The feeding efficiency and survival of flies that had four bloodmeals on mice that received anti-recTSGF 1–2 antibodies.

<p>(A) Mean engorged blood meal weights of flies measured before and after each blood meal. Eight cages of flies were allowed to feed on mice at Day 2, Day 5, Day 8 and Day 11 after the mice received rabbit anti-recTSGF treatment. Cages were weighed before and after each blood meal. Each data point corresponds to one cage, which includes the average blood engorgement data for all alive flies in that cage (n = 8 flies per cage at the beginning). (B) Percent survival of flies maintained on rec-TSGF antibody treated and control mice, respectively. The first day they were exposed to mice is considered Day 0. Both groups had 64 flies at the beginning of the experiment. (C) Average weight change in each cage between Day 1 and Day 12. Each data point represents the average weight change in each cage. As one mouse in the TSGF antibody treated group died before the 3^rd blood meal, 6 cages of flies from the TSGF antibody treated group and 8 cages from the control group were measured here. (D): Lipid levels determined from flies 72 h after their 4th blood meal. Each data point represents the average of all alive flies in each cage (n = 6 cages in TSGF antibody treated group and n = 8 cages in control group). Statistical analyses were performed using the Mann Whitney test in the GraphPad Prism 6 software package. No significant difference were detected between the two groups (P>0.1).</p

Immunoblot of sialome proteins from <i>Gmm</i>, <i>Gpd</i>, <i>Gff</i> and <i>Gpg</i>.

<p>Same amount of sialome proteins from <i>Gmm</i>, <i>Gpd</i>, <i>Gff</i> and <i>Gpg</i> were probed with (A) rec-<i>Gmm</i> Tsal1, (B) rec-<i>Gmm</i> TSGF-1 and (C) rec-<i>Gmm</i> TSGF-2 antibodies, respectively. (D) <i>Gpg</i> sialome components, equivalent to 0.2 pairs of salivary glands, are analyzed on Coomassie Blue stained SDS-PAGE (lane 2), and by immunoblot analysis using rec-<i>Gmm</i> TSGF-2 antibodies (lane 3) and anti-<i>Gff</i> saliva antibodies (lane 4). Lane 1 shows molecular marker. * indicates TSGF-2 corresponding protein band.</p

SDS PAGE analysis of saliva from different tsetse species.

<p>Lanes 1–4 show protein profiles of <i>Gmm</i>, <i>Gpd</i>, <i>Gff</i> and <i>Gpg</i> sialomes analyzed by SDS-PAGE analysis stained by Coomassie Blue. M indicates the Molecular Weight marker. Bands referred to in the sialome of each species are numbered from top to bottom. One representative image for the sialome data is shown. Additional results from SG extracts and replicate sialome samples are shown in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004038#pntd.0004038.s005" target="_blank">S1 Fig</a>.</p

Identification of the major immunogenic and non-immunogenic proteins in <i>Gmm</i> sialome.

<p>The most abundant protein bands extracted for LC-MS/MS analysis are shown boxed. Fractions 1, 4 and 5, as marked by “*”, correspond to protein bands that were noted as immunogenic in the analysis presented in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004038#pntd.0004038.g002" target="_blank">Fig 2</a>. Protein fractions 2 and 3 were combined representing non-immunogenic proteins.</p

Specific genes increased in BSF parasites from mouse blood.

<p>Specific genes increased in BSF parasites from mouse blood.</p

Sequence alignment comparison of <i>Gmm</i> TSGF family with <i>Drosophila</i> ADGF family.

<p>Note: Bold characters indicating the highest identity of TSGF with <i>Dm</i> ADGF sequences.</p><p>*: TSGF7 have a stop codon in 607–609 bp. Here the protein sequence included in the comparison is only 202 aa. But the nucleotide sequence is the whole transcripts corresponding to the ORF sequence of other TSGF and ADGFs in alignment.</p><p>Sequence alignment comparison of <i>Gmm</i> TSGF family with <i>Drosophila</i> ADGF family.</p

Insights into the Trypanosome-Host Interactions Revealed through Transcriptomic Analysis of Parasitized Tsetse Fly Salivary Glands

<div><p>The agents of sleeping sickness disease, <i>Trypanosoma brucei</i> complex parasites, are transmitted to mammalian hosts through the bite of an infected tsetse. Information on tsetse-trypanosome interactions in the salivary gland (SG) tissue, and on mammalian infective metacyclic (MC) parasites present in the SG, is sparse. We performed RNA-seq analyses from uninfected and <i>T. b. brucei</i> infected SGs of <i>Glossina morsitans morsitans</i>. Comparison of the SG transcriptomes to a whole body fly transcriptome revealed that only 2.7% of the contigs are differentially expressed during SG infection, and that only 263 contigs (0.6%) are preferentially expressed in the SGs (SG-enriched). The expression of only 37 contigs (0.08%) and 27 SG-enriched contigs (10%) were suppressed in infected SG. These suppressed contigs accounted for over 55% of the SG transcriptome, and included the most abundant putative secreted proteins with anti-hemostatic functions present in saliva. In contrast, expression of putative host proteins associated with immunity, stress, cell division and tissue remodeling were enriched in infected SG suggesting that parasite infections induce host immune and stress response(s) that likely results in tissue renewal. We also performed RNA-seq analysis from mouse blood infected with the same parasite strain, and compared the transcriptome of bloodstream form (BSF) cells with that of parasites obtained from the infected SG. Over 30% of parasite transcripts are differentially regulated between the two stages, and reflect parasite adaptations to varying host nutritional and immune ecology. These differences are associated with the switch from an amino acid based metabolism in the SG to one based on glucose utilization in the blood, and with surface coat modifications that enable parasite survival in the different hosts. This study provides a foundation on the molecular aspects of the trypanosome dialogue with its tsetse and mammalian hosts, necessary for future functional investigations.</p></div

<i>Gmm</i> ADGF family gene organization and transcript abundance.

<p>(A): Chromosomal organization of <i>Gmm</i> ADGF family on genome scaffolds. (B) Phylogenetic tree showing the relatedness of the <i>Drosophila</i> spp. and <i>Glossina</i> spp. ADGF family members inferred from amino acid sequences. The scale bar represents 0.100 substitutions per site. The numbers on top of the branches indicate the bootstrap values. (C) The expression of the four members of the ADGF family of genes from <i>Gmm</i>, <i>Gpd</i>, <i>Gpg and Gff</i> salivary gland. The RNAseq based transcriptomes of salivary glands in different tsetse species were analyzed and the transcript abundance values are shown as RPKM.</p

Overview of trypanosome transcriptome analysis.

<p>A. Number of reads mapping to the parasite protein coding genes from blood and parasite-infected salivary gland RNA-seq libraries. B. Reads per gene from parasite-infected blood RNA-seq library and parasite-infected salivary gland RNA-seq libraries. C. Genes expressed at significantly higher levels in parasite-infected blood and in salivary glands.</p

Specific salivary gland contigs and proteins suppressed during trypanosome infection.

<p>A. Fold change during salivary gland infection based on RNA-seq analysis from SG-enriched dataset that have a combined infected and control RPKM value of over 1000 from <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002649#pntd.0002649.s002" target="_blank">Table S1</a>. The combined infected and control RPKM value is shown next to each bar. B. Percent of RNA-seq reads represented by the contigs with decreased expression during trypanosome infection. C. Total protein profile analyzed by SDS-PAGE analysis from one pair of infected and control salivary glands, one representative sample is shown. D. Western blot analysis of Tsal1, TSGF-1, TSGF-2 as well as Tubulin from control and infected SG.</p