Sodalis Glossinidius and Wolbachia Infections in Wild Population of Glossina Morsitans Submorsitans Caught in the Area of Lake Iro in the South of Chad

Djoukzoumka Signaboubo University of Dschang Vincent Khan Payne University of Dschang Ibrahim Mahamat Alhaj Moussa University of Bremen Mahamat Hassane Mahamat Institut de Recherche en Elevage pour le Développement Sartrien Tagueu Kanté University of Dschang Youssouf Mouliom Mfopit Institute of Agricultural Research for development Petra Berger University of Bremen Soerge Kelm University of Bremen Gustave Simo (  gsimoca@yahoo.fr ) University of Dschang


Introduction
Tsetse ies are biological vectors of African trypanosomes that cause human and animal African trypanosomiases respectively in humans and animals. Human African trypanosomiasis (HAT) is caused by two subspecies of trypanosomes: Trypanosoma brucei rhodesiense which is responsible of the acute form of HAT in eastern and southern Africa, and T. b. gambiense that causes the chronic form of HAT in western and central Africa [1]. About 65 million people are at risk of HAT and currently, the number of reported cases is, for the rst time below 2000 new cases [2]. Control efforts undertaken in the last three decades have brought HAT under control and led to its inclusion into the WHO "roadmap for the interruption of transmission to human by 2030 [3].
Animal African trypanosomiases (AAT) are caused by several trypanosomes species and subspecies including for instance T. b. brucei, T. congolense, T. vivax and T. simiae. These diseases remain a constraint for animal production and agriculture development. In absence of vaccine for African trypanosomiases, the control strategies deployed against these infectious diseases rely mainly on the diagnosis and treatment of infected mammals and vector control. The development of drug-resistant trypanosomes could jeopardize the control measures aiming to eliminate the protozoan parasites through treatment of infected hosts. Moreover, the strategy relying on treatment of infected mammals cannot be applied on wild animals that could serve as reservoirs of different trypanosome species and subspecies. In such context, vector control remains a very important component for the management of African trypanosomiases [4]. Integrating vector control as key component of new control strategies is becoming crucial to achieve the complete interruption of HAT transmission and boost AAT control. In this light, several approaches including the setup of tsetse traps, screens or "tiny targets", the use of insecticide and the modi cation tsetse biotopes have been developed to ght tsetse ies [5]. Although the implementation of these approaches enabled to reduce tsetse populations in most settings, their sustainability remains challenging and some of these approaches have environmental impacts [6]. There is a need to develop innovative vector control methods that may not have such impacts. In recent decades, growing interests have been focused on factors enable to interfere with the vectorial competence of tsetse ies [7,8]. It is in this light that interactions between trypanosomes, tsetse and symbiotic microorganisms have been investigated [9,10]. Three symbiotic microorganisms including Wigglesworthia glossinidia, Sodalis glossinidius and Wolbachia have been associated with tsetse.
Wigglesworthia glossinidia is an obligate primary symbiont on which tsetse depend for their vital physiological functions such as host fertility and immune maturation [11,12,9]. Sodalis glossinidius is a secondary and non-essential symbiont. Although its biological function remains unknown [13,14], S. glossinidius has been suspected to play a role in the susceptibility of tsetse to trypanosome infections by favoring midgut establishment of trypanosomes through a complex biochemical mechanism [13,15,11]. Wolbachia are also non-essential symbionts found in a wide range of arthropods and nematodes [16,17,18]. They are transmitted from mother to offspring and can protect their hosts against viral pathogens [19,20]. Abundant in both male and female germ-cells as well as somatic tissues, Wolbachia are able to induce cytoplasmic incompatibility which leads to embryonic death in tsetse ies [21]. With such ability, investigations on Wolbachia could improve vector control through the development of transgenic tsetse that have the ability of releasing speci c molecules that can interfere with the establishment of trypanosomes.
In wild tsetse populations from several tsetse infested regions, S. glossinidius and Wolbachia have been reported in G. m. morsitans, G. m. centralis, G. f. fuscipes, G. austeni, G. pallidipes, G. p. palpalis, G. f. quanzensis and G. brevipalpis [7, 22, 23, 24, 20; 25, 26, 27]. Previous investigations on the tripartite association reported contrasting results in different ecosystems. A negative association has been reported between Wolbachia and trypanosomes in G. f. fuscipes, suggesting that the presence of Wolbachia could prevent trypanosome infections [27]. In tsetse of palpalis group, no association has been reported between Wolbachia and trypanosome infections [28]. For S. glossinidius, some authors reported a positive association between S. glossinidius and trypanosome infections [29] while others found that the presence of S. glossinidius does not seem to favor trypanosome infections in G. p. palpalis [25]. The tripartite association between tsetse, symbiotic microorganisms and trypanosome infections seems to vary according to tsetse species as well as ecological settings. Decrypting the role that each symbiotic microorganisms could play in the establishment and the development of trypanosomes in tsetse species of each ecological setting is importance for the understanding of vectorial competence of tsetse ies.
In the present study, Wolbachia and S. glossinidius were screened in wild populations of G. m. submorsistans caught in the area of Lake Iro in the south of Chad with the aim of generating data that may help to understand the in uence of these symbiotic microorganisms on the vectorial competence of G. m. submorsistans.  2) and sequencing, a DNA fragment of 120 bp was obtained. In addition, the obtained sequences must also show at least 98% of similarity with those of S. glossinidius available in the data base.

Discussion
Challenges limiting the appraisal of tsetse microbiome include the di culties to identify bacterial species in these ies. To ll these gaps in understudied ecological settings, PCR-based method was used to identify S. glossinidius and Wolbachia in wild population of G. m. submorsitans caught in the area of lake Iro in the south of Chad. The identi cation of S. glossinidius and Wolbachia in wild population of G. m. submorsitans of Lake Iro is in line with previous studies reporting these two symbiotic microorganisms in wild populations of G. m. morsitans, G. tachinoides, G. p. palpalis, G. pallidipes, G. f. quanzensis and G. brevipalpis [2010,30,31,25,26,22,23].
The S. glossinidius infetion rate of 9.0% obtained in the present study is similar to 9.3% reported in Liberia for G. p. palpalis [29]. This rate in higher than 1.4% reported in Zambia for wild populations of G. pallidipes [31], but lower than 15.65%, 17.5%, 54.9% and 93.7% previously reported respectively in the Democratic Republic of Congo for G. f. quanzensis, in Zambia for G. m. morsitans, in Cameroon for G. p. palpalis and in Zambia for G. brevipalpis [7,31,23]. These results con rm the high heterogeneity of S. glossinidius infection rates according to tsetse species [31,7]. Nevertheless, reliable comparisons between data from by different studies require to understand the study designs. In the present study, S. glossinidius was identi ed in tsetse body while in other studies, whole tsetse or parts of the insect such as the abdomen, the thorax and the legs were used. The heterogeneity observed in the S. glossinidius infection rates could be explained by some variations in methodological approaches, the intrinsic characters of each tsetse species and environmental factors (vegetation, humidity, temperature) encountered in different ecological settings. In natural conditions where environmental factors vary and have impacts on the biology of tsetse ies, the relationship between tsetse and its symbiotic microorganisms is affected. As the survival and the transmission of these symbiotic micro-organisms are linked to tsetse biology because of their limited metabolic capacities, each environmental factor affecting the biology of tsetse could change its interactions with symbiotic micro-organisms. In such a scenario, S. glossinidius could not undergo horizontal transmission with the same e ciency and in consequence, its infection rates could vary with environmental factors. When environmental variations are removed like in experimental studies or in insectarium [33], the symbiotic association between tsetse and its symbionts is not affected. As already reported in G. p. gambiense and G. m. morsitans, high vertical transmission and high infection rates of symbiotic micro-organisms are observed in tsetse ies [11].
The signi cant association (r = 4.992; P = 0.025; 95% CI= [0.178-5.012]) observed between S. glossinidius and trypanosome infections indicates that the presence of S. glossinidius seems to favor trypanosome infections in G. m. submorsitans of the area of lake Iro in the south of Chad. Although these results are not in agreement with those reporting no signi cant association between the presence of S. glossinidius and trypanosome infections in G. austeni [20], G. brevipalpis, G. m. morsitans and G. pallidipes [31], our ndings are in line with those reported in G. p. palpalis of some sleeping sickness foci of Cameroon [7] and other tsetse species [20,8,11]. The discrepancies observed in the tripartite association between tsetse, S. glossinidius and trypanosomes may result from differences in the biology of different tsetse species as well as the bioclimatic conditions impacting the relationship between tsetse and its symbiotic micro-organisms. Moreover, our results showing signi cant association (r = 3.147; P = 0.043; 95% CI= [0.178-5.012]) between S. glossinidius and T. simiae, but no association for other trypanosome species identi ed in this study suggest that the tripartite association between tsetse, S. glossinidius and trypanosomes could vary according to trypanosome species. Better understanding these tripartite associations requires more in-depth investigations on wild populations of different tsetse species of various tsetse infested areas.
The identi cation of Wolbachia in wild populations of G. m. submorsitans may have some implications in the development of new vector control strategies. On the basis of its capacity of inducing cytoplasmic incompatibility and to be transmitted from mother to offspring, Wolbachia can be genetically modi ed with the objective of producing biomolecules able to interfer with the establishment and/or the development of trypanosomes in tsetse ies. If that occurs, the vectorial competence of tsetse will be affected and disease transmission could be blocked through genetically modi ed Wolbachia strains that conferred resistance to tsetse y [26].
The overall Wolbachia infection rate of 14.5% obtained in the present study is lower than 25.32%, 44.3%, 88.8%, 98% and 100% reported respectively in G. p. palpalis [26], G. f. fuscipes [27], G. f. Quanzensis [23], G. austeni [33,20] and G. m. morsitans [34]. These results show a certain heterogeneity in the Wolbachia infection rates according to tsetse species. As already reported by Kante et al. [26], this heterogeneity could be related to speci c biological characteristics of each tsetse subspecies. For identical stimulus, it has been reported that interactions between tsetse y and its symbiotic micro-organisms vary according to speci c biological response of each tsetse species or subspecies [26]. Such variations affect not only the interactions between tsetse and its symbiotic micro-organism, but also the Wolbachia infection rates. Some discrepancies observed in the Wolbachia infection rates could be explained by some differences in the study design as well as the analytical methods. In the present study, Wolbachia was searched in tsetse body (whole tsetse without legs, wings and proboscis) while in other studies, investigations were undertaken on isolated tissues or whole tsetse y. In addition, the fact that one molecular marker was used to detect Wolbachia infections has probabily underestimated its infections rates. Indeed, in tsetse ies from the same ecological setting, Kante et al.
[26] reported signi cant differences in the Wolbachia infection rates when different molecular markers were used. In Camerooun for instance, the detection of wsp gene was two-fold more sensitive in tsetse from Campo while 16S rDNA showed higher sensitivity in ies from Fontem [26]. In addition to differences in the sensitivity of molecular markers, the technical approaches could also have impacts on the Wolbachia infection rates. If the density of Wolbachia in some G. m. submorsitans is below the detection threshold of standard PCR-based method, some infections could pass undetected. Wamwiri et al. [20] highlight a high density of Wolbachia in G. austeni populations from Kenya and a low density in the same tsetse species of South Africa. In addition, a low density of Wolbachia has been reported in Rhagoletis cerasi [35] and Drosophila paulistorum [36]. While searching for sensitive and reliable markers or tools for Wolbachia identi cation remains a goal to achieve, the use of one marker or standard PCR-based method may lead to an underestimation of the real Wolbachia infection rates.
The 9.8% of G. m. submorsitans harboring co-infections of Wolbachia and trypanosomes is low compared to 29.84% and 26% reported respectively in G. p. palpalis [26] and G. tachinoides in Cameroun [22]. Although the technical approach and the study design could partially explain this low co-infection rate, such co-infections are probably not common in G. m. submorsitans of lake Iro. The absence of signi cant association (r = 1.754; P = 0.185; 95% CI = [0.360-1.219]) between Wolbachia and trypanosome infections suggests that the presence of this bacterium does not seem to be an obstacle for the establishment of trypanosomes. These results are in agreement with those of Kante et al. [26] reporting no association in G. p. palpalis from in sleeping sickness foci of Cameroon. They contrast data of Alam et al. [27] showing a negative correlation between Wolbachia and trypanosome infections and suggesting that the presence of this bacterium prevent trypanosome infections in G. f. fuscipes. The tripartite association between tsetse, Wolbachia and trypanosomes seems to vary according to tsetse species or subspecies.
Results of the present study showing that only 2.31% of tsetse ies were co-infected by Wolbachia and S. glossinidius are in agreement with the 5.43% previously reported in G. f. quanzensis [23]. They indicate that co-infections between Wolbachia and S. glossinidius are rare in wild populations of G. m. submorsitans. The co-infection rate between S. glossinidius and Wolbachia is probably underestimated in the present study because the molecular markers used have been reported to be of low sensitiviety, especially when only one marker was used to detect symbiotic microorganisms. The low co-infection rate revealed between S. glossinidius and Wolbachia can be also explained by the biological effects of each of these bacteria. Indeed, association studies revealed that the presence of S. glossinidius seems to favor trypanosome infections while no association was reported between Wlobachia and trypanosome infections. In other studies, the negative correlation reported between trypanosomes and Wolbachia infections suggested that the presence of Wolbachia seems to prevent trypanosome infections [27,30]. These observations suggest that some antagonistic actions, resulting from different biological actions of Wolbachia and S. glossinidius could occur in tsetse y during trypanosome infections.
Our investigations on tripartite associations were based on presence/absence of trypanosome or S. glossinidius or Wolbachia. Instead of focusing on this presence/absence, the genetic characterization of S. glossinidius or Wolbachia strains could provide additional values to decript these associations. In previous investigations, it has been reported that the tripartite association could be affected by speci c genotypes of S. glossinidius and some trypanosome species such as T. b. gambiense and T. b. brucei [8]. For some trypanosome species, speci c S. glossinidius genotypes have been reported to affect the vectorial competence of G. p. gambiensis and G. m. morsitans [11]. Genetic characterization of bacteria populations could provide additionnal data to improve knowledge on this tripartite association, and also to better understand the real contribution of symbiotic microorganisms (S. glossinidius or Wolbachia) in the vectorial competence of tsetse ies. To obtain the real overview of the vector competence of tsetse ies, it is also important to take into consideration other factors such as the level of lectin in the tsetse gut at the time of parasite uptake, the y species, the age, the teneral status of tsetse and its rst blood meal on a non-infected host because these factors affect its ability to be infected and could mitigate the in uence of symbiotic micro-organisms. Such factors could play a signi cant role in the success or failure of parasite establishment because the processes leading to this establishment involve complex interactions between these factors [13].

Conclusion
This study revealed S. glossinidius and Wolbachia in wild population of G. m. submorsitans of Lake Iro in the south of Chad. It showed that few tsetse ies harbor co-infections of Wolbachia and S. glossinidius. Co-infections of Wolbachia and trypanosomes or S. glossinidius and trypanosomes are not common in G. m. submorsitans. No association was revealed between Wolbachia and trypanosomes while signi cant association was observed between the presence of S. glossinidius and trypanosome infections. Decrypting the tripartite association involving tsetse, symbionts and trypanosomes requires additional studies aiming to understand the relationship between haplotypes or genotypes of Wolbachia and/or S. glossiniduis and trypanosome infections.

Study area
Tsetse ies were caught in the area of Lake Iro, along the Salamat River in the Middle Chari region of the south of Chad (Fig. 1). This area is considered as a buffer zone of the Zakouma national park where domestic and wild animals can meet. It is located between latitude 09°59'N and longitude 019°26'E and has a climate of Sudano-Sahelian type with one dry season (November to April) and one rainy season (May to October). This locality has an average annual temperature and relative humidity of respectively 27°C and 50% [37]. The rainfall varies from 800mm to 1200mm per year [38] and the vegetation is dominated by oodplains and dense forests containing shrubs. The hydrographic network is mainly dominated by the Lake Iro and the Salamat River, which ows into the Chari River that feeds Lake Chad in the north. The population is estimated at 174,195 inhabitants who are mainly herdermen, farmers and shermen [39].

Entomological survey
Three entomological surveys were performed in November 2018, February 2019 and February 2020. During these surveys, biconical traps [40] were set up along the Salamat River, especially where the bioclimatic conditions were considered favourable for the development of tsetse ies. The geographical coordinates of each tsetse trap were recorded using a global positioning system (GPSMAP® 60CSx Garmin). The temperature and the relative humidity were recorded using a thermohygrometer (EasyLog TH, Lascar, Whiteparish, UK). Tsetse ies were collected each day at 9 am. The collected ies were morphologically identi ed and their sex and species determined.

Collection of tsetse y organs
From each tsetse y, the wing pairs (for morphometric analyses) as well as the legs (for genetic studies) were removed and introduced separately into dry microtubes. Thereafter, the proboscis (for the identi cation of trypanosome species) and the remaining body (for the identi cation of trypanosome species and symbiotic micro-organisms) of each tsetse were collected and each of them placed in a 1.5mL cryotube containing 200µl of nucleic acid preservative solution (25 mM sodium citrate, 10 mM EDTA and 70% ammonium sulphate). The dissecting tweezers were decontaminated in 5% solution of sodium chloride and then rinsed with distilled water after dissection of each tsetse y. In the eld, the samples were preserved at 4 degrees and once in the Laboratory, they were stored at -80 degree.
DNA extraction from the tsetse body DNA was extracted from the body of each tsetse y using 5% chelex-resin (Chelex 100, Bio-rad). Brie y, each tsetse body was removed and put into a new 1.5 mL microtube. This tsetse body was crushed using the tip of the Pasteur pipette. Thereafter, 100 µl of chelex 5% solution were added. Each microtube was vortexed and incubated at 56°C for 30 minutes in a Thermomixer. After this incubation, the microtubes were vortexed and reincubated at 95°C for 5 minutes. These microtubes were subsequently centrifuged at 10,000 rpm for 1 minute. The concentration of DNA extracts was determined using a Nanodrop 1000 (Thermo Scienti c-Germany). DNA extracts were stored at -20°C for molecular analysis.

Tsetse species identi cation
The con rmation of each tsetse y was performed by amplifying and sequencing the Cytochrome oxidase I (COI) gene. This ampli cation was done using CO1-sense (5' TTG ATTTTT TGG TCA TCC AGA AGT-3') and CO1-non-sense (5'-TGA AGC TTA AAT TCA TTG CAC TAA TC-3') premers designed by Dyer et al. [41]. Brie y, PCR reaction was performed in a nal volume of 25 µL containing 2.5 U of dream taq polymerase, 2.5µL of the dream taq buffer (10X), 0.2 mM of dNTPs (all provided by Thermo Scienti c, Dreieich, Germany), 2 µM of each primer and 1µL of DNA extract. The ampli cation program consisted of an initial denaturation step of 95°C for 5 min, followed by 35 cycles. Each of these cycles was made up of a denaturation step at 94°C for 1 min, an annealing step at 55°C for 1 min and an elongation step at 72°C for 1 min. This was followed by a nal elongation at 72°C for 5 min.
After each PCR reaction, 20 µL of PCR products were checked by electrophoresis on 1.5% agarose gel containing 3 µL G-stain (Serva, Heidelberg, Germany). The agarose gel was stained, visualized under ultraviolet light (UV) and photographed.
Each sample for which a DNA fragment of 930 bp was revealed by electrophoresis was selected and the remaining amplicons puri ed using GeneJet DNA puri cation kit (Thermo Scienti c, Dreieich, Germany). This puri cation was performed following the manufacturer's instruction. Each puri ed COI DNA fragment was sequenced by a commercial company (SeqLab, Göttin-gen, Germany). To identify each tsetse species or subspecies, each sequenced fragment of COI was compared to those available in the database (Genbank) of the National Center for Biotechnology Information (NCBI). This was done through a BLAST search.
Sodalis glossinidius was identi ed by PCR using the pSG2 primers (pSG2-sense: 5'-TGAAGTTGGGAATGTCG-3': pSG2-non-sense: 5'-AGTTGTAGCACAGCGTGTA-3') as previously described by Darby et al. [42]. The PCR reaction was performed in a nal volume of 15 µL containing 1.5 µL of Dream taq buffer, 0.5 µL of Dream taq polymerase (5U/µL), 0.3 µL of dNTPs (10 mM/µL), 0.5 µL of each primer (100mg/µL) and 0.5 µL of DNA extract. The ampli cation program consisted of an initial denaturation step at 94°C for 3 minutes followed by 30 cycles. Each of these cycles was made up of a denaturation step at 94°C for 30 sec, an annealing step at 51°C for 45 sec and an elongation step at 72°C for 1 min. The nal elongation was done at 72°C for 5 min. For each PCR reaction, positive and negative controls were used. In the positive control, puri ed genomic DNA of S. glossinidius was used while in the negative control, the DNA solution was replaced by nuclease free water.
After PCR reactions, amplicons were separated by electrophoresis on 2% agarose gel containing 3 µL of G-stain (Serva, Heidelberg, Germany). The electrophoresis was performed at 100 volt for 60 min. At the end of each electrophoresis, the gel was visualized under UV light. Samples were considered positive for S. glossinidius if a DNA fragment of 120 bp was observed.
The presence of S. glossinidius was con rmed by sequencing 5 positive randomly selected samples. For these 5 selected samples, the same PCR was performed in a nal volume of 50µL. After electrophoretic separation of PCR products of these 5 samples, amplicons were puri ed from agarose gel using the GenJet puri cation kit following the manufacturer's instructions. The puri ed PCR products were cloned into pJET 1.2 cloning vector (Thermo Scienti c) following the manufacturer's instructions. Recombinant clones or clones containing pSG2 sequences were identi ed by PCR using pSG2 primers. PCR reactions were carried out in the same conditions as described above.
Recombinant clones were picked up from petri dish and cultured overnight at 37°C into LB medium supplemented with ampicillin (100 µg/ml). The bacteria cultures were centrifuged at 4500 xg for 15 min at 4°C and the pellet was recorvered. Plasmid DNA was puri ed from each pellet using the GeneJET Plasmid MiniPrep Kit (Thermo Fischer Scienti c). The Plasmidic DNA was send for sequencing that was performed by a commercial company (institute SeqLab, Göttingen, Germany).

Molecular identi cation of Wolbachia
Wolbachia was identi ed by amplifying the wsp (Wolbachia surface protein) gene as described by Baldo et al. [43]. The ampli cation was done using wsp-sense (5'-GTCCAATARSTGATGARGAAAC-3') and wspnon-sense (CYGCACCAAYAGYRCTRTAAA-3') primers designed by Baldo et al. [43]. PCR reaction was performed in a total volume of 15 µL containing 1.5 µL of Dream taq buffer, 0.1 µL of Dream taq polymerase (5U/µL), 0.3 µL of dNTPs (10mM/µL) (all from Thermo Fischer Scienti c) and 0.5 µL of each primer (100mg/µL) (provided by Sigma-Aldrich, Darmstadt, Germany) and 0.5 µL of DNA extract. The ampli cation program consisted of an initial denaturation step for 5 minutes at 95°C followed by 35 cycles. Each cycle was made up of a denaturation step at 94°C for 30 sec, an annealing step at 53°C for 30 sec and an elongation step at 72°C. The nal elongation was done at 72°C for 10 minutes. During each PCR, positive and negative controls were used. The positive control was a puri ed genomic DNA of Wolbachia while in the negative control, the DNA solution was replaced by nuclease free water.
Amplicons of each PCR reaction were subjected to an electrophoresis that was carried out as described above. Samples were considered as having Wolbachia infections if a DNA fragment of 513 bp was identi ed after electrophoresis. As for S. glossinidius, the presence of Wolbachia was con rmed by sequencing amplicons of 5 positive randomly selected samples. For each of these samples, another PCR was performed in a volume of 50 µL. The amplicons were puri ed by the GenJET puri cation kit (Thermo Fischer Scienti c). The puri ed PCR product was directly sent to a commercial company (SeqLab, Göttingen, Germany) for sequencing.
Geneious Pro version 5.5.9 software was used to store, organize and analyze the sequences obtained from PSG2 and wsp genes of S. glossinidius and Wolbachia, respectively. The presence of S. glossinidius and Wolbachia was con rmed by BLAST searching respectively of pSG2 and wsp sequences at the data base (Genbank) of the National Center for Biotechnology Information (NCBI). After BLAST search, a sequence was considered as belonging to S. glossinidius if the sequenced DNA fragment of 120 bp had at least 98% of similarity with those of S. glossinidius available in the data base. For Wolbachia, the nucleotide sequence of the sequenced DNA fragment of 513 bp must also have at least 98% of similarity with the sequence of Wolbachia available in the database.

Statistical analysis
The R software was used for statistical analysis [44]. The Chi-square test was used to compare the infection rates of S. glossinidius and Wolbachia according to sex and sampling periods. The test was considered signi cant when the P value was below 0.05. Generalised linear modelling (glm) with 95% con dence intervals (CIs) was used to evaluate the association between symbiotic microorganisms and trypanosome infections. For association studies, data on trypanosome infections were retrieved for results of Djoukzoumka et al. (submitted). To carry out these association studies, T. vivax was excluded because its lifecycle is restricted to the mouthparts of tsetse ies.

Availability of data and materials
All data generated and/or analyzed during this study are included in this article.
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable. Tsetse ies were caught in the area of Lake Iro, along the Salamat River in the Middle Chari region of the south of Chad. Note: The designations employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors.