Serine proteinases of the human body louse (Pediculus humanus): sequence characterization and expression patterns.

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 sequences of Try2 and Chy1 show an identity of 33%-40% to trypsinogens and chymotrypsinogens of other insects. Considering previously published partial sequences, 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 introns 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 distensible anterior region of the midgut of lice but sometimes 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 differences have been verified in quantitative real-time PCR.

Isolation and characterization of a cDNA encoding for a lysozyme from the gut of the reduviid bug Triatoma infestans.

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.

The development of Blastocrithidia triatomae (Trypanosomatidae) in the reduviid bug Triatoma infestans (Insecta): influence of feeding.

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.

Differential display of mRNAs associated with blood feeding in the midgut of the bloodsucking bug, Triatoma 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.

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

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% to <3%. During this period, the proportion of dead from all epimastigotes increased from 34% to >99%. 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.