• Figure 2.
    Download figureOpen in new tabFigure 2. Tsetse odorant binding protein 6 does not mediate the development and function of phagocytic hemocytes.

    (A) Survival following systemic challenge of siOBP6 and siGFP adults with 5 × 102 CFU of E. coli K12. Fly survival was monitored every other day for the duration of the 14 day experimental period. Survival assays were performed in triplicate, using 25 flies per replicate. Red curve depicts a statistically significant difference in infection outcome (p<0.0001, log-rank test). (B) Hemocyte abundance in siOBP6 and siGFP adults was quantified microscopically using a hemocytometer (Figure 2—source data 1). (C) A representative micrograph of hemocyte-engulfed recE. coliGFP from siOBP6, siGFP and siOBP6R adults. Experiment was performed using hemolymph collected from four distinct flies per (Figure 2—source data 2). Hemolymph was collected 12 hpc and fixed on glass slides using 2% paraformaldehyde. Magnification is x400. (D) E. coli densities (CFU/μl of hemolymph) in the hemolymph of siOBP6, siGFP and siOBP6R adults at 2 and 6 dpc (Figure 2—source data 3). In (B) and (D), symbols represent one hemolymph sample per group, and bars represent the median hemocyte quantity (B) or bacterial density (D) per sample. Statistical analysis = ANOVA followed by Tukey’s HSD post-hoc analysis.

    DOI: http://dx.doi.org/10.7554/eLife.19535.006

    Figure 2—source data 1.Circulating hemocytes per microliter of hemolymph.

    DOI: http://dx.doi.org/10.7554/eLife.19535.007

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    Figure 2—source data 3.Colony forming units (CFU) per microliter of hemolymph.

    DOI: http://dx.doi.org/10.7554/eLife.19535.009

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    Figure 3.
    Download figureOpen in new tabFigure 3. Obp6 mediates the melanization cascade in adult tsetse.

    (A) Survival following administration of clean wounds to the thoracic cuticle of siOBP6, siGFP and siOBP6R adults. Survival assays were performed in triplicate, using 25 flies per replicate. Red curve depicts a statistically significant difference in infection outcome (p<0.0001, log-rank test). (B) A representative micrograph of the cuticle of siRNA treated adults 3 hr post-wounding (hpw) with a clean needle. Melanin deposited at the wound site of siGFP and siOBP6R controls, and hemolymph exudate from a siOBP6 treatment individual, are identified by black and red arrowheads, respectively. Scale bar = 500 μm. Experiment was performed using four distinct flies per group (Figure 3—source data 1). (C) Quantitation of PPO1 and PPO2 in the hemolymph of siOBP6, siGFP and siOBP6R adults three hpw with a clean needle. Shown is a representative Western blot analysis using Drosophila anti-PPO1 and anti-PPO2 antibodies. 8 μl of pooled hemolymph was run per gel lane. Hemolymph was collected and pooled from four individuals from each group. Western blots were repeated in triplicate [Figure 3—source data 2 (for PPO1 westerns) and Figure 3—source data 3 (for PPO2 westerns)]. (D) PO activity in the hemolymph of siOBP6, siGFP and siOBP6R adults at 0 and 3 hpw with a clean needle. n = 5 biological replicates per group per time point for pre-wound readings, and n = 8 biological replicates per group per time point for post-wound readings. Data are presented as mean ± SEM. Bars with different letters indicate a statistically significant difference between pre- and post-wound values (specific p values are listed in the Figure 3—source data 4). Statistical test = 2 way ANOVA followed by Tukey’s HSD post-hoc analysis.

    DOI: http://dx.doi.org/10.7554/eLife.19535.010

    Figure 3—source data 1.Melanin deposition at tsetse cuticular wound sites.

    DOI: http://dx.doi.org/10.7554/eLife.19535.011

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    Figure 3—source data 2.Tsetse prophenoloxidase 1 (PPO1) western blots.

    DOI: http://dx.doi.org/10.7554/eLife.19535.012

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    Figure 3—source data 3.Tsetse prophenoloxidase 2 (PPO2) western blots.

    DOI: http://dx.doi.org/10.7554/eLife.19535.013

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    Figure 4.
    Download figureOpen in new tabFigure 4. Obp6 expression in the gut of larval tsetse is an integral component of the systemic pathway that actuates crystal cell production.

    (A) Representative micrograph depicting spontaneous PPO activation in early third instar siGFP, siOBP6 and siOBP6R tsetse larvae following subjection to a 10 min heat shock at 65°C. Experiment was repeated using one larvae from five distinct moms from each group. Melanotic spots were quantitated microscopically. Statistical analysis = Kruskal-Wallis test followed by Dunn’s post-hoc analysis (Figure 4—source data 1). (B) RT-qPCR analysis of obp6, serpent and lozenge expression in embryos prior to maternal treatment with siRNA, and in siOBP6, siGFP and siOBP6R tsetse larvae from siRNA treated moms. Embryo replicates (n = 5) contain three embryos, larval replicates (n = 7 for siOBP6, n = 5 for siGFP and n = 6 for siOBP6R) contain a mixture of four first and second instar larvae. ND, not detectable. Data are presented as mean ± SEM. Bars with different letters indicate a statistically significant difference between samples (specific p values for larval samples are listed in the Figure 4—source data 2). Statistical analysis = 2 way ANOVA followed by Tukey’s HSD post-hoc analysis. (C) Representative image of obp6 and lozenge spatial expression patterns, determined using semi-quantitative RT-PCR, in the gut and carcass of second instar GmmWT larvae. Experiment was repeated using guts and carcasses from five distinct individuals (Figure 4—source data 3).

    DOI: http://dx.doi.org/10.7554/eLife.19535.015

    Figure 4—source data 1.Sessile crystal abundance in larval tsetse.

    DOI: http://dx.doi.org/10.7554/eLife.19535.016

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    Figure 4—source data 2.Relative obp6, serpent and lozenge gene expression in tsetse embryoes and larvae.

    DOI: http://dx.doi.org/10.7554/eLife.19535.017

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    Figure 4—source data 3.Tissue distribution of obp6 and lozenge expression in tsetse larvae.

    DOI: http://dx.doi.org/10.7554/eLife.19535.018

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    Figure 6.
    Download figureOpen in new tabFigure 6. Model illustrating the functional relationship between maternally-transmitted enteric symbionts and melanization in tsetse.

    GmmWT larvae imbibe enteric symbiotic-containing milk gland secretions throughout their intrauterine developmental program. These bacteria colonize larval gut-associated tissues, including the bacteriome, and in doing so, induce the expression of obp6. OBP6 is either secreted directly into the hemolymph, or acts locally to induce expression of another unknown, (also secreted) protein. One of these molecules then acts systemically in the larval hematopoietic niche (hn) to stimulate lozenge (lz) expression in a small proportion of serpent (srp) expressing prohemocytes. These cells then become PPO-producing crystal cells [remaining prohemocytes become phagocytes after expressing glial cells missing (gcm)]. Finally, crystal cells are expelled from the hn, where they circulate in the hemolymph and are available to produce wound-healing melanin. Larvae that develop in the absence of symbiotic bacteria (GmmApo) fail to produce any hemocytes, while those that develop in the presence of reduced obp6 transcript abundance (GmmOBP6-) fail to express lozenge and thus likely fail to generate crystal cells. dv, dorsal vessel; hc, hemocoel; w, wound; ep, epithelial cells of midgut; bc, bacteriome; pm, peritrophic matrix; gl, gut lumen.

    DOI: http://dx.doi.org/10.7554/eLife.19535.027

  • The following dataset was generated:

    Joshua B Benoit, Aurélien Vigneron, Nichole A Broderick, Yineng Wu, Jennifer S Sun, John R Carlson, Serap Aksoy, Brian L Weiss, 2016,Glossina morsitans strain: Yale Transcriptome or Gene expression: 1st instar tsetse fly larvae (whole organism), https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA309164, Publicly available at NCBI BioProject (accession no. PRJNA309164)

    The following previously published datasets were used:

    Scolari F, Benoit JB, Michalkova V, Aksoy E, Takac P, Abd-Alla AM, Malacrida AR, Aksoy S, Attadro GM, 2016,Glossina morsitans morsitans Male accessory gland and testes raw illumina reads, http://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA295435, Publicly available at NCBI BioProject (accession no. PRJNA295435)

    Ichalkova V, Krause TB, Bohova J, Zhang Q, Baumann AA, Mireji PO, Takac P, Denlinger DL, Ribeiro JM, Aksoy S, 2014,Glossina morsitans morsitans transcriptome during milk production, http://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA205861, Publicly available at NCBI BioProject (accession no. PRJNA205861)