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Home , Glossina fuscipes, Glossina morsitans, Paratransgenesis, Sodalis, Wolbachia

The ISME Journal (2015) 9, 1496–1507 & 2015 International Society for Microbial Ecology All rights reserved 1751-7362/15 www..com/ismej MINI REVIEW Adult blood-feeding tsetse , trypanosomes, microbiota and the fluctuating environment in sub-Saharan

Anne Geiger1, Fleur Ponton2,3 and Gustave Simo4 1UMR 177, IRD-CIRAD, CIRAD TA A-17/G, Campus International de Baillarguet, Montpellier Cedex 5, France; 2School of Biological Sciences, The University of Sydney, Sydney, New South Wales, Australia; 3The Charles Perkins Centre, The University of Sydney, Sydney, New South Wales, Australia and 4Molecular Parasitology and Entomology Unit, Department of Biochemistry, Faculty of Science, University of Dschang, Dschang,

The tsetse transmits the protozoan brucei, responsible for African , one of the most neglected tropical diseases. Despite a recent decline in new cases, it is still crucial to develop alternative strategies to combat this disease. Here, we review the literature on the factors that influence trypanosome transmission from the fly vector to its host (particularly ). These factors include climate change effects to and vector development (in particular climate warming), as well as the distribution of host reservoirs. Finally, we present reports on the relationships between vector nutrition, immune function, microbiota and , to demonstrate how continuing research on the evolving ecology of these complex systems will help improve control strategies. In the future, such studies will be of increasing importance to understand how vector-borne diseases are spread in a changing world. The ISME Journal (2015) 9, 1496–1507; doi:10.1038/ismej.2014.236; published online 12 December 2014

Introduction differentiation from the early procyclic form into the late procyclic form (which is established in the gut), African unicellular belonging to the followed by the mesocyclic form (a maturation step Trypanosoma are the causative agents of sleeping in the anterior midgut), followed by the proliferating sickness in humans, where it is known as Human epimastigote form (in the salivary glands or probos- (HAT), as well as in cis, depending on the trypanosome ), and , where it is known as African finally differentiation into the non-proliferating Trypanosomiasis (also known as Nagana). They metacyclic form (in the salivary glands or proboscis, infect successively two hosts during their life cycle depending on the trypanosome species) (Van den (thus they are called ‘digenetic’ parasites): an insect, Abbeele et al., 1999). This last form is the only one the (Glossina spp.) which is required for that is infective for mammals, and is transmitted their transmission to the second host, most often a from the fly’s saliva into a subsequent mammal mammal (and for the transmission from one mam- host’s bloodstream during ingestion of a new blood mal host to another). After the strictly hematopha- meal. If susceptible, this host will possibly become gous tsetse fly bites a trypanosome-infected mammal infected and develop sleeping sickness. Thus, in and takes an infected blood meal, the ingested addition to its role as a trypanosome ‘transporter’, trypanosomes reach the fly midgut, where they the tsetse fly is crucial in providing a milieu where differentiate from the bloodstream form (short the parasite can differentiate, multiply and become stumpy trypomastigotes—present in the mammal infective to mammals. The ability of the fly to blood) into the early procyclic form (Sbicego et al., acquire the parasite, favor its maturation, and 1999). The parasites then differentiate into several transmit it to a mammalian host is called ‘vector forms during their migration from the gut to the competence’, and depends on both the Glossina and salivary glands. This sequential process includes trypanosome species, among other factors. Interestingly, when flies are fed on trypanosome- Correspondence: A Geiger, UMR 177, IRD-CIRAD, CIRAD TA infected blood under optimal laboratory conditions, A-17/G, Campus International de Baillarguet, 34398 Montpellier less than 50% become infected. This demonstrates Cedex 5, France. E-mail: [email protected] that resistance (usually designated as ‘refractori- Received 17 May 2014; revised 3 November 2014; accepted 6 ness’) to trypanosome infection is the normal status November 2014; published online 12 December 2014 of the fly. Midgut infection rates rarely exceed 10% Tsetse fly–trypanosome– relationship A Geiger et al 1497 in natural fly populations. In addition, many The serious nature of this disease has led to its midgut-infected flies do not produce mature para- targeting for elimination by the WHO and PATTEC sites, indicating that they will never become (Pan-African Tsetse and Trypanosomiasis Eradica- infective (Moloo et al., 1986; Dukes et al., 1989; tion Campaign), and subsequently by the London Maudlin and Welburn, 1994). Declaration on Neglected Tropical Diseases. The Four species groups, morsitans, palpalis, austeni number of new cases has begun to decrease in recent and fusca, within tsetse flies are known to transmit years, mirroring a situation that was observed in the different species or subspecies of trypanosomes. The 1960s before the last heavy outbreak in the 1990s. ‘morsitans group’ is the major vector for trypano- So, in spite of this decrease, the severity of the somes of the subgenera Trypanozoon and Nanno- situation demands a deeper understanding of HAT monas, which respectively include Trypanosoma to improve current treatment approaches, as well as brucei brucei (Tbb) and Trypanosoma congolense to help design novel strategies to control the disease. (Tc; the savanah type being the most prevalent in These goals are in line with PATTEC and the WHO- ); these are the main nagana-causing parasites fixed objective to eliminate HAT. As sleeping in sub-Saharan Africa (Nyeko et al., 1990; sickness is a vector-borne disease, control strategies Reifenberg et al., 1997; Solano et al., 1999). This can be focused on the patient (by developing disease is responsible for dramatic losses in live- preventive and/or curative approaches) and/or on stock production, estimated at US$ 4.5 billion/year the tsetse fly vector (to eradicate or impede its vector (Reinhardt, 2002). Furthermore, the morsitans group competence). Unfortunately, these approaches are is the vector of rhodesiense hindered by a lack of vaccines and a limited drug (Tbr), the causative agent of the acute form of HAT toolbox that produces harmful side effects (Simarro that is endemic in 13 east African countries et al., 2008). To complicate matters, current drug (Welburn et al., 2009). On the other hand, the treatments have led to the emergence of resistant palpalis group, which poorly transmits Tbb and Tc trypanosome strains (Baker et al., 2013). (Kazadi, 2000), is the vector of Trypanosoma brucei Domestic (that is, ) and wild animals (that is, gambiense, which is responsible for the chronic diverse rodents, carnivores and primates) found form of HAT in 24 countries of western and central within HAT zones are a valuable nutritional asset Africa (Hoare, 1972; Kennedy, 2008; Welburn et al., for people living in these areas. At the same time, 2009). these animals present a risk to humans, since they HAT develops in two phases. During the first may harbor different trypanosome species, includ- stage (which is hematolymphatic), the parasite ing those specific to HAT. This unfortunately creates proliferates in the blood and the lymph. This may a situation where these animals act as trypanosome progress into the second stage (which is menin- reservoirs (Njiokou et al., 2006; Simo et al., 2006; goencephalitic) if trypanosomes cross the blood– Farikou et al., 2010a), from which their parasites are brain barrier and subsequently invade the central spread by tsetse flies that feed indifferently on nervous system. The second stage is usually humans or other domestic or wild mammals. characterized by severe neurological disorders The cyclical transmission of trypanosomes is and is frequently fatal if not treated. The signs highly dependent on the biochemical and physio- and symptoms are generally similar for the acute logical interactions that occur between the parasite and chronic form of HAT. They differ however in and its insect host. These in turn depend on a frequency, severity and kinetic appearance. Acute variety of biotic and abiotic factors including: form usually progresses to death within 6 months. climate change; geographical distribution and Chronic form has a more progressive course with environmental conditions of HAT foci; tsetse an average duration of almost 3 years (reviewed in fly population flow between foci; disease Dumas and Bouteille, 1996 and reviewed in Franco epidemiology; type and distribution of trypano- et al., 2014). some reservoirs; changes in tsetse fly nutritional HAT is one of the most neglected tropical diseases behavior; and finally the nature and diversity in the world (Brun et al., 2010), even though in of fly intestinal microbiota, including symbiotic terms of mortality it ranks ninth out of 25 human (Wigglesworthia glossinidia, glossinidius infectious and parasitic diseases in Africa (Welburn and Wolbacchia spp.) and diverse non-symbiotic et al., 2009). To this day, sleeping sickness is bacteria (Dale and Maudlin, 1999; Wang et al., responsible for major disruptions to social, agricul- 2013a). Investigations of these factors have already tural and economic development in Africa (Simarro beguninthepastseveralyears. et al., 2011). For instance, the disease was recently By revisiting the existing literature, the present estimated to cause the loss of 1.5 million disability- review on microbial ecology aspects of HAT aims to adjusted life years per year (Hotez et al., 2009). As advance research on how changes in environmental sleeping sickness mostly affects marginalized popu- conditions can affect trypanosome–tsetse fly–gut lations living in isolated rural areas, the disease is a microbiota interactions, and consequently, the severe burden to rural poor populations that often dynamics of the disease. Successful pursuits in do not have access to health facilities (Odiit et al., these areas will enable the design of novel strategies 2004). for disease control.

The ISME Journal Tsetse fly–trypanosome–bacteria relationship A Geiger et al 1498 Global changes and sleeping sickness vertebrate hosts and the trypanosome. Detailed transmission studies of these factors would improve our under- standing of how the disease is spread in environ- Global climate changes are of particular importance ments affected by socioeconomic, environmental to -borne diseases (Rogers and Randolph, and climatic changes. So, the increasing world 2006; Moore et al., 2012). The spread of sleeping population that will, soon, reach seven billion sickness is tripartite, involving the trypanosome, the people (Tollefson, 2011) requires an ever-increasing tsetse fly vector (and its symbionts) and the number of and new farmlands to satisfy vertebrate hosts. The perpetuation of the parasite our nutritional needs. Increasing the number of itself relies on two connected populations, the adult livestock in farmlands drastically alters the sub- tsetse flies as well as the mammals from which they Saharan African environment by modifying not only take their blood meal. Both the fly vectors and the livestock distribution, but also the distribution vertebrate hosts require specific climatic conditions of tsetse flies that feed on it (and possibly their (for example, temperature and humidity) for their nutritional behavior). Indeed, the importance of survival, and propagation (Dean et al., environmental factors to transmission intensity 1969). The ability of trypanosomes to establish in and trypanosome distribution is increasingly being the midgut, and then to migrate to and mature recognized (Van den Bossche et al., 2010; Bouyer within the salivary glands, depends on several biotic et al., 2013). Accordingly, a recent study of climate and abiotic factors. Modification of these factors change effects on the of African trypano- may affect vector competence, which may then somiasis predicts that 46–77 million additional impact trypanosome transmission to host verte- people will be at risk of sleeping sickness by 2090 brates and thus the spread of the disease (Moore et al., 2012). (Figure 1). There is clearly a need for interdisci- plinary investigations to determine how global changes (that is, changing temperature, rainfall patterns, increasing urbanization, deforestation, Impact of global changes on the grassland degradation and overgrazing) could affect developmental rates of trypanosomes and a variety of factors that include: the geographical tsetse flies distribution of trypanosome vertebrate-host reser- In vector-borne diseases, temperatures above 34 1C voirs; the nutritional behavior of tsetse flies, the frequently have a negative impact on the survival of development of trypanosome and tsetse flies; and both the insect vectors and the parasites (Rueda interactions between the tsetse fly vector, the et al., 1990). Thus, such high temperature may also impact unfavorably tsetse fly and trypanosome populations. Tsetse fly pupation and survival requires favorable environmental conditions, including moderate temperature (23–25 1C), high relative humidity (75–90%) with weak saturation deficit (to avoid high evaporation power) and shade (Ndegwa et al., 1992; Courtin et al., 2010; Pagabeleguem et al., 2012). Nevertheless, higher temperatures induce a more rapid blood meal digestion by the tsetse fly; consequently, the fly may feed more frequently, which can increase both the rate of trypanosome ingestion (when the fly feeds on an infected host) and transmission (when the fly have become trypanosome infected and feeds on a non trypanosome-infected host) (Terblanche et al., 2008). Thus, higher temperatures could have both positive and negative effects on HAT transmis- sion. Finally, there may exist an optimal tempera- ture that would favor an optimal balance between vector and parasite populations development rates Figure 1 Factors that may influence tsetse fly susceptibility for and the parasite transmission rate. trypanosome infection. For trypanosome transmission to occur, Moreover, seasonal alternation, local environmen- the parasite must first be established in the tsetse fly midgut following an infective blood meal taken from a mammal source tal changes and differences between geographic acting as a host reservoir of trypanosomes; the trypanosome must areas may modify the balance between the different then mature in the salivary glands or the mouthparts, depending species; this is particularly relevant among wild on the trypanosome species. Many abiotic and biotic factors may that are fed upon by tsetse flies (Staak affect the success or failure of trypanosome development. The role of these factors in vector competence depends on how they et al., 1986; Mukabana et al., 2002; Farikou et al., affect trypanosome development in the tsetse fly, and the fly’s 2010a). Any modification of tsetse fly nutritional susceptibility or resistance to trypanosome infection. behavior may impact trypanosome transmission, as

The ISME Journal Tsetse fly–trypanosome–bacteria relationship A Geiger et al 1499 well as the spread of HAT and Animal African As stated in the introduction, the rate of trypano- Trypanosomiasis. A fly’s nutritional behavior can some midgut colonization in the tsetse fly is be determined by identifying the origin of its generally low (Moloo et al., 1986; Maudlin and ingested blood meal and which residues are still Welburn, 1994). During this midgut colonization present in the gut. Such an investigation was step, tsetse flies employ mechanisms for eliminating performed in the Bipindi and Campo HAT foci of the trypanosomes, whereas the parasites attempt to south Cameroon in 2008, which established that evade the tsetse fly for their own the collected flies had taken their blood meals from survival (Aksoy et al., 2003). Several molecules can humans (46%), pigs (37%) and wild mammals be released during the time course of these interac- (17%). Notable differences between the two foci tions (Table 1), either by tsetse flies or by trypano- were nevertheless recorded: in Bipindi, 23% and somes. This release can be modified according to 67% of flies took their blood meal from humans fly-intrinsic (for example, sex of flies) and fly- and pigs, respectively, whereas these figures were extrinsic factors (for example, trypanosome species; respectively 63% and 23% for flies in Campo. starvation). Therefore, successful establishment There were also substantial differences observed (that is, midgut colonization) of trypanosomes in between years: in 2004, 45% and 7% of flies tsetse depends on their ability to adapt, transform, respectively from Bipindi and Campo took their survive and grow rapidly after their quick transition blood meal from pigs, versus 67% and 23%, from the vertebrate host blood to the different respectively, in 2008 (Simo et al., 2008, Farikou environment of the tsetse gut (Simo et al., 2010). et al., 2010a). These results illustrate how the Trypanosome invasion activates innate immune nutritional behavior of tsetse flies depends on the responses within tsetse flies (Hao et al., 2001; Hu geographical area and how quickly it can change and Aksoy, 2006) by inducing the tsetse production over a relatively short period. of several molecules including: antimicrobial pep- tide; glutamine/proline-rich (EP) ; reactive oxygen species; and several other molecules Developmental and immune responses in involved in the immunodeficiency (Imd) pathway the trypanosome–tsetse fly association (Hao et al., 2001; Lehane et al., 2003; Hu and Aksoy, 2006; Nayduch and Aksoy, 2007; MacLeod et al., Trypanosomes undergo several rounds of differen- 2007a; Haines et al., 2010). In fact, the balance tiation and proliferation during their life cycle. between the released molecules has an important Although the development cycle differs somewhat role in the success or failure of trypanosome between trypanosome species, the two main stages establishment within the tsetse fly midgut, as it is consist of establishment and maturation steps. Both crucial for creating a suitable environment for T. congolense (subgenus Nannomonas) and T. brucei their survival and development. For example, the (subgenus Trypanozoon) establish within the fly prevalence of trypanosomes increased in flies when midgut (midgut colonization step) but mature in the the expression of either the Imd pathway or the tsetse proboscis and salivary glands, respectively; downstream-expressed antimicrobial effec- the development cycle then culminates with the tor was downregulated by RNAi (Hao et al., 2001; metacyclic form that is infective for mammals Hu and Aksoy, 2006). MacLeod et al. (2007a) have (humans, wild or domestic animals) (Van den also shown that antioxidants promote the establish- Abbeele et al., 1999). ment of trypanosome in tsetse flies.

Table 1 Effects of various intrinsic and extrinsic factors on trypanosome development within the tsetse fly vector

Factors Produced by Effect References

Attacin Tsetse fly Trypanocidal activity Hao et al., 2001; Hu and Aksoy, 2006 Diptericin Tsetse fly Trypanocidal activity Hao et al., 2001; Hu and Aksoy, 2006 Glutamin/proline-rich (EP) protein Tsetse fly Inhibits trypanosome establishment Haines et al., 2010 Reactive oxygen species Tsetse fly Inhibits trypanosome establishment Hao et al., 2001; Lehane et al., 2003; Hu and Aksoy, 2006; Nayduch and Aksoy, 2007; MacLeod et al., 2007a Nitrogen monoxide (NO) Tsetse fly Promotes trypanosome migration to MacLeod et al., 2007b salivary glands and maturation L-Cysteine Tsetse fly Promotes trypanosome migration to MacLeod et al., 2007b salivary glands and maturation Purines Tsetse fly Promotes trypanosome survival Henriques et al., 2003 Heat-shock protein 70 Trypanosome Reaction against stress in fly midgut Simo et al., 2010 Heat-shock protein 83 Starvation Tsetse fly Increases establishment and/or Kubi et al., 2006 maturation of trypanosome in tsetse fly and offspring

The ISME Journal Tsetse fly–trypanosome–bacteria relationship A Geiger et al 1500 Subsequently, trypanosomes require signals such as 2013a). Wigglesworthia encodes vitamins that may L-cysteine and/or nitric oxide, as well as environ- promote host reproduction as well as fly nutrition mental stimuli, for their migration to the salivary throughout its development (Nogge, 1982; Rio et al., glands, where they mature (MacLeod et al., 2007b). 2012). In addition to the midgut, Sodalis develops in In the presence of trypanosomes, tsetse flies will several other tsetse fly (Glossina spp.) organs (Wang modify the expression of several of their . In et al., 2013a). The specific elimination of Sodalis response to this differential regulation, trypa- has been reported to result in reduced tsetse fly nosomes regulate the expression of their own genes longevity (Dale and Welburn, 2001; Wang et al., for their survival (Savage et al., 2012). For example, 2013b). T. b. gambiense and T. b. rhodesiense express genes Tsetse flies can also harbor a third symbiont, the associated with reactions against stress (Simo et al., a-proteobacterium (O’Neill et al., 1993), 2010), indicating that trypanosomes are exposed to which is a non-essential bacterium that infects many environmental stress within the tsetse fly midgut. different invertebrates (Werren et al., 2008). The Owing to the delicate equilibrium governing these presence of this bacterium is restricted to the molecular interactions, any internal or external reproductive organs of the tsetse fly and is trans- perturbation may impact the fly–trypanosome rela- mitted transovarially (Wang et al., 2013a). Although tionship. For instance, injecting tsetse flies with it is highly prevalent within laboratory-reared tsetse E. coli was shown to stimulate their immune system, fly colonies (Cheng et al., 2000), its prevalence resulting in a severe blocking of trypanosome in natural tsetse fly populations is variable establishment subsequent to any infected blood (Doudoumis et al., 2012). Wolbachia has also been meal (Hao et al., 2001). Therefore, the upregulation shown to induce strong cytoplasmic incompatibility of several immune responsive genes early in infec- in tsetse, as by the second gonotrophic cycle, none tion can act to block parasite transmission. These of the females in an incompatible cross yield any results have previously been discussed in the progeny (Alam et al., 2011). This phenomenon context of potentially using transgenic approaches occurs when a Wolbachia-infected male mates with to modulate tsetse fly vector competence (Hao et al., an uninfected female resulting in degeneration of 2001). A deficit in mammal blood meal availability, the future embryo. In contrast, when a Wolbachia- and other environmental factors, can also cause infected female mates with either an uninfected nutritional stress in a tsetse population; in addition male or a male infected with the same as the to making tsetse flies significantly more susceptible female, the female will produce viable Wolbachia- to midgut infection, these factors boost the matura- infected offspring. Furthermore, these offspring will tion of midgut infections (Akoda et al., 2009a). be more numerous than those produced by a non These examples illustrate how external factors, Wolbachia-infected female after with a non which do not depend on the trypanosome or tsetse Wolbachia-infected male (Alam et al., 2011). This fly, can modify their association. In this context, reproductive advantage for infected females has two environmental changes can impact the biochemis- implications. First, it results in the spread of try, physiology and even survival of tsetse flies and Wolbachia infections along with the other traits trypanosomes. Their interactions will also be (Sodalis) that the infected might display affected as a consequence, since both organisms (Hoffman et al., 1998; Dobson et al., 2002). Second, must first adapt their physiology to the modified it produces a progressive replacement of the initial environmental conditions. Very recent develop- fly population by a population of Wolbachia- ments in the genomics of the tsetse fly infected flies (Alam et al., 2011; Medlock et al., (International Glossina Genome Initiative, 2014) 2013). now provide novel ways to further investigate these Recent investigations of the midgut microbiota tsetse–trypanosome interactions through compara- composition in natural tsetse fly populations col- tive and functional genomics. lected from HAT foci in three African countries (, Cameroon and ) have revealed the presence of an unexpectedly diverse bacterial com- Impacts of the tsetse fly microbiome and munity (more than 10 bacteria species in Glossina nutrition on fly physiology and fuscipes fuscipes from Kenya (identified using Trypanosoma transmission culture-depending and non culture-depending methods), more than 5 bacteria species in G. p. Tsetse flies harbor three bacterial symbionts, includ- palpalis from Cameroon (using culture-depending ing the obligate primary (essential) symbiont Wig- methods)) (Geiger et al., 2009, 2011; Lindh and glesworthia glossinidia (Wang et al., 2013a) and the Lehane, 2011). Their diversity was shown to depend secondary (non-essential) symbiont Sodalis glossi- on the tsetse species or subspecies, as well as on the nidius (Dale and Maudlin, 1999). Both symbionts, geographic origin, although differences in environ- which belong to the Enterobacteriaceae family, mental conditions and food supply may also colonize the tsetse fly gut (Aksoy et al., 2013) and influence the diversity of the harbored bacterial are vertically transmitted to the intrauterine-devel- communities. Recently, Aksoy et al. (2014) investi- oping larvae via milk gland secretions (Wang et al., gated the different levels and patterns of gut

The ISME Journal Tsetse fly–trypanosome–bacteria relationship A Geiger et al 1501 microbial diversity among individuals from tsetse in contrast, flies carrying Wigglesworthia are fly populations in ( highly resistant (Wang et al., 2009). Furthermore, morsitans, G. f. fuscipes and Glossina pallidipes), comparisons between Wigglesworthia spp. from using multiple approaches such as deep sequencing G. morsitans morsitans and from G. brevipalpis have of the V4 hypervariable region of the 16S rRNA revealed metabolic variations. These differences gene, 16S rRNA gene clone libraries, and bacterium- involve the chorismate, phenylalanine and folate specific quantitative PCR. In contrast to the former, biosynthetic pathways, which are only present this study revealed an extremely limited microbiota in Wigglesworthia from G. morsitans morsitans. diversity in the investigated flies. The obligate African trypanosomes are auxotrophic for these Wigglesworthia was dominant in all molecules and salvage them exogenously. This samples (499%), and a wide prevalence of low- could explain the differences observed in trypano- density Sodalis infections (o0.05%) was also some susceptibility between these two tsetse species observed. However, 22% of the samples displayed (Rio et al., 2012). high Sodalis density colonization; they also carried Colonization with S. glossinidius has been shown co-infections with Serratia. The wild fly micro- to increase susceptibility for trypanosomes in tsetse biomes display more bacterial species than insec- flies (Welburn et al., 1993) through a mechanism tary-reared flies, where, by now, only one species, a involving the production of N-acetyl glucosamine novel one named Serratia glossinae, has been (Maudlin and Ellis, 1985; Welburn and Maudlin, previously identified using a culture-dependent 1999). This sugar results from the hydrolysis of method (Geiger et al., 2010). Finally, bacteria pupae chitin, by an endochitinase from S. glossini- diversity characterized in wild flies was very dius. Furthermore, this sugar is reported to inhibit a variable depending on fly species, geographical midgut lectin from the tsetse fly, which is lethal to origin as well as on the different microbiome trypanosome procyclic forms (Welburn and analysis techniques used. Thus, the need to pursue Maudlin, 1999; Dale and Welburn, 2001). More and extend such investigations. recently, it was reported that the ability of two Colonization of the gut by microbial communities different trypanosome subspecies to establish in the may or may not increase tsetse fly resistance against tsetse fly midgut is significantly linked to the trypanosome invasion. Underlying mechanisms presence of S. glossinidius-specific genotypes include competition for nutrients, niche occupation (Geiger et al., 2007). This suggests that different and stimulation of immune responses (Stecher and Sodalis genotypes might be associated with differing Hardt, 2011; Brestoff and Artis, 2013; Engel and capacities for trypanosome-establishment facilita- Moran, 2013; Furusawa et al., 2014). Recent data tion. In addition, susceptibility may increase in suggest that Sodalis and Wigglesworthia can response to a greater density of the symbiont in the modulate trypanosome development (Table 2). fly gut (Cheng and Aksoy, 1999). The favorable effect Wigglesworthia must be present during the of Sodalis on fly infection by trypanosomes has immature larval stages for the adult tsetse fly recently been assessed in large tsetse fly sampling immune system to develop and function properly campaigns conducted in two sleeping sickness foci (Weiss et al., 2011). The artificial elimination of in southern Cameroon (Farikou et al., 2010b). Wigglesworthia from flies does not only render Finally, additional diversity analyses have shown them sterile, but will also compromise their that the geographical isolation of the two foci may immune system development. This in turn increases have induced the independent evolution of Sodalis fly susceptibility to gut trypanosome infection; and tsetse fly populations, suggesting a probable

Table 2 Effects of various tsetse fly symbionts on tsetse fly susceptibility to trypanosome infection

Factors Symbiont Effect References

Unknown Wigglesworthia glossinidia Development of tsetse immune system and resis- Wang et al., 2009; Weiss et al., 2011 tance to trypanosome infection Chorismate, Wigglesworthia glossinidia Increases susceptibility of Glossina morsitans mor- Rio et al., 2012 phenylalanine, strain sitans species to trypanosomes folate Chitinase Increases susceptibility of tsetse to trypanosomes Welburn et al., 1993 Density Sodalis glossinidius Increases susceptibility of tsetse to trypanosomes Cheng and Aksoy, 1999 Genotypes Sodalis glossinidius Associated with tsetse fly infection by Geiger et al., 2007 different trypanosome species Unknown Sodalis glossinidius Effect on fly infection Farikou et al., 2010b Haplotypes Sodalis glossinidius Associated with prevalence of tsetse fly infection Farikou et al., 2011 Phage Sodalis glossinidius Associated with tsetse fly resistance to Hamidou Soumana et al., 2014 trypanosome infection Unknown Wolbachia sp. Inhibits development of trypanosome in Aksoy et al., 2013 tsetse fly

The ISME Journal Tsetse fly–trypanosome–bacteria relationship A Geiger et al 1502 coevolution between Sodalis and tsetse flies ingested blood can be just as important as the (Farikou et al., 2011). Taken together, these data quantity (Broderick et al., 2004; Chandler et al., could help explain reported epidemiological differ- 2011). Preference for tsetse fly mammalian hosts ences in HAT cases between HAT foci. Recently, (human, wild or domestic animals) can differ greatly the transcriptional signature of Sodalis hosted by according to Glossina species, wild life and geogra- trypanosome-infected flies was compared with that phical locations (Omolo et al., 2009; Farikou et al., of Sodalis hosted by refractory flies (that is, flies that 2010a; Muturi et al., 2011). Due to the difference in were not infected despite having taken a trypano- blood composition between different mammalian some-infected blood meal). Many of the modulated hosts (human, wild or domestic animals), it can be transcripts in the symbiont population within flies expected that blood meals taken from different host refractory to trypanosome infection cluster within types will differentially influence gut microbiota networks involving lysozyme activity, bacteriolytic composition in tsetse flies, which might explain enzymes, bacterial cytolysis and cell wall macro- some of the geographical variation previously molecule catabolic processes. These observations observed (Geiger et al., 2009, 2011). In addition, suggest the possible involvement of a Sodalis-hosted flies may ingest bacteria within the environment, prophage in tsetse Trypanosoma resistance particularly from the skin surface of hosts during (Hamidou Soumana et al., 2014). blood meals (Poinar et al., 1979; Simo et al., 2008, Several other mechanisms may be involved in the Farikou et al., 2010a). Blood composition and modulation of trypanosome infection by midgut sources may therefore be important factors that microbiota including the production of anti-para- modulate vector competence, through complex sitic molecules by the bacteria inhabiting the tsetse interactions between nutrients, immunity and bac- fly vector gut (reviewed in Azambuja et al., 2005). terial communities. For example, pigment-producing bacteria have already been identified in the tsetse fly midgut (Geiger et al., 2009, 2010, 2011; Lindh and Lehane, Implications for large-scale tsetse fly 2011) and the prodigiosin pigment is reported to be control toxic to (Lazaro et al., 2002) and Trypanosoma cruzi (Azambuja et al., 2004). Current strategies for insect pest control manage- Nutrition also affects the susceptibility of tsetse ment include the application of chemical insecti- flies to trypanosomal infections, since extreme cides, the dissemination of sterile male insects and starvation periods in teneral (young flies that have the introduction of natural predators (including lady never taken a blood meal) and non-teneral tsetse beetles) or parasites (including parasitic ) flies can increase the proportion of adult flies, and (Engel and Moran, 2013). Environmental factors their offspring, that will develop mature trypano- influence microbial interactions and the resilience some infections that can be transmitted to humans of a community, which is in turn influenced by (Kubi et al. 2006; Akoda et al., 2009b). Previous microbial diversity (Masurekar, 2008). These factors studies have suggested that immune function is will then influence vector competence. Previous affected by the nutritional state of the fly, as well insight on the interactions between tsetse flies, (Attardo et al., 2006). Bacterial populations may also trypanosomes, microbiota and the environment can vary in persistence, abundance and species compo- also be used to improve the control of tsetse flies, by sition within the tsetse fly host; the host environ- providing clues on how to manipulate tsetse fly gut ment and nutrition are major determinants in this . This approach may also be of case (Chandler et al., 2011). This underscores the practical value for generating novel modes of pest importance of defining the relationships between biocontrol. A number of bacteria-based approaches diet and the composition and function of gut have also been suggested, some of which have been microbiota (Ponton et al., 2011, 2013). For example, successfully implemented (Aksoy et al., 2008; Engel blood meals have been associated with massive and Moran, 2013). proliferation of bacteria residing in the digestive One recent review (Engel and Moran, 2013) has tract (Kumar et al., 2010; Oliveira et al., 2011; Wang reported that the composition of the gut microbiota et al., 2011) through the effects of reactive oxygen in invertebrate hosts could influence vector compe- species levels (Oliveira et al., 2011). This process tence via different approaches including the mod- has been demonstrated in the mosquito midgut, ulation of immune responses, niche competition or where a blood meal immediately decreases the level production of inhibitory molecules (Azambuja et al., of reactive oxygen species through a mechanism 2005; Dong et al., 2009; Hoffmann et al., 2011; involving heme-mediated protein kinase C activa- Cirimotich et al., 2011a, b). Therefore, it is likely that tion, creating a favorable environment for bacterial induced modifications of the gut microbiota proliferation (Oliveira et al., 2011). composition could impact the vector competence As a food source, blood contains a number of of flies. As mentioned above, natural tsetse flies components that can interfere with insect physiol- are colonized by a taxonomically diverse array ogy (Luckhart and Riehle, 2007; Kang et al., 2008; of microbiota (Geiger et al., 2009, 2011; Aksoy Pakpour et al., 2013), such that the quality of et al., 2014).

The ISME Journal Tsetse fly–trypanosome–bacteria relationship A Geiger et al 1503 One pest control method in particular, para- Similar experiments could be conducted on transgenesis (Aksoy et al., 2008), uses modified g-irradiated tsetse flies, an approach used to eradi- symbionts to express molecules that could increase cate isolated tsetse populations (Vreysen et al., tsetse fly resistance to trypanosomes, by stopping 2000), since irradiation may modify their intestinal the development of parasites. This method is bacterial community content and so decrease their suggested instead of fly transgenesis, since tsetse fitness and sexual competitiveness. flies are viviparous (Attardo et al., 2006): their embryos and larvae develop in utero,rendering microinjection of transgenes into the embryo very Concluding remarks and future difficult. The symbiont Sodalis may be used for perspectives paratransgenesis (Medlock et al., 2013), as it is cultivable and thus suitable for genetic manipula- In spite of the recent decline in cases, the possibility tion in vitro (Aksoy et al., 2008). Wigglesworthia, of an expansion of HAT and other diseases must not by contrast, cannot be used as it is uncultivable. be underestimated, as reflected by the recent out- Importantly, the Sodalis symbiont inhabits the break of Ebola virus disease in western Africa. tsetse gut in immediate proximity with trypano- Today, a broad range of data are available in disease somes, thereby directly exposing them to Sodalis fields concerned with sleeping sickness. However, effector (De Vooght et al., 2012). Sodalis is these data are dispersed and one major difficulty is vertically transmitted to tsetse offspring and can thus transmit the manipulated character from Box 1 Further issues to develop. one generation to the next. Finally, the Sodalis genome is rich in pseudogenes, making it suscep- Some proposals we provide have immediate tible to large-scale gene erosion (Toh et al., 2006). practical applications, whereas others will first Due to its reduced functional genome, Sodalis is require fundamental investigations. metabolically dependent on tsetse flies for survi- val; it has never been found associated with other 1. Realize an exhaustive cartography of the HAT insects. This makes Sodalis a potentially safe foci, especially in isolated bush areas. candidate for a paratransgenesis approach. In 2. Evaluate the asymptomatic HAT prevalence, practice, tsetse flies harboring a recombinant and the expansion of this disease. Sodalis strain encoding trypanosome resistance 3. Develop dispensaries in isolated areas. genes must be disseminated into natural fly 4. Generate a ‘sentinel’ network. populations, so as to replace the current suscep- 5. Develop multidisciplinary investigations to tible population (Alam et al., 2011). Wolbachia- improve our understanding of the environ- induced cytoplasmic incompatibility can be mental and human factors that favor the exploited to drive this rapid dissemination within maintaining of the endemic stage of the natural populations of tsetse fly vectors and disease, or epidemic outbreaks. progressively replace it, thereby allowing disease 6. On the basis of (5), develop predictive control (Alam et al., 2011). approaches to identify areas potentially at In addition to its role in the Sodalis/Wolbachia risk of sleeping disease following environ- couple (dissemination of flies harboring the recom- mental modifications. binant Sodalis), Wolbachia may be used alone. The 7. More extensive characterization of the com- embryonic death caused by Wolbachia-induced position of tsetse fly microbiota. cytoplasmic incompatibility can be applied to 8. Develop integrated investigations of the inter- suppress tsetse fly populations (Alam et al., 2011; actions between the fly, its microbiome and Aksoy et al., 2013). Another advantage of the the trypanosome, to identify genes involved Wolbachia symbiont is its capability to inhibit the in susceptibility/refractoriness of the fly to development of trypanosomes in tsetse (Table 2) trypanosome infection. (Aksoy et al., 2013). 9. Investigate the vertical and horizontal trans- One alternative to paratransgenesis could be to mission of the bacteria species hosted by the increase the prevalence of gut bacteria that are female fly. naturally present in tsetse flies and that are capable 10. Characterize the genetic diversity within popu- of reducing the trypanosome load in tsetse fly lations of the different fly species, as well as natural populations. their indigenous symbiont Sodalis glossinidius Finally, several examples of the effect of insect gut populations, to detect a possible relationship bacteria on the fitness or sexual competitiveness of between tsetse fly and Sodalis genotypes. insect vectors have been reported. One promising 11. Evaluate the feasibility of using genetically approach has been observed by feeding the tephritid engineered bacteria that would express trypa- fly Ceratitis capitata with a specific bacterial nocidal molecules in the fly’s gut or molecules diet that can improve the fitness and sexual capable of blocking the trypanosome develop- competitiveness of g-irradiated sterile male insects ment cycle, to block the fly’s vector competence. (Gavriel et al., 2011; Engel and Moran, 2013).

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