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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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Hemocyte-mediated phagocytosis is a critical component of tsetse'simmune response.(A) 8-d-old GmmWT were subjected to septicinfection with GFP-expressing E. coli K12. Twelve hourspost-infection hemolymph was collected, fixed on glass slides using 2%paraformaldehyde, and microscopically examined for the presence ofhemocyte-engulfed bacterial cells. Scale bar  = 10µm. (B) The process of hemocyte-mediated phagocytosis in tsetse wasblocked by micro-injecting polystyrene beads into the hemocoel of 8-d-old WTindividuals. In consecutive 12 h intervals following bead injection, flies wereinfected with GFP-expressing E. coli K12 and then hemolymphwas collected and fixed as described above. Hemocytes appear to have engulfedthe beads, thus prohibiting the subsequent uptake of bacterial cells. The insetin each panel shows a higher magnification image of one hemocyte, which isidentified by a white triangle in the left-most panel. Scale bar = 20 μm. (C) Tsetse flies that harbor hemocytesincapable of engulfing E. coli are susceptible to septicinfection with this bacterium while their wild-type counterparts are not. Thesusceptible phenotype is exhibited regardless of whether phagocytosis-inhibitedtsetse were inoculated with 103 or 106 CFU of E.coli. Beads alone had no effect on tsetse mortality. No significantdifference existed in survival outcome between matureGmmWT phagocytosis inhibited flies infected with103 versus 106 CFU of E. coli(p  = 0.47, log-rank analysis).Furthermore, no significant difference was present between matureGmmWgm− flies withuninhibited hemocytes (Figure1A, bottom panel) and mature GmmWTphagocytosis inhibited flies (p  = 0.11)infected with 106 CFU of E. coli.
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pbio-1000619-g003: Hemocyte-mediated phagocytosis is a critical component of tsetse'simmune response.(A) 8-d-old GmmWT were subjected to septicinfection with GFP-expressing E. coli K12. Twelve hourspost-infection hemolymph was collected, fixed on glass slides using 2%paraformaldehyde, and microscopically examined for the presence ofhemocyte-engulfed bacterial cells. Scale bar  = 10µm. (B) The process of hemocyte-mediated phagocytosis in tsetse wasblocked by micro-injecting polystyrene beads into the hemocoel of 8-d-old WTindividuals. In consecutive 12 h intervals following bead injection, flies wereinfected with GFP-expressing E. coli K12 and then hemolymphwas collected and fixed as described above. Hemocytes appear to have engulfedthe beads, thus prohibiting the subsequent uptake of bacterial cells. The insetin each panel shows a higher magnification image of one hemocyte, which isidentified by a white triangle in the left-most panel. Scale bar = 20 μm. (C) Tsetse flies that harbor hemocytesincapable of engulfing E. coli are susceptible to septicinfection with this bacterium while their wild-type counterparts are not. Thesusceptible phenotype is exhibited regardless of whether phagocytosis-inhibitedtsetse were inoculated with 103 or 106 CFU of E.coli. Beads alone had no effect on tsetse mortality. No significantdifference existed in survival outcome between matureGmmWT phagocytosis inhibited flies infected with103 versus 106 CFU of E. coli(p  = 0.47, log-rank analysis).Furthermore, no significant difference was present between matureGmmWgm− flies withuninhibited hemocytes (Figure1A, bottom panel) and mature GmmWTphagocytosis inhibited flies (p  = 0.11)infected with 106 CFU of E. coli.

Mentions: We investigated the role hemocytes might play in determining the susceptiblephenotype we observed in tsetse following infection with E. coli. Weinfected mature GmmWT individuals with GFP-expressingE. coli and were able to observe that hemocytes had engulfed alarge number of the introduced cells by 12 hpi (Figure 3A). We next inhibited phagocytosis byintroducing blue fluorescent microspheres directly into tsetse's hemocoel and 12h later infected the bead-treated individuals with GFP-expressing E.coli. Microscopic inspection of hemocytes harvested 12 hpi withE. coli revealed the presence of internalized microspheres andthe absence of engulfed E. coli. This observation indicated that wewere successful in blocking hemocyte phagocytosis (Figure 3B). We subsequently maintained ourmicrosphere-injected tsetse for 2 wk with the intention of determining the impact ofimpaired phagocytosis on host survival outcome. MatureGmmWT flies exhibiting impaired phagocytosis werehighly susceptible to infection with both 1×103 and1×106E. coli K12. In fact, by day 12 post-infection, all of these flieshad perished regardless of the initial dose used for infection (Figure 3C). This observation contrasts starkly withinfection outcome in mature GmmWT that exhibit normallyfunctioning hemocytes (Figure 1A,middle graph). Our results suggest that defects in phagocytosis severely compromisethe ability of these tsetse flies to overcome bacterial infection.


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

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

Hemocyte-mediated phagocytosis is a critical component of tsetse'simmune response.(A) 8-d-old GmmWT were subjected to septicinfection with GFP-expressing E. coli K12. Twelve hourspost-infection hemolymph was collected, fixed on glass slides using 2%paraformaldehyde, and microscopically examined for the presence ofhemocyte-engulfed bacterial cells. Scale bar  = 10µm. (B) The process of hemocyte-mediated phagocytosis in tsetse wasblocked by micro-injecting polystyrene beads into the hemocoel of 8-d-old WTindividuals. In consecutive 12 h intervals following bead injection, flies wereinfected with GFP-expressing E. coli K12 and then hemolymphwas collected and fixed as described above. Hemocytes appear to have engulfedthe beads, thus prohibiting the subsequent uptake of bacterial cells. The insetin each panel shows a higher magnification image of one hemocyte, which isidentified by a white triangle in the left-most panel. Scale bar = 20 μm. (C) Tsetse flies that harbor hemocytesincapable of engulfing E. coli are susceptible to septicinfection with this bacterium while their wild-type counterparts are not. Thesusceptible phenotype is exhibited regardless of whether phagocytosis-inhibitedtsetse were inoculated with 103 or 106 CFU of E.coli. Beads alone had no effect on tsetse mortality. No significantdifference existed in survival outcome between matureGmmWT phagocytosis inhibited flies infected with103 versus 106 CFU of E. coli(p  = 0.47, log-rank analysis).Furthermore, no significant difference was present between matureGmmWgm− flies withuninhibited hemocytes (Figure1A, bottom panel) and mature GmmWTphagocytosis inhibited flies (p  = 0.11)infected with 106 CFU of E. coli.
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Related In: Results  -  Collection

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pbio-1000619-g003: Hemocyte-mediated phagocytosis is a critical component of tsetse'simmune response.(A) 8-d-old GmmWT were subjected to septicinfection with GFP-expressing E. coli K12. Twelve hourspost-infection hemolymph was collected, fixed on glass slides using 2%paraformaldehyde, and microscopically examined for the presence ofhemocyte-engulfed bacterial cells. Scale bar  = 10µm. (B) The process of hemocyte-mediated phagocytosis in tsetse wasblocked by micro-injecting polystyrene beads into the hemocoel of 8-d-old WTindividuals. In consecutive 12 h intervals following bead injection, flies wereinfected with GFP-expressing E. coli K12 and then hemolymphwas collected and fixed as described above. Hemocytes appear to have engulfedthe beads, thus prohibiting the subsequent uptake of bacterial cells. The insetin each panel shows a higher magnification image of one hemocyte, which isidentified by a white triangle in the left-most panel. Scale bar = 20 μm. (C) Tsetse flies that harbor hemocytesincapable of engulfing E. coli are susceptible to septicinfection with this bacterium while their wild-type counterparts are not. Thesusceptible phenotype is exhibited regardless of whether phagocytosis-inhibitedtsetse were inoculated with 103 or 106 CFU of E.coli. Beads alone had no effect on tsetse mortality. No significantdifference existed in survival outcome between matureGmmWT phagocytosis inhibited flies infected with103 versus 106 CFU of E. coli(p  = 0.47, log-rank analysis).Furthermore, no significant difference was present between matureGmmWgm− flies withuninhibited hemocytes (Figure1A, bottom panel) and mature GmmWTphagocytosis inhibited flies (p  = 0.11)infected with 106 CFU of E. coli.
Mentions: We investigated the role hemocytes might play in determining the susceptiblephenotype we observed in tsetse following infection with E. coli. Weinfected mature GmmWT individuals with GFP-expressingE. coli and were able to observe that hemocytes had engulfed alarge number of the introduced cells by 12 hpi (Figure 3A). We next inhibited phagocytosis byintroducing blue fluorescent microspheres directly into tsetse's hemocoel and 12h later infected the bead-treated individuals with GFP-expressing E.coli. Microscopic inspection of hemocytes harvested 12 hpi withE. coli revealed the presence of internalized microspheres andthe absence of engulfed E. coli. This observation indicated that wewere successful in blocking hemocyte phagocytosis (Figure 3B). We subsequently maintained ourmicrosphere-injected tsetse for 2 wk with the intention of determining the impact ofimpaired phagocytosis on host survival outcome. MatureGmmWT flies exhibiting impaired phagocytosis werehighly susceptible to infection with both 1×103 and1×106E. coli K12. In fact, by day 12 post-infection, all of these flieshad perished regardless of the initial dose used for infection (Figure 3C). This observation contrasts starkly withinfection outcome in mature GmmWT that exhibit normallyfunctioning hemocytes (Figure 1A,middle graph). Our results suggest that defects in phagocytosis severely compromisethe ability of these tsetse flies to overcome bacterial infection.

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