Mean (± S.E.) prevalence of midgut infections in male <i>G. m. morsitans</i> (<i>Gmm</i>) or <i>G. p. palpalis</i> (<i>Gpp</i>) after RNAi knockdown using ds<i>tsetse EP</i>.

<p>Controls were either ds<i>Ampicillin</i>, ds<i>eGFP</i> or nuclease free water*. Flies were infected with either <i>T. b. brucei</i> TSW196 or <i>T. congolense</i> 1/148 (italics) blood stream forms in the indicated bloodmeal.</p

Immunoblot film lies below the nigrosine-stained PVDF.

<p>There is a decline in tsetse EP protein levels in midguts over a 7 day starvation period following the 5^th bloodmeal. 24 h  =  Flies starved for 7 days, fed a blood meal and then sacrificed 24 hours later. L  =  molecular mass ladder. Midgut proteins (1/2 midgut equivalent from pool of 5) were blotted with mAb 247.</p

dsRNA knockdown of putative tsetse serpins results in decreased trypanosome prevalence in the tsetse midgut.

<p>(A) Individual knockdown (KD) of putative tsetse midgut serpins by feeding dsRNA resulted in the suppression of the target <i>serpin</i> mRNA transcript. A degree of cross-reactivity was observed with two genes, with knockdown of <i>GmmSRPN9</i> leading to a significant decrease in transcript level of two other serpins (<i>GmmSRPN5</i> and <i>GmmSRPN10</i>) and knockdown of <i>GmmSRPN3</i> leading to a significant increase in <i>GmmSRPN9</i>. (B) Knocking down each putative tsetse midgut serpin resulted in a decrease in trypanosome infection rates in the tsetse midgut ten days post infection, but this decrease in infection rate was only significant when either <i>GmmSRPN9</i> or <i>GmmSRPN10</i> were knocked down compared to ds<i>EGFP</i>-treated controls (P<0.05, t-test and one-way ANOVA). Knockdowns were carried out in tandem with approximately 20 flies per group and each bar represents at 3 biological replicates.</p

HS complement is lethal to PFs.

<p>(A) The lethality of HS can be inactivated by pre-treating the serum with heat. PF mortality decreases significantly when HS is heat inactivated at 50°C for 1 h, and up to 88.5±5.67% survive when the temperature of inactivation was 60°C. (B) PF lysis by HS decreases when HS was pre-treated with CVF to exhaust complement cascade factors. (C) Tsetse infection experiments carried out using heat inactivated serum resulted in a significant (P = 0.01) increase in infected midguts in experimental groups where heat inactivated serum was used for infection and feeding. (D) A similar increase in infection rate was observed when the HS used in experiments was pre-treated with CVF, though this increase was not statistically significant compared to tsetse that were infected and maintained with HS pre-treated with boiled CVF after 4 experimental replicates.</p

Expression of His::GmmSRPN10 is temperature sensitive and the expressed protein inhibits killing of PF trypanosomes by HS <i>in vitro</i>.

<p>(A) Recombinant expression of His::GmmSRPN10 results in an intact (∼40 kDa) and truncated (∼37 kDa) protein fraction that were identified using mass spectrometry. Expression at 30°C enriches the intact fraction. (B) Supression of trypanocidal activity by His::GmmSRPN10 can be achieved by pre-treating HS with the recombinant protein. (C–D) His::GmmSRPN10 inhibits the activity of rC1s and rFactorD, recombinant human complement cascade serine proteases, in a concentration dependent manner. Inhibition of the complement cascade serine proteases at comparable concentrations were not observed in the controls performed in tandem using a commercially available soybean trypsin inhibitor.</p

GmmSRPN10 is secreted into the midgut lumen and is important for PF infection.

<p>(A) Western blot (using α-GmmSRPN10 antisera) analyses of midgut tissue and midgut lumen content from teneral (T) and fed (F) tsetse suggests that GmmSRPN10 (arrow) is secreted into the midgut lumen in teneral (newly emerged and yet unfed) flies in preparation for blood feeding. Lower panel, stained PVDF membrane with nigrosine. (B) Comparison of <i>β-tubulin</i> and <i>GmmSRPN10</i> transcript levels between <i>GmmSRPN10</i> knockdown and control tsetse at the point of dissection at ten days post trypanosome infection indicate that <i>β-tubulin</i> transcript levels remain relatively unchanged in knockdown flies while <i>GmmSRPN10</i> transcript is significantly decreased to less than 50% (bars represent 3 independent experiments). (C) Downregulation of GmmSRPN10 protein in midguts from dsGmmSRPN10 treated knockdown flies. (D) The knockdown of <i>GmmSRPN10</i> results in a small but significant decrease in PF midgut infection, suggesting that the expression of GmmSRPN10 is important for PF survival (each data point represents a biological replicate, with a total of >300 flies dissected scored for infection per treatment).</p

Investigated tsetse midgut serpins.

<p>Four putative serpins were initially identified from a tsetse midgut EST library. Subsequent sequencing and annotation of the genome has now assigned these serpins with GMOY IDs in the Invertebrate Vectors of Human Pathogens database, VectorBase (<a href="https://www.vectorbase.org" target="_blank">https://www.vectorbase.org</a>).</p><p>Investigated tsetse midgut serpins.</p

Putative tsetse midgut serpins show active site conservation with human complement cascade inhibitory serpin, but may not have evolved as a consequence of a haematophagous lifestyle.

<p>(A) Clustal-W alignment the putative tsetse serpins RCL region (GmmSRPN3, GmmSRPN5, GmmSRPN9, GmmSRPN10) with human SerpinG1 complement cascade inhibitor indicate conserved (highlighted in grey) key residues involved in the inactivation of serine proteases. (B) A phylogenetic tree (with bootstrap values shown) generated from neighbour-joining alignment of putative tsetse midgut serpins (denoted with *) and representative serpins from arthropod species with fully sequenced genomes (<i>Aedes aegypti</i>, <i>Anopheles gambiae</i>, <i>Apis mellifera</i>, <i>Bombyx mori</i>, <i>Drosophila melanogaster</i> and <i>Tribolium castaneum</i>). Distinct clustering of these putative midgut serpins with that of haematophagous species (<i>Aedes aegypti, Anopheles gambiae</i>) was not apparent, suggesting that the serpins may have either evolved following speciation of <i>Glossina morsitans morsitans</i> or from the last common arthropod ancestor.</p

A breakdown explaining the equivalent number of trypanosomes at different volumes used in the study.

<p>A breakdown explaining the equivalent number of trypanosomes at different volumes used in the study.</p

Details of five alternative field-friendly DNA extraction methods.

<p>Details of five alternative field-friendly DNA extraction methods.</p