The holobiont transcriptome of teneral tsetse fly species of varying vector competence

Abstract Background Tsetse flies are the obligate vectors of African trypanosomes, which cause Human and Animal African Trypanosomiasis. Teneral flies (newly eclosed adults) are especially susceptible to parasite establishment and development, yet our understanding of why remains fragmentary. The tsetse gut microbiome is dominated by two Gammaproteobacteria, an essential and ancient mutualist Wigglesworthia glossinidia and a commensal Sodalis glossinidius. Here, we characterize and compare the metatranscriptome of teneral Glossina morsitans to that of G. brevipalpis and describe unique immunological, physiological, and metabolic landscapes that may impact vector competence differences between these two species. Results An active expression profile was observed for Wigglesworthia immediately following host adult metamorphosis. Specifically, ‘translation, ribosomal structure and biogenesis’ followed by ‘coenzyme transport and metabolism’ were the most enriched clusters of orthologous genes (COGs), highlighting the importance of nutrient transport and metabolism even following host species diversification. Despite the significantly smaller Wigglesworthia genome more differentially expressed genes (DEGs) were identified between interspecific isolates (n = 326, ~ 55% of protein coding genes) than between the corresponding Sodalis isolates (n = 235, ~ 5% of protein coding genes) likely reflecting distinctions in host co-evolution and adaptation. DEGs between Sodalis isolates included genes involved in chitin degradation that may contribute towards trypanosome susceptibility by compromising the immunological protection provided by the peritrophic matrix. Lastly, G. brevipalpis tenerals demonstrate a more immunologically robust background with significant upregulation of IMD and melanization pathways. Conclusions These transcriptomic differences may collectively contribute to vector competence differences between tsetse species and offers translational relevance towards the design of novel vector control strategies.

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Colonization of the tsetse fly midgut with commensal Enterobacter inhibits trypanosome infection establishment

10.1101/472373 ◽

2018 ◽

Author(s):

Brian L. Weiss ◽

Michele A. Maltz ◽

Aurélien Vigneron ◽

Yineng Wu ◽

Katharine Walter ◽

...

Keyword(s):

Control Strategies ◽

Vector Competence ◽

Tsetse Fly ◽

Sub Saharan Africa ◽

Tsetse Flies ◽

Wild Type ◽

African Trypanosomes ◽

Control Programs ◽

Causative Agents ◽

Sub Saharan

AbstractTsetse flies (Glossina spp.) vector pathogenic trypanosomes (Trypanosoma spp.) in sub-Saharan Africa. These parasites cause human and animal African trypanosomiases, which are debilitating diseases that inflict an enormous socio-economic burden on inhabitants of endemic regions. Current disease control strategies rely primarily on treating infected animals and reducing tsetse population densities. However, relevant programs are costly, labor intensive and difficult to sustain. As such, novel strategies aimed at reducing tsetse vector competence require development. Herein we investigated whether an Enterobacter bacterium (Esp_Z), which confers Anopheles gambiae with resistance to Plasmodium, is able to colonize tsetse and induce a trypanosome refractory phenotype in the fly. Esp_Z established stable infections in tsetse’s gut, and exhibited no adverse effect on the survival of individuals from either group. Flies with established Esp_Z infections in their gut were significantly more refractory to infection with two distinct trypanosome species (T. congolense, 6% infection; T. brucei, 32% infection) than were age-matched flies that did not house the exogenous bacterium (T. congolense, 36% infected; T. brucei, 70% infected). Additionally, 52% of Esp_Z colonized tsetse survived infection with entomopathogenic Serratia marcescens, compared with only 9% of their wild-type counterparts. These parasite and pathogen refractory phenotypes result from the fact that Esp_Z acidifies tsetse’s midgut environment, which inhibits trypanosome and Serratia growth and thus infection establishment. Finally, we determined that Esp_Z infection does not impact the fecundity of male or female tsetse, nor the ability of male flies to compete with their wild-type counterparts for mates. We propose that Esp_Z could be used as one component of an integrated strategy aimed at reducing the ability of tsetse to transmit pathogenic trypanosomes.Author SummaryTsetse flies transmit pathogenic African trypanosomes, which are the causative agents of socio-economically devastating human and animal African trypanosomiases. These diseases are currently controlled in large part by reducing the population size of tsetse vectors through the use of insecticides, traps and sterile insect technique. However, logistic and monetary hurdles often preclude the prolonged application of procedures necessary to maintain these control programs. Thus, novel strategies, including those aimed at sustainably reducing the ability of tsetse to transmit trypanosomes, are presently under development. Herein we stably colonize tsetse flies with a bacterium (Enterobacter sp. Z, Esp_Z) that acidifies their midgut, thus rendering the environment inhospitable to infection with two distinct, epidemiologically important trypanosome strains as well as an entomopathogenic bacteria. In addition to inducing a trypanosome refractory phenotype, colonization of tsetse with Esp_Z exerts only a modest fitness cost on the fly. Taken together, these findings suggest that Esp_Z could be applied to enhance the effectiveness of currently employed tsetse control programs.

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Tsetse blood-meal sources, endosymbionts and trypanosome-associations in the Maasai Mara National Reserve, a wildlife-human-livestock interface

PLoS Neglected Tropical Diseases ◽

10.1371/journal.pntd.0008267 ◽

2021 ◽

Vol 15 (1) ◽

pp. e0008267

Author(s):

Edward Edmond Makhulu ◽

Jandouwe Villinger ◽

Vincent Owino Adunga ◽

Maamun M. Jeneby ◽

Edwin Murungi Kimathi ◽

...

Keyword(s):

Blood Meal ◽

Glossina Pallidipes ◽

Tsetse Fly ◽

Tsetse Flies ◽

African Buffalo ◽

African Trypanosomiasis ◽

African Trypanosomes ◽

Sodalis Glossinidius ◽

Vertebrate Hosts ◽

Blood Meals

African trypanosomiasis (AT) is a neglected disease of both humans and animals caused by Trypanosoma parasites, which are transmitted by obligate hematophagous tsetse flies (Glossina spp.). Knowledge on tsetse fly vertebrate hosts and the influence of tsetse endosymbionts on trypanosome presence, especially in wildlife-human-livestock interfaces, is limited. We identified tsetse species, their blood-meal sources, and correlations between endosymbionts and trypanosome presence in tsetse flies from the trypanosome-endemic Maasai Mara National Reserve (MMNR) in Kenya. Among 1167 tsetse flies (1136 Glossina pallidipes, 31 Glossina swynnertoni) collected from 10 sampling sites, 28 (2.4%) were positive by PCR for trypanosome DNA, most (17/28) being of Trypanosoma vivax species. Blood-meal analyses based on high-resolution melting analysis of vertebrate cytochrome c oxidase 1 and cytochrome b gene PCR products (n = 354) identified humans as the most common vertebrate host (37%), followed by hippopotamus (29.1%), African buffalo (26.3%), elephant (3.39%), and giraffe (0.84%). Flies positive for trypanosome DNA had fed on hippopotamus and buffalo. Tsetse flies were more likely to be positive for trypanosomes if they had the Sodalis glossinidius endosymbiont (P = 0.0002). These findings point to complex interactions of tsetse flies with trypanosomes, endosymbionts, and diverse vertebrate hosts in wildlife ecosystems such as in the MMNR, which should be considered in control programs. These interactions may contribute to the maintenance of tsetse populations and/or persistent circulation of African trypanosomes. Although the African buffalo is a key reservoir of AT, the higher proportion of hippopotamus blood-meals in flies with trypanosome DNA indicates that other wildlife species may be important in AT transmission. No trypanosomes associated with human disease were identified, but the high proportion of human blood-meals identified are indicative of human African trypanosomiasis risk. Our results add to existing data suggesting that Sodalis endosymbionts are associated with increased trypanosome presence in tsetse flies.

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

Applied and Environmental Microbiology ◽

10.1128/aem.01858-12 ◽

2012 ◽

Vol 78 (21) ◽

pp. 7760-7768 ◽

Cited By ~ 53

Author(s):

Michele A. Maltz ◽

Brian L. Weiss ◽

Michelle O'Neill ◽

Yineng Wu ◽

Serap Aksoy

Keyword(s):

Immune System ◽

Pathogenic Bacteria ◽

Tsetse Fly ◽

Symbiotic Bacteria ◽

Tsetse Flies ◽

Host Immune System ◽

Content Type ◽

African Trypanosomes ◽

Sodalis Glossinidius

ABSTRACTMany 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 (Glossinaspp.) house 3 maternally transmitted symbionts, one of which is a commensal (Sodalis glossinidius) found in several host tissues, including the gut. We determined thatSodalisforms biofilms in the tsetse gut and that this process is influenced by theSodalisouter membrane protein A (OmpA). MutantSodalisstrains that do not produce OmpA (SodalisΔOmpA mutants) fail to form biofilmsin vitroand are unable to colonize the tsetse gut unless endogenous symbiotic bacteria are present. Our data indicate that in the absence of biofilms,SodalisΔOmpA mutant cells are exposed to and eliminated by tsetse's innate immune system, suggesting that biofilms helpSodalisevade 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 promoteSodaliscolonization of the tsetse gut may enhance the development of novel disease control strategies.

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Tsetse blood-meal sources, endosymbionts, and trypanosome infections provide insight into African trypanosomiasis transmission in the Maasai Mara National Reserve, a wildlife-human-livestock interface

10.1101/2020.04.06.027367 ◽

2020 ◽

Cited By ~ 1

Author(s):

Edward Edmond Makhulu ◽

Jandouwe Villinger ◽

Vincent Owino Adunga ◽

Maamun M. Jeneby ◽

Edwin Murungi Kimathi ◽

...

Keyword(s):

Blood Meal ◽

Vector Competence ◽

Glossina Pallidipes ◽

Tsetse Fly ◽

Tsetse Flies ◽

African Buffalo ◽

African Trypanosomiasis ◽

Trypanosome Infection ◽

African Trypanosomes ◽

Blood Meals

AbstractBackgroundAfrican trypanosomiasis (AT) is a neglected disease of both humans and animals caused by Trypanosoma parasites, which are transmitted by obligate hematophagous tsetse flies (Glossina spp.). Understanding of AT transmission is hampered by limited knowledge on interactions of tsetse flies with their vertebrate hosts and the influence of endosymbionts on vector competence, especially in wildlife-human-livestock interfaces. We identified the tsetse species, their blood-meal sources, and the correlation between endosymbiont and trypanosome infection status in the trypanosome-endemic Maasai Mara National Reserve (MMNR) of Kenya.Methodology/Principal FindingsAmong 1167 tsetse flies (1136 Glossina pallidipes, 31 Glossina swynnertoni) collected from 10 sampling sites, 28 (2.4%) were positive by PCR for trypanosomes, majority (17/28) being Trypanosoma vivax. Blood-meal analyses based on high-resolution melting analysis of mitochondrial cytochrome c oxidase 1 and cytochrome b gene PCR products (n = 345) identified humans as the most common vertebrate host (37%), followed by hippopotamus (29.1%), African buffalo (26.3%), elephant (3.39%), and giraffe (0.84%). Trypanosome-infected flies had fed on hippopotamus and buffalo. Additionally, PCR analysis revealed that tsetse flies were more likely to be infected with trypanosomes if they were infected with the Sodalis glossinidius endosymbiont (P = 0.0022 Fisher’s exact test).Conclusions/SignificanceDiverse species of wildlife hosts may contribute to the maintenance of tsetse populations and/or persistent circulation of African trypanosomes in the MMNR. Although the African buffalo is known to be a key reservoir of AT, the higher proportion of hippopotamus blood-meals in trypanosomes-infected flies identified here indicates that other wildlife species may also be important to transmission cycles. No trypanosomes associated with human disease were identified, but the high proportion of human blood-meals identified are indicative of human African trypanosomiasis transmission risk. Furthermore, this work provides data showing that Sodalis endosymbionts can is associated with increased trypanosome infection rates in endemic ecologies.Author summaryHuman and animal African trypanosomiasis are neglected tropical diseases with potential to spread to new areas. Wild animals are important reservoirs for African trypanosomes and crucial in the emergence and re-emergence of AT. Vertebrate host-vector-parasite interactions are integral to trypanosome transmission. We investigated the vertebrate blood-meals and trypanosomes-endosymbionts co-infections in tsetse flies, which have been associated with reservoirs and vector competence, respectively, on AT transmission in Kenya’s Maasai Mara National Reserve. We identified tsetse fly diversity, trypanosome and endosymbiont infection status, and vertebrate blood-meal hosts to infer potential transmission dynamics. We found that Glossina pallidipes was the major tsetse fly vector and that Trypanosoma vivax was the main trypanosome species circulating in the region. Humans, hippopotamus, and buffalo were the most frequented for blood-meals. Buffalo and hippopotamus blood-meals were identified in trypanosome infected flies. Feeding of the flies on both humans and wildlife may potentiate the risk of the human trypanosomiasis in this ecology. Additionally, we found that the endosymbiont Sodalis glossinidius is associated with higher trypanosome infection rates in wild tsetse flies. These findings emphasize the importance of understanding the interaction of tsetse flies with vertebrate blood-meal sources and their endosymbionts in the transmission and control of AT.

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Genome Size Determination and Coding Capacity of Sodalis glossinidius, an Enteric Symbiont of Tsetse Flies, as Revealed by Hybridization to Escherichia coliGene Arrays

Journal of Bacteriology ◽

10.1128/jb.183.15.4517-4525.2001 ◽

2001 ◽

Vol 183 (15) ◽

pp. 4517-4525 ◽

Cited By ~ 41

Author(s):

Leyla Akman ◽

Rita V. M. Rio ◽

Charles B. Beard ◽

Serap Aksoy

Keyword(s):

Genome Size ◽

Tsetse Fly ◽

Whole Genome Sequence ◽

Tsetse Flies ◽

African Trypanosomes ◽

Sodalis Glossinidius ◽

Novel Approach ◽

Living Organisms ◽

Conserved Gene ◽

Coding Capacity

ABSTRACT Recent molecular characterization of various microbial genomes has revealed differences in genome size and coding capacity between obligate symbionts and intracellular pathogens versus free-living organisms. Multiple symbiotic microorganisms have evolved with tsetse fly, the vector of African trypanosomes, over long evolutionary times. Although these symbionts are indispensable for tsetse fecundity, the biochemical and molecular basis of their functional significance is unknown. Here, we report on the genomic aspects of the secondary symbiont Sodalis glossinidius. The genome size ofSodalis is approximately 2 Mb. Its DNA is subject to extensive methylation and based on some of its conserved gene sequences has an A+T content of only 45%, compared to the typically AT-rich genomes of endosymbionts. Sodalis also harbors an extrachromosomal plasmid about 134 kb in size. We used a novel approach to gain insight into Sodalis genomic contents, i.e., hybridizing its DNA to macroarrays developed for Escherichia coli, a closely related enteric bacterium. In this analysis we detected 1,800 orthologous genes, corresponding to about 85% of theSodalis genome. The Sodalis genome has apparently retained its genes for DNA replication, transcription, translation, transport, and the biosynthesis of amino acids, nucleic acids, vitamins, and cofactors. However, many genes involved in energy metabolism and carbon compound assimilation are apparently missing, which may indicate an adaptation to the energy sources available in the only nutrient of the tsetse host, blood. We present gene arrays as a rapid tool for comparative genomics in the absence of whole genome sequence to advance our understanding of closely related bacteria.

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Viviparity and habitat restrictions may influence the evolution of male reproductive genes in tsetse fly (Glossina) species

BMC Biology ◽

10.1186/s12915-021-01148-4 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Grazia Savini ◽

Francesca Scolari ◽

Lino Ometto ◽

Omar Rota-Stabelli ◽

Davide Carraretto ◽

...

Keyword(s):

Reproductive Behavior ◽

Selective Pressure ◽

Demographic History ◽

Reproductive Rate ◽

Tsetse Fly ◽

Sub Saharan Africa ◽

Comparative Genomic ◽

Tsetse Flies ◽

African Trypanosomes ◽

Reproductive Genes

Abstract Background Glossina species (tsetse flies), the sole vectors of African trypanosomes, maintained along their long evolutionary history a unique reproductive strategy, adenotrophic viviparity. Viviparity reduces their reproductive rate and, as such, imposes strong selective pressures on males for reproductive success. These species live in sub-Saharan Africa, where the distributions of the main sub-genera Fusca, Morsitans, and Palpalis are restricted to forest, savannah, and riverine habitats, respectively. Here we aim at identifying the evolutionary patterns of the male reproductive genes of six species belonging to these three main sub-genera. We then interpreted the different patterns we found across the species in the light of viviparity and the specific habitat restrictions, which are known to shape reproductive behavior. Results We used a comparative genomic approach to build consensus evolutionary trees that portray the selective pressure acting on the male reproductive genes in these lineages. Such trees reflect the long and divergent demographic history that led to an allopatric distribution of the Fusca, Morsitans, and Palpalis species groups. A dataset of over 1700 male reproductive genes remained conserved over the long evolutionary time scale (estimated at 26.7 million years) across the genomes of the six species. We suggest that this conservation may result from strong functional selective pressure on the male imposed by viviparity. It is noteworthy that more than half of these conserved genes are novel sequences that are unique to the Glossina genus and are candidates for selection in the different lineages. Conclusions Tsetse flies represent a model to interpret the evolution and differentiation of male reproductive biology under different, but complementary, perspectives. In the light of viviparity, we must take into account that these genes are constrained by a post-fertilization arena for genomic conflicts created by viviparity and absent in ovipositing species. This constraint implies a continuous antagonistic co-evolution between the parental genomes, thus accelerating inter-population post-zygotic isolation and, ultimately, favoring speciation. Ecological restrictions that affect reproductive behavior may further shape such antagonistic co-evolution.

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Extrachromosomal DNA of the Symbiont Sodalis glossinidius

Journal of Bacteriology ◽

10.1128/jb.187.14.5003-5007.2005 ◽

2005 ◽

Vol 187 (14) ◽

pp. 5003-5007 ◽

Cited By ~ 24

Author(s):

A. C. Darby ◽

J. Lagnel ◽

C. Z. Matthew ◽

K. Bourtzis ◽

I. Maudlin ◽

...

Keyword(s):

Tsetse Fly ◽

Tsetse Flies ◽

Extrachromosomal Dna ◽

Sodalis Glossinidius

ABSTRACT The extrachromosomal DNA of Sodalis glossinidius from two tsetse fly species was sequenced and contained four circular elements: three plasmids, pSG1 (82 kb), pSG2 (27 kb), and pSG4 (11 kb), and a bacteriophage-like pSG3 (19 kb) element. The information suggests S. glossinidius is evolving towards an obligate association with tsetse flies.

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

Applied and Environmental Microbiology ◽

10.1128/aem.00741-08 ◽

2008 ◽

Vol 74 (19) ◽

pp. 5965-5974 ◽

Cited By ~ 161

Author(s):

Roshan Pais ◽

Claudia Lohs ◽

Yineng Wu ◽

Jingwen Wang ◽

Serap Aksoy

Keyword(s):

Blood Meal ◽

Pcr Amplification ◽

Tsetse Fly ◽

Tsetse Flies ◽

Mutualistic Symbiosis ◽

Sodalis Glossinidius ◽

Specific Pcr ◽

Amplification Assay ◽

Blood Meal Digestion ◽

Vectorial Competence

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.

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Molecular identification of Wolbachia and Sodalis glossinidius in the midgut of Glossina fuscipes quanzensis from the Democratic Republic of Congo

Parasite ◽

10.1051/parasite/2019005 ◽

2019 ◽

Vol 26 ◽

pp. 5 ◽

Cited By ~ 1

Author(s):

Gustave Simo ◽

Sartrien Tagueu Kanté ◽

Joule Madinga ◽

Ginette Kame ◽

Oumarou Farikou ◽

...

Keyword(s):

Molecular Markers ◽

Democratic Republic ◽

Democratic Republic Of Congo ◽

Bacterial Flora ◽

Tsetse Flies ◽

Infection Rates ◽

Republic Of Congo ◽

African Trypanosomes ◽

Sodalis Glossinidius ◽

Aldolase Gene

During the last 30 years, investigations on the microbiome of different tsetse species have generated substantial data on the bacterial flora of these cyclical vectors of African trypanosomes, with the overarching goal of improving the control of trypanosomiases. It is in this context that the presence of Wolbachia and Sodalis glossinidius was studied in wild populations of Glossina fuscipes quanzensis from the Democratic Republic of Congo. Tsetse flies were captured with pyramidal traps. Of the 700 Glossina f. quanzensis captured, 360 were dissected and their midguts collected and analyzed. Sodalis glossinidius and Wolbachia were identified by PCR. The Wolbachia-positive samples were genetically characterized with five molecular markers. PCR revealed 84.78% and 15.55% midguts infected by Wolbachia and S. glossinidius, respectively. The infection rates varied according to capture sites. Of the five molecular markers used to characterize Wolbachia, only the fructose bis-phosphate aldolase gene was amplified for about 60% of midguts previously found with Wolbachia infections. The sequencing results confirmed the presence of Wolbachia and revealed the presence of S. glossinidius in the midgut of Glossina f. quanzensis. A low level of midguts were naturally co-infected by both bacteria. The data generated in this study open a framework for investigations aimed at understanding the contribution of these symbiotic microorganisms to the vectorial competence of Glossina fuscipes quanzensis.

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Vitamin B6Generated by Obligate Symbionts Is Critical for Maintaining Proline Homeostasis and Fecundity in Tsetse Flies

Applied and Environmental Microbiology ◽

10.1128/aem.01150-14 ◽

2014 ◽

Vol 80 (18) ◽

pp. 5844-5853 ◽

Cited By ~ 62

Author(s):

Veronika Michalkova ◽

Joshua B. Benoit ◽

Brian L. Weiss ◽

Geoffrey M. Attardo ◽

Serap Aksoy

Keyword(s):

Pyridoxal Phosphate ◽

Tsetse Fly ◽

Tsetse Flies ◽

Vitamin B ◽

Lactation Period ◽

Content Type ◽

African Trypanosomes ◽

Microbial Extracts ◽

Lactating Females ◽

Interfering Rna

ABSTRACTThe viviparous tsetse fly utilizes proline as a hemolymph-borne energy source. In tsetse, biosynthesis of proline from alanine involves the enzyme alanine-glyoxylate aminotransferase (AGAT), which requires pyridoxal phosphate (vitamin B6) as a cofactor. This vitamin can be synthesized by tsetse's obligate symbiont,Wigglesworthia glossinidia. In this study, we examined the role ofWigglesworthia-produced vitamin B6for maintenance of proline homeostasis, specifically during the energetically expensive lactation period of the tsetse's reproductive cycle. We found that expression ofagat, as well as genes involved in vitamin B6metabolism in both host and symbiont, increases in lactating flies. Removal of symbionts via antibiotic treatment of flies (aposymbiotic) led to hypoprolinemia, reduced levels of vitamin B6in lactating females, and decreased fecundity. Proline homeostasis and fecundity recovered partially when aposymbiotic tsetse were fed a diet supplemented with either yeast orWigglesworthiaextracts. RNA interference-mediated knockdown ofagatin wild-type flies reduced hemolymph proline levels to that of aposymbiotic females. Aposymbiotic flies treated withagatshort interfering RNA (siRNA) remained hypoprolinemic even upon dietary supplementation with microbial extracts or B vitamins. Flies infected with parasitic African trypanosomes display lower hemolymph proline levels, suggesting that the reduced fecundity observed in parasitized flies could result from parasite interference with proline homeostasis. This interference could be manifested by competition between tsetse and trypanosomes for vitamins, proline, or other factors involved in their synthesis. Collectively, these results indicate that the presence ofWigglesworthiain tsetse is critical for the maintenance of proline homeostasis through vitamin B6production.

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