Background 
Saliva of blood sucking arthropods contains compounds that antagonize their hosts' hemostasis, which include platelet aggregation, vasoconstriction and blood clotting; saliva of these organisms also has anti-inflammatory and immunomodullatory properties. Perhaps because hosts mount an active immune response against these compounds, the diversity of these compounds is large even among related blood sucking species. Because of these properties, saliva helps blood feeding as well as help the establishment of pathogens that can be transmitted during blood feeding.


Methodology/Principal Findings 
We have obtained 1,626,969 reads by pyrosequencing a salivary gland cDNA library from adult females Amblyomma maculatum ticks at different times of feeding. Assembly of this data produced 72,441 sequences larger than 149 nucleotides from which 15,914 coding sequences were extracted. Of these, 5,353 had >75% coverage to their best match in the non-redundant database from the National Center for Biotechnology information, allowing for the deposition of 4,850 sequences to GenBank. The annotated data sets are available as hyperlinked spreadsheets. Putative secreted proteins were classified in 133 families, most of which have no known function.


Conclusions/Significance 
This data set of proteins constitutes a mining platform for novel pharmacologically active proteins and for uncovering vaccine targets against A. maculatum and the diseases they carry.

/pdf/a-deep-insight-into-the-sialotranscriptome-of-the-gulf-coast-1ey2j1qgkg.pdf

A Deep Insight into the Sialotranscriptome of the Gulf Coast Tick, Amblyomma maculatum

The bloodsucking hemipteran Rhodnius prolixus is a vector of Chagas' disease, which affects 7–8 million people today in Latin America. In contrast to other hematophagous insects, the triatomine gut is compartmentalized into three segments that perform different functions during blood digestion. Here we report analysis of transcriptomes for each of the segments using pyrosequencing technology. Comparison of transcript frequency in digestive libraries with a whole-body library was used to evaluate expression levels. All classes of digestive enzymes were highly expressed, with a predominance of cysteine and aspartic proteinases, the latter showing a significant expansion through gene duplication. Although no protein digestion is known to occur in the anterior midgut (AM), protease transcripts were found, suggesting secretion as pro-enzymes, being possibly activated in the posterior midgut (PM). As expected, genes related to cytoskeleton, protein synthesis apparatus, protein traffic, and secretion were abundantly transcribed. Despite the absence of a chitinous peritrophic membrane in hemipterans - which have instead a lipidic perimicrovillar membrane lining over midgut epithelia - several gut-specific peritrophin transcripts were found, suggesting that these proteins perform functions other than being a structural component of the peritrophic membrane. Among immunity-related transcripts, while lysozymes and lectins were the most highly expressed, several genes belonging to the Toll pathway - found at low levels in the gut of most insects - were identified, contrasting with a low abundance of transcripts from IMD and STAT pathways. Analysis of transcripts related to lipid metabolism indicates that lipids play multiple roles, being a major energy source, a substrate for perimicrovillar membrane formation, and a source for hydrocarbons possibly to produce the wax layer of the hindgut. Transcripts related to amino acid metabolism showed an unanticipated priority for degradation of tyrosine, phenylalanine, and tryptophan. Analysis of transcripts related to signaling pathways suggested a role for MAP kinases, GTPases, and LKBP1/AMP kinases related to control of cell shape and polarity, possibly in connection with regulation of cell survival, response of pathogens and nutrients. Together, our findings present a new view of the triatomine digestive apparatus and will help us understand trypanosome interaction and allow insights into hemipteran metabolic adaptations to a blood-based diet.

https://www.arca.fiocruz.br/bitstream/icict/10228/2/fernando_gentaetal_IOC_2014.pdf

An insight into the transcriptome of the digestive tract of the bloodsucking bug, Rhodnius prolixus.

Triatominae-Trypanosoma cruzi/T. rangeli: Vector-parasite interactions.

https://www.cell.com/trends/parasitology/pdf/S1471-4922(10)00157-1.pdf

Interactions between intestinal compounds of triatomines and Trypanosoma cruzi

Triatomines are hemipteran bugs acting as vectors of the protozoan parasite Trypanosoma cruzi This parasite causes Chagas disease, one of the major parasitic diseases in the Americas Studies of triatomine genetics and evolution have been particularly useful in the design of rational vector control strategies, and are reviewed here The phylogeography of several triatomine species is now slowly emerging, and the struggle to reconcile the phenotypic, phylogenetic, ecological and epidemiological species concepts makes for a very dynamic field Population genetic studies using different markers indicate a wide range of population structures, depending on the triatomine species, ranging from highly fragmented to mobile, interbreeding populations Triatomines transmit T cruzi in the context of complex interactions between the insect vectors, their bacterial symbionts and the parasites; however, an integrated view of the significance of these interactions in triatomine biology, evolution and in disease transmission is still lacking The development of novel genetic markers, together with the ongoing sequencing of the Rhodnius prolixus genome and more integrative studies, will provide key tools to expanding our understanding of these important insect vectors and allow the design of improved vector control strategies

/pdf/genetics-and-evolution-of-triatomines-from-phylogeny-to-2ekjlc92sh.pdf

Genetics and evolution of triatomines: from phylogeny to vector control

Background Triatomines are vectors of Trypanosoma cruzi, the etiological agent of Chagas disease in Latin America. The most effective vector, Triatoma infestans, has been controlled successfully in much of Latin America using insecticide spraying. Though rarely undertaken, surveillance programs are necessary in order to identify new infestations and estimate the intensity of triatomine bug infestations in domestic and peridomestic habitats. Since hosts exposed to triatomines develop immune responses to salivary antigens, these responses can be evaluated for their usefulness as epidemiological markers to detect infestations of T. infestans. Methodology/Principal Findings T. infestans salivary proteins were separated by 2D-gel electrophoresis and tested for their immunogenicity by Western blotting using sera from chickens and guinea pigs experimentally exposed to T. infestans. From five highly immunogenic protein spots, eight salivary proteins were identified by nano liquid chromatography-electrospray ionization-tandem mass spectrometry (nanoLC-ESI-MS/MS) and comparison to the protein sequences of the National Center for Biotechnology Information (NCBI) database and expressed sequence tags of a unidirectionally cloned salivary gland cDNA library from T. infestans combined with the NCBI yeast protein sub-database. The 14.6 kDa salivary protein [gi|149689094] was produced as recombinant protein (rTiSP14.6) in a mammalian cell expression system and recognized by all animal sera. The specificity of rTiSP14.6 was confirmed by the lack of reactivity to anti-mosquito and anti-sand fly saliva antibodies. However, rTiSP14.6 was recognized by sera from chickens exposed to four other triatomine species, Triatoma brasiliensis, T. sordida, Rhodnius prolixus, and Panstrongylus megistus and by sera of chickens from an endemic area of T. infestans and Chagas disease in Bolivia. Conclusions/Significance The recombinant rTiSP14.6 is a suitable and promising epidemiological marker for detecting the presence of small numbers of different species of triatomines and could be developed for use as a new tool in surveillance programs, especially to corroborate vector elimination in Chagas disease vector control campaigns.

/pdf/immunogenic-salivary-proteins-of-triatoma-infestans-3rxqyg1zvy.pdf

Immunogenic salivary proteins of Triatoma infestans: development of a recombinant antigen for the detection of low-level infestation of triatomines.

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.

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

Blood trypomastigotes of Trypanosoma cruzi were isolated from infected athymic rnu/rnu rats and purified by an improved procedure of DEAE-Sephacel ion-exchange chromatography. Elution into a buffer supplemented with bovine serum albumin avoided column-induced changes on the surface of the parasites. Biotin-labelled bovine serum albumin, fluorescence microscopy, flow cytometry and Western blot analysis revealed a very intense binding of albumin to the parasite. Incubation and washing of cells without protein supplementation did not result in any damage or lysis of parasites but it did cause extensive shedding of cellular and surface proteins into the supernatant which could be prevented by using the protein-supplemented buffer. A decreasing yield of high molecular weight cellular proteins in relation to centrifugal force was a general phenomenon observed in scanning densitometry of SDS gels after isolation in either protein-supplemented buffer or protein-free buffer. The quantity of shed cellular components increased with increasing centrifugal force. In contrast, quantities of high molecular weight, biotin-labelled surface proteins increased with greater centrifugal force, indicating labelling of otherwise inaccessible residues. These data emphasize the importance of protein supplementation of buffers with proteins and of choosing low centrifugation forces (< 400 g) during investigations of T. cruzi.

Effects of the isolation methodology on protein profiles of blood trypomastigotes of Trypanosoma cruzi

The development and pathogenicity of a Trypanosoma cruzi strain (“Chile 5”) of low virulence were studied after infection of nude rats with different doses of blood trypomastigotes (10–107 parasites/rat). Peak parasitemias were correlated with the infection dose, which also influenced the mean survival times (26–36 days post-infection). Within 26 or 27 days, a subcutaneous injection of 107 blood trypomastigotes developed to about 8–20 × 107 parasites/ml.

Guenter A. Schaub

Papers

Immunogenic salivary proteins of Triatoma infestans: development of a recombinant antigen for the detection of low-level infestation of triatomines.

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

Effects of the isolation methodology on protein profiles of blood trypomastigotes of Trypanosoma cruzi

Development of high numbers of blood trypomastigotes of Trypanosoma cruzi in nude rats.