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

Weiss BL, Wang J, Aksoy S - PLoS Biol. (2011)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Epidemiology and Public Health, Division of Epidemiology of Microbial Diseases, Yale University School of Medicine, New Haven, Connecticut, United States of America. brian.weiss@yale.edu

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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The effect of age and symbiont status on the development of tsetse'scellular immune response.(A) Number of hemocytes per µl of hemolymph in young (3 d) and mature (8d) GmmWT andGmmWgm- tsetse (n = 5 individuals from each tsetse strain at both timepoints). (B) Quantitative analysis of sessile hemocyte abundance in young andmature GmmWT and matureGmmWgm- tsetse (n = 4 individuals from each tsetse strain and age point).All tsetse strains tested were subjected to hemocoelic injection with bluefluorescent microspheres. Twelve hours post-injection, flies were dissected toreveal tsetse's heart. The left-most panel is a Brightfield image of thethree chambers that make up the dorsal vessel (DV; scale bar = 350 µm). The anterior-most chamber is indicatedwithin a white circle. The three remaining panels are close-ups of the anteriorchamber (scale bar for all 3 panels  = 80 µm),visualized by excitation with UV light (365/415 nm). Relative fluorescence pertsetse group was determined using ImageJ software. (C) The presence ofWigglesworthia affects the expression of genes involved inhemocyte differentiation in immature larval tsetse. Target gene expression inGmmWT andGmmWgm− larval instars1–3 is indicated relative to the constitutively expressed tsetseβ-tubulin gene. Quantitative measurements were performed on threebiological samples in duplicate. All values in this figure are represented asmeans (±SEM). * p<0.05, **p<0.005 (Student' t-test).
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pbio-1000619-g005: The effect of age and symbiont status on the development of tsetse'scellular immune response.(A) Number of hemocytes per µl of hemolymph in young (3 d) and mature (8d) GmmWT andGmmWgm- tsetse (n = 5 individuals from each tsetse strain at both timepoints). (B) Quantitative analysis of sessile hemocyte abundance in young andmature GmmWT and matureGmmWgm- tsetse (n = 4 individuals from each tsetse strain and age point).All tsetse strains tested were subjected to hemocoelic injection with bluefluorescent microspheres. Twelve hours post-injection, flies were dissected toreveal tsetse's heart. The left-most panel is a Brightfield image of thethree chambers that make up the dorsal vessel (DV; scale bar = 350 µm). The anterior-most chamber is indicatedwithin a white circle. The three remaining panels are close-ups of the anteriorchamber (scale bar for all 3 panels  = 80 µm),visualized by excitation with UV light (365/415 nm). Relative fluorescence pertsetse group was determined using ImageJ software. (C) The presence ofWigglesworthia affects the expression of genes involved inhemocyte differentiation in immature larval tsetse. Target gene expression inGmmWT andGmmWgm− larval instars1–3 is indicated relative to the constitutively expressed tsetseβ-tubulin gene. Quantitative measurements were performed on threebiological samples in duplicate. All values in this figure are represented asmeans (±SEM). * p<0.05, **p<0.005 (Student' t-test).

Mentions: We observed that young GmmWT were markedly moresusceptible to infection with E. coli K12 than were their maturecounterparts. Furthermore, symbiont status also altered infection outcome, as matureGmmWgm− perished followingE. coli infection while age-matched WT individuals survived.These differential infection outcomes appeared to result from disparities in cellularimmune system function between the different tsetse lines we examined. Based on theseobservations we hypothesized that the obligate mutualistWigglesworthia plays a crucial role in regulating the developmentof cellular immunity in tsetse during immature stages. To test this hypothesis wequantified the number of circulating and sessile hemocytes present in young andmature adult GmmWT and mature adultGmmWgm−. Our results indicate thata 1.4-fold increase in circulating hemocyte number occurs between day 3 and day 8 inWT tsetse, while no significant change in circulating hemocyte number was observedbetween young and mature GmmWgm− (Figure 5A). Interestingly, matureGmmWT adults harbored 3.4× more circulatinghemocytes than did mature GmmWgm− adults(Figure 5A). We also looked atsessile hemocyte abundance as a further indicator ofWigglesworthia's impact on the development of cellularimmunity in tsetse. In Drosophila, this hemocyte subtypeconcentrates in large quantities around the anterior end of the fly's dorsalvessel [29].Thus, we indirectly quantified sessile hemocyte abundance immediately adjacent to theanterior-most chamber of tsetse's dorsal vessel by measuring the fluorescentemission of microspheres that were found engulfed in this region. YoungGmmWT adults engulfed 1.2× more microspheresthan their mature counterparts and 15.7× more than matureGmmWgm− adults. Furthermore,mature WT adults engulfed 13.2× more microspheres than did age-matchedGmmWgm− adults (Figure 5B).


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

Weiss BL, Wang J, Aksoy S - PLoS Biol. (2011)

The effect of age and symbiont status on the development of tsetse'scellular immune response.(A) Number of hemocytes per µl of hemolymph in young (3 d) and mature (8d) GmmWT andGmmWgm- tsetse (n = 5 individuals from each tsetse strain at both timepoints). (B) Quantitative analysis of sessile hemocyte abundance in young andmature GmmWT and matureGmmWgm- tsetse (n = 4 individuals from each tsetse strain and age point).All tsetse strains tested were subjected to hemocoelic injection with bluefluorescent microspheres. Twelve hours post-injection, flies were dissected toreveal tsetse's heart. The left-most panel is a Brightfield image of thethree chambers that make up the dorsal vessel (DV; scale bar = 350 µm). The anterior-most chamber is indicatedwithin a white circle. The three remaining panels are close-ups of the anteriorchamber (scale bar for all 3 panels  = 80 µm),visualized by excitation with UV light (365/415 nm). Relative fluorescence pertsetse group was determined using ImageJ software. (C) The presence ofWigglesworthia affects the expression of genes involved inhemocyte differentiation in immature larval tsetse. Target gene expression inGmmWT andGmmWgm− larval instars1–3 is indicated relative to the constitutively expressed tsetseβ-tubulin gene. Quantitative measurements were performed on threebiological samples in duplicate. All values in this figure are represented asmeans (±SEM). * p<0.05, **p<0.005 (Student' t-test).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3104962&req=5

pbio-1000619-g005: The effect of age and symbiont status on the development of tsetse'scellular immune response.(A) Number of hemocytes per µl of hemolymph in young (3 d) and mature (8d) GmmWT andGmmWgm- tsetse (n = 5 individuals from each tsetse strain at both timepoints). (B) Quantitative analysis of sessile hemocyte abundance in young andmature GmmWT and matureGmmWgm- tsetse (n = 4 individuals from each tsetse strain and age point).All tsetse strains tested were subjected to hemocoelic injection with bluefluorescent microspheres. Twelve hours post-injection, flies were dissected toreveal tsetse's heart. The left-most panel is a Brightfield image of thethree chambers that make up the dorsal vessel (DV; scale bar = 350 µm). The anterior-most chamber is indicatedwithin a white circle. The three remaining panels are close-ups of the anteriorchamber (scale bar for all 3 panels  = 80 µm),visualized by excitation with UV light (365/415 nm). Relative fluorescence pertsetse group was determined using ImageJ software. (C) The presence ofWigglesworthia affects the expression of genes involved inhemocyte differentiation in immature larval tsetse. Target gene expression inGmmWT andGmmWgm− larval instars1–3 is indicated relative to the constitutively expressed tsetseβ-tubulin gene. Quantitative measurements were performed on threebiological samples in duplicate. All values in this figure are represented asmeans (±SEM). * p<0.05, **p<0.005 (Student' t-test).
Mentions: We observed that young GmmWT were markedly moresusceptible to infection with E. coli K12 than were their maturecounterparts. Furthermore, symbiont status also altered infection outcome, as matureGmmWgm− perished followingE. coli infection while age-matched WT individuals survived.These differential infection outcomes appeared to result from disparities in cellularimmune system function between the different tsetse lines we examined. Based on theseobservations we hypothesized that the obligate mutualistWigglesworthia plays a crucial role in regulating the developmentof cellular immunity in tsetse during immature stages. To test this hypothesis wequantified the number of circulating and sessile hemocytes present in young andmature adult GmmWT and mature adultGmmWgm−. Our results indicate thata 1.4-fold increase in circulating hemocyte number occurs between day 3 and day 8 inWT tsetse, while no significant change in circulating hemocyte number was observedbetween young and mature GmmWgm− (Figure 5A). Interestingly, matureGmmWT adults harbored 3.4× more circulatinghemocytes than did mature GmmWgm− adults(Figure 5A). We also looked atsessile hemocyte abundance as a further indicator ofWigglesworthia's impact on the development of cellularimmunity in tsetse. In Drosophila, this hemocyte subtypeconcentrates in large quantities around the anterior end of the fly's dorsalvessel [29].Thus, we indirectly quantified sessile hemocyte abundance immediately adjacent to theanterior-most chamber of tsetse's dorsal vessel by measuring the fluorescentemission of microspheres that were found engulfed in this region. YoungGmmWT adults engulfed 1.2× more microspheresthan their mature counterparts and 15.7× more than matureGmmWgm− adults. Furthermore,mature WT adults engulfed 13.2× more microspheres than did age-matchedGmmWgm− adults (Figure 5B).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Epidemiology and Public Health, Division of Epidemiology of Microbial Diseases, Yale University School of Medicine, New Haven, Connecticut, United States of America. brian.weiss@yale.edu

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.

Show MeSH
Related in: MedlinePlus