sign in sign up
<<
Home , Glossina fuscipes, Sodalis, Wolbachia

View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Frontiers - Publisher Connector MINI REVIEW ARTICLE published: 29 October 2013 CELLULAR AND MICROBIOLOGY doi: 10.3389/fcimb.2013.00069 Tsetse fly microbiota: form and function

Jingwen Wang*, Brian L. Weiss and Serap Aksoy

Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT, USA

Edited by: Tsetse flies are the primary vectors of African trypanosomes, which cause Brice Rotureau, Institut Pasteur, and African in 36 countries in sub-Saharan Africa. These flies France have also established symbiotic associations with bacterial and viral . Reviewed by: Laboratory-reared tsetse flies harbor up to four vertically transmitted organisms—obligate Jan Van Den Abbeele, Institute of Tropical Medicine, Belgium Wigglesworthia, commensal , parasitic and Salivary Gland Hypertrophy Anne Geiger, Institut de Recherche Virus (SGHV). Field-captured tsetse can harbor these symbionts as well as environmentally pour le Développement (IRD), acquired commensal . This microbial community influences several aspects of France tsetse’s physiology, including nutrition, fecundity and competence. This review Steve Perlman, University of Victoria, Canada provides a detailed description of tsetse’s microbiome, and describes the physiology *Correspondence: underlying host-microbe, and microbe-microbe, interactions that occur in this fly. Jingwen Wang, Department of Epidemiology of Microbial Diseases, Keywords: tsetse fly, symbiont, wigglesworthia, Sodalis, wolbachia Yale School of Public Health, 60 College Street, LEPH 624, New Haven, CT 06510-3201, USA e-mail: [email protected]

INTRODUCTION milk gland) secretions that contain proteins, lipids and amino Tsetse flies (Glossina sp.) serve as hosts to numerous microor- acids (Attardo et al., 2008, 2012; Benoit et al., 2012; Wang ganisms. This is the primary vector of bru- and Aksoy, 2012). Several milk associated protein-encoding cei parasites that cause a chronic wasting disease in , including milk gland proteins (mgp) 1–10, transferrin (human , or HAT) and domesticated and acid sphingomyelinase 1, are differentially expressed dur- (animal African trypanosomiasis, or AAT) in 36 coun- ing pregnancy in the milk gland organ. All 10 milk proteins tries throughout sub-Saharan Africa. Both HAT and AAT are are up-regulated during lactation to support larval growth, pernicious diseases that inflict untold hardships upon their mam- and then down-regulated drastically following parturition. malian hosts. In fact, African trypanosomes are regarded as one Interference with the expression of milk proteins through a of the greatest constraints to Africa’s economic development double-stranded RNA interference approach has negative effects (Simarro et al., 2008; Welburn and Maudlin, 2012). on fecundity by extending larvagenesis and/or inducing pre- Tsetse also has a relationship with multiple bacterial mature abortions (Attardo et al., 2010; Yang et al., 2010;S. and at least one virus. Tsetse’s bacterial partners can include 3 Aksoy and J. Benoit, personal communication). Thus, com- maternally-transmitted as well as a taxonomi- pounds that interfere with milk production could be used cally diverse collection of commensals that the fly acquires from as novel tools to reduce vector populations. it’s environment. Additionally, many tsetse flies also harbor a Understanding the mechanism(s) that regulates the coordi- salivary gland-associated DNA virus. These microbes are inti- nated transcription of milk proteins may lead to the discov- mately associated with many important aspects of their host’s ery of key regulatory factors that could be exploited to reduce biology. Thus, increasing our fundamental knowledge regarding fecundity. how tsetse interacts with it’s microbiota will allow us to develop novel and innovative control strategies aimed at reducing tsetse THE BIOLOGY OF TSETSE populations and/or tsetse vector competence. Bacterial endosymbionts play vital roles in insect metabolic processes, the maintenance of fecundity and EXPLOITING TSETSE’S UNUSUAL REPRODUCTIVE development (Douglas, 2011). Tsetse flies house 3 endogenous PHYSIOLOGY FOR POPULATION CONTROL symbionts, Wigglesworthia, Sodalis and Wolbachia, which also Tsetse flies lead a relatively sterile existence when compared to the impact the physiology of their host (Figure 1). vast majority of other . Adult males and females feed exclu- sively on sterile blood. Also, unlike other oviparous WIGGLESWORTHIA insects, female tsetse produce only one egg per gonotrophic cycle Maternal milk is more than a source of nourishment for devel- (Tobe, 1978). Tsetse offspring develop in their mother’s . oping tsetse offspring. This substance also serves as a conduit 3rd larvae are deposited and immediately pupate. Adult through which vertically-transmitted symbiotic bacteria colo- flies emerge 30 days later. This mode of results in nize developing larvae. All laboratory reared tsetse flies exam- deposition of only 8–10 progeny per female over her lifespan. ined to date house up to 3 maternally transmitted symbiotic During larvagenesis, tsetse progeny receive nourishment via bacteria. The first of these symbionts is the obligate mutualist highly modified maternal accessory gland (referred to as the Wigglesworthia. Tsetse’s association with this bacterium began

Frontiers in Cellular and Infection Microbiology www.frontiersin.org October 2013 | Volume 3 | Article 69 | 1 Wang et al. Tsetse fly microbiota: form and function

FIGURE 1 | Localization of symbionts and SGHV in tsetse.

50-80 million years ago, and since this time Wigglesworthia has Wigglesworthia provides two well-documented functional ben- been subjected to strict vertical transmission within its species- efits to its tsetse host. The first benefit is nutritional, as in the specific tsetse host (Chen et al., 1999). This contiguous associ- absence of this bacterium, intrauterine larval development is ation accounts for Wigglesworthia’s extraordinarily streamlined stunted and progeny are aborted (Schlein, 1977; Nogge, 1978; chromosome, which is about 700 kb in size (Akman et al., Nogge and Gerresheim, 1982; Pais et al., 2008). Interestingly, 2002; Rio et al., 2012). Comparison of the structural organi- Wigglesworthia’s contracted genome encodes an unusually high zation and content of two distant Wigglesworthia species number of putative vitamin biosynthesis pathways (Akman et al., analyzed from Glossina brevipalpis and G. morsitans revealed 2002; Rio et al., 2012). More than 10% of the retained CDSs overall high synteny between the two genomes, similarly high are involved in the biosynthesis of cofactors, prosthetic groups AT biases (over 80%) and highly conserved coding capacity and carriers, supporting Wigglesworthia’s genetic contributions including the absence of dnaA-based DNA replication mecha- to de novo metabolism of biotin, thiazole, lipoic acid, FAD nisms. However, despite extensive conservation, unique genes (riboflavin, B2), folate, pantothenate, thiamine (B1), pyridoxine were identified between the two symbiont genomes that may (B6), and protoheme. This genotypic feature supports the theory result in divergent metabolomes impacting host physiology (Rio that Wigglesworthia supplements its tsetse host with nutritious et al., 2012). Analysis of the Wigglesworthia morsitans specific metabolites that are naturally present at low titers in vertebrate gene set reveals the presence of a complete shikimate biosyn- blood. Similarly, in mosquitoes, some bacteria, including Serratia, thetic pathway, in which 3-deoxyD-arabino-heptulosonate-7- Enterobacter, and Asaia, regulate blood digestion and supplement phosphate (DAHP) can be converted into chorismate. This their host with vitamins (Minard et al., 2013). These observa- pathway is degraded in the Wigglesworthia brevipalpis genome. tions indicate that depends on the presence of Downstream of the chorismate pathway, W. morsitans’ chro- gut-associated microbes that facilitate blood meal metabolism. mosome encodes pabA, pabB (aminodeoxychorismate synthase Wigglesworthia’s second function in tsetse is immunological. II and I, respectively) and pabC (4-amino-4-deoxychorismate More specifically, when flies undergo intrauterine larval lyase). These enzymes catalyze the formation of p-aminobenzoate development in the absence of this bacterium they present from chorismate–an essential component in folate biosynthe- a severely compromised immune system during adulthood. sis. The W. morsitans genome also contains an aspC homolog Under these conditions, Wigglesworthia-free tsetse are unusually that can be used following chorismate biosynthesis to pro- susceptible to infection with normally non-pathogenic E. coli duce phenylalanine. Interestingly, African trypanosomes are K12 and trypanosomes (Wang et al., 2009; Weiss et al., 2011, unable to synthesize phenylalanine and folate, yet their genome 2012, 2013). encodes transporters capable of salvaging both metabolites from Tsetse houses two distinct populations of Wigglesworthia.The their host environment (Berriman et al., 2005). It remains first resides intracellularly within bacteriocytes, which collectively to be seen whether genomic variations noted in the two form an organ called the bacteriome that is found immedi- Wigglesworthia species lead to different biosynthetic capabilities. ately adjacent to tsetse’s anterior midgut (Aksoy, 1995, 2000). Additionally, further investigations are required to determine if Tsetse’s second population of Wigglesworthia is found extracellu- these metabolic distinctions contribute to the differential try- larly in milk gland secretions (Attardo et al., 2008). Interestingly, panosome vector competences exhibited by G. morsitans and G. Wigglesworthia’s genome contains genes that encode a complete brevipalpis flies (Harley and Wilson, 1968; Moloo and Kutuza, flagellar structure (Akman et al., 2002). Flagella-associated genes 1988). (fliC, motA)arehighlyexpressedinWigglesworthia that reside

Frontiers in Cellular and Infection Microbiology www.frontiersin.org October 2013 | Volume 3 | Article 69 | 2 Wang et al. Tsetse fly microbiota: form and function

extracellularly in tsetse’s milk gland, but not in the intracellu- WOLBACHIA lar bacteriome population (Rio et al., 2012). Expression of a Some tsetse populations also harbor a parasitic bacterium functional flagellum in milk-associated Wigglesworthia may facil- from the Wolbachia. Wolbachia is a wide spread alpha- itate vertical transmission of this bacterium from the pregnant , infecting approximately 70% female to the developing intrauterine larvae, where the symbiont insects (Hilgenboecker et al., 2008). This bacterium manip- colonizes juvenile tissues. ulates the reproductive biology of its host through a vari- ety of mechanisms, including cytoplasmic incompatibility (CI), SODALIS male killing, feminization and (Werren et al., Tsetse’s second endosymbiont is the commensal Sodalis,whichis 2008). Expression of CI occurs when a Wolbachia infected a gram-negative organism closely related to free-living microbes male mates with an uninfected female, causing developmen- within the Enterobacteriacaea. In addition to tsetse, Sodalis- tal arrest during embryogenesis. In contrast, Wolbachia infected allied bacteria are present in several other insects, including females can mate with uninfected males, or with a male stinkbugs and weevils (Kaiwa et al., 2010; Toju et al., 2010). infected with the same Wolbachia , and produce viable Unlike Wigglesworthia, Sodalis exhibits a wide tissue tropism and offspring. In tsetse, Wolbachia is localized exclusively intra- can be found both intra and extra-cellularly in various tissues cellularly in germ line tissues and can be detected in early including midgut, fat body, milk gland, salivary glands and hemo- oocyte, embryo and larvae (Cheng et al., 2000; Balmand et al., coel (Cheng and Aksoy, 1999; Balmand et al., 2013). Sodalis’ 2013). Unlike Sodalis and Wigglesworthia,whicharetrans- 4.2 Mb chromosome is similar in size to those of its free liv- mitted via milk gland secretions, Wolbachia is transmitted ing ancestors. However, Sodalis’ genome exhibits a low coding transovarially via germ line cells. In G. morsitans laboratory capacity and an unusually high number of pseudogenes (over colonies, Wolbachia induces CI when Wolbachia-cured females 600 genes) in pathways that it likely no longer requires as a resi- mate with wild-type Wolbachia-infected males. Offspring from dent tsetse endosymbiont (Toh et al., 2006). Interestingly, Sodalis’ these crosses perish during early embryogenesis while the recip- genome contains features typically associated with pathogenic rocal cross survives and produces viable progeny (Alam et al., lifestyles, including 3 type three secretion systems (TTSS). The 2011). Sodalis TTSSs, which function during tsetse’s juvenile develop- mental stages, share high homology with those from Salmonella SALIVARY GLAND HYPERTROPHY VIRUS (SGHV) and Yersinia. Interestingly, while structural genes associated with Both colony-reared and natural tsetse populations harbor a rod- these systems are retained, effector protein genes, the products of shaped, enveloped DNA virus called Salivary Gland Hypertrophy which are pathogenic to host cells, are lacking (Toh et al., 2006). Virus (SGHV). In tsetse, SGHV can cause hypertrophy of the Sodalis also has retained the capacity to synthesize a complete salivary glands and gonadal lesions (Jaenson, 1978). SGHV can flagellar structure, which may be important for it to colonize be vertically transmitted via maternal milk gland secretions or intrauterine tsetse offspring. This bacterium can be cultured in horizontally during the feeding process (Abd-Alla et al., 2011). cell-free medium, which further indicates its recent association While the majority of SGHV-infected flies are asymptomatic with with tsetse and its intermediate status between free living and no apparent loss of host fitness, flies infected with high virus obligate intracellular bacteria. titers exhibit reduced fecundity and lifespan, and display hyper- Sodalis lacks a clearly defined functional role within its tsetse trophied salivary glands (Sang et al., 1999; Abd-Alla et al., 2011). host. More so, several natural tsetse populations lack this bac- In field populations, infection prevalence of SGHV varies in dif- terium, suggesting that it presents a truly commensal pheno- ferent species and locations (Alam et al., 2012; Malele et al., type within its tsetse host. However, several studies indicate that 2013). Sodalis may play a role in tsetse’s ability to vector pathogenic try- panosomes. Sodalis hasbeenreportedtoincreasetsetse’ssuscepti- METABOLIC INTERDEPENDENCE OF TSETSE’S MICROBIOTA bility to trypanosomes by obstructing the trypanocidal activity of Selective elimination of tsetse’s microbial partners via treat- host midgut lectins (Welburn et al., 1993). Specifically, a G. morsi- ment with antibiotics revealed that these microorganisms may tans line that harbored a high Sodalis infection prevalence exhib- be metabolically dependent on one another for their survival in ited greater susceptibility to trypanosomes than did a G. austeni the tsetse host (Belda et al., 2010; Snyder et al., 2010; Snyder line that harbored a low Sodalis infection prevalence (Welburn and Rio, 2013; Wang et al., 2013). For example, offspring from et al., 1993). Additionally, treatment of tsetse with the antibi- female tsetse that are fed a diet supplemented with ampicillin otic streptozotocin results in the elimination of Sodalis exclusively. lack obligate Wigglesworthia but still retain Sodalis and Wolbachia While the fecundity of these flies does not change, they become (Pais et al., 2008). In the absence of Wigglesworthia, Sodalis more resistant to trypanosome (Dale and Welburn, densities decline and are eventually eliminated from subsequent 2001). In G. palpalis populations collected in Cameroon, para- generations. Wigglesworthia was shown to provide thiamine to site infection prevalence positively correlated with the presence Sodalis, which lacks the ability to synthesize this metabolite but of Sodalis in examined flies (Farikou et al., 2010; Soumana et al., has retained a transporter to scavenge it from the environment 2013). These results suggest that in contrast to Wigglesworthia, (Snyder et al., 2010; Wang et al., 2013). The fate of SGHV in tsetse which increases tsetse refractoriness to trypanosomes, Sodalis also depends upon the presence of the fly’s endogenous bacte- appears to favor the establishment of trypanosome infections in rial symbionts. Specifically, virus proliferation and transmission tsetse. decreased in tsetse that lacked either Wigglesworthia or all 3

Frontiers in Cellular and Infection Microbiology www.frontiersin.org October 2013 | Volume 3 | Article 69 | 3 Wang et al. Tsetse fly microbiota: form and function

maternally transmitted microbes (Boucias et al., 2013; Wang 2013). For example, Wolbachia strains detected in Ugandan G. et al., 2013). Taken together these findings indicate that nutri- fuscipes belong to two different lineages. Furthermore, unusu- tional dependencies between tsetse’s endogenous symbionts ally high groEL sequence diversity was present both within and not only influence host fitness, but also regulate micro- between individuals. Thus, there appears to be high diversity bial density and transmission dynamics of the individual in infection prevalence, density and the circulating strains asso- partners. ciated with different tsetse natural populations. Interestingly, three Wolbachia genes, 16S rRNA, fbpA and wsp,haveinserted ESTABLISHMENT OF SYMBIONT INFECTIONS IN THE GUT into the G. m. morsitans host chromosome and the ongoing A complete understanding of the mechanisms that allow com- G. m morsitans genome project has revealed that insertions mensal bacteria to evade their host’s gut immune mechanisms from Wolbachia into host chromosome may be more extensive and enable them to colonize their host’s gut is vital for suc- (Doudoumis et al., 2012; S. Aksoy and K. Bourtzis, personal cessful paratransgenic applications. Recent work using the tsetse communication). model system indicates that bacterial surface coat modifica- Tsetse’s viviparous mode of reproduction serves as a rigor- tions may be intimately involved in determining symbiont infec- ous barrier between immature developmental stages and the tion outcomes. One such molecule, outer membrane protein microbe-rich external environment. However, several environ- A (OmpA), regulates Sodalis’ ability to form biofilms, which mentally acquired bacterial commensals were recently found in are essential for this bacterium to colonize tsetse’s gut. A thegutsofwildtsetse(Geiger et al., 2013). Bacteria belong-  mutant Sodalis (Sodalis ompA) line that does not express ompA ing to 3 phyla (23 species), the , Proteobacteria and wasunabletoformbiofilmsin vitro. In contrast, wild-type Actinobacteria,werecharacterizedfromeastAfricanG. fuscipes  Sodalis, and biofilm-defective E. coli (E. coli ompA) genetically (Lindh and Lehane, 2011). G. p. palpalis, G. pallicera, G. nigro- modified to express Sodalis ompA were able to produce these fusca,andG. caliginea collectedinCameroonandAngolawere structures (Maltz et al., 2012). OmpA also influences Sodalis’ found to harbor cultured bacteria from 9 distinct genera (Geiger in vivo colonization competency. Following per os inocula- et al., 2009, 2011). The relative densities of these infections and tion into tsetse that lack their endogenous microbiome (which their persistent association with the tsetse remains to be eluci-  serves as an alternative source of OmpA), Sodalis ompA are dated. Taxonomically diverse commensal bacteria housed in wild unable to colonize the fly gut. In contrast, wild-type cells tsetse flies may also influence their host’s physiology and vector persist in this niche (Maltz et al., 2012). This finding sug- competence. However, further studies are required to verify this gests that biofilm formation is essential for Sodalis to be able hypothesis. to colonize the host gut, as these structures may protect the bacteria from the hostile effects of the tsetse’s immune sys- CONCLUSIONS AND FUTURE DIRECTIONS tem. The identification and functional characterization of addi- Tsetse flies house several microorganisms, all of which interact tional commensal symbiont colonization factors will be useful with one another and their host to manipulate the physiology for enhancing the efficacy of paratransgenesis in insect disease of the system as a whole. With the aid of the Wigglesworthia, vectors. Sodalis, Wolbachia,SGHVandT. brucei sequenced and anno- tated genomes (the annotation of G. morsitans is almost com- TSETSE-MICROBE ASSOCIATIONS IN NATURAL plete), more in depth studies on the interactions between POPULATIONS tsetse’s multiple partners can be performed. Work to date has To date, all field-collected tsetse flies examined harbor obligate focused primarily on host-microbe interactions in laboratory- Wigglesworthia.Incontrast,Sodalis infection prevalence in tsetse reared tsetse. However, a growing number of studies using field populations varies from 0 to 85% (Maudlin et al., 1990; tsetse, and other insect disease vectors, indicate greater spa- Farikou et al., 2010; Lindh and Lehane, 2011; Alam et al., 2012). tial and temporal diversity of symbiotic composition, density Additionally, multiple Sodalis genotypes are present in some and prevalence in natural populations. Future work with natu- field-captured tsetse, suggesting that beyond prevalence Sodalis is ral populations will be important to understand the functional genetically distant between different tsetse species (Geiger et al., role of this diversity on host fitness and pathogen infection 2005; Farikou et al., 2011). Like Sodalis, Wolbachia infection outcomes. prevalence in field-captured tsetse differed significantly between different host species and different populations within the same ACKNOWLEDGMENTS species (Alam et al., 2012; Doudoumis et al., 2012). Based on We are grateful to many members of our laboratory who standard PCR assays, Wolbachia can be detected in G. m. mor- contributed to the data we describe in this short review. sitans, G. m. centralis and G. austeni populations, but not G. We especially thank Geoffrey Attardo for editorial assistance. tachinoides. Additionally, low density Wolbachia infections are This study received support from NIH AI051584 and Li reported in several species and populations, including G. fuscipes Foundation and Ambrose Monell Foundation awards to S.A. and G. morsitans subspecies (Alam et al., 2012; Schneider et al., We are also grateful to the CRPs organized through the 2013). In field G. m. morsitans, Wolbachia infection prevalence Joint FAO/International Atomic Energy Agency, Seibersdorf ranges from 10 to 100% depending on their resident habitats Austria for discussion and to Slovak Academy of Sciences, (Doudoumis et al., 2012). Some G. fuscipes individualsalsohar- Bratislava, Slovakia for providing tsetse puparia for colony main- bor infections with multiple Wolbachia strains (Symula et al., tenance.

Frontiers in Cellular and Infection Microbiology www.frontiersin.org October 2013 | Volume 3 | Article 69 | 4 Wang et al. Tsetse fly microbiota: form and function

REFERENCES endosymbionts. J. Invertebr. populations of different species of numbers of infective organisms Abd-Alla, A. M., Parker, A. G., Vreysen, Pathol. 112(Suppl.)S116–S122. doi: tsetse flies (genus Glossina). BMC extruded during feeding. Ann. Trop. M. J., and Bergoin, M. (2011). Tsetse 10.1016/j.jip.2012.04.002 Microbiology 12(Suppl.)S1–S3. doi: Med. Parasitol. 62, 178–187. salivary gland hypertrophy virus: Belda, E., Moya, A., Bentley, S., and 10.1186/1471-2180-12-S1-S3 Hilgenboecker, K., Hammerstein, hope or hindrance for tsetse control. Silva, F. J. (2010). Mobile genetic Douglas, A. E. (2011). Lessons from P., Schlattmann, P., Telschow, PLoS Negl. Trop. Dis. 5:e1220. doi: element proliferation and gene inac- studying insect symbioses. Cell A., and Werren, J. H. (2008). 10.1371/journal.pntd.0001220 tivation impact over the genome Host Microbe 20, 359–367. doi: How many species are infected Akman, L., Yamashita, A., Watanabe, structure and metabolic capabili- 10.1016/j.chom.2011.09.001 with Wolbachia?–A statistical H., Oshima, K., Shiba, T., Hattori, ties of ,thesec- Farikou, O., Njiokou, F., Mbida analysis of current data. FEMS M., et al. (2002). Genome sequence ondary endosymbiont of tsetse flies. Mbida, J. A., Njitchouang, G. R., Microbiol. Lett. 281, 215–220. doi: of the endocellular obligate sym- BMC Genomics 22, 449–465. doi: Djeunga, H. N., Asonganyi, T., 10.1111/j.1574-6968.2008.01110.x biont of tsetse flies, Wigglesworthia 10.1186/1471-2164-11-449 et al. (2010). Tripartite interac- Jaenson, T. G. (1978). Virus-like rods glossinidia. Nat. Genet. 32, 402–407. Benoit, J. B., Attardo, G. M., tions between tsetse flies, Sodalis associated with salivary gland doi: 10.1038/ng986 Michalkova, V., Takac, P., glossinidius and trypanosomes–an hyperplasia in tsetse, Glossina Aksoy, S. (1995). Wigglesworthia gen. Bohova, J., and Aksoy, S. (2012). epidemiological approach in two pallidipes. Trans. R. Soc. Trop. nov.andWigglesworthia glossini- Sphinomyelinase activity in historical human African try- Med. Hyg. 978, 234–238. doi: dia sp. nov., taxa consisting of the mother’s milk is essential panosomiasis foci in Cameroon. 10.1016/0035-9203(78)90200-6 the mycetocyte-associated, primary for juvenile development: a Infect. Genet. Evolut. 10, 115–121. Kaiwa, N., Hosokawa, T., Kikuchi, Y., endosymbionts of tsetse flies. Int. case from lactating tsetse flies. doi: 10.1016/j.meegid.2009.10.008 Nikoh, N., Meng, X. Y., Kimura, J. Syst. Bacteriol. 45, 848–851. doi: Biol. Reprod. 87, 1–10. doi: Farikou, O., Thevenon, S., Njiokou, N., et al. (2010). Primary gut 10.1099/00207713-45-4-848 10.1095/biolreprod.112.100008 F., Allal, F., Cuny, G., and Geiger, symbiont and secondary, Sodalis- Aksoy, S. (2000). Tsetse—a haven for Berriman, M., Ghedin, E., Hertz- A. (2011). Genetic diversity and allied symbiont of the Scutellerid microorganisms. Parasitol. Today Fowler, C., Blandin, G., Renauld, population structure of the sec- stinkbug . Appl. 16, 114–118. doi: 10.1016/S0169- H., Bartholomeu, D. C. (2005). ondary symbiont of tsetse flies, Environ. Microbiol. 76, 3486–3494. 4758(99)01606-3 The genome of the African Sodalis glossinidius, in sleeping doi: 10.1128/AEM.00421-10 Alam, U., Hyseni, C., Symula, R. E., trypanosome Trypanosoma bru- sickness foci in Cameroon. PLoS Lindh, J. M., and Lehane, M. J. Brelsfoard, C., Wu, Y., Kruglov, cei. Science 309, 416–422. doi: Negl. Trop. Dis. 5:e1281. doi: (2011). The tsetse fly Glossina O., et al. (2012). Implications 10.1126/science.1112642 10.1371/journal.pntd.0001281 fuscipes fuscipes (Diptera: of microfauna-host interactions Boucias, D. G., Kariithi, H. M., Geiger, A., Cuny, G., and Frutos, R. Glossina) harbours a surpris- for trypanosome transmission Bourtzis, K., Schneider, D. I., (2005). Two Tsetse fly species, ing diversity of bacteria other dynamics in Kelley, K., Miller, W. J., et al. Glossina palpalis gambiensis than symbionts. Antonie Van fuscipes in Uganda. Appl. Environ. (2013). Transgenerational trans- and morsi- Leeuwenhoek 99, 711–720. doi: Microbiol. 78, 4627–4637. doi: mission of the Glossina pallidipes tans, carry genetically distinct 10.1007/s10482-010-9546-x 10.1128/AEM.00806-12 hytrosavirus depends on the populations of the secondary sym- Malele, I. I., Manangwa, O., Nyingilili, Alam, U., Medlock, J., Brelsfoard, presence of a functional sym- biont Sodalis glossinidius. Appl. H. H., Kitwika, W. A., Lyaruu, E. C., Pais, R., Lohs, C., Balmand, biome. PLoS ONE 22:e61150. doi: Environ. Microbiol. 71, 8941–8943. A., Msangi, A. R., et al. (2013). S., et al. (2011). Wolbachia sym- 10.1371/journal.pone.0061150 doi: 10.1128/AEM.71.12.8941- Prevalence of SGHV among tsetse biont infections induce strong Chen, X., Li, S., and Aksoy, S. (1999). 8943.2005 species of economic importance cytoplasmic incompatibility in Concordant of a symbiont Geiger, A., Fardeau, M. L., Grebaut, in Tanzania and their implication the tsetse fly Glossina morsitans. with its host insect species: molec- P., Vatunga, G., Josénando, T., for SIT application. J. Invertebr. PLoS Pathog. 7:e1002415. doi: ular phylogeny of genus Glossina Herder, S., et al. (2009). First isola- Pathol. 112(Suppl.)S133–S137. doi: 10.1371/journal.ppat.1002415 and its bacteriome-associated tion of Enterobacter, Enterococcus, 10.1016/j.jip.2012.07.018 Attardo, G. M., Benoit, J. B., endosymbiont, Wigglesworthia and Acinetobacter spp. as inhab- Maltz, M. A., Weiss, B. L., O’Neill, Michalkova, V., Yang, G., Roller, L., glossinidia. J. Mol. Evol. 48, 49–58. itants of the tsetse fly (Glossina M., Wu, Y., and Aksoy, S. (2012). Bohova, J., et al. (2012). Analysis of doi: 10.1007/PL00006444 palpalis palpalis) midgut. Infect. OmpA-mediated biofilm formation lipolysis underlying lactation in the Cheng, Q., and Aksoy, S. (1999). Tissue Genet. Evol. 9, 1364–1370. doi: is essential for the commensal tsetse fly, Glossina morsitans. Insect tropism, transmission and expres- 10.1016/j.meegid.2009.09.013 bacterium Sodalis glossinidius to Biochem. Mol. Biol. 42, 360–370. sion of foreign genes in vivo in Geiger, A., Fardeau, M. L., Njiokou, F., colonize the tsetse fly gut. Appl. doi: 10.1016/j.ibmb.2012.01.007 midgut symbionts of tsetse flies. Joseph, M., Asonganyi, T., Ollivier, Environ. Microbiol. 78, 7760–7768. Attardo, G. M., Lohs, C., Heddi, A., Insect Mol. Biol. 8, 125–132. doi: B., et al. (2011). Bacterial diver- doi: 10.1128/AEM.01858-12 Alam, U. H., Yildirim, S., and Aksoy, 10.1046/j.1365-2583.1999.810125.x sity associated with populations Maudlin, I., Welburn, S. C., and S. (2008). Analysis of milk gland Cheng, Q., Ruel, T. D., Zhou, W., of Glossina spp.fromCameroon Mehlitz, D. (1990). The relationship structure and function in Glossina Moloo,S.K.,Majiwa,P.,O’Neill,S. and distribution within the Campo between -like-organisms morsitans: milk protein production, L., et al. (2000). Tissue distribution sleeping sickness focus. Microb. Ecol. and trypanosome infections in symbiont populations and fecun- and prevalence of Wolbachia infec- 62, 632–643. doi: 10.1007/s00248- natural populations of tsetse in dity. J. Insect Physiol. 54, 1236–1242. tions in tsetse flies, Glossina spp. 011-9830-y Liberia. Trop. Med. Parasitol. 41, doi: 10.1016/j.jinsphys.2008.06.008 Med. Vet. Entomol. 14, 44–50. doi: Geiger, A., Fardeau, M. L., Njiokou, F., 265–267. Attardo,G.M.,Ribeiro,J.M.,Wu, 10.1046/j.1365-2915.2000.00202.x and Ollivier, B. (2013). Glossina spp. Minard, G., Mavingui, P., and Moro, Y., Berriman, M., and Aksoy, S. Dale, C., and Welburn, S. C. Gut bacterial flora and their putative C. V. (2013). Diversity and func- (2010). Transcriptome analysis of (2001). The endosymbionts role fly-hosted trypanosome devel- tion of bacterial microbiota reproductive tissue and intrauterine of tsetse flies: manipulating opment. Cell. Infect. Microbiol. 3, in the mosquito holobiont. developmental stages of the tsetse host-parasite interactions. Int. 1–8. doi: 10.3389/fcimb.2013.00034 Parasit. Vectors 6, 146–157. doi: fly (Glossina morsitans morsitans). J. Parasitol. 1, 628–631. doi: Harley,J.M.B.,andWilson,A.J. 10.1186/1756-3305-6-146 BMC Genomics 11, 160–169. doi: 10.1016/S0020-7519(01)00151-5 (1968). Comparison between Moloo, S. K., and Kutuza, S. B. 10.1186/1471-2164-11-160 Doudoumis, V., Tsiamis, G., Wamwiri, Glossina morsitans, G. pallidipes (1988). Comparative study on the Balmand, S., Lohs, C., Aksoy, S., F., Brelsfoard, C., Alam, U., Aksoy, andG.fuscipesasvectorsoftry- susceptibility of different Glossina and Heddi, A. (2013). Tissue E., et al. (2012). Detection and panosomes of the Trypanosoma species to Trypanosoma brucei bru- distribution and transmis- characterization of Wolbachia infec- congolense group: the proportions cei infection. Trop. Med. Parasitol. sion routes for the tsetse fly tions in laboratory and natural infected experimentally and the 39, 211–213.

Frontiers in Cellular and Infection Microbiology www.frontiersin.org October 2013 | Volume 3 | Article 69 | 5 Wang et al. Tsetse fly microbiota: form and function

Nogge, G. (1978). Aposymbiotic flies and hybrids of Glossina morsi- N., et al. (2010). “Candidatus 299–337. doi: 10.1016/B978-0-12- tsetse flies, Glossina morsitans tans subgroup species. J. Invertebr. Curculioniphilus buchneri,” a novel 398457-9.00004-4 morsitans obtained by feed- Pathol. 112(Suppl.)S104–S115. doi: clade of bacterial endocellular Welburn, S. C., Arnold, K., Maudlin, ing on rabbits immunized 10.1016/j.jip.2012.03.024 symbionts from weevils of the I., and Gooday, G. W. (1993). specifically with symbionts. Simarro, P. P., Jannin, J., and Cattand, genus Curculio. Appl. Environ. Rickettsia-like organisms and chiti- J. Invertebr. Pathol. 24, 299–304. P. (2008). Eliminating human Microbiol. 76, 275–282. doi: nase production in relation to trans- doi: 10.1016/0022-1910(78)90026-4 African trypanosomiasis: where 10.1128/AEM.02154-09 mission of trypanosomes by tsetse Nogge, G., and Gerresheim, A. do we stand and what comes Wang, J., and Aksoy, S. (2012). flies. Parasitology 107, 141–145. doi: (1982). Experiments on the next. PLoS Med. 5:e55. doi: PGRP-LB is a maternally trans- 10.1017/S003118200006724X elimination of symbionts 10.1371/journal.pmed.0050055 mitted immune milk protein Werren, J. H., Baldo, L., and Clark, from the Tsetse-, Glossina- Snyder, A. K., and Rio, R. V. (2013). that influences symbiosis and M. E. (2008). Wolbachia:master Morsitans-Morsitans (Diptera, The interwoven biology of the in tsetse’s offspring. manipulators of invertebrate biol- Glossinidae), by antibiotics and tsetse holobiont. J. Bacteriol. Proc. Natl. Acad. Sci. U.S.A. 109, ogy. Nat. Rev. Microbiol. 6, 741–751. lysozyme. J. Invertebr. Pathol. 40, 195, 4322–4330. doi: 10.1128/ 10552–10557. doi: 10.1073/pnas. doi: 10.1038/nrmicro1969 166–179. doi: 10.1016/0022-2011 JB.00487-13 1116431109 Yang, G., Attardo, G. M., Lohs, C., (82)90112-4 Snyder, A. K., Deberry, J. W., Runyen- Wang, J., Brelsfoard, C., Wu, Y., and and Aksoy, S. (2010). Molecular Pais, R., Lohs, C., Wu, Y., Wang, J., Janecky, L., and Rio, R. V. (2010). Aksoy, S. (2013). Intercommunity characterization of two novel and Aksoy, S. (2008). The obligate Nutrient provisioning facilitates effects on microbiome and milk proteins in the tsetse fly mutualist Wigglesworthia glossinidia homeostasis between tsetse fly GpSGHV density regulation in (Glossina morsitans morsitans). influences reproduction, digestion, (Diptera: Glossinidae)symbionts. tsetse flies. J. Invertebr. Pathol. Insect Mol. Biol. 19, 253–262. doi: and immunity processes of its Proc. R. Soc. 7, 2389–2397. doi: 112(Suppl.)S32–S39. doi: 10.1016/j. 10.1111/j.1365-2583.2009.00987.x host, the tsetse fly. Appl. Environ. 10.1098/rspb.2010.0364 jip.2012.03.028 Microbiol. 74, 5965–5974. doi: Soumana, I. H., Simo, G., Njiokou, Wang, J., Wu, Y., Yang, G., and Aksoy, Conflict of Interest Statement: The 10.1128/AEM.00741-08 F., Tchicaya, B., Abd-Alla, A. M., S. (2009). Interactions between authors declare that the research Rio, R. V., Symula, R. E., Wang, Cuny, G., et al. (2013). The bacte- mutualist Wigglesworthia and tsetse was conducted in the absence J., Lohs, C., Wu, Y., Snyder, rial flora of tsetse fly midgut and peptidoglycan recognition protein of any commercial or financial A. K., et al. (2012). Insight its effect on trypanosome trans- (PGRP-LB) influence trypanosome relationships that could be con- into the transmission biology mission. J. Invertebr. Pathol. 112 transmission. Proc. Natl. Acad. strued as a potential conflict of and species-specific functional (Suppl.)S89–S93. doi: 10.1016/j.jip. Sci. U.S.A. 106, 12133–12138. doi: interest. capabilities of tsetse (Diptera: 2012.03.029 10.1073/pnas.0901226106 glossinidae)obligatesymbiontSymula, R. E., Alam, U., Brelsfoard, Weiss, B. L., Maltz, M., and Received: 13 August 2013; accepted: Wigglesworthia. MBio 14:e00240. C., Wu, Y., Echodu, R., Okedi, L. Aksoy, S. (2012). Obligate sym- 11 October 2013; published online: 29 doi: 10.1128/mBio.00240-11 M., et al. (2013). Wolbachia asso- bionts activate immune system October 2013. Sang, R. C., Jura, W. G., Otieno, L. ciation with the tsetse fly, Glossina development in the tsetse fly. Citation: Wang J, Weiss BL and Aksoy H., Mwangi, R. W., and Ogaja, P. fuscipes fuscipes, reveals high lev- J. Immunol. 188, 3395–3403. doi: S (2013) Tsetse fly microbiota: form and (1999). The effects of a tsetse DNA els of genetic diversity and complex 10.4049/jimmunol.1103691 function. Front. Cell. Infect. Microbiol. virus infection on the functions evolutionary dynamics. BMC Evol. Weiss, B. L., Wang, J., and Aksoy, S. 3:69. doi: 10.3389/fcimb.2013.00069 of the male accessory reproductive Biol. 13, 31–42. doi: 10.1186/1471- (2011). Tsetse immune system mat- This article was submitted to the jour- gland in the host fly Glossina morsi- 2148-13-31 uration requires the presence of nal Frontiers in Cellular and Infection tans centralis (Diptera; Glossinidae). Tobe, S. S. (1978). Reproductive obligate symbionts in larvae. PLoS Microbiology. Curr. Microbiol. 38, 349–354. doi: physiology of Glossina. Annu. Rev. Biol. 9:e1000619. doi: 10.1371/jour- Copyright © 2013 Wang, Weiss and 10.1007/PL00006815 Entomol. 23, 283–307. doi: 10.1146/ nal.pbio.1000619 Aksoy. This is an open-access article dis- Schlein, Y. (1977). Lethal effect of annurev.en.23.010178.001435 Weiss, B. L., Wang, J., Maltz, M. A., tributed under the terms of the Creative on tsetse flies follow- Toh, H., Weiss, B. L., Perkin, S. A., Wu, Y., and Aksoy, S. (2013). Commons Attribution License (CC BY). ing damage to bacterioid sym- Yamashita, A., Oshima, K., Hattori, Trypanosome infection estab- The use, distribution or reproduction bionts. Experientia 33, 450–451. doi: M., et al. (2006). Massive genome lishment in the tsetse fly gut in other forums is permitted, provided 10.1007/BF01922204 erosion and functional adaptations is influenced by microbiome- the original author(s) or licensor are Schneider, D. I., Garschall, K. I., Parker, provide insights into the symbi- regulated host immune barriers. credited and that the original publica- A. G., Abd-Alla, A. M., and Miller, otic lifestyle of Sodalis glossinidius PLoS Pathog. 9:e1003318. doi: tion in this journal is cited, in accor- W. J. (2013). Global Wolbachia in the tsetse host. Genome Res. 16, 10.1371/journal.ppat.1003318 dance with accepted academic practice. prevalence, titer fluctuations and 149–156. doi: 10.1101/gr.4106106 Welburn, S. C., and Maudlin, I. (2012). No use, distribution or reproduction is their potential of causing cyto- Toju, H., Hosokawa, T., Koga, R., Priorities for the elimination of permitted which does not comply with plasmic incompatibilities in tsetse Nikoh, N., Meng, X. Y., Kimura, sleeping sickness. Adv. Parasitol. 79, these terms.

Frontiers in Cellular and Infection Microbiology www.frontiersin.org October 2013 | Volume 3 | Article 69 | 6