Bacterial infection outcomes in tsetse’s midgut, and subsequent fly survival

We investigated the ability of Kco_Z and Sodalis (as a control) to colonize the gut of both wild-type (hereafter referred to as ‘Gmm^WT’) and symbiont-free tsetse (aposymbiotic, hereafter referred to as ‘Gmm^Apo’). Gmm^WT flies were used to interrogate the interaction between Kco_Z and the natural tsetse microbiota, while the use of Gmm^Apo individuals allowed us to correlate the presence of distinct, experimentally introduced bacterial taxa with specific fly phenotypes. We challenged all flies per os with 1x10³ CFU of either Kco_Z or Sodalis in their first blood meal and then monitored bacterial proliferation over a 28 day period. By 7 days post-inoculation, midgut bacterial load in Gmm^WT flies that housed Kco_Z (Gmm^WT/Kco_Z) and Sodalis (Gmm^WT/Sgm) was 1.9x10⁷ ± 6.4x10⁶ CFU and 4.5x10⁵ ± 6.4x10⁶ CFU, respectively, and Gmm^Apo/Kco_Z (9.3x10⁶ ± 5.3x10⁵ CFU) and Gmm^Apo/Sgm (1.4x10⁶ ± 3.9x10⁵ CFU) flies harbored a similar bacterial load at the same time point post-inoculation (Fig 1A). The midgut load of Kco_Z and Sodalis did not change significantly in any of the fly groups over the following 21 days (Fig 1A), thus suggesting that the bacteria had achieved stable-state infections within their fly hosts by one week post-acquisition.

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Fig 1. Bacterial colonization of tsetse’s midgut, and the effect on fly survival.

Distinct groups of newly emerged adult wild-type (Gmm^WT) and aposymbiotic (Gmm^Apo) females were colonized with 1x10³ CFU of either Kosakonia cowanii Zambiae (Kco_Z) or Sodalis (Sgm), and then bacterial load and fly survival was measured. (A) Average number (±SEM) of bacterial CFUs per tsetse gut per group per time point. n≥5 individuals per group per timepoint. (B) Kaplan-Meier plot depicting survival of Gmm^WT and Gmm^Apo females colonized with either Kco_Z or Sgm. Infection experiments were performed using three distinct biological replicates (n = 25 flies per replicate). No significant difference in survival was observed between any of the fly groups (p = 0.88; log-rank test).

https://doi.org/10.1371/journal.ppat.1007470.g001

We next examined whether midgut infections with Kco_Z or Sodalis impacted tsetse survival. We found that 76% of Gmm^WT/Kco_Z individuals, and 84% of Gmm^WT/Sgm individuals, survived for 28 days following bacterial inoculation. Similarly, 84% and 80% of Gmm^Apo/Kco_Z and Gmm^Apo/Sgm flies, respectively, survived the duration of the experiment (Fig 1B). Percent survival was not significantly different between any of these groups, indicating that Kco_Z and Sodalis both exhibit commensal phenotypes in wild-type and aposymbiotic tsetse.

Kco_Z is resistant to Peptidoglycan Recognition Protein-LB (PGRP-LB)

The midgut of adult tsetse expresses peptidoglycan recognition protein LB (pgrp-lb), which encodes a pattern recognition receptor that exhibits potent antimicrobial activity [14,15]. Thus, in order to colonize tsetse’s midgut, a microorganism must be resistant to this molecule. We investigated whether innate resistance to PGRP-LB represents one mechanism that allows Kco_Z to colonize tsetse’s gut. We found that 108% (±16) of Kco_Z cells were able to survive 1 h in the presence of recPGRP-LB, while only 2.3% (±1.0) of E. coli cells survived for the same time period. Additionally, 154% (±14) of Sodalis cells survived following a 12 h incubation with recPGRP-LB (Fig 2). These findings suggest that like native Sodalis, Kco_Z is resistant to the antimicrobial properties of PGRP-LB and is able to survive in the presence of this protein (as indicated by a slight increase in bacterial load compared to the initial inoculate). This phenotype may facilitate this bacterium’s ability to successfully colonize tsetse’s gut.

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Fig 2. Kco_Z is resistant to normally bactericidal Peptidoglycan Recognition Protein-LB.

Survival of cultured Kco_Z, Sodalis and E. coli following exposure (1 hr for Kco_Z and E. coli, and 24 hr for Sodalis) to recombinant (rec) PGRP-LB (10 μg/ml). Results are presented as % of initial inoculum, which was determined by dividing the number of bacterial CFU present after treatment with recPGRP-LB by the number of CFU present prior to inoculation. Each point on the graph represents a distinct bacterial culture. Statistical significance was determined using a one-way ANOVA followed by Tukey’s HSD post-hoc analysis.

https://doi.org/10.1371/journal.ppat.1007470.g002

Kco_Z colonized aposymbiotic tsetse present a trypanosome refractory phenotype

Kco_Z successfully colonizes the gut of Gmm^Apo flies, resides in the niche for at least 28 days, and has no impact on fly survival during that time period. Thus, we next evaluated whether colonization with this bacterium impacts trypanosome infection establishment in tsetse’s midgut. We began by challenging mature Gmm^Apo flies because they are highly susceptible to trypanosome infection (~50%) while their age-matched Gmm^WT counterparts are refractory (~3%) [16]. Distinct groups of eight day old Gmm^Apo/Sgm and Gmm^Apo/Kco_Z flies, which housed similar numbers of their respective exogenous bacteria (S1A Fig), were administered a meal supplemented with 1x10⁶ blood stream form (BSF) trypanosomes per ml of blood. Thereafter all flies were maintained on regular blood for two weeks, at which point their midguts were dissected and microscopically examined for the presence of parasites. An age-matched control cohort consisted of similarly challenged Gmm^Apo flies. We found that infection prevalence in the Gmm^Apo/Sgm group (57%) was similar to that of Gmm^Apo controls (52%), while infection prevalence in Gmm^Apo/Kco_Z individuals was significantly lower (19%) (Fig 3A). These data indicate that the presence of Kco_Z in tsetse’s gut interferes with the ability of trypanosomes to establish an infection in this niche. This parasite resistant phenotype is similar to that which occurs in the gut of Kco_Z colonized mosquitoes following exposure to malaria parasites [29,30].

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Fig 3. The trypanosome refractory phenotype exhibited by Gmm^Apo/Kco_Z flies is not directly caused by reactive oxygen intermediates.

(A) Percentage of Gmm^Apo, Gmm^Apo/Sgm and Gmm^Apo/Kco_Z flies harboring midgut infections with bloodstream form (BSF) YTAT 1.1 trypanosomes. Statistical analysis was performed using a GLM followed by multiple comparisons and Tukey contrasts, and different letters represents statistical significance between treatments and controls. (B) Mid-log phase cultures of Kco_Z and Sodalis synthesize similar quantities of H₂O₂ (p = 0.6; paired t-test). Measurements were taken from 7 distinct clonal populations of each bacterium. (C) Percentage of Gmm^Apo and Gmm^Apo/Kco_Z flies infected with BSF YTAT 1.1 trypanosomes after being fed blood meals containing antioxidant vitamin C over the course of the 14 day experiment. Despite exposure of both tsetse groups to the ROI-suppressing vitamin, Gmm^Apo/Kco_Z flies were still significantly more refractory to trypanosome infection than were Gmm^Apo individuals (p = 0.002; GLM Wald test). In (A), (B) and (C) different letters represents statistical significance between treatments and treatments and controls.

https://doi.org/10.1371/journal.ppat.1007470.g003

African trypanosomes are not susceptible to Kco_Z generated reactive oxygen intermediates

In A. gambiae, Kco_Z produces reactive oxygen intermediates (ROIs) that are directly toxic to Plasmodium [29,30]. ROIs have also been implicated as mediators of trypanosome infection outcomes in tsetse. Specifically, tsetse are rendered susceptible to trypanosome infection when the initial infectious blood meal is supplemented with the antioxidants vitamin C or cysteine [18,31]. These antioxidants detoxify ROIs that otherwise induce programmed cell death processes in trypanosomes [32]. In light of this information, we investigated the correlation between Kco_Z generated ROIs and the trypanosome refractory phenotypes we observed in adult Gmm^Apo/Kco_Z individuals. As an indicator of bacterial ROI production, we quantified H₂O₂ concentrations in supernatants from mid-log phase Kco_Z and Sodalis cultures. Kco_Z and Sodalis supernatants contained 127nM (±15) and 142nM (±13) of H₂O₂, respectively (Fig 3B).

We next tested whether ROIs produced by Kco_Z inhibit the ability of trypanosome to infect Gmm^Apo/Kco_Z flies. Individual groups of eight day old Gmm^Apo and Gmm^Apo/Kco_Z individuals were offered a blood meal containing infectious trypanosomes together with the antioxidant vitamin C. All trypanosome challenged flies were subsequently maintained on vitamin C supplemented blood for 14 days. Under these conditions, 74% of Gmm^Apo+vitC flies were infected with trypanosomes, while only 11% of their Gmm^{Apo/Kco_Z+vitC} counterparts housed parasite infections (Fig 3C). These results suggest that ROIs produced by Kco_Z that reside stably in tsetse’s gut are not the sole determinants of the fly’s susceptibility to infection with trypanosomes.

Kco_Z produces acid that is toxic to trypanosomes

We observed that Gmm^Apo/Kco_Z flies are significantly more refractory to infection with trypanosomes than are Gmm^Apo/Sgm individuals, despite the fact that Sodalis and Kco_Z produce similar amounts of H₂O₂. This outcome implies that Kco_Z modulates trypanosome infection outcomes in tsetse via a mechanism other than ROI production. Members of the genus Kosakonia [33–35], as well as several enteric commensals including Enterobacter spp. [36–38], produce organic acids, and these products can inhibit pathogen growth by creating an acidic environment [39,40]. Because many trypanosomatids, including members of the genera Trypanosoma and Leishmania, are highly sensitive to environmental pH [41,42], we investigated whether Kco_Z creates an acidic environment that prohibits T. brucei growth in vitro. Specifically, we heat killed (HK) Kco_Z (1x10⁶ log-phase in 500 μl of LB media) and added the solution to trypanosome cultures maintained in vitro. This medium includes phenol red, which is a pH-sensitive dye that when in solution turns from red-pink to yellow as the quantity of acid in the environment increases. Addition of this HK Kco_Z extract immediately turned the Beck’s media yellow, and the pH measured at 5.8 (± 0.39). This value was significantly lower than trypanosome cultures that were supplemented with 500 μl of 1x10⁶ log-phase HK trypanosomes (pH 7.3 ± 0.28), HK Sodalis (pH 7.4 ± 0.39) or LB (Kco_Z growth media; pH 7.1 ± 0.28) or MM media (Sodalis growth media; pH 7.2± 0.29) alone (Fig 4A). We subsequently monitored trypanosome growth in cultures that received the above-mentioned supplements. We observed that trypanosomes failed to replicate in Beck’s medium that contained HK Kco_Z extracts, while trypanosomes multiplied in all of the other culture conditions (Fig 4B).

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Fig 4. Kco_Z produces a low pH environment that is toxic to trypanosomes.

(A) Early log phase trypanosomes (T. b. brucei YTAT 1.1), cultured in 10ml of Beck’s medium containing the pH sensitive dye phenol red, exposed to 1ml of heat treated LB media (Kco_Z culture medium), 1ml of heat treated BHI media (Sodalis culture medium), heat killed (HK) Kco_Z (5x10⁶ cells) in 1ml of LB media and HK Sodalis (5x10⁶ cells) in 1ml of BHI media. Controls are trypanosomes alone (tryps). All heated treatments and controls were allowed to cool to room temperature prior to adding them to the trypanosome cultures. Two hours post-treatment, culture pH was measured. HK Kco_Z significantly reduced the pH of the trypanosome culture (p<0.001). The experiment was repeated using 6 distinct clonal trypanosome populations (the image represents one of the six replicates). (B) Density of trypanosomes in culture 2h, 5h and 24h after addition of the treatments described in (A) above. At the 24h time point, all trypanosomes exposed to HK Kco_Z extracts were dead while those from the other groups were replicating similarly to controls. (C) Density of trypanosomes cultured in normal (pH 7.2) and artificially produced (via the addition of 0.1N HCl) acidic (pH 5.8) environments. Artificial acidic conditions kill all trypanosomes with 24 h. (D) The density of cultured trypanosomes exposed to HK Kco_Z extracts buffered to pH 7.2 (via the addition of 0.1N NaOH). The buffering treatment rescues parasite growth. In (A), (B) and (C), statistical significance was determined using a one-way ANOVA followed by Tukey’s HSD post-hoc analysis in (A), and a two-way ANOVA followed by Tukey’s HSD post-hoc analysis in (B) and (C). Different letters represent statistical significance between treatments and controls. In (B), (C) and (D), experiments were performed using 5 or 6 distinct clonal trypanosome populations.

https://doi.org/10.1371/journal.ppat.1007470.g004

Heat-killed Kco_Z extracts create an acidic environment when added to trypanosome cultures, and trypanosomes fail to replicate in this environment. These findings do not rule out the possibility that trypanosomes are capable of surviving Kco_Z-induced acidic conditions, and instead, some other unknown component of the medium [e.g., a bacterium-derived trypanocidal molecule(s)] exhibits toxic properties. To address this possibility, we monitored trypanosome growth in Beck’s medium, the pH of which was artificially decreased to 5.8 (the same as that achieved by adding HK Kco_Z extracts) via the addition of exogenous acid. Under these conditions trypanosomes failed to replicate (Fig 4C). Furthermore, when we buffered Beck’s medium containing HK Kco_Z extracts back up to pH 7.2, trypanosomes replicated normally (Fig 4D). Taken together, these data indicate that Kco_Z produces an acidic environment that is toxic to trypanosomes, thus impeding their growth in vitro.

Kco_Z acidifies tsetse’s gut

We observed that trypanosomes are unable to multiply when cultured in medium supplemented with acidic Kco_Z extracts. Thus, we next investigated whether Kco_Z produces acid in vivo in tsetse’s gut. To do so we colonized teneral, aposymbiotic flies with either Kco_Z or Sodalis, and 5 days later fed them a meal containing 2.5% sucrose and 0.04% phenol red solubilized in water. Twenty-four hours later, midguts from a sample of flies (n = 8 per group) were excised and plated on solid medium containing phenol red. Gmm^Apo/Kco_Z and Gmm^Apo/Sgm flies housed similar densities of the introduced bacteria (S1B Fig), and their respective mediums changed color to reflect corresponding pH shifts (S1C Fig). The remaining flies were dissected to expose their midgut in situ, and the color of the gut contents was visualized microscopically. We observed that the gut contents of Gmm^Apo/Kco_Z flies were yellow in color (Fig 5), thus indicating that the environment had become acidified. Conversely, the gut contents of Gmm^Apo/Sgm individuals were red, which is similar to the more alkaline environment present in the gut of Gmm^WT tsetse (Fig 5).

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Fig 5. Kco_Z acidifies tsetse’s gut.

Midgut pH of Gmm^Apo/Kco_Z, Gmm^Apo/Sgm, Gmm^WT/Kco_Z and Gmm^WT flies. Distinct groups of teneral Gmm^Apo flies were inoculated per os with either Kco_Z or Sodalis, while Gmm^WT individuals received Kco_Z (all flies received 5x10⁴ CFU of bacteria per ml of blood) or no bacteria. Five days post-inoculation, all individuals were offered a meal containing sucrose (2.5%) and phenol red (0.04%) dissolved in water. Twenty-four hours later, fly abdomens were excised and the color of the solution found within the midgut was observed. Each image represents one of five flies monitored for each treatment.

https://doi.org/10.1371/journal.ppat.1007470.g005

Finally, we investigated whether Kco_Z also produces acid in the gut Gmm^WT tsetse by inoculating teneral individuals with 1x10³ CFU of the bacterium (these flies were designated Gmm^WT/Kco_Z). Five days later a cohort of Gmm^WT/Kco_Z females (these flies housed 1.27x10⁶ ± 8.6x10⁴ Kco_Z at this time point; S1D Fig), as well as age matched Gmm^WT controls, were fed a sugar meal containing phenol red (as described above) to observe gut pH. Similar to our results noted in Gmm^Apo/Kco_Z flies, we observed that the gut of Gmm^WT/Kco_Z individuals was yellow, thus indicative of an acidified environment. Conversely, the gut environment of Gmm^WT flies was red and thus comparatively alkaline (Fig 5). Thus, the presence of indigenous symbionts does not impede the ability of Kco_Z to acidify the gut of wild-type flies.

Gmm^WT/Kco_Z are highly refractory to infection with trypanosomes and entomopathogenic bacteria

We hypothesized that exogenous microorganisms would be unable to successfully infect Gmm^WT/Kco_Z due to their acidified midgut environment. To test this hypothesis we first co-inoculated teneral Gmm^WT males with Kco_Z and T. congolense parasites. Two weeks post-challenge we observed no significant difference in the percentage of Gmm^WT/Kco_Z (15%) and control Gmm^WT (23%) flies that harbored trypanosome infections in their midguts (Fig 6A). We next inoculated teneral Gmm^WT males with Kco_Z and then three days later (5 day old adults) challenged Gmm^WT/Kco_Z individuals with either T. congolense or T. brucei parasites (both of these parasite species are naturally transmitted by G. m. morsitans; [43,44]). Under these conditions we observed that Gmm^WT/Kco_Z males were significantly more refractory to infection with both parasite species (T. congolense, 6%; T. brucei, 32%) than were their age-matched Gmm^WT counterparts (T. congolense, 36%; T. brucei, 70%; Fig 6B). Thus, tsetse must house an established Kco_Z infection in its gut at the time of trypanosome challenge in order to present a refractory phenotype. Finally, we found that trypanosome infected Gmm^WT/Kco_Z house similar densities of Kco_Z as do age-matched individuals that eliminated their trypanosome infection (S1E Fig), again indicating that exogenous Kco_Z appears to be resistant to tsetse’s trypanocidal immune response. Additionally, the presence of tsetse’s indigenous, enteric microbiota does not interfere with Kco_Z mediated obstruction of trypanosome infection establishment.

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Fig 6. Gmm^WT/Kco_Z flies are significantly more refractory to infection with parasitic trypanosomes and entomopathogenic S. marcescens.

(A) Percentage of Gmm^WT and Gmm^WT/Kco_Z flies infected with T. congolense 14 days after they were co-inoculated with Kco_Z and parasites in their first (teneral) blood meal. (B) Percentage of Gmm^WT and Gmm^WT/Kco_Z flies infected with T. congolense (left graph) and T. brucei (right graph). For these experiments Gmm^WT/Kco_Z flies housed the exogenous bacteria for five days prior to challenge with trypanosomes. In (A) and (B) Statistical analysis was performed using a GLM followed by multiple comparisons and Tukey contrasts, and different letters represents statistical significance between treatments and controls. (C) Kaplan-Meier plot depicting survival of Gmm^WT and Gmm^WT/Kco_Z flies following challenge (three days after administering Kco_Z or Sodalis) with S. marcescens. Infection experiments were performed using three distinct biological replicates (n = 25 flies per replicate). Gmm^WT/Kco_Z flies were also significantly more refractory to S. marcescens infection than were their Gmm^WT counterparts (p = 0.001; log-rank test).

https://doi.org/10.1371/journal.ppat.1007470.g006

Finally, we investigated whether Kco_Z also protects tsetse against infection with an entomopathogenic bacteria. To do so teneral Gmm^WT males were fed 1x10³ CFU of Kco_Z, and then three days later, the same dose of Serratia marcescens strain db11, which is highly virulent to wild-type tsetse [17,18,45]. Five day old Gmm^WT males infected with the same dose of S. marcescens were used as controls. Fly survival following Serratia inoculation was monitored over a 14 day period in both fly groups. We observed that 51% and 9% of Gmm^WT/Kco_Z and Gmm^WT individuals, respectively, survived their infection with S. marcescens (Fig 6C).

Taken together, our results detailed above indicate that wild-type tsetse present a parasite and entomopathogen refractory phenotype when they house an established Kco_Z infection in their gut. This phenotype like occurs because the acidified nature of the gut environment is inhospitable to the development of exogenous microbes.

Kco_Z infection exerts a minimal fitness cost on tsetse

Kco_Z produces acid in tsetse’s gut such that the environment becomes inhospitable to trypanosomes. To address whether decreased midgut pH adversely impacts tsetse fitness, we quantified several fitness parameters in Gmm^WT/Kco_Z flies (a sample of these individuals housed 1.39x10⁶ ± 1.1x10⁵ Kco_Z at the time they were used for experimentation; S1F Fig).

We began by measuring midgut weight, which reflects over all digestive health. We observed no significant difference in midgut weight between 8 day old Gmm^WT/Kco_Z and Gmm^WT males (3.8 ± 1.1 mg and 3.2 ± 1.2 mg, respectively) and females (12.8 ± 1.8 mg and 13.0 ± 1.8 mg, respectively) 24 hrs after acquiring their last blood meal (Fig 7A). We next measured fecundity parameters in female and male Gmm^WT/Kco_Z and Gmm^WT flies to determine if stable infection with this bacterium would alter their reproductive capacity. We began by measuring gonotrophic cycle (GC) duration of Gmm^WT/Kco_Z and Gmm^WT females. The length of the 1^st GC was not significantly different between Gmm^WT/Kco_Z (24.0 ± 1.2 days) and Gmm^WT females (24.0 ± 0.9 days) (Fig 7B). However, the 2^nd and 3^rd GCs of Gmm^WT/Kco_Z females (13.0 ± 1.1 and 14.0 ± 1.1 days, respectively) were significantly longer than those of their age-matched WT counterparts (11.7 ± 1.0 and 11.5 ± 1.1 days, respectively) (Fig 7B). We also determined that pupal weight from all three GCs was similar between both fly groups (GC1, Gmm^WT/Kco_Z = 23.1 ± 1.5 mg, Gmm^WT = 22.9 ± 1.4 mg; GC2, Gmm^WT/Kco_Z = 23.4 ± 1.7 mg, Gmm^WT = 24.2 ± 1.8 mg; GC3, Gmm^WT/Kco_Z = 24.7 ± 1.7 mg, Gmm^WT = 24.4 ± 1.6 mg) (Fig 7C). Thus, Kco_Z infection impacts the reproductive physiology of female tsetse by increasing GC duration and hence the number of offspring infected individuals are able to produce over the course of their lifespan. However, despite this effect, infection with this bacterium does not impact pupal weight.

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Fig 7. Kco_Z infection impacts specific tsetse fitness parameters.

(A) Midgut weight, as an indicator of blood meal digestion proficiency, in six day old Gmm^WT/Kco_Z and Gmm^WT males and females (guts were weighed 24 h after the flies had consumed their last blood meal). Each point on the graph represents one individual, and statistical significance was determined via multiple t-tests. (B). Gonotrophic cycle (GC) length of Gmm^WT/Kco_Z and Gmm^WT females. Age-matched, pregnant females from each group (n = 35 per group) were housed in individuals cages and monitored daily to observe frequency of pupal deposition. Statistical significance was determined via log-rank test. (C) Weight of pupae from Gmm^WT/Kco_Z and Gmm^WT females over three GCs. Each point on the graph represents one individual, and statistical significance was determined via multiple t-tests. (D) Mating competitiveness of Gmm^WT/Kco_Z compared to Gmm^WT males. Matings were setup in individual cages (n = 80). Each cage housed a virgin female, to which one virgin Gmm^WT/Kco_Z and Gmm^WT male was added. Males and females were age-matched, and each had fed twice prior to exposure. Statistical significance was determined via Chi-squared test. (E) Sperm abundance in the reproductive tracts of three and 14 day old (fed twice) virgin Gmm^WT/Kco_Z and Gmm^WT males. Sperm quantity is a reflection of sperm-specific dynein intermediate chain (sdic) transcript abundance. Absolute sdic transcript abundance was determined by comparing experimental sample cycle threshold (C_t) values to those derived from an sdic internal standard curve. Each point on the graph represents one individual, and statistical significance was determined via multiple t-tests. (F) Quantitation of Wigglesworthia and Sodalis in Gmm^WT/Kco_Z and Gmm^WT male and female tsetse. Abundance of symbiont specific gene transcripts (Wigglesworthia, thiC; Sodalis, fliC) was used as a proxy to quantify bacterial load. This was performed by comparing thiC and fliC cycle threshold (C_t) values in Gmm^WT/Kco_Z and Gmm^WT flies to those derived from symbiont gene-specific internal standard curves. Wigglesworthia and Sodalis can be polyploid (Rio et al., 2006; Weiss et al., 2006), and as such, we normalized symbiont genome copy number to constitutively expressed tsetse gapdh copy number. Each point on the graph represents one individual, and statistical significance was determined via multiple t-tests.

https://doi.org/10.1371/journal.ppat.1007470.g007

We also investigated the effect of Kco_Z infection on the reproductive fitness of male tsetse by comparing the mating competitiveness of Gmm^WT/Kco_Z and Gmm^WT individuals. To do so we set up 80 individual cages, each of which contained one sexually mature virgin female. We subsequently placed one sexually mature Gmm^WT/Kco_Z and Gmm^WT male in each cage and monitored the arena to determine which of the two males successfully mated with the female. We observed that 47.5% of matings occurred between Gmm^WT/Kco_Z males and females (neither male mated with the female in two of the cages) (Fig 7D), thus indicating that Kco_Z infection does not significantly alter male mating competitiveness (χ² = 0.1025641, df = 1, p = 0.748774). Next we compared the number of sperm present in three and 14 day old Gmm^WT/Kco_Z and Gmm^WT males by quantifying transcript abundance of sperm-specific dynein intermediate chain (sdic). The Drosophila homologue of this gene is transcribed exclusively in sperm cells [46] and is used to quantify sperm abundance [47]. We observed no significant difference in sdic transcript abundance between three day old or 14 day old Gmm^WT/Kco_Z and Gmm^WT males (Fig 7E).

Finally, we examined whether the presence of Kco_Z impacts the load of endosymbiotic Wigglesworthia and Sodalis. These measurements, which were taken at 14 days post-inoculation with Kco_Z, are important because tsetse’s microbiota impact many aspects of their host’s fitness, including fecundity and immune system development and function [48,49]. We observed that infection with Kco_Z did not significantly alter the load of tsetse’s midgut (bacteriome) population of obligate Wigglesworthia in Gmm^WT/Kco_Z males or females (Fig 7F). Conversely, midguts from Gmm^WT/Kco_Z males and females housed significantly fewer Sodalis than did midguts from their Gmm^WT counterparts (Fig 7F). Taken together, these data indicate that Kco_Z significantly impacts specific fitness parameters in both female and male flies.

Ethical consideration

This work was carried out in strict accordance with the recommendations in the Office of Laboratory Animal Welfare at the National Institutes of Health and the Yale University Institutional Animal Care and Use Committee. The experimental protocol was reviewed and approved by the Yale University Institutional Animal Care and Use Committee (Protocol 2011–07266).

Tsetse, bacteria and trypanosomes

Tsetse flies (Glossina morsitans morsitans) were maintained in Yale University’s insectary at 24°C with 55% relative humidity. Flies received defibrinated (via mechanical agitation for 20 minutes) bovine blood (Quad Five, Ryegate, MT, USA) through an artificial membrane feeding system every 48 h [72]. Aposymbiotic tsetse (Gmm^Apo) were generated and maintained as described previously [73]. Throughout the manuscript, flies referred to as ‘teneral’ were unfed adults recently eclosed (≤ 24h) from their pupal case. All tsetse lines used in this study are described in S1 Table.

Sodalis were isolated from tsetse pupae as described previously [74], and subsequently maintained in liquid brain heart infusion (BHI) media (Becton Dickinson). When necessary, Sodalis were plated on either Brain Heart Infusion agar supplemented with 10% defibrinated bovine blood (BHIB) or Mitsuhashi-Maramorosch (MM)-agar plates. Kosakonia cowanii Zambiae [previously designated Enterobacter sp Zambiae (Esp_Z); GenBank accession number CP022690.1; 28], isolated from the gut of the mosquito, Anopheles gambiae [25]), and Serratia marcescens (strain db11; isolated from a moribund Drosophila sp.) [75] were grown in liquid LB media or on LB-agar plates at 30°C.

In vivo Trypanosoma congolense and T. brucei brucei (YTAT 1.1) were expanded in rats and harvested from infected blood at peak parasitemia. Rat blood containing blood stream form (BSF) parasites was aliquoted and cryopreserved (in the liquid nitrogen vapor phase, approximately -150°C) for subsequent tsetse challenge experiments.

Recombinant PGRP-LB antibacterial assays

Antibacterial activity of recombinant (rec) PGRP-LB was determined as described previously [15], with minor modification. Specifically, recPGRP-LB was added (10 μg/ml of media) to early log-phase (OD = 0.2–0.4) cultures of Kco_Z, Sodalis and E. coli. Controls consisted of bacterial cultures exposed to bovine serum albumin. Using a plate-based quantification assay [58], E. coli and Kco_Z load was subsequently measured 1 hr. later, while Sodalis load was measured 24 hr. later. Results are presented as % of initial inoculum, which was determined by dividing the number of bacterial CFU present after treatment with recPGRP-LB by the number of CFU present prior to inoculation.

Microbial infection assays

Per os bacterial challenge of wild-type (Gmm^WT) and Gmm^Apo flies was performed by feeding teneral adults a heat inactivated (HI; 56°C for 1 hr) blood meal (to inactivate the vertebrate complement system) inoculated with 5x10⁴ colony forming units (CFU) of each bacterial strain per ml of blood. Because tsetse flies consume approximately 20 μl of blood per feeding, each fly is inoculated with 1x10³ bacterial cells. Gmm^Apo flies colonized with either Sodalis or Kco_Z are designated Gmm^Apo/Sgm and Gmm^Apo/Kco_Z, respectively, and Gmm^WT flies colonized with Kco_Z are designated Gmm^WT/Kco_Z. For all experiments that employed tsetse flies inoculated with either Sodalis or Kco_Z, bacterial midgut load was determined by homogenizing microscopically dissected gut tissue in 0.85% NaCl and serially diluting and plating the samples on LB-agar (E. coli, Kco_Z and Serratia) or BHIB or MM (Sodalis) plates supplemented with antibiotics [58]. CFU per plate were counted manually, and counts are presented in the corresponding Results subsections.

For trypanosome infections, all flies received infectious blood meals containing 1x10⁶/mL BSF T. congolense or T. b. brucei parasites. Gmm^Apo, Gmm^Apo/Sgm and Gmm^Apo/Kco_Z were challenged as eight day old adults (3^rd blood meal), while Gmm^WT and Gmm^WT/Kco_Z flies were challenged as five day old adults (2^nd blood meals). For Kco_Z/trypanosome co-infection experiments, distinct groups of mature Gmm^WT individuals were inoculated with 1x10⁶/mL BSF T. congolense parasites and 5x10⁴ CFU/ml of Kco_Z. Two weeks post-trypanosome challenge, all flies were dissected and their midguts microscopically examined to determine parasite infection status.

Detection and inhibition of tsetse reactive oxygen intermediates

Kco_Z and Sodalis cultures were grown to mid-log phase (OD = 0.25), and cell-free supernatants were generated via centrifugation. Hydrogen peroxide (H₂O₂) concentrations in bacterial culture supernatants were determined using an Amplex Red Hydrogen Peroxide/Peroxidase assay kit according to the manufacturer’s (Invitrogen) protocol. In brief, supernatants were incubated for 30 min. with the assay reagent, and resulting fluorescence units were quantified using a Bio-Tek plate reader.

Antioxidants were used to inhibit tsetse ROI activity in vivo. The assay used was similar to those described previously [18,29,30]. In brief, treated flies were offered a blood meal inoculated with trypanosomes [1x10⁶/mL BSF T. b. brucei (YTAT 1.1)] and supplemented with vitamin C (10mM) or cysteine (10μM). All subsequent meals also contained antioxidant supplements.

Determination of bacterial acid production in vitro

Sodalis and Kco_Z were grown in their respective liquid media to an O.D. of 1.0. Subsequently, 5x10⁶ cells (this value represents the approximate maximum load to which these bacteria grow in tsetse’s gut; see Fig 1A) were diluted to a volume of 1 ml (again in respective liquid media) and heat-killed (80°C for 1.5 hr). Conditioned media containing dead cells was added to early log growth phase T. b. brucei YTAT 1.1 grown in a Beck’s medium (GE Hyclone), which contains phenol red. When in solution this compound serves as a pH-sensitive colorimetric indicator that changes from pink-red to yellow as environmental pH drops. Other treatment groups were inoculated with 1 ml of heated, clean LB (Kco_Z growth medium) or clean BHI (Sodalis growth medium), while the control group consisted of trypanosomes alone. Two hours after exposing T. b. brucei to treatment conditions, cultures were assayed to determine pH using a Mettler Toledo pH meter. The pH of trypanosome containing Beck’s medium was experimentally reduced (to pH 5.8) via the addition of 0.1N HCl, while HK Kco_Z extracts were buffered to pH 7.2 via the addition of 0.1N NaOH. Trypanosome load in all treatment and control groups was determined at 2, 5 and 24 hour time points by counting live parasites using a Brite-Line hemocytometer.

Determination of bacterial acid production in vivo

Microbial regulation of pH in tsetse’s midgut was determined by feeding teneral Gmm^Apo flies a HI blood meal inoculated with either Sodalis or Kco_Z (5x10⁴ CFU/ml of blood). Additionally, teneral Gmm^WT flies received the same quantity of Kco_Z. Five days post-bacterial challenge, colonized individuals were administered a meal composed of sucrose (2.5%) and phenol red (0.04%) solubilized in water. Twenty-four hours later, the color of the solution contained in the midgut was determined by incising the fly abdomen and observing the intact gut using a dissecting microscope (Zeiss Discovery) equipped with a digital camera (Zeiss AxioCam MRc 5). Remaining flies were dissected and their guts were harvested, homogenized in 0.85% NaCl, serially diluted and plated onto MM-agar plates (prepared as described in [56]) supplemented with phenol red (0.025 g/L) and sucrose (a 2.5% sucrose solution was spread onto plates immediately prior to applying tsetse gut extracts). CFU per plate was counted manually, and the growth medium was monitored to observe pH-induced changes in color.

Fitness assays

For all fitness assays, Gmm^WT teneral females and males were infected with Kco_Z during their first blood meal. To determine midgut weight, midguts were dissected from 9 day old Gmm^WT/Kco_Z and Gmm^WT females and males (24 h after consuming their last blood meal) and weighed using a Mettler Toledo (AL104) balance. The effect of Kco_Z infection on female fecundity was measured by quantifying the length of three gonotrophic cycles (GC) and by weighing pupal offspring. To measure GC length, Gmm^WT/Kco_Z and Gmm^WT females were mated as 5 day old adults and thereafter maintained in individual cages. All females were monitored daily to determine when they deposited larvae, and all deposited larvae were weighed.

The effect of Kco_Z infection on male reproductive fitness was measured by quantifying the mating competitiveness and sperm abundance of individuals that housed the bacterium versus those that did not. Mating competitiveness assays were performed in individual tubular cages (height, 6 cm; diameter 12.7 cm), each of which housed one 5 day old virgin female (fed twice). Subsequently, one age-matched Gmm^WT/Kco_Z and Gmm^WT male (also fed twice) was added to each cage. These males were distinguished from one another by removing the proximal tarsus of the right foreleg from one of the individuals. The arena was observed until one of the males had successfully mounted the female, at which point the cage was submerged in ice and the free male identified. To eliminate any bias associated with removal of the foreleg tarsus, the experiment was repeated twice (n = 40 cages per experiment), each time with either Gmm^WT/Kco_Z or Gmm^WT males receiving the distinguishing procedure. Sperm abundance was measured by RT-qPCR quantification of sperm-specific dynein intermediate chain (sdic) expression in the reproductive tracts of three and 14 day old (fed twice) virgin Gmm^WT/Kco_Z and Gmm^WT males. Absolute sdic transcript abundance was determined by comparing experimental sample cycle threshold (C_t) values to those derived from an sdic internal standard curve.

Sodalis fliC and Wigglesworthia thiC gene specific primers were used to quantify the absolute abundance of these bacteria. This was performed by comparing Sodalis fliC and Wigglesworthia thiC cycle threshold (C_t) values in Gmm^WT/Kco_Z and Gmm^WT females and males to those derived from symbiont gene-specific internal standard curves. Because Wigglesworthia and Sodalis can be polyploid [76,77], we normalized symbiont genome copy number to constitutively expressed tsetse gapdh copy number. All RT-qPCR primers are listed in S2 Table. All RT-qPCR assays were carried out in duplicate, and replicates were averaged for each sample. Negative controls were included in all amplification reactions.

Statistical analyses

For trypanosome infection experiments, statistical analyses were carried out using the R software for macOS (version 3.3.2) or GraphPad Prism (v.6). A generalized linear model (GLM) was generated using binomial distribution with a logit transformation of the data. The binary infection status (infected or recovered) was analyzed as a function of the bacterium used to colonize the insects (or its absence). For experiments requiring a pairwise comparison, we performed a Wald test on the individual regression parameter (nature of the bacterium used to colonize) to test its statistical difference. For experiments requiring multiple comparisons, multiple pairwise tests were generated using Tukey contrasts on the generalized linear model (GLM) using glht function of "multcomp" package in R. For the mating assay, under the null hypothesis "Kco_Z does not change the attractiveness of the male", the probability that a female will chose either of the males is p = 0.5. We tested the validity of the null hypothesis by using a Chi-square goodness of fit test. Details of the statistical tests described above are indicated in S1 Dataset. All statistical tests used, and statistical significance between treatments, and treatments and controls, are indicated on the figures or in their corresponding legends. All samples sizes are provided in corresponding figure legends or are indicated graphically as points on dot plots. Biological replication implies distinct groups of flies were collected on different days, and where applicable, treated with distinct treatments (e.g., different bacterial cultures or individual aliquots of trypanosomes).