SODALIS GLOSSINIDIUS AND VECTORIAL COMPETENCE OF GLOSSINA: A POSSIBLE APPROACH TO CONTROL TRYPANOSOME TRANSMISSION

SODALIS GLOSSINIDIUS ET COMPETENCE VECTORIELLE DESGLOSSINES: UNE APPROACHE POSSIBLE POUR CONTROLER LA TRANSMISSION DES TRYPANOSOMES 

Running title: Sodalis glossinidius and vectorial competence of Glossina

Anne Geiger, Sophie Ravel, Roger Frutos & Gérard Cuny

¹ IRD, UR035, Laboratoire de Recherche et de Coordination sur les Trypanosomoses. IRD-CIRAD, TA      207 / G, Campus International de Baillarguet, 34398 Montpellier cedex 5 (FRANCE)
² Cirad-Emvt, TA30/G, Campus International de Baillarguet, 34398 Montpellier cedex 5

* Corresponding Author, Tel : + 33 4 67 59 39 25 ; Fax: + 33 4 67 59 39 20 ;
E-mail : Anne.Geiger@mpl.ird.fr

Résumé

            Trypanosoma congolense est le principal agent causal de la trypanosomose animale en Afrique subsaharienne. Malgré les progrès réalisés, la maladie persiste avec des conséquences économiques dramatiques. Dans l’optique des nouvelles stratégies de lutte, notamment antivectorielles, la connaissance préalable des mécanismes de transmission du parasite s’avère indispensable et le rôle des symbiotes dans la compétence vectorielle des glossines doit être élucidé. Nous avons donc tenté d'établir une corrélation entre présence du symbiote et compétence vectorielle de Glossina morsitans morsitans et Glossina palpalis gambiensis (espèces respectivement bonnes et faibles vectrices de T. congolense), en recherchant le symbiote dans les intestins moyens, infectés et non infectés, et dans les trompes de mouches portant des formes matures ou immatures du parasite.

            S. glossinidius a été détecté dans tous les intestins moyens, infectés et non infectés, des deux espèces de glossines, ainsi que dans toutes les trompes de G. p. gambiensis portant des infections matures ou immatures, mais jamais dans celles de G. m. morsitans, pourtant vecteur principal du parasite. Le symbiote ne serait donc pas impliqué dans la maturation du parasite mais pourrait toutefois l’être dans le processus d’établissement.

            L'existence d'une diversité génétique chez les populations de S. glossinidius, susceptible de moduler l'étape d'acquisition du parasite, a été recherchée chez des isolats provenant des deux espèces de glossines. Les isolats issus de G. p. gambiensis et de G. m. morsitans forment des groupes génétiquement séparés. Ceci pourrait expliquer les différences de capacité vectorielle des deux hôtes.

Summary

            Trypanosoma congolense, transmitted by tsetse fly, is the main causing agent of animal trypanosomiasis (nagana) in sub-Saharan Africa. Despite progress in the understanding of the disease, nagana persists with dramatic economic consequences. With the prospect of the new strategies of fight, in particular vector control, the preliminary knowledge of the transmission mechanisms of the parasite remains essential, and the role of the symbiontes in the vectorial competence of Glossina needs to be elucidated. We thus investigated the presence/absence of Sodalis glossinidius in T. congolense-infected and non infected midguts of Glossina palpalis gambiensis and Glossina morsitans morsitans, respectively poor and major vectors of the parasite, as well as in probosces of flies displaying mature or immature infection.

            S. glossinidius was detected in all midguts, infected or not, from both Glossina species. It was also detected in all probosces from G. p. gambiensis displaying mature or immature infection, but never in probosces from G. m. morsitans, the major vector of the parasite. These results suggest that S. glossinidius might not be involved in the T. congolense maturation process. However, they do not exclude that the symbiont could participate in the parasite establishment.

            Presence of a genetic diversity among S. glossinidius populations which could modulate this step was investigated on S. glossinidius sampled on hemolymph from both Glossina species. Isolates from G. p. gambiensis and G. m. morsitans are genetically distinct and group into clearly separate clusters. This may explain the differences in the vectorial abilities of these two Glossina species.

Introduction

            Tsetse flies are the insect vectors of African trypanosomes among which are the causive agents of Human African trypanosomiasis or sleeping sickness, and nagana in animals. Trypanosoma (Nannomonas) congolense is the main disease causing agent of nagana in sub-Saharan Africa. The savannah type is the most prevalent in cattle [20, 24, 29] and is responsible for dramatic losses in livestock production. Despite progress in the understanding of the disease, nagana persists in sub-Saharan Africa. There is no foreseeable progress in developing mammalian vaccines or new effective and affordable drugs for chemotherapy, while drug resistance is increasing [9, 19]. Novel strategies must be investigated and among them are better risk-management strategies and alternative vector-based strategies such as the engineering of insects capable of blocking the transmission of the parasite [27].

            These alternative strategies require a clear and full understanding of the various steps and mechanisms involved in the transmission of the parasite. To be transmitted to the mammalian host, Trypanosoma congolense must first be established in the insect midgut and, upon migration to the mouthparts, undergo a maturation process. Parasites present in the infected mammalian bloodstream enter fly midgut during a blood meal and rapidly differentiate into procyclic forms. Then they either die in the midgut of refractory individuals or survive to yield persistent procyclic infections in susceptible insects. Factors involved in this establishment step are still largely unknown. Once established, parasites migrate to the mouthparts where they differentiate into epimastigote forms and, finally, into infectious metacyclic forms (maturation step) which can then be transmitted to mammals by the fly during its blood feeding [12, 33].

            However, only a small part of tsetse flies showing midgut infection develop mature infection and are capable of transmitting the disease [16]. This ability to acquire the trypanosome, favor its maturation, and transmit it to a mammalian host is known as the "vectorial competence" of the fly. A large variability in vectorial competence has been recorded between different species of Glossina. The morsitans group was shown to be a good vector of T. congolense, whereas the palpalis group was a poor vector [11, 13, 17, 25, 32].

            Sodalis glossinidius, member of the Enterobacteriaceae family [3], is a secondary-symbiont (S-endosymbiont) of Glossina [4], and was reported to be involved in the vectorial competence of Glossina [15]. Although S. glossinidius has been associated with maternally inherited factors of Glossina susceptibility for trypanosome transmission [15, 35, 37], its actual role in the ability of tsetse flies to acquire and transmit the parasite is still controversial [15, 18, 28, 34, 36].

            In order to investigate whether the presence of S. glossinidius could be significantly correlated with the vectorial competence of tsetse flies for T. congolense savannah type, we conducted a detection analysis of S. glossinidius in various tissues of artificially T. congolense infected flies of two species of Glossina : Glossina palpalis gambiensis (G. p. gambiensis)and Glossina morsitans morsitans (G. m. morsitans).

            In this article, we report that there is no straight correlation between the presence of S. glossinidius and the ability of the insect to acquire the parasite. We also report that the ability of T. congolense savannah type to maturate in the mouthparts does not seem to be influenced by the symbiont. Finally, we report that the tissue tropism of S. glossinidius is species dependent and inversely correlated with vectorial competence.

Materials and Methods

Insects and parasites.

            Populations of G. p. gambiensis and G. m. morsitans were maintained in a level-2 containment insectary, at Cirad-Emvt in Montpellier, France, at 23°C and 80% relative humidity. These colonies originate from flies field-collected in Burkina Faso and Zimbabwe, respectively. T. congolense clone E325 (Savannah type) was isolated from wild infected Glossina pallidipes individuals collected in Uganda [31].

Tsetse flies infected and non-infected organs.

            G. p. gambiensis and G. m. morsitans individuals were artificially infected with T. congolense savannah type (clone E 325) as previously described [23]. Sixty five G. p. gambiensis and 67 G. m. morsitans individuals were dissected 48 days post infection. The midgut and proboscis of each fly were collected separately, suspended in 30 µl of sterile distilled water and incubated a) for 1 h at 56°C, and b) for 30 min at 95°C in 30 µl of a 5% Chelex 100 resin (Biorad, CA). Finally, suspensions were centrifuged for 5 min at 15000 g. Samples were frozen and stored at -20°C until use. The presence of T. congolense savannah type was detected by polymerase chain reaction (PCR) using TCS1 and TCS2 primers [14].

PCR detection of S. glossinidius.

            PCR analyses were performed on chelex-extracted DNA using primers GPO1-F (5'-TGAGAGGTTCGTCAATGA) and GPO1-R (5'-ACGCTGCGTGACCATTC) [6, 8, 21]. These primers drive the amplification of a 1.2-kb fragment from an extrachromosomal element. PCR was conducted in 50 µl thermostable polymerase buffer containing 1 µl of DNA template, 1.5 mM MgCl2, 0.2 mM of each dNTP, 20 pmol of each primer and 0.5 units of Taq DNA polymerase (QBIOgene, Ilkirch, France). Reaction conditions were as previously described [6, 8, 21]. Twenty µl of each sample were analyzed in 1.2% agarose gel, stained with ethidium bromide and photographed under UV light.

Cloning and sequencing of S. glossinidius 16S rDNA.

            16S rDNA from S. glossinidius was cloned by PCR using the conserved primers 61 F (5'GCTTAACACATGCAAG) and 1227R (5'-CCATTGTAGCACGTGT) which amplify a 1100-bp fragment of 16S rDNA from Eubacteria [21]. DNA was extracted from haemolymph of G. p. gambiensis and G. m. morsitans individuals. PCR was performed in 50 µl thermostable polymerase buffer containing 5 µl of DNA template, 1.5 mM MgCl2, 0.2 mM dNTP, 20 pmol of each primer and 0.5 units of Taq DNA polymerase (QBIOgene, Ilkirch, France). Reaction conditions comprised an initial 5-min denaturation step at 94°C, followed by 35 cycles of denaturation (94°C, 1 min), annealing (55°C, 1 min) and extension (72°C, 1 min) followed by a 10-min final elongation step at 72°C. The 1100-bp PCR product was cloned into pGEM-T Easy Vector (Promega) and recombinant plasmids were sequenced (Genome Express, Grenoble, France). Sequences were compared to reference sequences from two different strains of S. glossinidius isolated from Glossina palpalis palpalis (GenBank accession number U64867) (belonging to the palpalis group as G. p. gambiensis) and from Glossina pallidipes (belonging to the morsitans group as G. m. morsitans) (GenBank accession number M99060), respectively [5]

Results

Detection of S. glossinidius in the midgut of T. congolense savannah type-infected and non-infected tsetse flies.

            Out of 65 dissected G. p. gambiensis midguts, 19 (29 %), were bearing T. congolense and 46 (71%) did not have any T. congolense (Table 1). With respect to G. m. morsitans, out of 67 dissected midguts, 12 (17.9 %) were infected with T. congolense whereas 55 (82.1 %) did not display any infection (Table 1). Using the S. glossinidius-specific GPO1 F / GPO1 R primers, the expected 1.2-kb PCR fragment was detected for both G. p. gambiensis and G. m. morsitans flies in all T. congolense-infected midguts as well as in all non-infected samples (Table 1 ; Fig. 1). No PCR product was obtained with the GPO1 F / GPO1 R primers from Escherichia coli DNA used as a negative control (Fig. 1).

            The presence of S. glossinidius in G. p. gambiensis and G. m. morsitans was further confirmed by amplification and sequencing of part of the 16S rDNA. The partial 16S rDNA sequence obtained from G. m. morsitans was identical to that previously described for S. glossinidius from Glossina pallidipes, the reference sequence of the morsitans group (GenBank accession number M99060). Similarly, the sequence of the partial 16S rDNA amplified from G. p. gambiensis was identical to that described for the S. glossinidius strain isolated from Glossina palpalis palpalis, the type species of the palpalis group (GenBank accession number U64867).

Table 1. Distribution of Sodalis glossinidius in midguts and probosces from Glossina morsitans morsitans and Glossina palpalis gambiensis individuals displaying T. congolense savannah type in their midgut.

Species          group of vectorial                          Presence of  Sodalis glossinidius in %

                     competence                  (PCR detection using specific GPO1 F/ GPO1 R primers)

                                                                       Midgut                                       Proboscis  

                                                       T. congolense                       T. congolense

                                    infected            non infected           mature           immature

                                                                                               infections          infections

G. p. gambiensis a   Low                   100 (19 c)          100 ( 46 d )          100 (9 e)         100 (10 f)

G. m. morsitans b    High                   100 (12 c)          100 ( 55 d)               0 (9 e)              0 (3 f)

a Midguts and probosces of 65 individuals were dissected 48 days post infection.

b Midguts and probosces of 67 individuals were dissected 48 days post infection.

( c) Number of dissected midguts with positive T. congolense savannah type detection

( d) Number of dissected midguts with negative T. congolense savannah type detection

( e) Number of dissected probosces with positive T. congolense savannah type detection

( f) Number of dissected probosces with negative T. congolense savannah type detection

( e and f) Probosces were obtained from the G. p. gambiensis (19) and G. m. morsitans (12)

Fig. 1. Detection of S. glossinidius from T. congolense- infected and non infected midguts.

DNA isolated from midgut of flies was subjected to PCR amplification using Sodalis specific primer set, GPO1 F / GPO1 R

Lanes 2 – 11 : amplification on DNA extracted from 5 T. congolense-infected midgut of G. m. morsitans flies(2-6) and from 5 non-infected midgut of G. m. morsitans flies(7-11). Lanes 12 – 21 : amplification on DNA extracted from 5 T. congolense-infectedmidgut of G. p. gambiensis flies(12-16) and from 5 non-infected midgut of G. p. gambiensis flies(17-21). Lane 22 : negative control. Lane 1 and 23 : molecular size markers.

Detection of S. glossinidius in probosces from flies displaying mature and non mature T. congolense savannah type infection.

            Probosces from all individuals displaying T. congolense midgut infection, i.e. 19 G. p. gambiensis individuals and 12 G. m. morsitans individuals (Table 1), were examined for the presence of T. congolense. Out of 19 G. p. gambiensis flies, 9 (47.4 %) displayed T. congolense in the proboscis, indicating that the parasite has reached the maturation stage. The other 10 T. congolense-infected G. p. gambiensis flies (52.6 %) did not bear any T. congolense in the proboscis and were thus displaying an immature infection. With respect to G. m. morsitans, out of 12 individuals, 9 (75 %) showed T. congolense in the proboscis whereas 3 (25 %) were characteristic of an immature infection with no parasite in the proboscis (Table 1). Although T. congolense could be detected in the probosces from both species, the distribution of S. glossinidius was found to be drastically different depending on the species. S. glossinidius was found in the proboscis of all the G. p. gambiensis individuals analyzed regardless of the status, mature or immature, of the T. congolense infection (Table 1., Fig. 2). Surprisingly, S. glossinidius was never detected in any proboscis from G. m. morsitans (Table 1 ; Fig. 2). Running the same PCR detection on an increasing amount of DNA yielded the same negative result with respect to detection of S. glossinidius in G. m. morsitans probosces. G. p. gambiensis individuals found negative for the presence of T. congolense in the midgut showed the presence of S. glossinidius in the proboscis (data not shown). However, S. glossinidius was not present in the proboscis of the G. m. morsitans flies with no T. congolense-infected midgut.

            Control PCR reactions were conducted on midguts and probosces from healthy insectary tsetse flies of  both G. p. gambiensis and G. m. morsitans. The same results as for artificially infected flies were obtained. S-endosymbionts were present in midguts from both fly species and in probosces from G. p. gambiensis. S. glossinidius was absent in probosces from G. m. morsitans

Fig. 2. Detection of S. glossinidius from probosces of G. p. gambiensis and G. m. morsitans flies displaying mature or immature T. congolense infection.

DNA isolated from probosces of flies was subjected to PCR amplification using Sodalis specific primer set, GPO1 F / GPO1 R.

Lane 2-7 : amplification on DNA extracted from the probosces of 3 G. m. morsitans flies with mature T. congolense infection (2-4) and of 3 G. m. morsitans flies with immature T. congolense infection (5-7). Lane 8-13 : amplification on DNA extracted from the probosces of 3 G. p. gambiensis flies with mature T. congolense infection (8-10) and of 3 G. p. gambiensis flies with immature T. congolense infection (11-13). Lane 14 : negative control. Lane 1 and 15 : molecular size markers.

Discussion

            The objective of this work was to determine whether the presence of S. glossinidius in organs was critical for establishment, maturation and transmission of T. congolense savannah type, i.e. midgut and proboscis, could be correlated to the vectorial competence of Glossina. Previous studies have suggested that the susceptibility of tsetse flies for trypanosome transmission was dependent on the presence of S. glossinidius in midgut epithelial cells [35, 37]. S. glossinidius was considered to allow the installation of the parasite in the insect midgut through the production of N-acetyl glucosamine (NAG) resulting from the hydrolysis of pupae chitin by a Sodalis-produced endochitinase. NAG was reported to inhibit a tsetse midgut lectin lethal for the procyclic forms of the trypanosome. The presence of S. glossinidius would thus allow the trypanosome to establish in the midgut [35, 37]. One could therefore expect the presence of S. glossinidius to be directly related to the ability of the trypanosome to infect the insect vector.

            Results reported here show that such a direct correlation was not observed. S. glossinidius was detected in the midgut of all parasite-infected and non-infected individuals of G. p. gambiensis and G. m. morsitans. A similar observation was reported on Glossina morsitans centralis, in which, after T. congolense artificial infection, Rickettsia-Like Organisms (RLO) were observed in all infected and non-infected midguts [18]. The authors concluded on the absence of any relationship between the presence of RLO in the midgut and either the susceptibility of a laboratory-bred Glossina morsitans centralis to T. congolense infection, had no effect on the ability of T. congolense infection to undergo full cyclical development in the vector. While all tsetse flies were found to carry Wigglesworthia, considered an obligatory endosymbiont, microscopic observations revealed marked differences with respect to levels of S. glossinidius populations in midgut tissues of different Glossina species [28]. Furthermore, the symbiont was not present in every individual analyzed [28]. In other experiments, Welburn and Maudlin [34] established a quantitative relationship between the number of RLO present in the midgut and susceptibility to trypanosome infection.

            Nevertheless, the persistent presence of S. glossinidius in either T. congolense-infected and non-infected midguts does not mean the symbiont is not involved in vectorial competence. S. glossinidius might be involved in the establishment of infection, but the interaction mechanisms are most likely more complex than previously thought. Other factors might be involved beyond the simple direct inhibition of an insect lectin. Such factors could act in subsequent key steps following lectin inhibition or facilitate the inhibition of the lectin by S. glossinidius. The persistent presence of S. glossinidius in midguts is well in line with the involvement of other factors or steps in T. congolense savannah-type establishment. The symbiont might therefore play a necessary, but not sufficient, role in this process. Furthermore, the genetic diversity of S. glossinidius has not been investigated yet and one might well consider the possible existence of populations of S. glossinidius differing in their ability to modulate/mediate vectorial competence. However, one can also consider that the presence of S. glossinidius is a mere coincidence masking the actual mechanisms. The data reported here do not allow a conclusion on these various hypotheses and further research is clearly needed to analyze functional interactions between the symbiont, the parasite and the vector. Further research is also required to investigate the genetic diversity of S. glossinidius and its potential correlation with the vectorial competence of Glossina.

            The presence of mature T. congolense in probosces of G. m. morsitans individuals whereas S. glossinidius is absent, also indicates that S. glossinidius plays no role in the maturation steps of T. congolense savannah typeat least in G. m. morsitans. Although, S. glossinidius was absent in probosces from G. m. morsitans -infected individuals, this species was nevertheless fully capable of secreting and transmitting infectious forms of T. congolense savannah type to rabbits [23], thus indicating that the symbiont plays no role either in the ability of the flies to secrete infectious forms of T. congolense savannah type. The situation is more complex with respect to G. p. gambiensis.

            Our data are in agreement with previous reports from Cheng and Aksoy [6] who, investigating on the tissue tropism of S. glossinidius, only found the S-endosymbionts in the salivary glands of flies from the palpalis group. The presence of S. glossinidius in the salivary glands of G. palpalis was also reported from ultrastructural analyses [38]. Surprisingly, this very similar G. p. gambiensis population harboring mature T. congolense savannah type and S. glossinidius in probosces was shown to be unable to secrete the parasite [23], whereas it could secrete infectious forms of Trypanosoma brucei gambiense [22]. This presence of S. glossinidius in probosces of palpalis flies of low vectorial competence, with respect to T. congolense, and their absence in probosces of morsitans flies of high vectorial competence, further confirms that S. glossinidius seems to play no role in the maturation process of T. congolense savannah type.

            This is also an indication that more complex mechanisms involving tripartite interactions might be at work beyond the putative permissive role of S. glossinidius in the establishment of T. congolense savannah type in the insect midgut. Investigations were reported earlier on the possible role of components expressed in the salivary glands [7, 26, 30]. Studies are under progress to characterize proteins produced by S. glossinidius [1, 2, 10].

            Nevertheless these results did not exclude the possibility for the symbiont to participate in the parasite establishment since correlation between S. glossinidius and vectorial competence might be related to the genetic diversity of the bacteria harbored by different species of Glossina rather than a mere presence / absence.

            A first approach to test this hypothesis consisted to demonstrate the existence of genetic diversity among S. glossinidius strains harbored by the two Glossina species. This was performed using the AFLP approach. A total of 165 markers were analyzed to investigate the genetic diversity of 39 S. glossinidius strains. Twenty samples were isolated from non infected  G. palpalis gambiensis whereas 19 were obtained from non infected G. morsitans morsitans individuals. Our results (Fig. 3) clearly evidenced the existence of a genetic diversity within S. glossinidius populations. Furthermore, it appeared that S. glossinidius strains from G. palpalis gambiensis and G. morsitans morsitans are grouped in clearly separated clusters. S. glossinidius strains from G. palpalis gambiensis are strongly structured into three clusters associated to high bootstrap values, whereas bacteria isolated from G. morsitans morsitans are distributed within three different weak clusters characterized by low bootstrap values. Cluster IV of S. glossinidius from G. morsitans morsitans is more closely related to the G. palpalis gambiensis strains than to the other G. morsitans morsitans groups. This differential structuring of the bacterial populations may reflect different host-related selection pressures. Furthermore, the presence of genetically distinct populations of S. glossinidius in G. palpalis gambiensis and G. morsitans morsitans is in agreement with the suspected role of this bacterial symbiont in vectorial competence.

Fig. 3. Unweighted Neighbor-Joining tree representation of the
genetic diversity of S. glossinidius strains from G. palpalis
gambiensis and G. morsitans morsitans

            Scale indicates genetic distance. Numbers at nodes represent bootstrap values (1000 replicates). Genetic similarities were calculated using Jaccard coefficient based on the 29 polymorphic bands positions observed for 5 AFLP primer pairs. The tree was constructed with DARwin 4.0.  Numbers represent S. glossinidius strains : 1-20: strains from G. palpalis gambiensis ; 21-39 : strains from G. morsitans morsitans ; M1 : S. glossinidius reference strain M1.

            Further research is nevertheless needed to clearly establish the correlation between a given genotype of S. glossinidius and vectorial competence. However, the demonstration of the existence of genetic diversity in S. glossinidius is a first step towards a better understanding of the tripartite Glossina – Sodalis – Trypanosoma interactions most likely involved in the transmission of this deadly reemerging disease.

Acknowledgments

     The authors are particularly grateful to B. Tchicaya and J. Janelle for maintenance and management of the tsetse colonies.

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