Yineng Wu

The Obligate Mutualist Wigglesworthia glossinidia Influences Reproduction, Digestion, and Immunity Processes of Its Host, the Tsetse Fly

ABSTRACT Tsetse flies (Diptera: Glossinidae) are vectors for trypanosome parasites, the agents of the deadly sleeping sickness disease in Africa. Tsetse also harbor two maternally transmitted enteric mutualist endosymbionts: the primary intracellular obligate Wigglesworthia glossinidia and the secondary commensal Sodalis glossinidius. Both endosymbionts are transmitted to the intrauterine progeny through the milk gland secretions of the viviparous female. We administered various antibiotics either continuously by per os supplementation of the host blood meal diet or discretely by hemocoelic injections into fertile females in an effort to selectively eliminate the symbionts to study their individual functions. A symbiont-specific PCR amplification assay and fluorescence in situ hybridization analysis were used to evaluate symbiont infection outcomes. Tetracycline and rifampin treatments eliminated all tsetse symbionts but reduced the fecundity of the treated females. Ampicillin treatments did not affect the intracellular Wigglesworthia localized in the bacteriome organ and retained female fecundity. The resulting progeny of ampicillin-treated females, however, lacked Wigglesworthia but still harbored the commensal Sodalis. Our results confirm the presence of two physiologically distinct Wigglesworthia populations: the bacteriome-localized Wigglesworthia involved with nutritional symbiosis and free-living Wigglesworthia in the milk gland organ responsible for maternal transmission to the progeny. We evaluated the reproductive fitness, longevity, digestion, and vectorial competence of flies that were devoid of Wigglesworthia. The absence of Wigglesworthia completely abolished the fertility of females but not that of males. Both the male and female Wigglesworthia-free adult progeny displayed longevity costs and were significantly compromised in their blood meal digestion ability. Finally, while the vectorial competence of the young newly hatched adults without Wigglesworthia was comparable to that of their wild-type counterparts, older flies displayed higher susceptibility to trypanosome infections, indicating a role for the mutualistic symbiosis in host immunobiology. The ability to rear adult tsetse that lack the obligate Wigglesworthia endosymbionts will now enable functional investigations into this ancient symbiosis.

Interactions between mutualist Wigglesworthia and tsetse peptidoglycan recognition protein (PGRP-LB) influence trypanosome transmission

Tsetse flies, the sole vectors of African trypanosomes, have coevolved with mutualistic endosymbiont Wigglesworthia glossinidiae. Elimination of Wigglesworthia renders tsetse sterile and increases their trypanosome infection susceptibility. We show that a tsetse peptidoglycan recognition protein (PGRP-LB) is crucial for symbiotic tolerance and trypanosome infection processes. Tsetse pgrp-lb is expressed in the Wigglesworthia-harboring organ (bacteriome) in the midgut, and its level of expression correlates with symbiont numbers. Adult tsetse cured of Wigglesworthia infections have significantly lower pgrp-lb levels than corresponding normal adults. RNA interference (RNAi)-mediated depletion of pgrp-lb results in the activation of the immune deficiency (IMD) signaling pathway and leads to the synthesis of antimicrobial peptides (AMPs), which decrease Wigglesworthia density. Depletion of pgrp-lb also increases the hosts susceptibility to trypanosome infections. Finally, parasitized adults have significantly lower pgrp-lb levels than flies, which have successfully eliminated trypanosome infections. When both PGRP-LB and IMD immunity pathway functions are blocked, flies become unusually susceptible to parasitism. Based on the presence of conserved amidase domains, tsetse PGRP-LB may scavenge the peptidoglycan (PGN) released by Wigglesworthia and prevent the activation of symbiont-damaging host immune responses. In addition, tsetse PGRP-LB may have an anti-protozoal activity that confers parasite resistance. The symbiotic adaptations and the limited exposure of tsetse to foreign microbes may have led to the considerable differences in pgrp-lb expression and regulation noted in tsetse from that of closely related Drosophila. A dynamic interplay between Wigglesworthia and host immunity apparently is influential in tsetses ability to transmit trypanosomes.

Trypanosome Infection Establishment in the Tsetse Fly Gut Is Influenced by Microbiome-Regulated Host Immune Barriers

Tsetse flies (Glossina spp.) vector pathogenic African trypanosomes, which cause sleeping sickness in humans and nagana in domesticated animals. Additionally, tsetse harbors 3 maternally transmitted endosymbiotic bacteria that modulate their hosts physiology. Tsetse is highly resistant to infection with trypanosomes, and this phenotype depends on multiple physiological factors at the time of challenge. These factors include host age, density of maternally-derived trypanolytic effector molecules present in the gut, and symbiont status during development. In this study, we investigated the molecular mechanisms that result in tsetses resistance to trypanosomes. We found that following parasite challenge, young susceptible tsetse present a highly attenuated immune response. In contrast, mature refractory flies express higher levels of genes associated with humoral (attacin and pgrp-lb) and epithelial (inducible nitric oxide synthase and dual oxidase) immunity. Additionally, we discovered that tsetse must harbor its endogenous microbiome during intrauterine larval development in order to present a parasite refractory phenotype during adulthood. Interestingly, mature aposymbiotic flies (Gmm Apo) present a strong immune response earlier in the infection process than do WT flies that harbor symbiotic bacteria throughout their entire lifecycle. However, this early response fails to confer significant resistance to trypanosomes. Gmm Apo adults present a structurally compromised peritrophic matrix (PM), which lines the fly midgut and serves as a physical barrier that separates luminal contents from immune responsive epithelial cells. We propose that the early immune response we observe in Gmm Apo flies following parasite challenge results from the premature exposure of gut epithelia to parasite-derived immunogens in the absence of a robust PM. Thus, tsetses PM appears to regulate the timing of host immune induction following parasite challenge. Our results document a novel finding, which is the existence of a positive correlation between tsetses larval microbiome and the integrity of the emerging adult PM gut immune barrier.

An insect symbiosis is influenced by bacterium-specific polymorphisms in outer-membrane protein A

Beneficial bacterial symbioses are ubiquitous in nature. However, the functional and molecular basis of host tolerance to resident symbiotic microbes, in contrast to resistance to closely related bacteria that are recognized as foreign, remain largely unknown. We used the tsetse fly (Glossina morsitans), which depends on symbiotic flora for fecundity and has limited exposure to foreign microbes, to investigate the tolerance phenomenon exhibited during symbiosis. We examined the potential role of bacterium-specific polymorphisms present in the major bacterial surface protein, outer-membrane protein A (OmpA), on host infection outcomes. Tsetse were successfully superinfected with their mutualistic facultative symbiont, Sodalis glossinidius, whereas infections with Escherichia coli K12 were lethal. In contrast, tsetse were resistant to an E. coli OmpA mutant strain, whereas recombinant Sodalis expressing E. coli OmpA became pathogenic. Profiling of tsetse immunity-related gene expression incriminated peptidoglycan recognition protein (pgrp)-lb as a determinant of the infection outcomes we observed. RNAi-induced knockdown of tsetse pgrp-lb significantly reduced host mortality after infection with otherwise lethal E. coli K12. Our results show that polymorphisms in the exposed loop domains of OmpA represent a microbial adaptation that mediates host tolerance of endogenous symbiotic bacteria.

OmpA-Mediated Biofilm Formation Is Essential for the Commensal Bacterium Sodalis glossinidius To Colonize the Tsetse Fly Gut

ABSTRACT Many bacteria successfully colonize animals by forming protective biofilms. Molecular processes that underlie the formation and function of biofilms in pathogenic bacteria are well characterized. In contrast, the relationship between biofilms and host colonization by symbiotic bacteria is less well understood. Tsetse flies (Glossina spp.) house 3 maternally transmitted symbionts, one of which is a commensal (Sodalis glossinidius) found in several host tissues, including the gut. We determined that Sodalis forms biofilms in the tsetse gut and that this process is influenced by the Sodalis outer membrane protein A (OmpA). Mutant Sodalis strains that do not produce OmpA (Sodalis ΔOmpA mutants) fail to form biofilms in vitro and are unable to colonize the tsetse gut unless endogenous symbiotic bacteria are present. Our data indicate that in the absence of biofilms, Sodalis ΔOmpA mutant cells are exposed to and eliminated by tsetses innate immune system, suggesting that biofilms help Sodalis evade the host immune system. Tsetse is the sole vector of pathogenic African trypanosomes, which also reside in the fly gut. Acquiring a better understanding of the dynamics that promote Sodalis colonization of the tsetse gut may enhance the development of novel disease control strategies.

The Peritrophic Matrix Mediates Differential Infection Outcomes in the Tsetse Fly Gut following Challenge with Commensal, Pathogenic, and Parasitic Microbes

The insect gut is lined by a protective, chitinous peritrophic matrix (PM) that separates immunoreactive epithelial cells from microbes present within the luminal contents. Tsetse flies (Glossina spp.) imbibe vertebrate blood exclusively and can be exposed to foreign microorganisms during the feeding process. We used RNA interference–based reverse genetics to inhibit the production of a structurally robust PM and then observed how this procedure impacted infection outcomes after per os challenge with exogenous bacteria (Enterobacter sp. and Serratia marcescens strain Db11) and parasitic African trypanosomes. Enterobacter and Serratia proliferation was impeded in tsetse that lacked an intact PM because these flies expressed the antimicrobial peptide gene, attacin, earlier in the infection process than did their counterparts that housed a fully developed PM. After challenge with trypanosomes, attacin expression was latent in tsetse that lacked an intact PM, and these flies were thus highly susceptible to parasite infection. Our results suggest that immunodeficiency signaling pathway effectors, as opposed to reactive oxygen intermediates, serve as the first line of defense in tsetse’s gut after the ingestion of exogenous microorganisms. Furthermore, tsetse’s PM is not a physical impediment to infection establishment, but instead serves as a barrier that regulates the fly’s ability to immunologically detect and respond to the presence of these microbes. Collectively, our findings indicate that effective insect antimicrobial responses depend largely upon the coordination of multiple host and microbe-specific developmental factors.

Implications of microfauna-host interactions for trypanosome transmission dynamics in Glossina fuscipes fuscipes in Uganda.

ABSTRACT Tsetse flies (Diptera: Glossinidae) are vectors for African trypanosomes (Euglenozoa: kinetoplastida), protozoan parasites that cause African trypanosomiasis in humans (HAT) and nagana in livestock. In addition to trypanosomes, two symbiotic bacteria (Wigglesworthia glossinidia and Sodalis glossinidius) and two parasitic microbes, Wolbachia and a salivary gland hypertrophy virus (SGHV), have been described in tsetse. Here we determined the prevalence of and coinfection dynamics between Wolbachia, trypanosomes, and SGHV in Glossina fuscipes fuscipes in Uganda over a large geographical scale spanning the range of host genetic and spatial diversity. Using a multivariate analysis approach, we uncovered complex coinfection dynamics between the pathogens and statistically significant associations between host genetic groups and pathogen prevalence. It is important to note that these coinfection dynamics and associations with the host were not apparent by univariate analysis. These associations between host genotype and pathogen are particularly evident for Wolbachia and SGHV where host groups are inversely correlated for Wolbachia and SGHV prevalence. On the other hand, trypanosome infection prevalence is more complex and covaries with the presence of the other two pathogens, highlighting the importance of examining multiple pathogens simultaneously before making generalizations about infection and spatial patterns. It is imperative to note that these novel findings would have been missed if we had employed the standard univariate analysis used in previous studies. Our results are discussed in the context of disease epidemiology and vector control.

Analysis of Multiple Tsetse Fly Populations in Uganda Reveals Limited Diversity and Species-Specific Gut Microbiota

ABSTRACT The invertebrate microbiome contributes to multiple aspects of host physiology, including nutrient supplementation and immune maturation processes. We identified and compared gut microbial abundance and diversity in natural tsetse flies from Uganda using five genetically distinct populations of Glossina fuscipes fuscipes and multiple tsetse species (Glossina morsitans morsitans, G. f. fuscipes, and Glossina pallidipes) that occur in sympatry in one location. We used multiple approaches, including deep sequencing of the V4 hypervariable region of the 16S rRNA gene, 16S rRNA gene clone libraries, and bacterium-specific quantitative PCR (qPCR), to investigate the levels and patterns of gut microbial diversity from a total of 151 individuals. Our results show extremely limited diversity in field flies of different tsetse species. The obligate endosymbiont Wigglesworthia dominated all samples (>99%), but we also observed wide prevalence of low-density Sodalis (tsetses commensal endosymbiont) infections (<0.05%). There were also several individuals (22%) with high Sodalis density, which also carried coinfections with Serratia. Albeit in low density, we noted differences in microbiota composition among the genetically distinct G. f. fuscipes flies and between different sympatric species. Interestingly, Wigglesworthia density varied in different species (104 to 106 normalized genomes), with G. f. fuscipes having the highest levels. We describe the factors that may be responsible for the reduced diversity of tsetses gut microbiota compared to those of other insects. Additionally, we discuss the implications of Wigglesworthia and Sodalis density variations as they relate to trypanosome transmission dynamics and vector competence variations associated with different tsetse species.

Transcriptome analysis of reproductive tissue and intrauterine developmental stages of the tsetse fly (Glossina morsitans morsitans)

BackgroundTsetse flies, vectors of African trypanosomes, undergo viviparous reproduction (the deposition of live offspring). This reproductive strategy results in a large maternal investment and the deposition of a small number of progeny during a females lifespan. The reproductive biology of tsetse has been studied on a physiological level; however the molecular analysis of tsetse reproduction requires deeper investigation. To build a foundation from which to base molecular studies of tsetse reproduction, a cDNA library was generated from female tsetse (Glossina morsitans morsitans) reproductive tissues and the intrauterine developmental stages. 3438 expressed sequence tags were sequenced and analyzed.ResultsAnalysis of a nonredundant catalogue of 1391 contigs resulted in 520 predicted proteins. 475 of these proteins were full length. We predict that 412 of these represent cytoplasmic proteins while 57 are secreted. Comparison of these proteins with other tissue specific tsetse cDNA libraries (salivary gland, fat body/milk gland, and midgut) identified 51 that are unique to the reproductive/immature cDNA library. 11 unique proteins were homologus to uncharacterized putative proteins within the NR database suggesting the identification of novel genes associated with reproductive functions in other insects (hypothetical conserved). The analysis also yielded seven putative proteins without significant homology to sequences present in the public database (unknown genes). These proteins may represent unique functions associated with tsetses viviparous reproductive cycle. RT-PCR analysis of hypothetical conserved and unknown contigs was performed to determine basic tissue and stage specificity of the expression of these genes.ConclusionThis paper identifies 51 putative proteins specific to a tsetse reproductive/immature EST library. 11 of these proteins correspond to hypothetical conserved genes and 7 proteins are tsetse specific.

Symbiont-induced odorant binding proteins mediate insect host hematopoiesis

Symbiotic bacteria assist in maintaining homeostasis of the animal immune system. However, the molecular mechanisms that underlie symbiont-mediated host immunity are largely unknown. Tsetse flies (Glossina spp.) house maternally transmitted symbionts that regulate the development and function of their host’s immune system. Herein we demonstrate that the obligate mutualist, Wigglesworthia, up-regulates expression of odorant binding protein six in the gut of intrauterine tsetse larvae. This process is necessary and sufficient to induce systemic expression of the hematopoietic RUNX transcription factor lozenge and the subsequent production of crystal cells, which actuate the melanotic immune response in adult tsetse. Larval Drosophila’s indigenous microbiota, which is acquired from the environment, regulates an orthologous hematopoietic pathway in their host. These findings provide insight into the molecular mechanisms that underlie enteric symbiont-stimulated systemic immune system development, and indicate that these processes are evolutionarily conserved despite the divergent nature of host-symbiont interactions in these model systems. DOI: http://dx.doi.org/10.7554/eLife.19535.001