Sequence characterization of an unusual lysozyme gene expressed in the intestinal tract of the reduviid bug Triatoma infestans (Insecta).

Antibacterial proteins like lysozyme are important components of the insect non-specific immune response against bacteria. The complementary deoxyribonucleic acid (cDNA) encoding a new lysozyme from Triatoma infestans, named lysozyme2, has been amplified by polymerase chain reaction and the rapid amplification of cDNA ends technique. The gene is expressed in the small intestine of the insect. The deduced protein sequence shows up to 70% similarity to lysozymes from other species. Furthermore, the protein exhibits significant structural concordance to other insect lysozymes. A striking feature of the lysozyme2 protein is the replacement of the conserved amino acid residues of the active site of classical c-type lysozymes, glutamate and aspartate, by valine and tyrosine.

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.

Cloning and characterization of a trypsin-encoding cDNA of the human body louse Pediculus humanus.

From a cDNA library of the whole insect, a trypsin gene of Pediculus humanus has been cloned and sequenced. The 908 bp clone has an open reading frame of 759 bp, which encodes a pre-proenzyme with 253 amino acid residues. A sixteen-residue N-terminal signal peptide is followed by a twelve-residue activation peptide with putative cleavage sites at Gly16 and Tyr28. The deduced amino acid sequence has several features typical of trypsin proteases and an overall identity of 35-43% with the trypsins of several haematophagous Diptera. The 1.0 kb genomic trypsin gene contains three introns of 102, 79 and 80 nucleotides following the codons for Gly16, Gln74 and Ala155, respectively. Only a single gene seems to be present. In Northern blot analysis, unfed first instar larvae have an identical or slightly lower level of trypsin mRNA than fed adult lice, and in adults 2-24 h after the bloodmeal this gene shows a constitutive expression. After in vitro transcription and translation, the activation peptide is cleaved by chymotrypsin, a so far unreported phenomenon in trypsin activation.

Ionic composition of the rectal contents and excreta of the reduviid bug Triatoma infestans.

We have investigated the chemical composition of the rectal contents, faeces and urine of the blood-sucking bug Triatoma infestans. This is the environment in which the important disease-causing organism, Trypanosoma cruzi, lives. Directly after feeding of Triatoma infestans, the pH of the excreta switched from an acidic to an alkaline pH and, 1 day later, back to a slightly acidic pH. The osmolality varied in the initial excreta and in the rectal contents on the day following the meal between 300 and 460 mosmol/kg H(2)O, but after an additional day it increased to 350-970 mosmol/kg H(2)O. Determinations by ion capillary electrophoresis showed that sulphate and phosphate dominated the rectal contents in unfed bugs. After feeding, the first four drops of fluid excreta were mainly a sodium chloride solution (>150 mM for each). One to 10 days after feeding strong individual variations in the concentrations of individual ions were evident, especially for potassium and sodium. Mean concentrations of chloride remained at about 70 mM; sulphate and phosphate showed an increase within the first 1 or 2 days and then reached a level of about 160 and 210 mM, respectively. The rectal contents of long-term starved bugs contained high concentrations of phosphate and potassium; sulphate and sodium were slightly lower.

Rhodnius prolixus: effects of the neolignan burchellin on in vivo and in vitro diuresis.

Supplementation of blood with the neolignan burchellin (100 microg/ml), a compound from the arboreous Lauraceae Aniba burchelli, affected the course of excretion of fourth-instar larvae of Rhodnius prolixus, especially directly after feeding, and reduced the volume of feces/urine excreted within 6 h of feeding to about 18% and, on the simultaneous addition of the diuretic hormone analogue 5-hydroxytryptamine (5-HT), about 71% of that observed in untreated bugs. In the latter, 5-HT induced a significant 60% increase in excretion. Regardless of whether Malpighian tubules originating from unfed, untreated or fed, burchellin-treated bugs were incubated in vitro in the hemolymph of these bugs or in physiological saline supplemented with 5-HT with or without burchellin or in homogenates of thoracic ganglionic masses of untreated and treated bugs, burchellin was consistently found to affect the secretion rates. Therefore, burchellin not only depresses the release of the diuretic hormone or induces the release of antidiuretic factors but also directly affects the Malpighian tubules.

The development of Trypanosoma cruzi in triatominae.

Trypanosoma cruzi multiplies and differentiates in the digestive tract of triatomine insects. These insects ingest an enormous amount of blood, with ingestion followed very rapidly by a strong diuresis, slow digestion and occasionally long periods of starvation. Resulting changes in the intestinal environment induce the development of dominant stages of T. cruzi--epimastigotes and metacyclic trypomastigotes--and can be correlated with the appearance of specific developmental stages--spheromastigotes and giant cells--which otherwise are only rarely seen. Here, Astrid Kollien and Günter Schaub outline recent research on these developmental steps of T. cruzi in the vector, and the effects of different compounds acting against the parasite in the vector.

The effect of azadirachtin on Blastocrithidia triatomae and Trypanosoma cruzi in Triatoma infestans (Insecta, Hemiptera).

The effect of azadirachtin on Blastocrithidia triatomae and Trypanosoma cruzi, which colonise the intestinal tract of the blood-sucking bug Triatoma infestans, was investigated. In established infections of controls without azadirachtin treatment, the small intestine of fifth-instar T. infestans contained up to 7 x l0(6) B. triatomae and the rectum 3 x 10(6). In comparison to this homoxenous flagellate, the population densities of T. cruzi in the respective regions were 99.3 and 76% lower. Treatment with azadirachtin (1 microg ml(-1)) via a blood meal and a concurrent infection with B. triatomae resulted in an increase of the population density (3 weeks p.i.), caused mainly by the mastigote stages in the rectum. In an established B. triatomae infection (12 weeks p.i.), feeding of azadirachtin did not affect the population density and composition. In an optimal T. cruzi-vector system, i.e. parasite and bug originate from the same locality, the treatment with azadirachtin at 20 weeks p.i. strongly reduced the population density in the small intestine of all bugs up to 100 days after treatment, but only in a minor percentage of the bugs in the rectum. Trypanosoma (cruzi incubated for up to 24 h in faeces of azadirachtin-treated bugs were not affected, indicating that the rectum of these bugs contained no toxic substances. The importance of these findings is that investigations of the mechanisms of action of azadirachtin offer a possibility to identify vector-derived compounds, which are necessary for the development of T. cruzi, thereby, giving us a possible new strategy to combat Chagas' disease.

Modes of association of Trypanosoma cruzi with the intestinal tract of the vector Triatoma infestans.

The interface between Trypanosoma cruzi and two regions of the intestinal tract of reduviid bugs, the small intestine and the rectum, was investigated by electron microscopy. The mode of association of the trypanosomes with the midgut surface differs fundamentally from that in the rectum, the preferred site of colonisation by T. cruzi. The parasites caused no detrimental changes in the extracellular membrane layers, microvilli or epithelial cells. Parasites resided mainly at the border of the gut contents, also regularly showing parasite-parasite interdigitations. In regions in which the extracellular membrane layers were absent or only weakly developed, trypanosome bodies or flagella occasionally could be found inserted shallowly between the tips of the microvilli. Since there were usually no ultrastructural modifications of the cell body and/or flagellum associated with attachment, there is apparently no strong attachment of the flagellates to the wall of the midgut. In the rectal lumen the flagellates also interdigitated with each other and on the rectal wall T. cruzi was intimately attached to the rectal cuticle lining. At the attachment site flagella were enlarged and sometimes contained electron-dense, hemidesmosome-like material beneath the plasma membrane.

Trypanosoma cruzi in the rectum of the bug Triatoma infestans: effects of blood ingestion by the starved vector.

To follow the developmental effects of feeding of the insect host after long starvation periods, the population density and composition of an established infection of Trypanosoma cruzi in the rectum of Triatoma infestans were determined 60 days after the last feeding (daf) and then at different intervals after feeding. The original population decreased and then increased up to the 10th daf. In starved bugs, about 30% were spheromastigotes (including intermediate forms), 20% epimastigotes, and 50% trypomastigotes, but one daf, these forms represented 2%, 70%, and 10%, respectively. In addition, one daf there were about 10% giant cells, i.e., a multiple division stage. In the following two days, this form represented on average 30-50% of the total population, but it then disappeared nearly completely. Thus, giant cells evidently develop by rapid growth of epimastigotes, if conditions become optimal after long starvation periods of the vector.

The effect of azadirachtin on fresh isolates of Trypanosoma cruzi in different species of triatomines.

The effect of azadirachtin was investigated using three different fresh isolates of Trypanosoma cruzi and five different triatomine species which were infected as third-instar larvae. The two T. cruzi strains which originated from sylvatic Triatoma vitticeps showed a high prevalence after the molt to the fifth instar in Panstrongylus megistus and Rhodnius neglectus and a low prevalence in Triatoma infestans and Rhodnius robustus. The third T. cruzi strain originating from a patient in Piaui showed a high prevalence in P. megistus, R. neglectus and T. infestans and a low prevalence in Triatoma sordida. Feeding the infected fifth instars with azadirachtin-supplemented blood (1 microg/ml) resulted 20 days later in some parasite/vector combinations in an increase, in others a decrease or an unchanged number of T. cruzi in comparison to bugs fed with unsupplemented blood.

The development of Trypanosoma cruzi (Trypanosomatidae) in the reduviid bug Triatoma infestans (Insecta): influence of starvation.

Fifth instars of Triatoma infestans with established Trypanosoma cruzi infections were dissected after different periods of starvation to determine the population density and the percentage of different developmental stages of T. cruzi in the small intestine and rectum of the bugs. After a short starvation period of 20 days, the population density in the small intestine was 20% (about 60,000) of the rectal population. The population in the small intestine was strongly reduced after an additional ten days of starvation, and no flagellates could be found there 60, 90 and 120 days after the last feeding. In the rectum, this reduction went down to 1% of the initial population, but a total elimination never occurred. Usually the remaining population contained more live than dead flagellates. Starvation also resulted in an increase in the rectum in the number and percentage of drop-like forms, intermediates between sphero- and epi- or trypomastigotes, from 1% initially to about 10% after 90 days of starvation. The percentage of spheromastigotes increased from 2% at 20 days after the last feeding to about 20% after an additional 40 and 70 days. Therefore, the spheromastigotes of T. cruzi seem to be induced by stress conditions.

Hydrophobic attachment of Trypanosoma cruzi to the rectal cuticle of Triatoma infestans and its influence on metacyclogenesis - a review.

Trypanosoma cruzi colonizes mainly the rectum of the vector, especially the rectal glands. We investigated the basic architecture of the rectal cuticle of Triatoma infestans and the mode of attachment of T. cruzi in the small intestine and the rectum. In addition, we determined the capacity of culture-derived epimastigotes to attach to artificial substrates and the influence of attachment on metacyclogenesis. After incubation of the rectum with wheat germ lectin (WGA) coupled to gold particles, the procuticle contained chitin, but the two layers of the epicuticle and the superficial layer bordering the rectal lumen did not. The specific fluorochrom Nile Red stained the entire rectal cuticle green, indicating the waxy composition of the superficial layer. In electron microscopic analysis the parasites were attached to the hydrophobic superficial wax layer but not to the epithelium of the midgut. In vitro culture-derived epimastigotes attached with a high affinity to all hydrophobic substrates tested, whereas hydrophilic substrates did not permit attachment. Emulsified hexadecane localized the attachment molecules to the terminal part of the flagellum. Inhibition of attachment by coating the culture tubes with hydrophilic agarose and constant agitation decreased the rate of epimastigote to trypomastigote transformation, whereas wax coating enhanced metacyclogenesis.

Development of Trypanosoma cruzi after starvation and feeding of the vector - a review.

Trypanosoma cruzi develops in the intestinal tract of reduviid bugs and may be affected by changes in the nutritional state of the vector. In regularly fed Triatoma infestans the population of T. cruzi in the rectum consists mainly of equal amounts of epi- and trypomastigotes. Starvation of the bug reduces the total number of flagellates and the number and percentage of trypomastigotes. The number and the percentage of drop-like forms and of resulting spheromastigotes, however, increases up to 30% 60 days after the last feeding (daf). Feeding of starved bugs (60 daf) reduces the original population density, which then increases again. In starved bugs 1 daf spheromastigotes (including intermediate forms) have almost disappeared and epimastigotes dominate. In addition "giant cells" (a multiple division stage) comprise about 10% of the population and in the following two days this form represents on average 30-50% of the total population, before disappearing nearly completely. Feeding the vector at 40 daf; a) induces the appearance of pure populations of trypomastigotes in immediately deposited drops of bug urine; b) induces metacyclogenesis in epimastigotes, and c) reduces metacyclogenesis in spheromastigotes. Incubating isolated recta together with the Malpighian tubules in Drosophila Ringer's solution and initiating the excretion with 5-hydroxy-tryptamine also induces metacylogenesis in epimastigotes.

Trypanosoma cruzi in the rectum of the bug Triatoma infestans: effects of blood ingestion of the vector and artificial diuresis.

The population density and the percentage of different developmental stages of an established infection of Trypanosoma cruzi were determined at 40 days after the last feeding of the fourth instar in the rectum (lumen, anterior and posterior wall) of fed and unfed groups of fifth instars of Triatoma infestans. Additionally, the rectum and the Malpighian tubules were incubated in saline, inducing diuresis by addition of the diuretic hormone. The rectum contained an average of 200,000-400,000 T. cruzi. After feeding the percentages of spheromastigotes and drop-like intermediate stages were reduced from < 7% and 15%, respectively, to < 3%, but those of slender intermediate stages increased statistically significantly from < 7% to 10%. After 4 h of diuresis the in-vitro-incubated isolated rectum with the four Malpighian tubules showed the same trends, indicating that diuresis rather than factors of the hemolymph or digestive products induces the development of metacyclic trypomastigotes of T. cruzi originating from epimastigotes.