The Tsetse Fly Obligate Mutualist Wigglesworthia morsitans Alters Gene Expression and Population Density via Exogenous Nutrient Provisioning

Abstract

The obligate mutualist Wigglesworthia morsitans provisions nutrients to tsetse flies. The symbiont's response to thiamine (B₁) supplementation of blood meals, specifically towards the regulation of thiamine biosynthesis and population density, is described. Despite an ancient symbiosis and associated genome tailoring, Wigglesworthia responds to nutrient availability, potentially accommodating a decreased need.

TEXT

The basis of many microbial symbioses, particularly those within a host, involves nutrient provisioning (29). The spatial cooccurrence of host-associated microbes has been demonstrated to involve several levels of intimacy, including the complementation of biosynthetic capabilities (24, 25, 65). An example of such metabolic interdependency is the provisioning of vitamins by one symbiont species, whereby a second produces essential amino acids, as described in insects within the Auchenorrhyncha suborder, such as cicadas and sharpshooters (24, 65), that subsist solely on a diet consisting of plant xylem. An even more extensive level of genetic complementation is exemplified through the integration of gene products from different symbiont species within a single metabolic pathway used in either cross-feeding (8) or the production of nutrients (20, 26). For example, within the mealybug, the synthesis of the essential amino acid phenylalanine requires metabolic cooperation between two different bacterial symbiont species (26). A similar partnership has also been described for tryptophan production within the cedar aphid (20). Additionally, precursors provided by the host may regulate the biosynthetic capacity of their microbial partner, ultimately influencing the amount of nutrients produced and released (22, 40, 51). Natural selection favoring metabolic integration may be a mechanism by which species avoid antagonism within their host while also optimizing energy efficiency, particularly if other essential products are provided by each partner.

The Black Queen Hypothesis (BQH) (30) highlights requisites for the evolution of cooperation between species despite selection generally favoring selfishness. The BQH states that the foundation of cooperative community evolution may involve the production of a leaky product by one species, inadvertently providing a public resource, followed by relaxed selection on these biosynthetic pathways within the genome of a beneficiary, thus driving interspecies dependency. Although not explicit to the BQH model, an extension of the hypothesis, stating that these dependencies can favor the development of even tighter associations, such as those of obligate coevolved partnerships exhibiting genomic signatures of cooperation involving the complementary loss of shared diffusible functions, has also been proposed (44).

The tsetse fly (Diptera: Glossinidae) provides an applicable symbiosis model to study microbial interactions and the evolution of mutualism. The tsetse microbial community is composed predominantly of two Gammaproteobacteria, an anciently associated obligate mutualist, Wigglesworthia species (2), and a more recently established commensal, Sodalis glossinidius (12). Tsetse flies are of significant medical and socioeconomic importance as the obligate vectors of parasitic African trypanosomes. A unique feature of tsetse biology is their reproductive strategy, referred to as adenotrophic viviparity, in which the majority of larval development occurs in utero. Nutritious lipids and proteins are provisioned to the developing larva through female accessory glands known as milk glands in a mechanism reminiscent of mammalian lactation (3, 7). In addition, Wigglesworthia and Sodalis are vertically transmitted through these milk gland secretions (4, 21). The strict blood-feeding lifestyle of tsetse flies, coupled with the relatively sterile intrauterine development of larvae, is believed to contribute to the retention of a simple community within tsetse flies (50).

Tsetse symbionts have been shown to impact host biology and have undergone genome adaptations resulting from host association (1, 42, 50). Both annotated Wigglesworthia genomes (Wigglesworthia morsitans, isolated from Glossina morsitans [42], and Wigglesworthia brevipalpis, isolated from Glossina brevipalpis [1]) have reduced sizes (∼0.7 Mb) due to significant population bottlenecks that occur during vertical transmission, contributing to high levels of genetic drift as well as the relaxed selection and purging of loci no longer necessary due to an obligate host association (61) dating back 50 million to 80 million years (9). The loss of the Wigglesworthia association results in significant detriment to tsetse flies, notably the reduction in reproductive output within females (32, 38) that can only be partially restored upon provisioning B vitamins to the blood diet (32, 33). Wigglesworthia has also been shown to be essential for symbiont-based maturation of host immunity (53, 58–60). Larvae that lack this symbiont are immunologically compromised as adults, with low numbers of hemocytes compared to that for age-matched controls (60). Intracellular Wigglesworthia strains are housed within bacteriocyte cells localized to the bacteriome organ in the tsetse anterior midgut. While Sodalis has a wider tissue tropism (5, 10), it is harbored primarily within the midgut (10). Relative to Wigglesworthia, the Sodalis genome is greater in size (∼4.2 Mb), yet it shows massive decay with a high number of pseudogenes (6, 50). Functional contributions toward tsetse biology by Sodalis are much less understood.

Wigglesworthia and Sodalis exhibit parallel growth patterns through tsetse host development (43), supporting coordinated activities. Comparative genomic analyses reveal that the majority of Wigglesworthia genes (∼90%) have homologs within the Sodalis genome. An exception is in thiamine (vitamin B₁) biosynthetic capability, which appears to be exclusive to W. morsitans (42, 46) and not possible by Sodalis (6). A recent study demonstrates significant transcriptional regulation of the thiamine biosynthesis locus thiC by W. morsitans through tsetse development (42). Examples of fine-tuned transcriptional regulation, particularly at the single-locus level, that suggest functional and adaptive responses are lacking in other obligate symbionts, such as Buchnera in aphids (28, 41, 62, 63) and Blochmannia in ants (48).

When grown in minimal medium with or without the presence of thiamine derivatives, Sodalis proliferation was shown to require this vitamin, specifically in the form of thiamine monophosphate (TMP) (46). TMP, a physiologically active thiamine derivative, is capable of being produced by W. morsitans. Moreover, intracellular invasion and multiplication, essential features of Sodalis persistence within tsetse flies (13), are also impacted by the availability of exogenous TMP (46). To complement a thiamine biosynthesis deficiency, TMP may be imported by Sodalis through a concentration-dependent thiamine ABC transporter (tbpAthiPQ) (46). Within tsetse flies, the expression of Sodalis tbpA, the thiamine binding protein component of the ABC transporter, was inversely correlated with TMP concentrations, similar to homologs of free-living relatives (55). Furthermore, genetic manipulation aimed at the disruption of the Sodalis tbpA locus has proven unsuccessful, suggesting a lethal phenotype (R. V. M. Rio, unpublished data). In addition, tbpA transcription exhibits developmental regulation relative to the tsetse life cycle (46), with the highest expression occurring at the conclusion of adult metamorphosis when nutrient supplies are low. Metamorphosis, particularly with holometabolous insects, is a metabolically expensive period during development when adult morphological features are generated without the intake of nutrients. Concordantly, the expression of W. morsitans thiC was shown to be highest at this stage in host development (42), potentially indicating this symbiont's response to accommodate a low-nutrient environment.

Here, we aim to further understand the dynamics of this symbiont nutrient-provisioning role by examining whether W. morsitans remains capable of responding to a lower functional necessity despite a drastically reduced genome and ancient host habitation. This paper details the effects of exogenous vitamin administration of the tsetse blood meal on W. morsitans transcriptional regulation and population proliferation through the use of gene expression analyses, quantitative PCR (qPCR), and cell viability assays.

W. morsitans alters gene expression with TMP supplementation.

The obligate mutualism of W. morsitans currently hinders isolation in pure culture and downstream applications such as genetic manipulation and subsequent host recolonization. Therefore, to circumvent this barrier, we supplemented tsetse blood meals with TMP, the thiamine derivative putatively synthesized by W. morsitans and previously demonstrated to affect Sodalis proliferation and insect cell invasion (46). Male and virgin female Glossina morsitans morsitans flies were maintained in the insectary at the Department of Biology at West Virginia University on a 12-h-light/12-h-dark schedule at 24 ± 1°C under 50 to 55% relative humidity. Flies were fed defibrinated bovine blood (Hemostat, Dixon CA) supplemented with 0 to 500 μM TMP every 48 h using an artificial membrane feeding system (27). No significant differences in tsetse fly survival, with three independent trials performed, were observed between treatment and control groups for the duration of the study period (log rank test; P = 0.43 for males; P = 0.69 for females).

Thiamine biosynthesis involves the condensation of thiazole and pyrimidine moieties (18). To assess the expression of W. morsitans loci involved in thiamine biosynthesis in response to exogenous TMP administration, we examined the thiamine biosynthesis gene thiC, involved in synthesis of the pyrimidine moiety, and thiI, involved in the formation of the thiazole moiety (18). At 4 weeks after the initial blood meal, semiquantitative reverse transcription (RT)-PCR was performed to assess the expression of thiC and thiI. Tsetse bacteriomes were dissected from each treatment group, and RNA was isolated following the TRIzol protocol (Invitrogen, Carlsbad, CA), verified free of DNA contamination, and used for first-strand cDNA synthesis with Superscript II reverse transcriptase (Invitrogen) and a 3′ end gene primer cocktail of WgthiCrev (5′-TGC AGC TCC AAT TCC TGA AGT-3′), WgthiIrev (5′-TCC TTT TTG GTA TAA ATA TAT CGC TTG-3′), and WggapArev (5′-TTG CAT GAA TTG CCC ATC TA-3′). The cDNA was then used as the template for second-strand synthesis using PCR with WgthiCfor (5′-GAG ATG GTT TGA GAC CTG GAT C-3′; T_a [annealing temperature] = 51°C; 45 cycles; amplicon = 272 bp) and WgthiCrev, WgthiIfor (5′-CGC TGA AAT ACC ATA TTT TCA AGA-3′; T_a = 55°C; 45 cycles; amplicon = 253 bp) and WgthiIrev, and WggapAfor (5′-GCA CCT CCA CAT GAC AAC AC-3′; T_a = 55°C; 45 cycles; amplicon = 216 bp) and WggapArev for primer sets. W. morsitans gapA served both to validate RNA integrity and as a loading control.

Semiquantitative RT-PCR results demonstrate that as the supplemented TMP concentration increases, W. morsitans thiC expression correspondingly decreases (Fig. 1A), a transcriptional profile similar to what has been described in free-living bacteria (57). The decrease in thiC expression was more prominent in tsetse males, with reduction first observed with 50 μM TMP, in contrast to 500 μM in females, a finding which may be indicative of the greater need for Wigglesworthia TMP provisioning within females and the insufficiency of lower TMP concentrations to completely fulfill this demand. In support, the removal of W. morsitans has previously been shown to result in female sterility, which can be partially restored upon the provisioning of B vitamins (33). Tsetse female reproductive biology is associated with significant energy demands necessary for oogenesis and other aspects of obligate viviparity (i.e., live birth). In contrast to the dynamics associated with thiC expression, no changes in thiI expression in the same individual virgin female samples were observed in response to TMP supplementation (Fig. 1B). A similar lack of transcriptional regulation of thiI by exogenous thiamine has also been observed with Salmonella enterica serovar Typhimurium (56), possibly due to its bifunctional role. In addition to its role in thiazole synthesis, ThiI is also involved in 4-thiouridine modification of tRNA (31). Although tRNA modification domains have been lost from Wigglesworthia ThiI, the transcription of thiI may still be conserved in an unregulated manner. In support, the transcription of W. morsitans thiI has also been observed in the teneral (i.e., newly emerged) tsetse adult stage (46).

Fig 1.

Expression of the W. morsitans thiamine biosynthesis genes thiC and thiI with exogenous TMP supplementation. Semiquantitative RT-PCR analyses of W. morsitans thiC (A) and thiI (B) expression in 4-week-old adults maintained with or without TMP supplementation. The constitutively expressed W. morsitans gapA was used to verify RNA integrity and as a loading control. At least 3 individual bacteriomes were examined per treatment group.

The ThiI protein is composed of three structural motifs, a THUMP domain, an adenylation domain, and a C-terminal rhodanese-like domain (54). It has recently been demonstrated that the C-terminal rhodanese domain of ThiI is sufficient for thiazole synthesis (23). Interestingly, W. morsitans thiI is truncated (∼260 nucleotides [nt]) and had previously been classified as a pseudogene (42). Alignment of W. morsitans (genome coordinates 670466 to 670200) and W. brevipalpis (genome coordinates 172250 to 172543) ThiI amino acid sequences with the rhodanese domains from Escherichia coli K-12 (NCBI accession no. YP_001729329) demonstrates 73.3% and 72.8% similarity levels, respectively, indicating the retention of the sole domain required for thiamine biosynthesis. In further support of their functional conservation, both annotated Wigglesworthia genomes contain the critical Arg414 and Cys456 residues in their rhodanese domains (39).

Symbiont population density is impacted by exogenous TMP supplementation.

The exogenous administration of TMP in tsetse blood meals for 2 weeks has previously been associated with a decrease in W. morsitans population density within female hosts, yet the trend was not significant (46). Therefore, we aimed to explore the effects on cell density by maintaining tsetse on control or 500 μM TMP-supplemented blood meals for a greater temporal period to determine if there is a response observed at the highest TMP concentration previously used to examine tsetse symbiont responses (46). Bacteriomes were dissected from 4-week-old adult tsetse flies, and DNA was isolated using the Holmes-Bonner method (17). qPCR was performed to determine W. morsitans genome numbers using the single-copy thiC gene (43) in a CFX96 real-time PCR detection system (Bio-Rad, Hercules, CA) using SsoFast EvaGreen Supermix (Bio-Rad), 4.0 μM primers (WgthiCfor and WgthiCrev), and 1 μl DNA as the template. The quantification of samples relative to standards was analyzed with Bio-Rad CFX Manager software and normalized to the single-copy G. morsitans chitinase gene (66) (NCBI accession no. AF337908; using primers GmchiF [5′-GAG ACA ACA ACT AAT TGG CAC TAC-3′] and GmchiR [5′-GCG TTC ATC GTC ATA ACC TAT CC-3′]; amplicon = 97 bp; T_a = 50.3°C) to determine W. morsitans genome numbers per host cell, as symbiotic organisms have been shown to contain multiple genomes per cell (19, 43). The assay was performed with ≥5 samples per treatment group, and triplicates were averaged for each sample. In concordance with previous studies (43, 46), our results demonstrate a significantly higher density of W. morsitans in females than in males (analysis of variance [ANOVA]; P = 0.0125; data not shown). The greater density in females further supports the significant nutrient-provisioning role of this symbiont for this sex (16, 32, 33). The density of W. morsitans within the bacteriomes of tsetse flies maintained on TMP-supplemented blood meals was significantly lower in males (Student's t test; P = 0.009) (Fig. 2) than in an age-matched control group. A similar trend in the reduction of W. morsitans density given TMP supplementation was also observed within the bacteriomes of treated females, yet it lacked statistical significance. Lower W. morsitans genome numbers, representative of symbiont population density, with TMP administration may indicate the capability to recognize and respond to a decreased functional necessity within the tsetse host, although more-probable host modulation cannot be discounted.

Fig 2.

Dietary thiamine supplementation impacts W. morsitans density. W. morsitans densities were compared in 4-week-old hosts maintained on control or 500 μM TMP-supplemented blood meals. Mean density values are shown, with error bars signifying 1 standard error of the mean (SEM). Sample sizes are provided below the treatment groups.

To further explore the trend of decreased symbiont density observed via qPCR, which quantifies the abundance of nucleic acids but does not represent cell counts or viability, a LIVE/DEAD BacLight bacterial viability assay (Invitrogen, Eugene, OR) was performed on the bacteriomes of tsetse flies maintained on control or 500 μM TMP-supplemented blood. This assay uses 2 nucleic acid stains: SYTO 9 dye stains all cells and fluoresces green, and propidium iodide (PI) enters cells with compromised membranes (i.e., dead) and emits a red fluorescence. When PI binds to nucleic acids, there is a displacement of SYTO 9 by PI and the consequential quenching of SYTO 9 by fluorescence resonance energy transfer (FRET) (47).

At 4 weeks after the initial blood meal, individual bacteriomes were dissected, placed into 1 ml 0.85% NaCl, and gently homogenized to release W. morsitans cells (43). The bacteria were then centrifuged at 4°C for 5 min at 5,000 rpm. The majority of the supernatant was removed, leaving 100 μl solution containing W. morsitans. A 1:1 combination of 3.34 mM SYTO 9 dye in dimethyl sulfoxide (DMSO) and 20 mM PI in DMSO was mixed thoroughly and maintained away from light. Subsequently, 0.3 μl of the dye mixture was mixed with the W. morsitans solution and incubated in the dark at 24°C for 15 min. A slide was then prepared with 5 μl stained bacterial suspension and visualized with fluorescence microscopy, and picture capture was performed using an Olympus FluoView FV1000 confocal laser-scanning microscope (Olympus, Tokyo, Japan). The detection channels were set to Alexa Fluor 488 (excitation, 488 nm; emission, 520 nm) to view SYTO 9 fluorescence and to PI (excitation, 543 nm; emission, 619 nm) to capture PI fluorescence. At least 3 bacteriome samples from each treatment group were examined, with ≥5 random fields of view recorded for each sample, and three independent assays were performed. The live (green) and dead (red) cells were visualized separately and as an overlay of the filters and quantified directly on microphotography (Fig. 3A). The procedure was also repeated with unstained W. morsitans or stained 0.85% NaCl lacking W. morsitans, both as negative controls, to verify that there was no auto- or background fluorescence, respectively (data not shown). A two-tailed Fisher exact test was performed using JMP 7.0 software (SAS Institute, Cary, NC) to compare the numbers of live and dead cells between treatments within each sex. Statistically significant differences were found in the numbers of dead W. morsitans cells when comparing bacteriome isolates from both treated female (the Fisher exact test; P < 0.0001) and male (the Fisher exact test; P < 0.0001) tsetse hosts with age-matched controls (Fig. 3B), with TMP-supplemented groups harboring a higher quantity of dead symbionts. The decrease in the ratio of live to dead cells within bacteriomes provides additional biological evidence that W. morsitans populations decrease in light of a lower functional demand. Evidence of altered proliferation due to lower functional necessity can be seen in other symbioses to prevent harmful effects toward the host. For example, the green hydra, Chlorohydra viridissima, actively expels its endosymbiotic algae during feeding when an alternative form of nutrition is available (14). In addition, the facultative endosymbiont of aphids, Hamiltonella defensa, harbors a toxin-encoding bacteriophage shown to be instrumental in killing developing parasitoid wasp larvae within infected aphids (35) and remains at intermediate frequencies in natural populations due to costly infection (34, 36). But upon the increased prevalence of parasitoid wasps in the host environment, selection favors the spread of these symbionts as a defense mechanism (34).

Fig 3.

Viability of W. morsitans strains isolated from the bacteriomes of 4-week-old tsetse flies maintained on TMP-supplemented blood. (A) Representative confocal fluorescence images of W. morsitans isolates stained with SYTO 9 and PI. (B) Proportions of green (live) and red (dead) cells were quantified in TMP-treated and age- and sex-matched control bacteriomes. Percentages of cells are depicted, with error bars signifying 1 SEM. A total of ≥3 individuals were analyzed per treatment, with ≥5 random frames per individual analyzed, and 3 independent assays were performed. A two-tailed Fisher exact test was performed to compare the numbers of live and dead cells between treatments. *, P < 0.0001. n, average number of cells counted per sample.

Conclusions.

These results demonstrate that although W. morsitans has been involved in symbiosis with tsetse flies for a historically significant amount of time (9) and has consequently undergone massive genome shrinkage to accommodate this lifestyle, it appears to still be capable of acclimating to changes in nutrient availability. More specifically, W. morsitans not only appears to retain thiamine biosynthetic capability, it is also able to respond to exogenous TMP administration by regulating the transcription of the thiamine biosynthetic locus thiC, used in production of the pyrimidine moiety, accordingly. Reductions in W. morsitans population density were also observed following vitamin administration, possibly either due to symbiont recognition of a decreased need or, more likely, particularly in light of the drastic genome reduction of W. morsitans, through host modulation. In support of more-probable host-mediated control, components of the tsetse immune system, notably the peptidoglycan recognition protein (PGRP-LB), have been shown to control the abundance of the W. morsitans symbiont (52, 53). In further support of host influence, modeling and experimental studies of the Buchnera and aphid symbiosis suggest that alterations in nutritional phenotypes may be directed by variation in the host's capacity to supply precursors to symbionts rather than differences in symbiont genomic capabilities (15, 22, 51). Unlike many other anciently associated obligate mutualists that are spatially segregated within bacteriocytes by a host membrane, W. morsitans lies free within the cytosol of host cells (2), perhaps enabling a greater ability to sense and respond to metabolic fluctuations within the host. Within many bacteria, thiamine biosynthesis is regulated through a riboswitch, in which the binding of an effector molecule (i.e., thiamine derivatives) causes allosteric control by feedback inhibition (64). Furthermore, within this riboswitch is a conserved thi-box nucleotide sequence, located in the 5′ untranslated leader region, which is maintained among a taxonomically wide range of organisms (37). Upon the in silico examination of these regions (Riboswitch Explorer; http://132.248.32.45/cgi-bin/ribex.cgi) within both available Wigglesworthia genomes, no identifiable evidence of a riboswitch is apparent. The involvement of more “global regulators” or small RNAs, which remain to be discovered in the Wigglesworthia genomes, may also be responsible for the dynamics in the transcriptional profile.

The reductive genome evolution of partners, as described in the BQH (30), can be applied to the tsetse fly symbiosis to further understand observed changes leading toward metabolic dependency among partners. Applying this theory, W. morsitans provisions thiamine to supplement the host blood diet, of which some is utilized by Sodalis for its growth within tsetse flies. Therefore, if TMP is synthesized as a leaky product by W. morsitans, there may be relaxed selection on thiamine biosynthesis loci within the Sodalis genome. In support of this, a loss of the Sodalis genomic components of this biosynthetic pathway has occurred through evolutionary time, consequently resulting in a metabolic dependency on W. morsitans for fitness.

The ability of W. morsitans to adapt to nutrient availability within its environment may be vital for maintaining homeostasis within tsetse flies, preventing symbiont populations from becoming too large, which may skew the relationship towards pathogenesis. Thiamine is a crucial cofactor in amino acid and carbohydrate metabolism; therefore, it is necessary for proper cell growth and physiology in all living organisms (45). Within insects, thiamine deficiency results in the degeneration of the fat body, stunted larval growth, and reduced fertility (reviewed in references 11 and 49). Our results support the hypothesis that TMP is a key metabolite in the maintenance of tsetse symbiont homeostasis, as it has been shown to impact Sodalis proliferation (46) and also various aspects of W. morsitans biology. Moreover, although a massive reduction in W. morsitans genomic content has occurred, thiamine production appears to remain intact within its biosynthetic capabilities. Future studies should examine how these modifications are being recognized, especially given the paucity of environmental sensing capabilities with a small to nonexistent repertoire of one- and two-component signal transduction systems within the Wigglesworthia genomes.