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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.

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.

An arbitrary-primed RNA PCR differential display strategy was used to identify midgut genes of the reduviid bug Triatoma infestans that were differentially expressed after a blood meal. From interesting bands, 33 distinct cDNAs were cloned and sequenced. Although many had long open reading frames, most of the transcripts were unrelated to any other sequences in any databases. Only 14 Triatoma sequences had strong homologies to those from other organisms, including genes encoding for 2-oxoglutarate dehydrogenase, CAD protein, NADH-ubiquinone-oxoreductase, epidermal growth factor, plectin, aminopeptidase, heat-shock-related 70-kDa protein, golgin, mitochondrial carrier protein and high-density lipoprotein. RT-PCR was used to demonstrate constitutive expression in four of five of these sequences. Northern hybridisation was difficult due to the very low expression levels of most of the genes. However, a gene-fragment highly homologous to a heat-shock-related 70-kDa protein was strongly expressed in starved bugs, down-regulated after feeding and again expressed later, suggesting a role for a heat-shock protein in starvation survival.

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 H2O, but after an additional day it increased to 350–970 mosmol/kg H2O. 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.

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 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 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

The echinostome cercaria,Cercaria patellae Lebour, 1911, which develops in the limpetPatella vulgata (Prosobranchia, Diotocardia, Patellidae), was investigated by light and scanning microscopy. The highest prevalence of limpets with rediae/cercariae occurred on bare rocks on the upper part of the shore. The prevalence was higher in larger snails and in those which had an orange foot-sole. The cercariae penetrated into other gastropods,Aplysia punctata (Opisthobranchia, Cephalaspidea, Aplysiidae),Acanthodoris pilosa (Opisthobranchia, Nudibranchiata, Lamellidorididae) andP. vulgata. No differences in size were found between metacercariae which naturally infected onlyP. vulgata and five-day-old metacercariae from experimental infections. Inin vitro experiments excystation of metacercariae from natural infections took place as a passive process after a double treatment with pepsin and trypsin which caused the dissolution of the cyst wall. After metacercariae from natural infections were fed to one-day-old chickens, the young adults developed. Detailed morphometrical data on rediae, cercariae and metacercariae are compared with previously published measurements of living specimens. Chaetotaxy demonstrated a distinct distribution of tegumental papillae of cercariae, resembling that of the Echinostomatidae or Philophthalmidae. Scanning electron microscopy of adults revealed short, blunt spines anteriorly, while flattened serrated spines occurred on the posterior tegument. Morphometry revealed no major differences to previously published data. Considering all ecological data and comparing the morphological data with those of previous publications by different authors, we conclude thatC. patellae Lebour, 1911, the cercaria in the limpet, is a developmental stage of a digenean in the intestine of the oystercatcherHaematopus ostralegus and should be referred toEchinostephilla patellae (Lebour, 1911) n. comb.

Two species of lice are ectoparasites of humans, Pediculus humanus with the two subspecies P. humanus capitis (head louse), living in head hair, and P. humanus humanus (body or clothing louse), staying in clothes and approaching the skin only to feed, and Pthirus pubis (crab louse), colonising mainly the hairs of the genital region. Besides humans, only some monkeys are hosts of these obligate parasites that spend the entire life cycle on the host. The strong association with the host is reflected in morphological peculiarities: very short antennae, reduced eyes, and no wings. Since they are hemimetabolous insects, all postembryonic stages suck blood. Symbionts are located in mycetomes and are transmitted during egg development. Only P. humanus humanus is relevant as a vector of Rickettsia prowazekii (louse-borne epidemic typhus), Bartonella quintana (trench or 5-day fever), and Borrelia recurrentis (louse-borne relapsing fever). Since economic instability and wars cause large migrations of humans, louse infestations become more prevalent, increasing the risk of transmission of neglected diseases.

After the previous characterization of one trypsin gene (Try1) of the human body louse Pediculus humanus, genes encoding a second trypsin (Try2) and a chymotrypsin (Chy1) have been cloned using degenerate serine proteinase primers and 5¢- and 3¢-RACE, and sequenced. The deduced 259 and 267 amino acid se- quences of Try2 and Chy1 show an identity of 33%- 40% to trypsinogens and chymotrypsinogens of other insects. Considering previously published partial se- quences, P. humanus possesses at least one Try1 gene, five variants/isoforms of Try2 and six variants/isoforms of Chy1. The genomic DNA of Try2 contains three in- trons and Chy1 contains five introns. Using whole mount in situ hybridization, gene expression of Try1, Try2 and Chy1 has been localized not only in the dis- tensible anterior region of the midgut of lice but some- times also in the area following the distensible region. The Try2 gene was always expressed at much lower levels than Try1 or Chy1. This lower expression, the constitutive expression of Try1 and Chy1 at 1, 2, 6, 12 and 24 h after feeding of adults and the regional differ- ences have been verified in quantitative real-time PCR.

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.

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.

We have isolated and characterised a Triatoma infestans cDNA encoding a lysozyme. A 174-bp fragment was amplified by PCR using degenerate oligodeoxyribonucleotide primers derived from the known amino acid sequences of lysozyme from other insects. This PCR fragment was used to screen a cDNA gut library of T. infestans. A clone containing the 3′-end of the lysozyme cDNA (219 bp) was isolated and sequenced. RACE was used to amplify the 5′-end of the lysozyme cDNA. After sequencing the complete lysozyme cDNA, the deduced 417 amino acid sequence showed high identity (40–50%) with other chicken-type lysozymes. The amino acid residues responsible for the catalytic activity and the binding of the substrate were essentially conserved. The expression pattern of the lysozyme gene in bugs at different molting and feeding states showed that this gene was upregulated in the digestive tract directly after the molt and after feeding. Additionally, this lysozyme gene was expressed differently in the different regions of the digestive tract, strongly in the cardia and stomach, the anterior regions of the midgut, and only traces of lysozyme mRNA could be detected in the small intestine, the posterior region of the midgut. Arch. Insect Biochem. Physiol. 53:134–145, 2003. © 2003 Wiley-Liss, Inc.

The population density and composition of an established infection of Blastocrithidia triatomae in the intestinal tract of fifth instars of Triatoma infestans were compared in unfed bugs, at 4 h and up to 15 days after feeding, and also in feces and urine deposited in the first 4 h after feeding. In unfed bugs, about 1-2 million B. triatomae colonized the small intestine and rectum, mainly epimastigotes (85% and 80%, respectively). During excretion, the percentage of cysts increased within the first two drops (from 15% to 35%) and then decreased slowly, indicating a washing-out of these unattached stages. The initial reduction in the B. triatomae population lasted up to 6 days after feeding. By 15 days after feeding, the populations had strongly increased in the small intestine and rectum, to 22 million and 2 million flagellates, respectively, as had cysts, comprising some 50% of the total population in the rectum.

Fifth instars of Triatoma infestans with established Blastocrithidia triatomae infections were dissected after different periods of starvation. After a short starvation period of 30 days, 60% of the total population (2,700,000 flagellates) occurred in the small intestine. Within the following 3 months, the numbers of living flagellates there (epimastigotes, cysts) were reduced by about 70% and the percentage of dead mastigotes increased to 30% of the respective total population. Epimastigotes always dominated (about 90%), followed by cysts and only up to 3% spheromastigotes. These relations were only slightly changed by starvation. In the rectum, at 30-120 days after feeding, the total population of living epimastigotes was reduced by 90% and the percentage of those attached to the rectal wall decreased from 10% to3%. During this period, the proportion of dead from all epimastigotes increased from 34% to99%. In the rectum, the percentage of cysts from the total population of living parasites increased from 41% to 88% at 30-60 days after feeding and remained at this percentage and total numbers, showing that especially the early phase of starvation strongly induced the encystment of B. triatomae.

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 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.

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.

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.

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.

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.