the depletion of serotonin-like immunoreactive processes on the body wall. Enhancement of cell body and axon immunostaining was also observed in preparations in which the abdominal nerves had been transected 24 hours previously. The location and morphology of these DUM neurons were studied in further detail by using cobalt backfilling of the abdominal nerves and intracellular injection of Lucifer yellow followed by immunohistochemistry. The five serotonin-like immunoreactive DUM neurons were found to represent a subpopulation in a group of ten DUM neurons located in the mesothoracic ganglionic mass and associated with the abdominal nerves. Each abdominal nerve received projections from two DUM neurons, only one of which was serotonin-like immunoreactive. Electrophysiological recordings revealed that the serotonin-like immunoreactive DUM neurons of Rhodnius had similar properties to previously described octopaminergic DUM neurons of insects. The five DUM neurons in Rhodnius appear to represent a subpopulation of DUM neurons containing serotonin.

Cytological evidence for serotonin-containing fibers in an abdominal neurohemal organ in a hemipteran.

The abdominal neurohemal organs of the hemipteran Rhodnius prolixus contain an extensive serotonin containing arborization. Endogenous serotonin within fibers and terminals in the neurohemal area were detected with histochemical and immunocytological techniques. The abdominal nerves which contain the neurohemal organs selectively sequester exogenous serotonin. Serotonin and its metabolites are biochemically detected within the mesothoracic ganglion, which is a known source of projections into the neurohemal organ. However, the source of the cell bodies which might send serotonergic fibers to the neurohemal organ remains undetermined because no correspondence was found between immunocytological maps of serotonin-containing cells in the ganglion, and projection maps into neurohemal organ (determined by cobalt back-filling).

Serotonin modulation of the release of sequestered [3H]serotonin from nerve terminals in an insect neurohemal organ in vitro.

Exogenous tritiated serotonin ([3H]5-HT) is taken up by and released from serotonin-containing fibers within abdominal nerves of Rhodnius prolixus during in vitro incubations. Sequestered [3H]5-HT behaves as expected of an endogenous neurosecretory product in this system. Release is Ca2+-dependent during both K+-induced and physiologically induced secretory episodes. The kinetics of the release of sequestered label parallels the kinetics of release of endogenous neurohormones. Preloaded preparations which are washed with unlabeled 5-HT release label in two fashions; 5-HT itself induces a release of label and, at lower concentrations, 5-HT facilitates the release of label induced by high K+ washes. Facilitatory effects appear to be mediated through receptors within the neurohemal organ (NHO).

Role of salivary antihemostatic components in blood feeding by triatomine bugs (Heteroptera).

Salivary gland homogenates from 4 genera of triatomine bugs were assayed for anticlotting, apyrase, and vasodilatory activities, and these activities were correlated with the efficiency of each bug species to initiate a blood meal. Antihemostatic activities spanned a large range of values. Apyrase activity in members of the genus Rhodnius was markedly different from that in other genera with respect to their sensitivity to divalent cation activators. Apyrase and vasodilatory activities, but not anticlotting activity, correlated with feeding efficiency of bugs taking a blood meal on a rat. Results are discussed within the context of the evolution of blood-feeding by insects.

Biochemical changes in the transition from vitellogenesis to follicular atresia in the hematophagous Dipetalogaster maxima (Hemiptera: Reduviidae).

In this work, we have explored the biochemical changes characterizing the transition from vitellogenesis to follicular atresia, employing the hematophagous insect vector Dipetalogaster maxima as a model. Standardized insect rearing conditions were established to induce a gradual follicular degeneration stage by depriving females of blood meal during post-vitellogenesis. For the studies, hemolymph and ovaries were sampled at representative days of pre-vitellogenesis, vitellogenesis and early and late follicular atresia. When examined by scanning electron microscopy, ovarioles at the initial stage of atresia were small but still showed some degree of asynchronism, a feature that was lost in an advanced degeneration state. At late follicular atresia, in vivo uptake assays of fluorescently labeled vitellogenin (Vg-FITC) showed loss of competitiveness of oocytes to uptake vitellogenin. Circulating vitellogenin levels in atresia were significantly higher than those registered at pre-vitellogenesis, most likely to maintain appropriate conditions for another gonotrophic cycle if a second blood meal is available. Follicular atresia was also characterized by partial proteolysis of vitellin, which was evidenced in ovarian homogenates by western blot. When the activity of ovarian peptidases upon hemoglobin (a non-specific substrate) was tested, higher activities were detected at early and late atresia whereas the lowest activity was found at vitellogenesis. The activity upon hemoglobin was significantly inhibited by pepstatin A (an aspartic peptidase inhibitor), and was not affected by E64 (a cysteine peptidase inhibitor) at any tested conditions. The use of specific fluorogenic substrates demonstrated that ovarian homogenates at early follicular atresia displayed high cathepsin D-like activity, whereas no activity of either, cathepsin B or L was detected. Mass spectrometry analysis of the digestion products of the substrate Abz-AIAFFSRQ-EDDnp further confirmed the presence of a cathepsin D-like peptidase in ovarian tissue. In the context of our findings, the early activation of cathepsin D-like peptidase could be relevant in promoting yolk protein recycling and/or enhancing follicle removal.