Tsetse Immune System Maturation Requires the Presence of Obligate Symbionts in Larvae

Tsetse harbors an obligate symbiont, Wigglesworthia glossinidia, that must be present during larval maturation for the fly's immune system to develop and function properly during adulthood.


Introduction
Bacteria comprise the most abundant and diverse life form on earth. The ubiquity of bacteria means they have colonized virtually every ecological niche, including habitation within more evolutionarily sophisticated multi-cellular animals. Co-evolution over millions of years has provided an opportunity for beneficial symbiotic associations to develop between phylogenetically distant taxa. Such affiliations are often mutualistic, meaning both partners benefit so that each can successfully inhabit diverse environments that neither could survive in on its own [1,2]. Deciphering the mutualistic relationships between prokaryotic bacteria and multicellular eukaryotic animals is a rapidly advancing field of research.
Performing detailed investigations into the relationships between symbiotic bacteria and higher eukaryotes often involves costly and complex procedures. However, insects have welldocumented symbioses that are attractive to study because they have relatively short generation times and are easy and less costly to rear. One insect that harbors multiple symbionts is the tsetse fly, Glossina morsitans. These microbes include the commensal Sodalis, the parasite Wolbachia, and the obligate mutualist Wigglesworthia glossinidia [3]. Molecular phylogenetic analysis indicates that tsetse's symbiosis with Wigglesworthia is ancient, dating back 50-80 million years [4]. The concordant nature of tsetse's obligate association with Wigglesworthia has driven the co-evolution of biological adaptations that are beneficial to both partners. For example, the localization of Wigglesworthia cells within host bacteriocytes provides a protective and metabolically favorable niche for this bacterium [5]. In return tsetse derives benefit from Wigglesworthia in at least two distinct ways. First, tsetse feeds exclusively on vertebrate blood, which is deficient in vitamins essential for survival. In accordance, a large proportion of Wigglesworthia's streamlined (700 kB) genome encodes vitamin biosynthesis pathways that presumably supplement tsetse's restricted diet [5,6]. Second, more recent studies indicate that Wigglesworthia may serve an immunologic role in tsetse. These flies are the sole vector of pathogenic African trypanosomes, the causative agent of sleeping sickness in humans [7]. In laboratory experiments infection with immunogenic trypanosomes results in a decrease in tsetse fecundity [8]. Furthermore, parasite infection prevalence is higher in flies that lack Wigglesworthia when compared to age-matched wild-type (WT) individuals [9]. Wigglesworthia is thought to influence tsetse's vectorial competence by modulating its host's humoral immune system [10]. Thus, by preventing energetically costly parasite infections, this obligate symbiont may indirectly benefit the reproductive fitness of tsetse.
Symbiotic bacteria are rapidly gaining recognition for their important contributions to host development and immunity. The most well-known example of this type of interaction involves the mammalian microbiome, which modulates gut development during early postnatal life and subsequently shapes our mucosal and systemic immune systems [11,12]. Symbionts also serve similar functions in invertebrate hosts. For example, light organ morphogenesis in juvenile bobtail squid (Euprymna scolopes) initiates only after symbiotic Vibrio fischeri cells have stably colonized this tissue [13]. The pea aphid, Acyrthosiphon pisum, harbors a secondary symbiont, Hamiltonella defensa, which can be infected with a lysogenic bacteriophage (A. pisum secondary endosymbiont; APSE). A. pisum that harbor both H. defensa and APSE are protected from being consumed by a parasitic wasp larvae through the action of phage-encoded toxins [14].
To the best of our knowledge no evidence exists that demonstrates insect symbionts can confer an impending protective phenotype in their adult host by directing immune system development during immature stages. In the present study we investigate the mechanism by which Wigglesworthia contributes to the development and function of cellular and humoral immune responses in adult tsetse. Our results show that the maturation and normal function of adult cellular immune responses in tsetse are severely compromised when Wigglesworthia is absent during larval development. This study reveals an important new facet that further anchors the obligate relationship between tsetse and Wigglesworthia and may serve as a useful model to understand the highly integrated and dynamic relationship between hosts and their beneficial bacterial fauna.

Tsetse's Susceptibility to E. coli Infection Is Dependent Upon Age and Symbiotic Status
Insects are normally capable of mounting an immune response that combats infection with various groups of bacteria. Interestingly, in comparison to Drosophila, tsetse flies are uniquely susceptible to septic infection with 10 3 colony-forming units (CFU) of normally non-pathogenic Escherichia coli (E. coli) K12 [15]. In the present study we further investigated tsetse's unique susceptibility to E. coli infection by subjecting wild-type (Gmm WT ) and adults from two age groups to hemocoelic infections with varying quantities of E. coli K12. Three-day-old Gmm WT individuals (flies from this age group are hereafter referred to as ''young'') were highly susceptible to this treatment, as 10 3 CFU resulted in the death of all flies by 8 d post-infection (dpi; Figure 1A, top graph). In contrast, 77% and 55% of 8-d-old WT individuals (flies 8 d old and older are hereafter referred to as ''mature'') survived for 14 dpi with 10 3 and 10 6 CFU of E. coli K12, respectively ( Figure 1A, middle graph).
We demonstrated previously that feeding pregnant female tsetse the antibiotic ampicillin results in the generation offspring that lack Wigglesworthia (Gmm Wgm2 ) but still harbor Sodalis [9] and presumably Wolbachia. In the present study we determined the survival outcome of mature Gmm Wgm2 inoculated with 10 6 CFU of E. coli K12 (the dose required to kill ,50% of mature WT flies; Figure 1A, middle graph). In comparison to age-matched WT tsetse, Gmm Wgm2 flies were highly susceptible to this infection. In fact, at 14 dpi, only 1% of these individuals remained ( Figure 1A, bottom graph). We reasoned that the dramatic susceptibility of mature Gmm Wgm2 flies to infection with E. coli could result from one of two scenarios. In the first scenario Wigglesworthia may directly contribute to the adult immune response to a foreign micro-organism. In the second scenario, the absence of Wigglesworthia during larvagensis may give rise to Gmm Wgm2 adults with compromised immune functions. To determine if the first scenario is correct we treated mature WT tsetse with the antibiotic tetracycline to eliminate all of their microbiota, including bacteriome-associated Wigglesworthia ( Figure 1B), and subsequently challenged these adult flies (Gmm WT/Wgm2 ) with 10 6 CFU of tetracycline resistant E. coli K12. We found that, similar to their WT counterparts (Gmm WT ; Figure 1A, middle panel), about 70% of Gmm WT/Wgm2 individuals survived this infection ( Figure 1C). Conversely, Gmm Wgm2 adults were highly susceptible ( Figure 1A, bottom graph). This result suggests that when Wigglesworthia is absent from tsetse during larval development subsequent adults are severely immuno-compromised.
We demonstrated that when larval tsetse lacks exposure to Wigglesworthia in utero their immune system is highly compromised during adulthood. This finding does not exclude the possibility that treatment of female flies with ampicillin to produce Gmm Wgm2 offspring induces perturbations in other constituents of tsetse's microbiota. Because tsetse's other two known endosymbionts, Sodalis and Wolbachia, are present at similar densities in Gmm WT and Gmm Wgm2 adults ( Figure 1D), we looked for the presence of uncharacterized endosymbionts or digestive tract-associated microbes that are potentially passed on to developing intra-uterine larvae where they subsequently impact immunity. We generated a clone library containing 16s rRNA gene sequences PCR amplified from 3 rd instar Gmm Wgm2 and Gmm WT larvae and then sequenced multiple clones from each tsetse line. Our results indicate that the

Author Summary
Beneficial bacterial symbionts, which are ubiquitous in nature, are often characterized by the extent to which they interact with the host. In the case of mutualistic symbioses, both partners benefit so that each one can inhabit diverse ecological niches where neither could survive on its own. Unfortunately, little is known about the functional mechanisms that underlie mutualistic relationships. Insects represent a group of advanced multi-cellular organisms that harbor well-documented symbiotic associations. One such insect, the tsetse fly, harbors a maternally transmitted bacterial mutualist called Wigglesworthia that provides its host with essential metabolites missing from its vertebrate blood-specific diet. In this study, we further examine the relationship between tsetse and Wigglesworthia by investigating the interaction between this bacterium and its host's immune system. We have found that when Wigglesworthia is absent from tsetse during the maturation of immature larval stages, subsequent adults are characterized by an underdeveloped cellular immune system and thus highly susceptible to infection with a normally non-pathogenic foreign microbe. These findings represent an additional adaptation that further anchors the steadfast relationship shared between tsetse and its obligate symbiont.
proportion of Wigglesworthia, Sodalis, and Wolbachia in 3 rd instar Gmm WT larvae is 19%, 75%, and 6%, respectively. As expected no Wigglesworthia 16s rRNA sequences were present in Gmm Wgm2 larvae, and the proportion of Sodalis and Wolbachia sequences present was 71% and 29%, respectively ( Figure 1E). No uncharacterized endosymbionts or gut-associated environmental microbes were present in any sample. Taken together, these results suggest that the presence of Wigglesworthia specifically is responsible for enabling immune system maturation in WT tsetse.
Our host survival curves indicate that mature Gmm WT and Gmm WT/Wgm2 can survive infection with E. coli while young Gmm WT and mature Gmm Wgm2 perish. To determine a cause for the variation in survival we observed between the four groups following infection with E. coli, we monitored bacterial growth dynamics over the course of the experiment in each group. Bacterial number within mature Gmm WT and Gmm WT/Wgm2 peaked at 3.8610 4 and 1.9610 4 E. coli, respectively, over the 2wk period, indicating that these groups appeared able to control their infections. In contrast, E. coli increased exponentially in young Gmm WT and mature Gmm Wgm2 and reached a maximum density at 6 dpi of 4.2610 6 and 8.8610 6 , respectively ( Figure 1F). These results implicate bacterial sepsis as the cause of high mortality observed in young Gmm WT and mature Gmm Wgm2 . All of the above-mentioned results taken together indicate that mature Gmm WT are considerably more resistant to infection with a foreign microbe than are their younger counterparts. Furthermore, tsetse's obligate mutualist, Wigglesworthia, must be present during the development of immature stages so that mature adults are able to overcome infection with E. coli.

Expression of Immunity-Related Genes in Adult Tsetse Varies Depending on Symbiont Status
To understand the basis of the compromised immunity we observed in mature Gmm Wgm2 flies, we evaluated the expression profile of a set of immunity-related genes from age-matched mature adult Gmm WT and Gmm Wgm2 flies that were either uninfected or 3 dpi with E. coli K12. We included the antimicrobial peptides (AMPs) attacin, cecropin, and defensin, which distinctly target gram-negative (attacin and cecropin) and grampositive bacteria (defensin) [16]. Our analysis also included thioester-containing proteins (tep2 and tep4), prophenoloxidase (PPO), and inducible nitric oxide synthase (iNOS). In insects TEPs likely function as pathogen-specific opsonins that bind to bacteria or parasites and promote their phagocytosis/encapsulation [17], while PPO initiates a proteolytic cascade that results in melanin deposition [18]. iNOS catalyzes synthesis of the signaling molecule nitric oxide (NO), which plays a role in humoral and cellular immunity in Anopheline mosquitoes [19], reduvid bugs [20], and Drosophila [21,22] by inducing AMP expression and recruiting hemocytes to the site of infection.
Our expression results indicate that symbiont status plays little or no role in the expression of immunity-related genes in uninfected adults. In fact, with the exception of the AMP defensin, no significant differences in immunity-related gene expression between mature uninfected Gmm WT and Gmm Wgm2 adults ( Figure 2A). However, we observed a considerably different profile of immunity-related gene expression when these different fly strains were infected with E. coli K12. Under these circumstances, all of the genes evaluated (with the exception of defensin) were expressed at significantly higher levels in Gmm WT compared to Gmm Wgm2 individuals ( Figure 2B). Particular striking was the fact that the induction of pathways associated with cellular immunity, such as pathogen recognition (tep2 and tep4) and melanization (PPO), were significantly compromised in mature Gmm Wgm2 adults. The absence of a robust cellular immune response is likely the cause of high mortality among these individuals following E. coli infection. This analysis indicates that Wigglesworthia must be present during the development of immature tsetse in order for immune-related genes to subsequently be expressed in mature E. coli-infected adults.

Hemocytes Play an Integral Role in Controlling E. coli Infection Outcomes in Tsetse
Our analysis of immunity-related gene expression in tsetse suggests that cellular immune pathway functions in adult tsetse are particularly compromised when Wigglesworthia is absent during immature development. The most prominent cellular immune mechanisms include melanization and phagocytosis. These processes, which ultimately result in the removal of foreign invaders in Drosophila [23] and A. aegypti [24], both arise from distinct crystal cell and plasmatocyte hemocyte lineages, respectively [25].
We investigated the role hemocytes might play in determining the susceptible phenotype we observed in tsetse following infection with E. coli. We infected mature Gmm WT individuals with GFP-expressing E. coli and were able to observe that hemocytes had engulfed a large number of the introduced cells by 12 hpi (Figure 3A). We next inhibited phagocytosis by introducing blue fluorescent microspheres directly into tsetse's hemocoel and 12 h later infected the bead-treated individuals with GFP-expressing E. coli. Microscopic inspection of hemocytes harvested 12 hpi with E. coli revealed the presence of internalized microspheres and the absence of engulfed E. coli. This observation indicated that we were successful in blocking hemocyte phagocytosis ( Figure 3B). We subsequently maintained our microsphere-injected tsetse for 2 wk with the intention of determining the impact of impaired phagocytosis on host survival outcome. Mature Gmm WT flies exhibiting impaired phagocytosis were highly susceptible to infection with both 1610 3 and 1610 6 E. coli K12. In fact, by day 12 post-infection, all of these flies had perished regardless of the initial dose used for infection ( Figure 3C). This observation contrasts starkly with infection outcome in mature Gmm WT that exhibit normally functioning hemocytes ( Figure 1A, middle graph). Our results suggest that defects in phagocytosis severely compromise the ability of these tsetse flies to overcome bacterial infection.
A notable result of our immunity-related gene expression analysis was a 37-fold decrease in PPO levels in Gmm Wgm2 flies. This enzyme is an essential component of the melanization pathway, and its expression ultimately results in host wound The process of hemocyte-mediated phagocytosis in tsetse was blocked by micro-injecting polystyrene beads into the hemocoel of 8-d-old WT individuals. In consecutive 12 h intervals following bead injection, flies were infected with GFP-expressing E. coli K12 and then hemolymph was collected and fixed as described above. Hemocytes appear to have engulfed the beads, thus prohibiting the subsequent uptake of bacterial cells. The inset in each panel shows a higher magnification image of one hemocyte, which is identified by a white triangle in the left-most panel. Scale bar = 20 mm. (C) Tsetse flies that harbor hemocytes incapable of engulfing E. coli are susceptible to septic infection with this bacterium while their wild-type counterparts are not. The susceptible phenotype is exhibited regardless of whether phagocytosis-inhibited tsetse were inoculated with 10 3 or 10 6 CFU of E. coli. Beads alone had no effect on tsetse mortality. No significant difference existed in survival outcome between mature Gmm WT phagocytosis inhibited flies infected with 10 3 versus 10 6 CFU of E. coli (p = 0.47, log-rank analysis). Furthermore, no significant difference was present between mature Gmm Wgm2 flies with uninhibited hemocytes ( Figure 1A, bottom panel) and mature Gmm WT phagocytosis inhibited flies (p = 0.11) infected with 10 6 CFU of E. coli. doi:10.1371/journal.pbio.1000619.g003 healing and the melanization, encapsulation, and subsequent removal of foreign microorganisms [26][27][28]. In conjunction with the remarkable variation in PPO expression observed between Gmm WT and Gmm Wgm2 , we were also able to visually observe the absence of a melanization response to E. coli infection in flies lacking Wigglesworthia. In fact, 30 min post-injection with E. coli, hemolymph was still actively exuding from the inoculation wound of Gmm Wgm2 flies. Conversely, in WT individuals no hemolymph was detectable and melanin was deposited at the wound site ( Figure 4). These results further suggest that hemocyte-mediated cellular immunity provides an imperative defense against the establishment of bacterial infections in tsetse's hemocoel. Furthermore, the absence of this response in Gmm Wgm2 individuals was likely responsible for the compromised host survival phenotype we observed following infection with E. coli.

Symbiotic Bacteria Impact the Maturation of Tsetse's Cellular Immune Response During Development
We observed that young Gmm WT were markedly more susceptible to infection with E. coli K12 than were their mature counterparts. Furthermore, symbiont status also altered infection outcome, as mature Gmm Wgm2 perished following E. coli infection while age-matched WT individuals survived. These differential infection outcomes appeared to result from disparities in cellular immune system function between the different tsetse lines we examined. Based on these observations we hypothesized that the obligate mutualist Wigglesworthia plays a crucial role in regulating the development of cellular immunity in tsetse during immature stages. To test this hypothesis we quantified the number of circulating and sessile hemocytes present in young and mature adult Gmm WT and mature adult Gmm Wgm2 . Our results indicate that a 1.4-fold increase in circulating hemocyte number occurs between day 3 and day 8 in WT tsetse, while no significant change in circulating hemocyte number was observed between young and mature Gmm Wgm2 ( Figure 5A). Interestingly, mature Gmm WT adults harbored 3.46 more circulating hemocytes than did mature Gmm Wgm2 adults ( Figure 5A). We also looked at sessile hemocyte abundance as a further indicator of Wigglesworthia's impact on the development of cellular immunity in tsetse. In Drosophila, this hemocyte subtype concentrates in large quantities around the anterior end of the fly's dorsal vessel [29]. Thus, we indirectly quantified sessile hemocyte abundance immediately adjacent to the anterior-most chamber of tsetse's dorsal vessel by measuring the fluorescent emission of microspheres that were found engulfed in this region. Young Gmm WT adults engulfed 1.26 more microspheres than their mature counterparts and 15.76 more than mature Gmm Wgm2 adults. Furthermore, mature WT adults engulfed 13.26 more microspheres than did age-matched Gmm Wgm2 adults ( Figure 5B).
Our results demonstrate that Wigglesworthia must be present during the development of immature tsetse in order for cellular immunity to develop and function properly in adults. Thus, we speculate that the absence of hemocytes in adult tsetse should reflect a lack of blood cell differentiation during the development of immature stages. In Drosophila the process of blood cell differentiation, or hematopoiesis, begins in the embryo and proceeds through all larval stages [30]. During Drosophila embryogenesis early hematopoiesis can be distinguished by the expression of the zinc finger transcription factor ''Serpent.'' Subsequently, another transcription factor, ''Lozenge,'' directs the differentiation of serpent-expressing precursor cells into a specific lineage of hemocytes called crystal cells. To address the relationship between the presence of Wigglesworthia and early hematopoiesis in tsetse, we used qPCR to evaluate the relative number of serpent and lozenge transcripts present in 1 st , 2 nd , and 3 rd instar larvae dissected from pregnant Gmm WT and Gmm Wgm2 females. Larval instars L1, L2, and L3 from WT females expressed 1.7, 2.1, and 1.9 times more serpent transcripts, and 4, 4.4, and 3.9 times more lozenge transcripts, respectively, than did their counterparts from females that lacked Wigglesworthia ( Figure 5C). This observed attenuated expression of both serpent and lozenge may account for the depleted hemocyte population, and compromised cellular immune function, we observed in adult Gmm Wgm2 individuals.

Discussion
In the present study we demonstrate that Wigglesworthia is intimately involved in regulating the maturation and function of tsetse's cellular immune system during immature larval development. We present a model that links the presence of Wigglesworthia in larval progeny with host immune system maturation during development and the subsequent ability of adult tsetse to overcome infection with foreign microbes (Figure 6). Obligate symbioses between intracellular bacteria and multi-cellular eukaryotes represent millions of years of co-evolution during which time both partners have adapted to increase each other's overall fitness. The association between tsetse and Wigglesworthia is an example of this reciprocal relationship in that neither organism can survive in the absence of the other.
We present several lines of evidence indicating that tsetse's resistance to E. coli positively correlates with fly age and symbiont infection status during juvenile stages. Specifically, our results signify that mature Gmm WT adults are resistant to E. coli infection, while young Gmm WT adults are susceptible. In contrast, both young and old adult flies that lack Wigglesworthia throughout all developmental stages are killed by E. coli infections. Like their WT counterparts, Gmm Wgm2 larvae acquire both Sodalis and Wolbachia while in utero [31]. Although the intrauterine larval environment is otherwise aseptic, adult Gmm Wgm2 can be exposed to a wide range of environmental microbes during adulthood. However, neither the presence of other symbiotic bacteria (Sodalis and Wolbachia) in larvae, nor environmental microbes acquired during adulthood, appear to be sufficient to induce immunity in Gmm Wgm2 adults. Additionally, we show that mature Gmm WT adults treated with antibiotics to eliminate Wigglesworthia (Gmm WT/Wgm2 ) remain resistant to E. coli infections. The resistant phenotype of Gmm WT/Wgm2 further signifies that obligate Wigglesworthia is not directly responsible for the ability of mature adult Gmm WT to overcome septic bacterial infection.
Instead, it appears that Wigglesworthia's presence during the maturation of larval progeny stimulates the development of host immunity in adults.
Our quantitative analysis of gene expression indicates that Gmm WT and Gmm Wgm2 adults exhibit no significant difference in the expression of genes that serve as hallmarks of humoral and cellular immunity in the absence of microbial challenge. However, following infection with E. coli, all pathways were significantly compromised in Gmm Wgm2 versus Gmm WT adults. The most notable discrepancy observed between the two tsetse lines involved the expression of prophenoloxidase. Following E. coli infection, the expression of this gene increased 37-fold in WT tsetse but remained virtually unchanged in individuals that lacked Wigglesworthia. In WT insects, PPO, which is an inactive zymogen, is proteolytically cleaved to produce phenoloxidase upon mechanical injury or the presence of foreign pathogens. Phenoloxidase then facilitates the process of melanin deposition [18]. A functional melanotic pathway would likely increase tsetse's resistance to E. coli infection in several ways. First, sequestration of E. coli in a melanotic capsule at the site of inoculation would likely help prevent their dissemination into adjacent host tissues. This phenomenon has been observed in both the hawk moth, Manduca sexta, and Drosophila following infection with pathogenic bacteria (Photorhabdus luminescens) and parasitic wasp eggs, respectively [32,33]. In both cases the lack of melanization at the wound site severely compromised the host's ability to subsequently overcome the foreign invader. Second, the melanization cascade results in the production of reactive oxygen intermediates that are directly toxic to foreign pathogens. In the flesh fly Sarcophaga peregrina and M. sexta, melanization intermediates such as DOPA exhibit direct antimicrobial activity [34,35]. Finally, melanin at the site of inoculation expedites wound healing that could prevent the spread of secondary infections [26].
The E. coli-susceptible phenotype of mature Gmm Wgm2 adults is likely reflective of their blood cell (hemocyte) deficit in comparison to E. coli-resistant WT flies. In Drosophila 90%-95% of the total hemocyte population is composed of sessile and circulating plasmatocytes, which are a distinct hemocyte lineage predominantly responsible for engulfing and digesting foreign pathogens [36]. The inability of mature Gmm Wgm2 tsetse to survive infection with E. coli may result from their significantly reduced population of phagocytic hemocytes available to engulf bacteria injected into the hemocoel. In fact, by injecting polystyrene beads as a means of blocking this physiological process, we demonstrate that phagocytosis is a critical component of tsetse's ability to manage septic infection with E. coli in WT flies. Similarly, mutant Drosophila that contain a depleted plasmatocyte population, or have been injected with beads to prohibit phagocytosis, exhibit a remarkable susceptibility to a variety of gram positive and negative bacteria [37,38]. Interestingly, Drosophila that lack functional plasmatocytes also exhibit a reduced capacity to activate humoral immune responses, further inhibiting their ability to fight bacterial infection [29].
In Drosophila the crystal cell hemocyte lineage controls the humoral melanization cascade via the release of PPO stored in large cytoplasmic inclusion bodies [30,39]. In addition to the dramatic reduction in PPO expression in Gmm Wgm2 tsetse, two further lines of evidence indicate that this fly strain harbors a significantly reduced population of hemocytes that function in a homologous manner to Drosophila crystal cells. First, lozenge expression in all three larval instars from Gmm Wgm2 females is significantly lower than in their Gmm WT counterparts. In Drosophila larval crystal cells fail to form in the absence of lozenge expression, yet the differentiation of other hemocyte types proceeds normally [40]. Second, we observed that hemocyte-deficient Gmm Wgm2 flies were unable to produce a viable clot at the site of bacterial inoculation. In mutant Drosophila strains that lack crystal cells, PPO is absent from the hemolymph. Consequently, the melanization cascade fails to initiate and hard clots do not form at wound sites [41].
We discovered that young Gmm WT adults harbor significantly less circulating hemocytes than do mature WT adults. This observation implies that circulating hemocyte number in adult WT tsetse increases as a function of age, although the specific mechanism underlying this process in tsetse is currently unknown. In adult Drosophila intact lymph glands are absent and no evidence exists for the de novo synthesis of hemocytes following metamorphosis [30,40]. Interestingly, we did observe more subepidermal sessile hemocytes in young compared to mature Gmm WT adults, although the number was not statistically significant between the two groups. We speculate that the increased abundance of circulating hemocytes we observed in mature compared to young Gmm WT may reflect a shift in the proportion of sessile to circulating cells instead of de novo production of new hemocytes. A comparable process occurs in Drosophila larvae and adults, where the proportion of sessile to circulating hemocytes changes following immune stimulation [42,43]. Furthermore, after larval Drosophila receive an epidermal wound, circulating hemocytes are rapidly recruited to the site of injury. These circulating cells are phagocytically active and likely function as a front-line surveillance system against tissue damage and microbial infection [44]. In the mosquito malaria vector Anopheles gambiae, exposure to Plasmodium parasites stimulated an increase in the number of granulocytes circulating in the hemolymph. These primed mosquitoes were subsequently more resistant to infection with pathogenic bacteria than their wild-type counterparts [45]. We propose that young Gmm WT adults and mature Gmm Wig2 adults may be susceptible to E. coli infection in part because they harbor significantly less circulating hemocytes than do older WT individuals that are resistant. Our previous studies indicated that Gmm Wig2 adults are highly susceptible to trypanosome infection [9,10] and that this phenotype may be modulated by tsetse's humoral immune system [46][47][48]. Studies are ongoing to determine if cellular immunity also modulates tsetse's trypanosome vectorial competence. Figure 6. Wigglesworthia modulates the development and function of tsetse's immune system. Through a currently unknown mechanism, the presence of Wigglesworthia in Gmm WT larvae stimulates hemocyte differentiation in a specialized organ that is homologous to Drosophila's lymph gland. Upon metamorphosis, specialized hemocyte subtypes are released from the lymph gland and carried over in a functional state to the adult. In the absence of Wigglesworthia, Gmm Wgm2 larvae produce significantly less hemocytes than their WT counterparts. Several innate immunity pathways are activated upon inoculation of E. coli into the hemocoel of mature adult Gmm WT . Tsetse's preliminary line of defense against E. coli infection likely involves melanization at the wound site. This process is initiated by localized crystal cells, which instigate the melanization cascade by secreting prophenoloxidase (PPO) into the hemolymph. Pathogens that circumvent the wound site then encounter phagocyte-mediated cellular and humoral immune responses. Soluble TEPs likely opsonize bacterial cells, thus tagging them for engulfment by phagocytes. Lysis of engulfed bacteria causes the release of bacterial peptidoglycan PGN that subsequently stimulates the production of AMPs by the fat body. AMPs, which are also generated by hemocytes, are then secreted into the hemolymph where they further abrogate microbial proliferation. Hemocytes also produce reactive oxygen intermediates, such as iNOS, that exhibit direct bacterial toxicity and further stimulate humoral immunity. In the case of Gmm Wgm2 adults, incapacitated hematopoiesis during larval stages results in severely compromised immunity that renders these flies highly susceptible as adults to bacterial infection. doi:10.1371/journal.pbio.1000619.g006 Neonatal humans (and presumably other mammals as well) acquire probiotic gut microbes through the act of breast feeding [49]. These bacteria subsequently stably colonize the naïve intestine where they promote immune system maturation and enhance defense against infection with pathogenic microbes [50]. Most lower eukaryotes, including insects, hatch from an egg deposited into the environment and thus rely mainly on environmental microbes to stimulate their innate immune systems during the development of immature stages. For example, Anopheline mosquitoes and Drosophila are exposed to environmental microbes throughout all stages of their development. Under these natural conditions subsequent adults exhibit potent innate immunity [51,52]. However, when these insects are reared under germ-free conditions, they exhibit a severely compromised humoral immune response [51,53]. Tsetse flies are unique among other insects because they lead a relatively aseptic existence. Not only do they feed exclusively on sterile vertebrate blood, but they also exhibit a unique viviparous reproductive strategy. During this type of reproduction, all embryonic and larval stages develop within the female's uterus. The protected in utero environment in which viviparous offspring develop limits their exposure to environmental microbes. However, throughout tsetse larvagenesis maternal milk gland secretions provide the developing offspring with nourishment as well as Wigglesworthia, Sodalis, and Wolbachia [31]. Interestingly, recently established Sodalis and Wolbachia appear unable to influence their host's physiology to the same extent as mutualist Wigglesworthia despite their presence throughout larval development. Future studies on this system will focus on determining the chemical and/or metabolic elements provided by Wigglesworthia that stimulate the maturation of its host's immune system during larval development.
The results of this study provide evidence of a novel functional role for obligate insect symbionts-host immune system activation during immature developmental stages to ensure robust function during adulthood. We demonstrate that the obligate Wigglesworthia provides tsetse offspring the stimuli necessary for immune system development, a process that exhibits functional parallels with the mammalian system following the transfer of beneficial microbes from mothers to their neonates during that act of breast feeding. This phenomenon represents an adaptation that further anchors the steadfast relationship shared between tsetse and its obligate mutualist. The essential nature of tsetse's dependence on Wigglesworthia provides a potentially exploitable niche to experimentally modulate host immunity, with the intention of diminishing this insect's capacity to vector deadly trypanosomes.

Tsetse and Septic Infections
Throughout the text, 3-d-old and 8-d-old tsetse are referred to as ''young'' and ''mature,'' respectively. Wild-type G. m. morsitans (Gmm WT ) were maintained in Yale's insectary at 24uC with 50%-55% relative humidity. These flies received defibrinated bovine blood every 48 h through an artificial membrane feeding system [54]. Two Wigglesworthia-free tsetse lines were generated for use in our experiments. The first, Gmm Wgm2 , was generated by supplementing the diet of pregnant females with 25 mg of ampicillin per ml of blood. Gmm Wgm2 (offspring of ampicillin-fed females) adult are devoid of Wigglesworthia throughout all developmental stages [9]. PCR using Wigglesworthia thiamine Cspecific primers (forward, 59-TGAAAACATTTGCAAAATTTG-39; reverse, 59-GGTGTTACATAGCATAACAT-39) confirmed that this tsetse strain lacked Wigglesworthia. The second Wiggle-sworthia-free tsetse line, Gmm WT/Wgm2 , was generated by feeding mature WT adults three blood meals supplemented with 40 mg/ml tetracycline. Gmm WT/Wgm2 individuals thus harbored Wigglesworthia, Sodalis, and Wolbachia throughout the development of all immature stages and early adulthood, but lacked these symbionts thereafter. The absence of Wigglesworthia from mature Gmm WT/ Wgm2 adults was confirmed microscopically by comparing the bacteriome (Wigglesworthia-harboring organ) contents of mature WT and Gmm WT/Wgm2 adults.
Septic infection of tsetse was achieved by anesthetizing flies with CO 2 and subsequently injecting individuals with live bacterial cells using glass needles and a Narashige IM300 micro-injector. The methods used to produce luciferase-expressing E. coli K12 (recE. coli pIL ), and the assay used to quantify luciferase expression in vivo, were described previously [15]. GFP-expressing and tetracyclineresistant E. coli K12 were produced via electroporation with pGFP-UV (Clontech, Mountain View, CA) and pBR322 (Promega, Madison, WI) plasmid DNA, respectively. All flies treated with tetracycline were subsequently infected with tetracycline resistant E. coli K12.

Real-Time Quantitative PCR (qPCR)
For analysis of immunity-related gene expression, sample preparation and qPCR were performed as described previously [15]. Quantitative measurements were performed on three biological samples in duplicate and results were normalized relative to tsetse's constitutively expressed b-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.
For symbiont quantification, total RNA was prepared from 40d-old adult Gmm WT and Gmm Wgm2 flies. Symbiont genome numbers were quantified using single-copy Sodalis fliC and Wolbachia groEL. Relative symbiont densities were normalized to tsetse b-tubulin. All qPCR was performed with an icycler iQ real time PCR detection system (Bio-Rad). Values are represented as the mean (6SEM). qPCR primer sequences are shown in Table  S1.

Bacterial 16s rRNA Clone Libraries
Universal bacterial 16S rRNA gene primers 27F (59-AGAGTTTGATCCTGGCTCA G-39) and 1492R (59-GGT-TACCTTGTTACGACTT-39) [55] were used to produce a clone library of 16s rRNA gene sequences found in 3 rd instar Gmm WT and Gmm Wgm2 larvae. Five individual larvae from each tsetse line were dissecting under sterile conditions and washed in DNase (Ambion, Austin, TX) to remove any surface contamination prior to DNA extraction. Genomic DNA was isolated using Holmes-Bonner buffer (0.1 mol/L Tris-HCl, pH 7.5; 0.35 mol/L NaCl; 10 mmol/L EDTA, pH 8.0; 2% SDS; 7 mol/L Urea), purified via phenol-chloroform extraction and precipitated in 100% EtOH. PCR was performed under standard reaction conditions [15], and the resulting products were cloned in the pGEM-T vector (Promega, Madison, WI). Twenty clone inserts from each larvae (100 in total from each tsetse line) were sequenced using the T7 vector specific primer, and homology to previously described 16s rRNAs was determined using the blastn database.

Hemolymph Collection and Hemocyte Quantification
Depending on the subsequent experiment, tsetse hemolymph was collected using one of two methods. For hemocyte quantification, 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. Hemocytes were quantified microscopically using a Bright-Line hemocytometer, and hemocyte numbers are represented as cells per ml of hemolymph.
When hemocytes were required for microscopic visualization, hemolymph was collected by employing a modified version of the high injection/recovery method previously developed for use in mosquitoes [56]. In brief, tsetse flies were sedated on ice and injected with 25 ml 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] between the last two abdominal schlerites using a glass needle and a Narashige IM300 micro-injector. Following a 30 min incubation on ice, a front leg was removed at the joint most proximal to the thorax. At this point internal pressure forced hemolymph diluted with anticoagulant buffer to be expelled from the wound site (more liquid could be recovered by applying gentle pressure to the distal end of the abdomen). Liquid was collected using a pipette and either placed into a chilled microfuge tube or directly into a 24-well cell culture plate. In the latter case, cells were allowed to adhere to the plate bottom, after which anticoagulant buffer was replaced with MM media.
Sessile hemocytes were observed by intra-thoracically injecting young and mature Gmm WT , and mature Gmm Wgm2 (n = 5 of each strain), with 2 ml of blue fluorescent (365/415 nm) 0.2 mm carboxylate-modified beads (Invitrogen corp.). Prior to use, beads were washed once in PBS and resuspended in 100% of their original volume. Flies were dissected 12 h post-injection to reveal their dorsal vessel and surrounding tissue, which was gently washed 3 times with PBS to remove any potentially contaminating circulating (non-adherent) hemocytes. Engulfed microspheres were visualized using a Zeiss steriomicroscope (Discovery v8) equipped with a coaxial fluorescence module. Semi-quantitative comparison of sessile hemocyte number between young and mature Gmm WT adults, and mature Gmm WT and Gmm Wgm2 adults, was performed by quantifying fluorescent signal intensity (n = 4 individuals from each group) using ImageJ software (http://rsbweb.nih.gov/ij/).

Phagocytosis and Melanization Assays
Phagocytosis by circulating tsetse hemocytes was observed by intra-thoracically infecting mature Gmm WT flies (n = 10) with 1610 6 GFP-expressing E. coli K12. Twelve hours post-infection, hemolymph was collected and hemocytes monitored to determine if they had engulfed the GFP-expressing bacterial cells. Hemo-lymph samples were fixed on glass microscope slides via a 2 min incubation in 2% PFA. Prior to visualization using a Zeiss Axioscope microscope, slides were overlayed with VectaShield hard set mounting medium containing DAPI (Vector Laboratories, Burlingame, CA).
Phagocytosis by tsetse hemocytes was inhibited with blue fluorescent (365/415 nm) 0.2 mm carboxylate-modified beads (Invitrogen Corp.). Prior to use, beads were washed once in PBS and resuspended in 100% of their original volume. Inhibition assays were performed by inoculating 8-d-old Gmm WT with 2 ml of beads via their thoracic compartment. Twelve hours later, these flies were similarly infected with 1610 3 and 1610 6 GFPexpressing E. coli K12 (experiment was performed in triplicate; n = 25 flies per replicate). Finally, 12 hours post-infection with E. coli, hemolymph was collected and processed as described above (these samples were overlayed with VectaShield hard set mounting medium that lacked DAPI).
Melanization assays were performed by intra-thoracically inoculating mature Gmm WT and Gmm Wgm2 (n = 10 of each strain) with 1610 3 E. coli K12. Subsequently, three individuals from each group were monitored microscopically every 10 min for the presence of melanin at the wound site. The remaining seven flies from each group were maintained for 2 wk in order to observe infection outcome.

Statistics
Statistical significance of survival curves was determined by logrank analysis using JMP (v8.02) software (www.jmp.com). Statistical analysis of qPCR data was performed by Student's t test using JMP (v8.02) software. Statistical significance between various treatments, and treatments and controls, is indicated in corresponding figure legends.