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Chagas disease, or American trypanosomiasis, is a parasitic disease caused by the protozoan Trypanosoma cruzi and is transmitted by insects from the Triatominae subfamily. To identify components involved in the protozoan-vector relationship, we constructed and analyzed cDNA libraries from RNA isolated from the midguts of uninfected and T. cruzi-infected Triatoma infestans, which are major vectors of Chagas disease. We generated approximately 440 high-quality Expressed Sequence Tags (ESTs) from each T. infestans midgut cDNA library. The sequences were grouped in 380 clusters, representing an average length of 664.78 base pairs (bp). Many clusters were not classified functionally, representing unknown transcripts. Several transcripts involved in different processes (e.g., detoxification) showed differential expression in response to T. cruzi infection. Lysozyme, cathepsin D, a nitrophorin-like protein and a putative 14 kDa protein were significantly upregulated upon infection, whereas thioredoxin reductase was downregulated. In addition, we identified several transcripts related to metabolic processes or immunity with unchanged expressions, including infestin, lipocalins and defensins. We also detected ESTs encoding juvenile hormone binding protein (JHBP), which seems to be involved in insect development and could be a target in control strategies for the vector. This work demonstrates differential gene expression upon T. cruzi infection in the midgut of T. infestans. These data expand the current knowledge regarding vector-parasite interactions for Chagas disease.

Proteases from the midgut gland of the Farfantepenaeus paulensis juveniles were assessed. Enzyme activity was determined using protease substrates and inhibitors. The effect of pH, temperature and calcium on proteolytic activity was assayed. Caseinolytic activity was analysed in substrate‐sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Trypsin, chymotrypsin and leucine aminopeptidase activity was detected. Proteolytic activity was strongly inhibited by the specific trypsin inhibitors. Tosyl‐phenylalanine chloromethyl ketone inhibited 59.3% of chymotrypsin activity. The greatest trypsin‐like activity occurred at pH 8.0 and 45 °C. Chymotrypsin‐like activity reached maximal values at alkaline pH (7.2–9.0) and 55 °C. CaCl2 did not increase trypsin‐like activity, but rather inhibited it at concentrations of 30 (20%), 50 (30%) and 100 mM (50%). The substrate‐SDS‐PAGE zymogram revealed eight proteinase bands. Two possibly thermal‐resistant (85 °C, 30 min) chymotrypsin isoforms were found, which were inhibited by phenyl‐methyl‐sulphonyl‐fluoride. Aminopeptidase activity of enzyme extracts (Arg, Leu, Lys, Phe and Val) and the recommended concentrations of these essential amino acids in penaeid shrimp diets were positively correlated (P<0.05). Beause protein digestion involves the combined action of different enzymes, adequate knowledge of shrimp digestion and enzyme characteristics is required for the assessment of the digestive potential of different feed sources and development of in vitro digestibility protocols.

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The aim of this study was to examine proteinases and peptidases from the hepatopancreas of two sub-adult stages of Farfantepenaeus subtilis: SAS 6 (5.93 ± 0.69 g wet weight) and SAS 13 (13.26 ± 0.60 g wet weight). Trypsin and chymotrypsin activity was higher in the extract from the SAS 6 individuals (P < 0.05). The highest activity among aminoacyl-b-naphthylamide substrates was found using alanine-, arginine-, leucine-and lysine-b-naphthylamide. There was a positive correlation between the recommended concentration of essential amino acids in penaeid shrimp feed and aminopeptidase activity in both sub-adult stages. Proteolytic activity of F. subtilis was strongly inhibited by specific trypsin inhibitors. The optimal temperature for trypsin, chymotrypsin and leucine aminopeptidase activity was between 45 and 55°C. Six and seven bands were found in caseinolytic zymograms for SAS 6 and SAS 13 respectively. All bands were inhibited by phenylmethylsulfonyl fluoride in both sub-adult stages. The use of tosyl-lysine-chloromethyl-ketone and benzamidine caused strong inhibition of the proteolytic bands. Trypsin and chymotrypsin activity was the main difference observed between the protease pattern of SAS 6 and SAS 13 F. subtilis.

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Pacifastin-like protease inhibitors belong to a recent classified protease inhibitor family and they are the smallest protease inhibitors described in animals. In this work, we purified and characterized, for the first time, two neutrophil elastase inhibitors belonging to the pacifastin family from the blood sucking insect Triatoma infestans eggs. The inhibitors showed the same N-terminal sequences, molecular masses of 4257 and 4024Da by MALDI-TOF mass spectrometry and dissociation constants (Ki) for neutrophil elastase of 0.52 and 0.29nM, respectively. Using a fat body cDNA library, we cloned a pacifastin precursor containing two protease inhibitor domains similar to locust pacifastins. The first pacifastin domain translated to T. infestans purified protein, named TIPI1. Recombinant TIPI1 expressed in Pichia pastoris system showed similar inhibitory activities compared to the native inhibitor. Its precursor, called TiPP1, is mainly expressed in fat body, and it is up-regulated after blood feeding. The immune challenges of 1(a) instar T. infestans nymph with bacteria or dsRNA strongly stimulated TiPP1 expression in fat body, suggesting a possible role of TiPP1 in T. infestans immunity. This work is the first to characterize a blood feeding insect pacifastin inhibitor.