G. A. Schaub

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.

Hydrophobic attachment of Trypanosoma cruzi to a superficial layer of the rectal cuticle in the bug Triatoma infestans

Abstract In the vector Triatoma infestans the human pathogenic flagellate Trypanosoma cruzi colonizes mainly the rectum. The rectal cuticle and associated trypanosomes were examined by electron microscopy. The cuticle of the rectum consisted of a superficial wax layer, which was not retained by conventional preparation, an outer and inner epicuticle, and a procuticle. Epimastigotes of T. cruzi attached to the superficial layer with a specialized area of the flagellum. The composition of the cuticle was analyzed by cytochemistry to determine constituents relevant for parasite attachment. Intense staining with the fluorochrome Nile red indicated the presence of lipids, and measurements of contact angles formed by test fluids with the rectal wall revealed that the luminal surface is hydrophobic. The mechanism of attachment of T. cruzi was found to be based on a hydrophobic interaction. The flagellates bound to lipids extracted from the cuticle and to saturated hydrocarbons. Chitin, which has been presumed to be the natural binding substrate of T. cruzi, was localized using gold-labeled wheat-germ lectin. Chitin occurred in the procuticle but was absent from the superficial and epicuticular layers and, thus, is not accessible for binding by T. cruzi. In addition, it could not be confirmed that galactose-specific lectins or heparin receptors mediate flagellate attachment to the rectum.

Changes of the abundance of Culicoides obsoletus s.s. and Culicoides scoticus in Southwest Germany identified by a PCR-based differentiation

The outbreak of bluetongue disease in Central Europe necessitates new approaches in the identification of vectors to follow-up changes of populations of species and not of complexes. Since females of species of the complex of Culicoides obsoletus are difficult to be identified according to morphological criteria, we applied a polymerase chain reaction (PCR)-based strategy targeting the mitochondrial cytochrome oxidase subunit I to differentiate between the species Culicoides obsoletus s.s. and Culicoides scoticus. Catches of culicoids obtained from May to November 2007 in an ultraviolet lamp trap at a cattle farm in Rhineland-Palatinate, Southern Germany were surveyed for changes of the abundance of both species. Only in May 2007, the samples contained similar proportions of both species. Afterwards, C. scoticus dominated with up to 88%. Calculating the number of specimens of both species within the total catches of culicoids, the numbers of C. obsoletus s.s. slightly decreased from May to July and increased to a little maximum in August. C. scoticus seemed to have three maxima in this period of time, the strongest one in August, presumably due to different generations and not to climatic conditions. These results indicate that the applied PCR strategy can be used for a detailed analysis of culicoids as basis for the estimation of the transmission risk of the bluetongue virus by different species of the Obsoletus complex.

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

Abstract 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 Piauí 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 μg/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.

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

Abstract Supplementation of blood with the neolignan burchellin (100 μg/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.

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.