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. 2011 May;9(5):e1000619.
doi: 10.1371/journal.pbio.1000619. Epub 2011 May 31.

Tsetse immune system maturation requires the presence of obligate symbionts in larvae

Brian L Weiss  1 , Jingwen Wang, Serap Aksoy
Affiliations

Tsetse immune system maturation requires the presence of obligate symbionts in larvae

Brian L Weiss et al. PLoS Biol. 2011 May.

Abstract

Beneficial microbial symbionts serve important functions within their hosts, including dietary supplementation and maintenance of immune system homeostasis. Little is known about the mechanisms that enable these bacteria to induce specific host phenotypes during development and into adulthood. Here we used the tsetse fly, Glossina morsitans, and its obligate mutualist, Wigglesworthia glossinidia, to investigate the co-evolutionary adaptations that influence the development of host physiological processes. Wigglesworthia is maternally transmitted to tsetse's intrauterine larvae through milk gland secretions. We can produce flies that lack Wigglesworthia (Gmm(Wgm-) yet retain their other symbiotic microbes. Such offspring give rise to adults that exhibit a largely normal phenotype, with the exception being that they are reproductively sterile. Our results indicate that when reared under normal environmental conditions Gmm(Wgm-) adults are also immuno-compromised and highly susceptible to hemocoelic E. coli infections while age-matched wild-type individuals are refractory. Adults that lack Wigglesworthia during larval development exhibit exceptionally compromised cellular and humoral immune responses following microbial challenge, including reduced expression of genes that encode antimicrobial peptides (cecropin and attacin), hemocyte-mediated processes (thioester-containing proteins 2 and 4 and prophenoloxidase), and signal-mediating molecules (inducible nitric oxide synthase). Furthermore, Gmm(Wgm-) adults harbor a reduced population of sessile and circulating hemocytes, a phenomenon that likely results from a significant decrease in larval expression of serpent and lozenge, both of which are associated with the process of early hemocyte differentiation. Our results demonstrate that Wigglesworthia must be present during the development of immature progeny in order for the immune system to function properly in adult tsetse. This phenomenon provides evidence of yet another important physiological adaptation that further anchors the obligate symbiosis between tsetse and Wigglesworthia.

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Conflict of interest statement

Serap Aksoy, coauthor on this manuscript, is also Editor-in-Chief of PLoS Neglected Tropical Diseases.

Figures

Figure 1
Figure 1. Host survival correlates with symbiont status following septic infection with E. coli K12.
(A) The effects of age and symbiont status on the survival of tsetse following systemic infection with E. coli K12. Mature adult GmmWgm- flies were significantly more susceptible to infection with 106 CFU of E. coli than were their wild-type counterparts (bottom and middle panels; p<0.001). (B) Gmm WT/Wgm− flies harbored Wigglesworthia during immature development but lacked the bacteria as mature adults. (C) Gmm WT/Wgm− adults were infected with tetracycline resistant E. coli 1 d after their last antibiotic-supplemented blood meal. Unlike their counterparts that lacked Wigglesworthia throughout immature development, Gmm WT/Wgm− was able to survive infection with E. coli. No significant difference in survival was observed between mature adult Gmm WT versus Gmm WT/Wgm− adults infected with 106 CFU of E. coli (Figure 1A middle panel and Figure 1C; p  = 0.07). (D) Relative Sodalis and Wolbachia densities in 40-d-old Gmm WT and GmmWgm − adults (n  = 5 of each) were normalized against host β-tubulin copy number. (E) Analysis of bacterial 16s rRNA clone libraries indicates that Gmm WT larvae harbored Wigglesworthia, Sodalis, and Wolbachia, while their counterparts from ampicillin treated females harbored only Sodalis and Wolbachia. No other bacteria were identified from either fly line. (F) Average number (±SEM) of recE. coli pIL per tsetse strain over time (n  = 3 individuals per strain per time point) following septic infection with 103 CFU of bacteria. Values shown in red represent lethal infections.
Figure 2
Figure 2. The effect of symbiont status on the expression of immunity-related genes in adult tsetse.
(A) Target gene expression in uninfected Gmm WT and GmmWgm − adults is indicated relative to the constitutively expressed tsetse β-tubulin gene. (B) Fold-change in the expression of immunity-related genes in Gmm WT and GmmWgm − tsetse 3 dpi with E. coli K12. All values for both tsetse strains are represented as a fraction of average normalized gene expression levels in bacteria-infected flies relative to expression levels in PBS-injected controls. In (A) and (B), quantitative measurements were performed on three biological samples in duplicate. Values are represented as means (±SEM). * p<0.05, ** p<0.005 (Student's t-test).
Figure 3
Figure 3. Hemocyte-mediated phagocytosis is a critical component of tsetse's immune response.
(A) 8-d-old Gmm WT were subjected to septic infection with GFP-expressing E. coli K12. Twelve hours post-infection hemolymph was collected, fixed on glass slides using 2% paraformaldehyde, and microscopically examined for the presence of hemocyte-engulfed bacterial cells. Scale bar  = 10 µm. (B) 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 μm. (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 103 or 106 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 103 versus 106 CFU of E. coli (p  = 0.47, log-rank analysis). Furthermore, no significant difference was present between mature GmmWgm − flies with uninhibited hemocytes (Figure 1A, bottom panel) and mature Gmm WT phagocytosis inhibited flies (p  = 0.11) infected with 106 CFU of E. coli.
Figure 4
Figure 4. The effect of symbiont status on melanization in tsetse.
Mature Gmm WT and GmmWgm − tsetse (n  = 10 of each strain) were intra-thoracically inoculated with 1×103 E. coli K12. Thirty minutes post-inoculation the wound site on individuals from each strain was inspected microscopically for the presence of hemolymph clotting and melanin deposition. Thirty minutes post-inoculation, neither hemolymph clotting nor melanin were observed at the wound site of GmmWgm − individuals (indicated by a red arrow). In contrast, within the same amount of time, hemolymph no longer exuded from the wound of WT flies and melanin was present surrounding the site (indicted by a white arrow).
Figure 5
Figure 5. The effect of age and symbiont status on the development of tsetse's cellular immune response.
(A) Number of hemocytes per µl of hemolymph in young (3 d) and mature (8 d) Gmm WT and GmmWgm - tsetse (n  = 5 individuals from each tsetse strain at both time points). (B) Quantitative analysis of sessile hemocyte abundance in young and mature Gmm WT and mature GmmWgm - tsetse (n  = 4 individuals from each tsetse strain and age point). All tsetse strains tested were subjected to hemocoelic injection with blue fluorescent microspheres. Twelve hours post-injection, flies were dissected to reveal tsetse's heart. The left-most panel is a Brightfield image of the three chambers that make up the dorsal vessel (DV; scale bar  = 350 µm). The anterior-most chamber is indicated within a white circle. The three remaining panels are close-ups of the anterior chamber (scale bar for all 3 panels  = 80 µm), visualized by excitation with UV light (365/415 nm). Relative fluorescence per tsetse group was determined using ImageJ software. (C) The presence of Wigglesworthia affects the expression of genes involved in hemocyte differentiation in immature larval tsetse. Target gene expression in Gmm WT and GmmWgm − larval instars 1–3 is indicated relative to the constitutively expressed tsetse β-tubulin gene. Quantitative measurements were performed on three biological samples in duplicate. All values in this figure are represented as means (±SEM). * p<0.05, ** p<0.005 (Student' t-test).
Figure 6
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, GmmWgm − 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 GmmWgm − adults, incapacitated hematopoiesis during larval stages results in severely compromised immunity that renders these flies highly susceptible as adults to bacterial infection.
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