Obligate symbionts activate immune system development in the tsetse fly

Abstract

Many insects rely on the presence of symbiotic bacteria for proper immune system function. However, the molecular mechanisms that underlie this phenomenon are poorly understood. Adult tsetse flies (Glossina spp.) house 3 symbiotic bacteria that are vertically transmitted from mother to offspring during this insect's unique viviparous mode of reproduction. Larval tsetse that undergo intrauterine development in the absence of their obligate mutualist, Wigglesworthia, exhibit a compromised immune system during adulthood. In this study we characterize the immune phenotype of tsetse that develop in the absence of all of their endogenous symbiotic microbes. Aposymbiotic tsetse (Gmm^Apo) present a severely compromised immune system that is characterized by the absence of phagocytic hemocytes and atypical expression of immunity-related genes. Correspondingly, these flies quickly succumb to infection with normally non-pathogenic E. coli. The susceptible phenotype exhibited by Gmm^Apo adults can be reversed when they receive hemocytes transplanted from wild-type donor flies prior to infection. Furthermore, the process of immune system development can be restored in intrauterine Gmm^Apo larvae when their moms are fed a diet supplemented with Wigglesworthia cell extracts. Our finding that molecular components of Wigglesworthia exhibit immunostimulatory activity within tsetse is representative of a novel evolutionary adaptation that steadfastly links an obligate symbiont with it's host.

INTRODUCTION

All metazoan life forms interact with prokaryotic organisms on a perpetual basis. These associations often result in a fitness advantage for one or both partners involved (1, 2). Insects represent a group of higher eukaryotes that harbor a well-defined bacterial microbiota. Unlike their mammalian counterparts, insects house less complex bacterial communities, are relatively inexpensive to maintain and produce large numbers of offspring in a short period of time. Several studies have demonstrated the importance of symbiotic bacteria as they relate to the proper function of their insect host's immune system. For example, Drosophila naturally infected with Wolbachia are protected (through an unknown mechanism) from several otherwise harmful RNA viruses (3). The malaria vector, Anopheles gambiae, is unusually susceptible to infection with Plasmodium parasites when they lack their commensal microbiota. In this case, symbiotic bacteria appear to mediate anti-Plasmodium immunity by activating basal expression of AMPs, inducing the production of phagocytic granulocytes, and directly generating anti-malarial reactive oxygen species (4, 5, 6).

Tsetse flies (Glossina spp.) harbor 3 symbiotic bacteria that regulate important aspects of their host's physiology. Two of these microbes, obligate Wigglesworthia and commensal Sodalis, are transferred to developing intrauterine progeny via maternal milk gland secretions (7). Tsetse's third symbiont, Wolbachia, is transferred via the germline (8). Tsetse that undergo intrauterine larval development in the absence of Wigglesworthia are immuno-compromised during adulthood. This phenotype is characterized by a significantly reduced population of phagocytic sessile and circulating hemocytes, and an unusual susceptibility to infection with pathogenic trypanosomes and normally non-pathogenic E. coli K12 (9, 10, 11). Further studies on the tsetse/Wigglesworthia symbiosis as it relates to host immunity have been obstructed by our inability to reconstitute symbiont-free flies with this bacterium.

In the present study we investigated the intimate relationship between immunity and symbiosis in tsetse by producing flies that underwent larval development in the absence of all endogenous microbes. We analyzed the immune system phenotype of aposymbiotic tsetse (Gmm^Apo) following microbial challenge, and investigated whether loss of immunity in Gmm^Apo flies could be rescued through either transfer of immune cells from healthy individuals or symbiont provisioning. We obtain results that reinforce the obligate nature of tsetse's relationship with Wigglesworthia, and provide further insights into the basic molecular mechanisms that underlie symbiont-induced maturation of host immunity.

MATERIALS AND METHODS

Tsetse and bacteria

G. morsitans morsitans were maintained in Yale's insectary at 24°C with 50–55% relative humidity. These flies received defibrinated bovine blood (Hemostat Laboratories) every 48 hours through an artificial membrane feeding system (12). Designations of all tsetse cohorts used in this study, the composition of their symbiont populations, and the treatments they received are described in Table 1.

Table 1.

Designation of tsetse cohorts used in this study, their symbiont status, and the treatment they received.

Tsetse designation	Symbiont statusa	Origin/treatmentb	Reference/source
Gmm WT	Wgm, Sgm, Wol	none	none
Gmm Wgm-	Sgm, Wol	offspring of moms treated with Amp, yeast extract	9
Gmm Apo	apo	offspring of moms treated with Tet, yeast extract	16
Gmm Apo/WT	apo	received hemolymph transplant from GmmWT donors	this study
Gmm Apo/Apo	apo	received hemolymph transplant from GmmApo donors	this study
Gmm Apo/Sol	apo	received soluble fraction of GmmWT donor hemolymph	this study
Gmm Apo/Cell	apo	received cellular fraction of GmmWT donor hemolymph	this study
Gmm Apo/Wgm	apo	offspring of symbiont-cured moms complimented with Wgm cell extracts	this study
Gmm Apo/Sgm	apo	offspring of symbiont-cured moms complimented with Sgm cell extracts	this study
Gmm Apo/NB	apo	offspring of symbiont-cured moms that received no bacterial compliment	this study

^aWgm, Wigglesworthia; Sgm, Sodalis; Wol, Wolbachia; apo, aposymbiotic

^bAmp, ampicillin; Tet, tetracycline

Luciferase-expressing E. coli K12 (recE. coli_pIL) were produced via transformation with construct pIL, which encodes the firefly luciferase gene under transcriptional control of Sodalis' insulinase promoter (13). The assay used to quantify recE. coli_pIL cells in vivo was performed as described previously (13). GFP-expressing E. coli K12 (recE. coli_GFP) were produced via electroporation with pGFP-UV plasmid DNA (Clontech). Sodalis were isolated from surface-sterilized G. m. morsitans pupae and cultured on Aedes albopictus C6/36 cells as described previously (14). Sodalis, which has a doubling time of approximately 24 h, were subsequently maintained cell-free in vitro at 25°C in Mitsuhashi-Maramorosch medium (1 mM CaCl2, 0.2 mM MgCl2, 2.7 mM KCl, 120 mM NaCl, 1.4 mM NaHCO3, 1.3 mM NaH2PO4, 22 mM D (+) glucose, 6.5 g/L lactalbumin hydrolysate and 5.0 g/L yeast extract) supplemented with 5% heat-inactivated fetal bovine serum (14).

Tsetse infections

Systemic challenge of tsetse was achieved by anesthetizing flies with CO₂ and subsequently injecting individuals with live bacterial cells using glass needles and a Narashige IM300 micro-injector. Per os bacterial challenges were performed by adding 500 colony forming units (CFU) of E. coli per 20 μl (the approximate amount consumed by a fly) of the total blood meal. The vertebrate host compliment system was heat-inactivated (56°C for 1 hr) prior to inoculating blood meals with bacterial cells. The number of bacterial cells injected or fed, control group designations, and sample size for all infection experiments are indicated in the corresponding figures and their legends.

Hemolymph collection and hemocyte quantification

Hemolymph collection from Gmm^WT and Gmm^Apo flies was performed using the high injection/recovery method as described previously (15). Subsequent determination of circulating hemocyte abundance was performed using a Bright-Line hemocytomter (11). Sessile hemocyte abundance was quantified by subjecting Gmm^WT and Gmm^Apo flies (n=3) to hemocoelic injection with blue fluorescent microspheres. 12 hr post-injection, flies were dissected to reveal tsetse's dorsal vessel (DV). Exposed tissue was rinsed 3 times with PBS to remove contaminating circulating hemocytes or any beads not engulfed by sessile hemocytes. The left-most panel is a Brightfield image of the 3 chambers that make up the DV (scale bar = 400 μm). The anterior-most chamber is indicated within a white circle, and the 2 remaining panels are the anterior chamber at higher magnification (scale bar = 80 μm). Engulfed beads were visualized microscopically by excitation with UV light (365/415 nm). Relative fluorescence, which was quantified using ImageJ software, represents the average amount of light emitted from 3 Gmm^WT and Gmm^Apo individuals.

Quantitative analysis of immunity-related gene expression

For quantitative real-time PCR (qPCR) analysis of immunity-related gene expression, whole flies were homogenized in liquid nitrogen and total RNA was extract using Trizol reagent (Invitrogen). Randomly-primed cDNAs were generated with Superscript II reverse transcriptase (Invitrogen), and qPCR analysis was performed using SYBR Green supermix and a Bio-Rad C1000 thermal cycler. Amplification primers are listed in Supplemental Table 1. Quantitative measurements were performed on 3 biological samples in duplicate and results were normalized relative to tsetse's constitutively expressed β-tubulin gene (determined from each corresponding sample). Fold-change data are represented as a fraction of average normalized gene expression levels in bacteria-infected flies relative to expression levels in corresponding uninfected controls. Values are represented as the mean (±SEM).

Hemolymph transplantation

Undiluted hemolymph was collected by removing one front fly leg at the joint nearest the thorax and then applying gentle pressure to the distal tip of the abdomen. Hemolymph exuding from the wound was collected using a glass micro-pipette and placed into a microfuge tube on ice. Four cohorts of newly emerged aposymbiotic recipient flies were used, 2 of which were designated Gmm^Apo/WT or Gmm^Apo/Apo based on whether they received hemolymph transplanted from WT or aposymbiotic donors, respectively. Gmm^Apo/WT or Gmm^Apo/Apo recipient flies received 1 μl of donor hemolymph (this volume represents approximately 1/3 of the total volume collected from donor flies). On day 8 post-transplantation, 3 of these flies were sacrificed to quantify hemocyte number using a Bright-Line hemocytometer. To separate Gmm^WT donor hemolymph into soluble and cellular fractions, samples were centrifuged at 3000 × g for 5 min. The cellular component was resuspended in a volume of chilled anticoagulant buffer [70% MM medium, 30% anticoagulant citrate buffer (98 mM NaOH, 186 mM NaCl, 1.7 mM EDTA and 41 mM citric acid, buffer pH 4.5), vol/vol; 15] equal to the total amount of hemolymph from which they were collected. The remaining 2 cohorts of Gmm^Apo recipient flies were injected with either 1 μl of cellular suspension (these flies are designated Gmm^Apo/Cell), or 1 μl of the soluble hemolymph fraction (these flies are designated Gmm^Apo/Sol).

All aposymbiotic recipient flies were challenged with either 10³ CFU of live recE. coli_pIL or recE. coli_GFP. Injections were performed using glass needles and a Narashige IM300 micro-injector. Quantification of recE. coli_pIL in recipient tsetse was performed as described above. Phagocytic capacity of transplanted hemocytes was determined by infecting Gmm^Apo/Apo recipient flies with 10³ CFU of live recE. coli_GFP. Twelve hours post-challenge, hemolymph was collected from 3 individuals and hemocytes monitored for the presence of engulfed GFP-expressing bacterial cells. Hemolymph samples were fixed on glass microscope slides via a 2 min incubation in 2% paraformaldehyde. Prior to visualization using a Zeiss Axioscope microscope, slides were overlayed with VectaShield hard set mounting medium containing DAPI (Vector Laboratories).

Bacterial complementation experiments

A cartoon illustrating in detail how bacterial compliment experiments were performed is shown in Supplemental Fig. 1. Three cohorts (n= 120 individuals/group) of pregnant female tsetse were fed a diet containing tetracycline (40 μg/ml of blood) every other day for 10 days. Additionally, throughout the course of the entire experiment, all blood meals (3 per week) also contained vitamin-rich yeast extract (1% w/v) to restore fertility associated with the absence of Wigglesworthia (16). Ten days post-copulation, 2 cohorts of symbiont-cured females were regularly fed a diet supplemented with Wigglesworthia and Sodalis cell extracts. By timing treatments in this manner, larvae from the 1^st gonotrophic cycle (GC) went through most of their development in the absence of bacterial compliment while those from the 2^nd and 3^rd GCs developed in the presence of bacterial compliment. Offspring of these females were designated Gmm^Apo/Wgm and Gmm^Apo/Sgm, respectively. Wigglesworthia was obtained by dissecting tsetse bacteriomes (an organ immediately adjacent to the midgut that houses this bacteria) from Gmm^WT females, while Sodalis was maintained in culture as describe above. Gmm^Apo/Wgm females were fed 1 bacteriome equivalent per 4 females, and Gmm^Apo/Sgm females were fed 4×10⁷ Sodalis per ml of blood (these flies thus ingested ~1×10⁶ Sodalis each time they fed). A 3^rd control cohort of symbiont-cured females received no bacterial compliment (their offspring are designated Gmm^Apo/NB), and a 4^th cohort of wild-type offspring (Gmm^WT) served as another control. To confirm the aposymbiotic status of offspring from symbiont-cured moms (Supplemental Fig. 2), genomic DNA was extracted from larval offspring (3^rd instar; n= 3) of all experimental cohorts using the Holmes-Bonner method (17). PCR (20μl reactions) was performed in an MJ Research thermalcycler using bacteria-specific primers (Supplemental Table 1) and the following cycle program: 95°C for 5 min. followed by 30 cycles at 95°C, 55°C and 72°C, each for 1 min, and a final 7 min elongation/extension at 72°C.

To determine whether complimenting symbiont-cured moms with bacterial cell extracts impacted the immune system phenotype of their offspring, qPCR was used (as described above) to monitor the expression of serpent and lozenge in larvae (1^st, 2^nd and 3^rd instar) from each of 3 gonotrophic cycles (GC; n=3 individuals per group per GC). All remaining offspring were allowed to mature to adulthood. At this time 3 individuals from each cohort and GC were taken to determine circulating hemocyte abundance (as described above). Furthermore, qPCR was used to compare immunity-related gene expression in E. coli-challenged Gmm^Apo/Wgm and Gmm^Apo/Sgm individuals (n=3) from the 2^nd GC of symbiont-cured moms. Finally, all remaining mature adult offspring were challenged with 10³ CFU of live recE. coli_GFP. Twelve hours post-challenge, hemolymph was collected and monitored to determine if hemocytes had engulfed GFP-expressing bacterial cells (n=3 individuals per group per GC). Hemolymph samples were fixed and visualized as described above.

Stats

Statistical significance between various treatments, and treatments and controls, is indicated in the corresponding figure legends. Survival curve comparisons were made by log-rank analysis using JMP (v9.0) software (www.jmp.com). Statistical analysis of qPCR data and hemocyte abundance was performed by Student's t test using Microsoft Excel software.

RESULTS

Aposymbiotic tsetse exhibit atypical hallmarks of cellular and humoral immunity

A positive correlation exists between the proper function of an insect's immune system and the dynamics of it's microbiome (18). In an effort to better define the relationship between symbiosis and immunity in tsetse, we fed pregnant female's on a diet supplemented with tetracycline and yeast. This antibiotic treatment clears all symbionts from the flies, while the vitamin-rich yeast extract rescues the loss of fertility associated with the absence of obligate Wigglesworthia (9, 16). We then investigated whether offspring that underwent intrauterine development in the absence of all symbiotic bacteria (Gmm^Apo) exhibited an immune system phenotype during adulthood that was different from that of their WT counterparts that developed in the presence of their complete microbiome. To do so, we began by quantifying the number of circulating and sessile hemocytes present in 8 day old adult (hereafter referred to as `mature) Gmm^WT and Gmm^Apo flies. Our results indicate that mature WT tsetse harbor 113× more circulating hemocytes per μl of hemolymph than do their aposymbiotic counterparts (Gmm^WT, 793 ± 34 hemocytes per μl of hemolymph; Gmm^Apo, 7 ± 1 hemocytes per μl of hemolymph; Fig. 1A). To determine the functional relationship between symbiont status and sessile hemocyte abundance, we thoracically micro-injected WT and aposymbiotic adults with fluorescent microspheres. In both tsetse and Drosophila sessile hemocytes concentrate in large quantities around the anterior chamber of the fly's dorsal vessel (11, 19). Thus, we indirectly quantified sessile hemocyte number by measuring the fluorescent emission of injected microspheres that were found engulfed in this region. We observed that mature Gmm^WT flies engulfed 16× more microspheres than did age-matched Gmm^Apo individuals (Fig. 1B and Supplemental Fig. 3).

Figure 1. Aposymbiotic tsetse display atypical hallmarks of cellular and humoral immunity.

(A) Number of circulating hemocytes per •l of hemolymph in mature Gmm^WT and Gmm^Apo flies (n=3 individuals from each tsetse line). (B) Quantitative analysis of sessile hemocyte abundance adjacent to the anterior chamber of the dorsal vessel of mature Gmm^WT and Gmm^Apo flies (n=3 individuals from each tsetse line). Relative fluorescence is proportional to the number of microspheres engulfed by sessile hemocytes and thus the number of these cells present in the region examined. (C) The effect of symbiont status and route of infection on the expression of selected immunity-related genes. Gene expression in uninfected Gmm^Apo and Gmm^WT individuals is normalized relative to constitutively-expressed tsetse β-tubulin (left panel). Fold-change in the expression of immunity-related genes in Gmm^Apo and Gmm^WT tsetse 3 d after per os (middle panel) and intra-thoracic (right panel) challenge with E. coli K12. All fold-change values are represented as a fraction of average normalized gene expression levels in bacteria-challenged flies relative to expression levels in PBS-injected controls. All quantitative measurements were performed on 3 biological samples in duplicate. Genes without a corresponding bar did not exhibit a fold-change in expression between samples compared, or their expression was undetectable via qPCR. Values are represented as means. * = p < 0.05, ** = p < 0.005 (Student's t-test).

Previously we determined that several genes associated with humoral, cellular and epithelial immune pathways, including those that encode the antimicrobial peptides (AMPs) attacin and cecropin, as well as thioester-containing proteins (tep2 and tep 4), prophenoloxidase and inducible nitric oxide synthase (iNOS), were expressed at significantly lower levels in Gmm^Wgm− compared to Gmm^WT flies following infection with E. coli (11). In the present study we monitored expression of these same genes in age-matched Gmm^WT and Gmm^Apo flies that were either unchallenged or 3 days post-challenge (dpc) with E. coli K12. Furthermore, we also evaluated the expression of peptidoglycan recognition protein LB (PGRP-LB), caudal, domeless and dual oxidase (DUOX). In tsetse and closely-related Drosophila, PGRP-LB and caudal serve as negative regulators of NF-kappaB-dependent antimicrobial peptide expression (10, 20, 21), while domeless is a cytokine receptor that regulates expression of tep4 through the `Janus Kinase Signal Transduction and Activator of Transcription' signaling pathway (22, 23). Finally, in Drosophila and mosquitoes, DUOX is involved in generating infection-induced antimicrobial reactive oxygen species (24, 25, 26).

Our expression analysis indicates that the presence of symbiotic bacteria during larval development induce basal immunity in tsetse. Specifically, we observed that DUOX, domeless and caudal are expressed at significantly lower levels in mature unchallenged Gmm^Apo compared to Gmm^WT flies (Fig. 1C, left graph). Following per os challenge with E. coli, no significant difference in immunity-related gene expression (with the exception of iNOS) was observed between Gmm^WT and Gmm^Apo flies (Fig. 1C, middle graph). However, systemic challenge resulted in a significant difference in the expression of all the genes we analyzed. Most notably, pathways associated with cellular immunity were significantly down-regulated in Gmm^Apo compared to Gmm^WT individuals, while those associated with humoral immune responses were significantly up-regulated (Fig. 1C, right graph). These findings indicate that tsetse's symbiotic bacteria are closely associated with the development of their host's immune system during larval maturation, and it's subsequent proper function in unchallenged and E. coli challenged adults.

Aposymbiotic tsetse are highly susceptible to normally non-pathogenic E. coli

We next determined whether Gmm^Apo individuals are more susceptible to challenge with E. coli than are WT tsetse or tsetse that lack only Wigglesworthia (Gmm^Wgm−). To do so we compared percent survival of mature adults from these 3 tsetse lines following systemic challenge with E. coli K12. We determined that 67% of mature Gmm^WT individuals, and 59% of mature Gmm^Wgm− individuals, survived systemic challenge with 10³ CFU of E. coli (Fig. 2A, top and middle panels). In contrast, all age-matched Gmm^Apo individuals perished by 12 dpc (Fig. 2A, bottom panel). We next challenged Gmm^WT and Gmm^Apo flies per os with 10³ and 10⁶ CFU of E. coli, and found that all individuals survived this challenge (Fig. 1A, top and bottom panels). This finding suggests that mature Gmm^Apo flies are considerably more susceptible to systemic challenge with a foreign microbe than are age-matched Gmm^WT and Gmm^Wgm− individuals. Furthermore, tsetse's ability to overcome per os challenge with E. coli appears to be independent of symbiont status.

Figure 2. Symbiont status mediates tsetse's ability to survive challenge with E. coli K12.

(A) The effect of symbiont status on the survival of tsetse following systemic and per os challenge with E. coli K12. Mature adult Gmm^Apo flies were significantly more susceptible to challenge with 10³ CFU of E. coli than were age-matched Gmm^WT (bottom and top panels, p < 0.001) and Gmm^Wgm− flies (bottom and middle panels, p < 0.001). Both Gmm^WT and Gmm^Apo flies survived per os challenge with E. coli. Infection experiments were performed in triplicate, using 25 flies per replicate. (B) Average number (±SEM) of recE. coli_pIL per tsetse cohort over time (n=3 individuals per cohort per time point) following systemic and per os challenge with 10³ CFU of bacteria. Values shown in grey represent lethal infections. By 8 dpc, not enough E. coli-injected Gmm^Apo flies remained to quantify bacterial density.

To determine a cause for the variation in survival we observed between Gmm^WT, Gmm^Wgm− and Gmm^Apo individuals following challenge with E. coli, we monitored the dynamics of bacterial growth in each of these fly groups over time. When fed E. coli, both mature aposymbiotic and WT individuals cleared all E. coli. Following systemic challenge with 10³ CFU of E. coli, bacterial densities within mature Gmm^WT flies reached 8.3×10³ cells before being cleared. Interestingly, Gmm^Wgm− flies, which perish following challenge with 10⁶ CFU of E. coli (11), were able to clear all exogenous bacterial cells following challenge with this lower dose. On the other hand, bacterial density in Gmm^Apo flies peaked at 7.8×10⁶ on day 6 post-challenge, after which all flies soon perished (Fig. 2B). This observation suggests that aposymbiotic tsetse were unable to control systemic infection with E. coli and thus likely perished as a result of their inability to tolerate high densities of this bacterium in their hemolymph. These findings taken together indicate that Gmm^Apo flies are significantly more susceptible to challenge with E. coli than are WT flies and flies that lack only Wigglesworthia.

Hemocyte transfer from WT tsetse restores the ability of Gmm^Apo adults to overcome infection with E. coli

We next set out to provide a definitive correlation between tsetse hemocytes and the fly's ability to overcome challenge with a foreign microbe. To do so we transplanted hemolymph from mature Gmm^WT and Gmm^Apo individuals (donor flies) into the hemocoel of susceptible Gmm^Apo flies (recipient flies are hereafter designated Gmm^Apo/WT and Gmm^Apo/Apo, respectively). Five days after this procedure we determined that Gmm^Apo/WT flies harbored 330 (± 20.4) hemocytes per μl of hemolymph, while Gmm^Apo/Apo flies harbored 5 (± 3.8) hemocytes per μl of hemolymph (Fig. 3A). We next investigated whether our hemolymph transplantation procedure was able to rescue the E. coli-susceptible phenotype exhibited by Gmm^Apo flies. To do so we challenged Gmm^Apo/WT and Gmm^Apo/Apo individuals with 10³ CFU of E. coli 3 days post-hemolymph transplantation, and subsequently monitored their survival over time. Our results indicate that 72% of Gmm^Apo/WT flies survived for 14 days following challenge. In comparison, only 2% of Gmm^Apo/Apo flies survived their challenge (Fig. 3B, top panel). These results demonstrate that Gmm^Apo flies are able to clear a systemic challenge with E. coli after they receive a transplant of hemolymph from WT donors.

Figure 3. Hemocytes modulate tsetse's ability to overcome challenge with E. coli K12.

(A) Hemolymph was collected from Gmm^Apo and Gmm^WT donor flies and immediately transplanted into Gmm^Apo recipients. Five days post-hemolymph transplantation, hemocyte abundance in Gmm^Apo/WT and Gmm^Apo/Apo recipient flies was quantified microscopically using a hemocytometer. Gmm^Apo/WT flies housed significantly more circulating hemocytes than did Gmm^Apo/Apo flies (** = p < 0.005). (B) Gmm^Apo/WT and Gmm^Apo/Apo recipient flies were challenged with E. coli 3 days after receiving a hemolymph transplant. Significantly more Gmm^Apo/WT individuals survived E. coli challenge than did their Gmm^Apo/Apo counterparts (top panel; p < 0.001). Donor hemolymph was then divided into cellular and soluble fractions via centrifugation. Significantly more Gmm^Apo/Cell individuals survived E. coli challenge than did their Gmm^Apo/Sol counterparts (bottom panel; p < 0.001). (C) Average number (±SEM) of recE. coli_pIL per Gmm^Apo/WT and Gmm^Apo/Apo recipient fly over time (n=3 individuals per treatment per time point) following systemic challenge with 10³ CFU of bacteria. Values shown in grey represent lethal infections. (D) 12 hr post-challenge with recE. coli_GFP, hemolymph was collected, fixed on glass slides using 2% paraformaldehyde and microscopically examined for the presence of hemocyte-engulfed bacterial cells. Gmm^Apo/WT recipient flies harbor engulfed bacterial cells.

We next investigated whether hemocytes, or a soluble antimicrobial or signaling molecule present in the transplanted hemolymph was responsible for restoring the resistant phenotype exhibited by recipient individuals. To address this issue we collected hemolymph from WT donors, separated it into soluble and cellular fractions by centrifugation, and then transplanted the separate fractions into 2 distinct groups of Gmm^Apo flies. Finally, 3 days later we systemically challenged both groups of recipient flies with 10³ CFU of E. coli K12. All aposymbiotic flies that received the soluble fraction of hemolymph from Gmm^WT donors (Gmm^Apo/Sol) perished by day 12 post-challenge. In comparison, 62% of Gmm^Apo recipients that received the cellular fraction of hemolymph from Gmm^WT donors (Gmm^Apo/Cell) survived for 14 days following bacterial challenge (Fig. 3B, bottom panel). These host survival curves indicate that Gmm^Apo flies survive challenge with E. coli when they had previously received a transplant of hemocytes, as opposed to soluble hemolymph molecules, from WT tsetse.

To determine a cause for the variation in survival we observed between these 2 groups, we monitored the dynamics of bacterial growth in each group over the course of the experiment. E. coli within Gmm^Apo/Apo flies replicated exponentially until a peak density of 2.1×10⁷ was reached at 6 dpc. This finding suggests that bacterial sepsis was the cause of high mortality we observed in this group of flies. In contrast, aposymbiotic recipients were able to clear all E. coli by 8 dpc when they had previously received a hemolymph transplant from Gmm^WT donors (Fig. 3C). More so, microscopic examination of hemolymph from Gmm^Apo/WT flies showed that transplanted hemocytes engulfed the introduced E. coli (Fig. 3D). Our results demonstrate that immune resistance can be restored in adult aposymbiotic tsetse if they harbor hemocytes transplanted from their WT counterparts.

Supplementation of Wigglesworthia to symbiont-cured females restores immune system development in aposymbiotic offspring

Previous experiments revealed that the milk gland population of tsetse's obligate symbiont, Wigglesworthia, must be present during the development of immature stages in order for subsequent adults to exhibit a functional cellular immune system (11). To date we have been unable to culture Wigglesworthia and thus can not recolonize aposymbiotic flies with this bacterium. To circumvent this impediment we tested whether we could restore the process of immune system development in Gmm^Apo offspring by supplementing the diet of pregnant, symbiont-cured females with Wigglesworthia-containing extracts of bacteriome tissue collected from WT females. A detailed description of the experimental design we used to test this theory is provided in the Materials and Methods and Supplemental Fig. 1.

In brief, two treatment cohorts of pregnant Gmm^WT females were fed a diet supplemented with tetracycline and yeast extract (16). Ten days post-copulation, these symbiont-cured females began receiving either Wigglesworthia or Sodalis cell extracts in every blood meal. The immune system phenotype of offspring from these females (Gmm^Apo/Wgm and Gmm^Apo/Sgm, respectively) was compared to that of control cohort offspring from symbiont-cured moms that received no bacterial supplement (Gmm^Apo/NB) and offspring from Gmm^WT moms. We first evaluated the relative abundance of transcripts that encode the transcription factors `Serpent' and `Lozenge'. In Drosophila, these molecules direct hemocyte differentiation, or hematopoiesis, during embryogenesis and early larvagenesis (27). In tsetse, larvae that develop in the absence of Wigglesworthia express significantly less serpent and lozenge than do their WT counterparts (11). In the present study we found that Gmm^Apo/Wgm, Gmm^Apo/Sgm and Gmm^Apo/NB larva from the 1^st gonotrophic cycle (GC) expressed significantly less serpent and lozenge than did Gmm^WT larva. However, after the onset of bacterial supplementation, Gmm^Apo/Wgm and Gmm^WT larva from the 2^nd and 3^rd GCs expressed comparable levels of serpent and lozenge, while Gmm^Apo/NB and Gmm^Apo/Sgm larva expressed less (Fig. 4A).

Figure 4. Dietary supplementation of Wigglesworthia cell extracts to symbiont-cured female tsetse induces immune system development in their aposymbiotic offspring.

Three groups of pregnant female tsetse were provided 4 blood meals supplemented with the antibiotic tetracycline to clear all of their endogenous microbiota. Two cohorts of these symbiont-cured females then received diets supplemented with either Wigglesworthia or Sodalis cell extracts to compliment the absence of these bacteria. The third group of symbiont-cured females received no bacterial compliment. Finally, a fourth group of WT females received no tetracycline or bacterial complementation. Offspring of these females, which are designated Gmm^Apo/Wgm, Gmm^Apo/Sgm, Gmm^Apo/NB and Gmm^WT, respectively, were collected from 3 gonotrophic cycles (GC) and subsequently monitored to determine their immune system phenotype. GC1 is indicated in grey to signify that bacterial compliment of Gmm^Apo/Wgm and Gmm^Apo/Sgm moms began after their first larval offspring were fully developed. (A) qPCR was performed on larval offspring (n=3 larva per cohort per GC) to determine their levels of serpent and lozenge expression. (B) Circulating hemocyte abundance in adult offspring (n=3 flies per cohort per GC) was quantified microscopically using a Bright-Line hemocytometer. In (A) and (B), bars with different letters indicate a statistically significant difference (p < 0.05) between samples. (C) Fold-change in the expression of immunity-related genes in Gmm^Apo/Wgm and Gmm^Apo/Sgm adults challenged with E. coli. Adult flies used for this experiment were from the 2^nd GC of symbiont-cured moms. All fold-change values are represented as a fraction of average normalized gene expression levels in bacteria-challenged flies relative to expression levels in PBS-injected controls. Genes without a corresponding bar did not exhibit a fold-change in expression between samples compared, or their expression was undetectable via qPCR. All quantitative measurements were performed on 3 biological samples in duplicate. Values are represented as means. * = p < 0.05, ** = p < 0.005.

Because serpent and lozenge expression can be indicative of hematopoiesis, we next compared the number of hemocytes present in Gmm^Apo/Wgm adults to that found in age-matched Gmm^WT, Gmm^Apo/NB and Gmm^Apo/Sgm flies. We found that the provisioning of Wigglesworthia extracts to symbiont-cured females resulted in an increase in the number of circulating hemocytes present in their offspring. Specifically, hemocyte density in Gmm^Apo/Wgm adults from GCs 2 and 3 was significantly greater (113 ± 33 and 127 ± 21 hemocytes/μl of hemolymph, respectively) than that found in age-matched Gmm^Apo/NB (7 ± 3 and 9 ± 4 hemocytes/μl of hemolymph, respectively) and Gmm^Apo/Sgm flies (10 ± 4 and 4 ± 1 hemocytes/μl hemolymph, respectively), but significantly less than that of Gmm^WT adults (733 ± 104 and 681 ± 68 hemocytes/μl hemolymph, respectively; Fig. 4B). Correspondingly, we observed that prophenoloxidase and tep4, which are expressed predominantly by hemocytes (28, 29), are found at significantly higher levels in adult Gmm^Apo/Wgm compared to Gmm^Apo/Sgm adults (from GC2) following systemic challenge with E. coli (Fig. 4C). A similar pattern was observed with genes involved in the generation of reactive oxygen species (DUOX and iNOS). Interestingly, humoral immunity-associated genes (AMPs and their regulators) were expressed at similar levels in E. coli-challenged Gmm^Apo/Wgm and Gmm^Apo/Sgm adults.

Our results suggest that feeding symbiont-cured moms a diet supplemented with Wigglesworthia cell extracts induces a physiological response that partially restores immune system development in their aposymbiotic offspring. Specifically, Gmm^Apo/Wgm larvae exhibit increased expression of the hematopoietic transcription factors serpent and lozenge, and as adults these flies present a functional immune system characterized by the presence of circulating phagocytic hemocytes. Furthermore, the expression of genes involved in epithelial and cellular immunity is enhanced in Gmm^Apo/Wgm adults.

Gmm^Apo/Wgm flies are resistant to E. coli challenge

We observed that Gmm^Apo/Wgm offspring exhibit hallmarks of enhanced immunity. Thus, we next tested whether mature Gmm^Apo/Wgm adults would be resistant to systemic challenge with E. coli K12, while age-matched Gmm^Apo/Sgm and Gmm^Apo/NB flies, would not. To this end, we observed that 38% and 43% of Gmm^Apo/Wgm adults from GCs 2 and 3, respectively, survived challenge with 10³ E. coli (Fig. 5A). Correspondingly, microscopic inspection of hemolymph from E. coli-resistant Gmm^Apo/Wgm adults revealed the presence of phagocytic hemocytes that harbored internalized E. coli cells (Fig. 5B). In contrast, Gmm^Apo/NB and Gmm^Apo/Sgm flies were highly susceptible to E. coli challenge, and like their Gmm^Apo counterparts, all perished within the 14 day experimental period (Fig. 5A). This susceptible phenotype likely resulted from the fact that Gmm^Apo/NB and Gmm^Apo/Sgm adults are devoid of phagocytic hemocytes (Figs. 4B and 5B). These findings suggest that aposymbiotic tsetse can survive infection with an otherwise lethal dose of E. coli if they complete intrauterine development while their moms were fed a diet containing Wigglesworthia cell extracts. This immuno-competent phenotype exhibited by Gmm^Apo/Wgm adults likely results from the presence of phagocytic hemocytes in their hemolymph.

Figure 5. Gmm^Apo/Wgm flies exhibit resistance to challenge with E. coli.

(A) Percent survival of mature Gmm^Apo/Wgm, Gmm^Apo/Sgm, Gmm^Apo/NB and Gmm^WT adults from 3 GCs following challenge with 10³ CFU of E. coli K12. Significantly more Gmm^Apo/Wgm flies from the 2^nd GC survived this challenge than did age-matched Gmm^Apo/Sgm and Gmm^Apo/NB individuals (p < 0.01). However, significantly fewer Gmm^Apo/Wgm flies from these GCs survived this challenge than did their WT counterparts (p < 0.01). Values shown in grey represent lethal infections. Sample sizes are as follows: GC1 (n=25 flies per replicate for all tsetse cohorts) and GC2 (n=25 flies per replicate for Gmm^WT and Gmm^Apo/Wgm flies; n=20 for Gmm^Apo/Sgm and Gmm^Apo/NB flies) infection experiments were performed in triplicate for all tsetse groups. GC3 is denoted with an asterisk because not enough Gmm^Apo/Sgm and Gmm^Apo/NB offspring were produced to perform the experiment in triplicate (even in the presence of yeast extract, the fecundity of symbiont-cured females drops over time). Thus, statistical comparisons between these two groups were not performed. (B) Twelve hours post- challenge with recE. coli_GFP, hemolymph was collected from all individuals (n=3 flies per group per GC) to monitor for the presence of phagocytic hemocytes. Samples were processed as previously described. In (A) and (B), GC1 is indicated in grey to signify that bacterial compliment of Gmm^Apo/Wgm and Gmm^Apo/Sgm moms began after their first intrauterine larval offspring were approximately mid-way through their 3^rd developmental instar.

DISCUSSION

Symbiotic bacteria are gaining increased recognition as potent modulators of insect immunity (18, 30). In the present study we provide evidence that tsetse's symbiotic bacteria are intimately associated with the maturation of their host's immune system during juvenile development and it's subsequent proper function during adulthood. We determined that aposymbiotic (Gmm^Apo) flies derived from symbiont-cured moms present a severely compromised cellular immune system, and as such are highly susceptible to systemic infection with normally non-pathogenic E. coli. This immuno-compromised phenotype can be reversed when Gmm^Apo adults receive hemocytes transplanted from WT individuals. Furthermore, the process of immune system development in Gmm^Apo larvae can be restored when their symbiont-cured moms are fed a diet supplemented with Wigglesworthia cell extracts. Our results demonstrate that evolutionary time has stably anchored the obligate association between tsetse and Wigglesworthia such that this bacterium directly engenders immunity, and thus ultimately the fecundity, of it's host. In return, tsetse provides Wigglesworthia with a protective and metabolite-rich niche that has enabled this bacterium to survive in this environment for at least 50 million years (31).

Tsetse that undergo intrauterine larval development in the absence of only Wigglesworthia (Gmm^Wgm−) exhibit a compromised immune system that, when compared to WT flies (Gmm^WT), is characterized by a 70% reduction in the number of phagocytic hemocytes (11). In the present study we found that eliminating all symbiotic bacteria from female tsetse markedly enhances the immuno-compromised phenotype of their offspring. In fact, Gmm^Apo adults harbor virtually no circulating (99% less than Gmm^WT adults) or sessile hemocytes and are correspondingly more susceptible to systemic infection with E. coli than are WT tsetse and tsetse that lack only Wigglesworthia. Gmm^Wgm− flies, which undergo intrauterine maturation in the presence of Sodalis and Wolbachia, house approximately 40× more circulating hemocytes than do their aposymbiotic counterparts and are more tolerant to E. coli challenge. (11). The enhanced immunity exhibited by Gmm^Wgm− individuals in comparison to their aposymbiotic counterparts suggests that the presence of Sodalis and Wolbachia during intrauterine development may induce a limited degree of immune system maturation in their tsetse host. Although no experimental evidence exists that demonstrates a functional role of this nature for Sodalis, Wolbachia exhibits immuno-modulatory properties in other insect models. For example, Drosophila treated with antibiotics to clear their Wolbachia infections are significantly more susceptible to a range of RNA viruses (32, 33). Furthermore, the mosquito Aedes aegypti can be stably transinfected with an exogenous strain of Wolbachia (wMelPop; 34). The presence of wMelPop appears to activate the immune system of offspring from transinfected females, which subsequently exhibit enhanced immunity against a range of pathogens (35, 36). Interestingly, unlike our laboratory colony, many natural populations of tsetse do not harbor Sodalis and/or Wolbachia, but are apparently still immuno-competent (37). It remains to be seen whether these symbionts play a role in stimulating immune system development in natural populations of tsetse.

Many insects, including Drosophila, Anopheles and Manduca, likely rely on their cellular immune systems as a potent first line of defense against systemic infection with pathogenic bacteria (38, 39, 40, 41). Similarly, tsetse become susceptible to infection with E. coli after their hemocyte function is abrogated via the uptake of polystyrene microspheres (11). In this study we provide further evidence that tsetse's ability to overcome systemic infection with E. coli also depends on the presence of a functional cellular immune system. First, E. coli kills Gmm^Apo adults despite the fact that they express dramatically more of the AMPs cecropin and attacin than do resistant WT flies. This finding suggests that AMPs alone are insufficient for tsetse to overcome systemic infection with E. coli. Secondly, Gmm^Apo adults survive this same infection if they had previously received hemolymph transplanted from WT donors. However, when WT donor hemolymph was separated into cellular and soluble fractions prior to transplantation, only Gmm^Apo recipients that received the cellular fraction (hemocytes) exhibited an E. coli-resistant phenotype. Thus, hemolymph-soluble factors such as an AMPs, hematopoeitic molecules or reactive oxygen species, presumably do not induce E. coli resistance when transplanted into Gmm^Apo flies. Instead resistance appears fixed to the cellular immunity-related activity of hemocyte-mediated phagocytosis.

Beneficial microbes in the human gut produce symbiosis factors that, unlike disease-causing virulence factors produced by pathogenic microbes, promote favorable health-related outcomes (42). For example, the human commensal, Bacteroides fragalis, produces one such molecule called polysaccharide A (PSA). Colonization of germ-free mice with this bacterium restores CD4⁺ T cell populations to levels conventionally found in mice that house their native microbiota. This process is consistent with B. fragalis PSA-induced development of secondary lymphoid tissues. B. fragalis PSA mutants fail to induce these systemic responses in germ-free animals (43). Similarly, mouse intestinal microbiota serve as a source of peptidoglycan (PGN) that enhances the efficacy of phagocytic neutrophils against pathogenic bacteria (44). To date no immuno-modulatory symbiosis factors have been characterized from insect-associated microbes. In this study we demonstrated that immune system development in Gmm^Apo larvae was activated when their moms were fed a diet supplemented with extracts of Wigglesworthia cells. This finding suggests that a molecular component of this obligate bacterium can actuate a trans-generational priming response in the intrauterine larvae of symbiont-cured females. This response restores the process of immune system maturation in larvae in the absence of milk gland-associated Wigglesworthia. Tsetse houses 2 distinct populations of Wigglesworthia, the first of which is found extracellularly in female milk gland secretions. These bacterial cells presumably colonize developing intrauterine larvae, which receive maternal milk for nourishment during tsetse's unique mode of viviparous reproduction (7). Tsetse's second population of Wigglesworthia resides within the cytosol of specialized bacteriocytes that collectively compromise an organ located immediately adjacent to midgut called the bacteriome (45). Interestingly, Gmm^Wgm− adults, which arise from female tsetse that house bacteriome-associated Wigglesworthia but lack their milk gland population, are immuno-compromised (11). Thus, this population of Wigglesworthia is insufficient to stimulate immune system development in intrauterine Gmm^Wgm− larvae. However, Wigglesworthia-containing bacteriome extracts supplemented in the diet of symbiont-cured moms can stimulate immune system development in Gmm^Apo larvae. Bacteriome-associated Wigglesworthia appear to produce the molecule(s) required to actuate immune system development in Gmm^Apo offspring, but they are concealed within the cytosol of bacteriocytes.

The mechanism by which Wigglesworthia extracts induce immune system development in Gmm^Apo larvae is currently unknown. In the mammalian system, symbiosis factors are translocated from the gut lumen to peripheral target immune tissues. In the mouse model B. fragalis cells, or B. fragalis PSA, is presumably taken up by gut-associated dendritic cells, which subsequently migrate to outlying lymphoid tissues where they signal for the differentiation of T cell lineages (43). In a similar manner, PGN shed by mouse intestinal microbiota is translocated from the luminal side of the gut epithelia into the circulatory system. A positive correlation exists between the concentration of PGN present in host sera and neutrophil function (44). Further experiments are required in the tsetse system to determine if immuno-stimulatory Wigglesworthia molecules are transported to the developing larvae where they exhibit direct activity, or if they act locally in the gut to induce a maternally-derived systemic response that subsequently induces larval immune system development.

Nutritional symbioses between bacteria and insects are well-documented (46, 47). The relationship between tsetse and Wigglesworthia presumably also has a nutritional component, as flies that lack this bacterium are reproductively sterile (48, 9). In fact, Wigglesworthia's highly reduced genome encodes many vitamins and cofactors that are missing from tsetse's vertebrate blood-specific diet (49). In this study we demonstrate that the tsetse-Wigglesworthia symbiosis is multi-dimensional in that this microbe is also intimately involved in activating the development of it's host's immune system. As such tsetse may be exploitable as relatively simple and efficient model for deciphering the basic molecular mechanisms that underlie symbiont-induced maturation of host immunity.

Abbreviations

AMP

antimicrobial peptide

DV

dorsal vessel

qPCR

real-time quantitative PCR

WT

wild-type

GC

gonotrophic cycle

PGRP-LB

peptidoglycan recognition protein LB

tep

thioester-containing protein

iNOS

inducible nitric oxide synthase

DUOX

dual oxidase

dpc

days post-challenge