Sodalis glossinidius (Enterobacteriaceae) and Vectorial Competence ofGlossina palpalis gambiensis and Glossina morsitans morsitans forTrypanosoma congolense Savannah Type

Anne Geiger,1 Sophie Ravel,1 Roger Frutos,2 G�rard Cuny1

1IRD, UR035, Laboratoire de Recherche et de Coordination sur les Trypanosomoses, IRD-CIRAD, TA 207/G, Campus International de Baillarguet,34398 Montpellier cedex 5, France2Cirad-Emvt, TA30/G, Campus International de Baillarguet, 34398 Montpellier cedex 5, France

Received: 24 November 2004 / Accepted: 15 January 2005

Abstract. Sodalis glossinidius is an endosymbiont of Glossina palpalis gambiensis and Glossinamorsitans morsitans, the vectors of Trypanosoma congolense. The presence of the symbiont wasinvestigated by PCR in Trypanosoma congolense savannah type-infected and noninfected midguts ofboth fly species, and into the probosces of flies displaying either mature or immature infection, toinvestigate possible correlation with the vectorial competence of tsetse flies. Sodalis glossinidius wasdetected in all midguts, infected or not, from both Glossina species. It was also detected in proboscesfrom Glossina palpalis gambiensis flies displaying mature or immature infection, but never in proboscesfrom Glossina morsitans morsitans. These results suggest that, a) there might be no direct correlationbetween the presence of Sodalis glossinidius and the vectorial competence of Glossina, and b) thesymbiont is probably not involved in Trypanosoma congolense savannah type maturation. It couldhowever participate in the establishment process of the parasite.

Tsetse flies are the insect vectors of African trypano-somes, among which are found the causing agents ofhuman African trypanosomiasis or sleeping sickness,and nagana in animals. Trypanosoma (Nannomonas)congolense is the main causing agent of nagana in sub-Saharan Africa. The savannah type is the most prevalentin cattle [20, 24, 29] and is responsible for dramaticlosses in livestock production. Despite progress in theunderstanding of the disease, nagana persists in sub-Saharan Africa. In the near future, there is no foresee-able progress in processing mammalian vaccines or neweffective and affordable drugs for chemotherapy, whiledrug resistance is increasing [9, 19]. Novel strategiesmust be investigated and among them are better risk-management strategies and alternative vector-basedstrategies such as the engineering of insects capable ofblocking the transmission of the parasite [27].

These alternative strategies require a clear and fullunderstanding of the various steps and mechanisms in-volved in the transmission of the parasite. To be trans-mitted to the mammalian host, Trypanosoma congolense(T. congolense) must first establish in the insect midgutand, upon migration to the mouthparts, undergo a mat-uration process. Parasites present in the infected mam-malian bloodstream enter fly midgut during a bloodmeal and rapidly differentiate into procyclic forms. Thenthey either die in the midgut of refractory individuals orsurvive to yield persistent procyclic infections in sus-ceptible insects. Factors involved in this establishmentstep are still largely unknown. Once established, para-sites migrate to the mouthparts where they differentiateinto epimastigote forms and, finally, into infectiousmetacyclic forms (maturation step), which can then betransmitted to mammals by the fly during its bloodfeeding [12, 33].

However, only a small part of tsetse flies showingmidgut infection develop mature infection and areCorrespondence to: A. Geiger; email: Anne.Geiger@mpl.ird.fr

CURRENT MICROBIOLOGY Vol. 51 (2005), pp. 35–40DOI: 10.1007/s00284-005-4525-6 Current

MicrobiologyAn International Journal

ª Springer Science+Business Media, Inc. 2005

capable of transmitting the disease [16]. This ability toacquire the trypanosome, favor its maturation, andtransmit it to a mammalian host is known as the ��vec-torial competence.�� A large variability in vectorialcompetence has been recorded between different speciesof Glossina. The morsitans group was shown to be agood vector of T. congolense, whereas the palpalisgroup was a poor vector [11, 13, 17, 25, 32].

Sodalis glossinidius (S. glossinidius), member of theEnterobacteriaceae family [3], is a secondary-symbiont(S-endosymbiont) of Glossina [4], and was reported asbeing involved in the vectorial competence of Glossina[15]. Although S. glossinidius has been associated withmaternally inherited factors of Glossina susceptibilityfor trypanosome transmission [15, 35, 37], its actual rolein the ability of tsetse flies to acquire and transmit theparasite is still controversial [15, 18, 28, 34, 36].

In order to investigate whether the presence ofS. glossinidius could be significantly correlated with thevectorial competence of tsetse flies for T. congolensesavannah type, we conducted a detection analysisof S. glossinidius in various tissues of artificiallyT. congolense–infected flies of two species of Glossina:Glossina palpalis gambiensis (G. p. gambiensis) andGlossina morsitans morsitans (G. m. morsitans).

In this article, we report that there is no straightcorrelation between the presence of S. glossinidius andthe ability of the insect to acquire the parasite. We alsoreport that the ability of T. congolense savannah typeto maturate in the mouthparts does not seem to beinfluenced by the symbiont. Finally, we report that thetissue tropism of S. glossinidius is species-dependentand inversely correlated with vectorial competence.S. glossinidius is present in both midgut and proboscisof G. p. gambiensis (poor vector), whereas it is notfound in the proboscis of G. m. morsitans (majorvector), which harbors mature forms of T. congolensesavannah type.

Materials and Methods

Insects and parasites. Populations of G. p. gambiensis andG. m. morsitans were maintained in a level-2 containment insectary,at Cirad-Emvt in Montpellier, France, at 23�C and 80% relativehumidity. These colonies originate from flies field-collected in BurkinaFaso and Zimbabwe, respectively. T. congolense clone E325 (savannahtype) was isolated from wild infected Glossina pallidipes individualscollected in Uganda [31].

Tsetse flies infected and noninfected organs. G. p. gambiensis andG. m. morsitans individuals were artificially infected withT. congolense savannah type (clone E 325) as previously described[23]. Sixty-five G. p. gambiensis and 67 G. m. morsitans individualswere dissected 48 days postinfection. The midgut and proboscis ofeach fly were collected separately, suspended in 30 lL of sterile

distilled water, and incubated a) for 1 h at 56�C, and b) for 30 min at95�C in 30 lL of a 5% Chelex 100 resin (Biorad, CA). Finally,suspensions were centrifuged for 5 min at 15,000 g. Samples werefrozen and stored at )20�C until use. The presence of T. congolensesavannah type was detected by polymerase chain reaction (PCR) usingTCS1 and TCS2 primers [14].

PCR detection of S. glossinidius. PCR analyses were performed onchelex-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 froman extrachromosomal element. PCR was conducted in 50 lLthermostable polymerase buffer containing 1 lL of DNA template,1.5 mM MgCl2, 0.2 mM of each dNTP, 20 pmol of each primer, and 0.5units of Taq DNA polymerase (QBIOgene, Ilkirch, France). Reactionconditions were: 5-min denaturation at 94�C followed by 35 cycles ofdenaturation (94�C, 1 min), annealing (55�C, 1 min) and extension(72�C, 1 min) and a final 10-min elongation step at 72�C. Twentymicroliters of each sample were analyzed in 1.2% agarose gel, stainedwith ethidium bromide, and photographed under UV light.

Cloning and sequencing of S. glossinidius 16S rDNA. 16S rDNAfrom S. glossinidius was cloned by PCR using the conserved primers61 F (5¢-GCTTAACACATGCAAG) and 1227R (5¢-CCATTGTAGCACGTGT), which amplify a 1100-bp fragment of 16S rDNA fromEubacteria [21]. DNA was extracted from haemolymph of G. p.gambiensis and G. m. morsitans individuals. PCR was performed in50-lL thermostable polymerase buffer containing 5 lL of DNAtemplate, 1.5 mM MgCl2, 0.2 mM dNTP, 20 pmol of each primer, and0.5 units of Taq DNA polymerase (QBIOgene, Ilkirch, France).Reaction conditions comprised an initial 5-min denaturation step at94�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 finalelongation step at 72�C. The 1100-bp PCR product was cloned intopGEM-T Easy Vector (Promega) and recombinant plasmids weresequenced (Genome Express, Grenoble, France). Sequences werecompared to reference sequences from two different strains of S.glossinidius isolated from Glossina palpalis palpalis (GenBankaccession number U64867) (belonging to the palpalis group as G. p.gambiensis) and from Glossina pallidipes (belonging to the morsitansgroup as G. m. morsitans) (GenBank accession number M99060),respectively [5].

Results

Detection of S. glossinidius in the midgutof T. congolense savannah type–infected andnoninfected tsetse flies. Sixty-five G. p. gambiensisand 67 G. m. morsitans individuals were dissected 48days postinfection and midguts were collected. Thepresence of T. congolense savannah type in the dissectedmidguts was detected by PCR using the TCS1 and TCS2primers [23]. Out of 65 dissected G. p. gambiensismidguts, 19 (29%) were bearing T. congolense and 46(71%) did not have any T. congolense (Table 1). Withrespect to G. m. morsitans, out of 67 dissected midguts,12 (17.9%) were infected with T. congolense, whereas55 (82.1%) did not display any infection (Table 1).Using the S. glossinidius-specific GPO1 F/GPO1 Rprimers, the expected 1.2-kb PCR fragment was detected

36 CURRENT MICROBIOLOGY Vol. 51 (2005)

for both G. p. gambiensis and G. m. morsitans flies inall T. congolense-infected midguts as well as in allnoninfected samples (Table 1; Fig. 1). No PCR productwas obtained with the GPO1 F/GPO1 R primers fromEscherichia coli DNA used as a negative control(Fig. 1).

The presence of S. glossinidius in G. p. gambiensisand G. m. morsitans was further confirmed by amplifi-cation and sequencing of part of the 16S rDNA. Thepartial 16S rDNA sequence obtained from G. m. mors-itans was identical to that previously described forS. glossinidius from Glossina pallidipes, the referencesequence of the morsitans group (GenBank accessionnumber M99060). Similarly, the sequence of the partial16S rDNA amplified from G. p. gambiensis was iden-tical to that described for the S. glossinidius strain iso-lated from Glossina palpalis palpalis, the type species ofthe palpalis group (GenBank accession numberU64867).

Detection of S. glossinidius in probosces from fliesdisplaying mature and nonmature T. congolensesavannah-type infection. Probosces from allindividuals displaying T. congolense midgut infection,i.e., 19 G. p. gambiensis individuals and 12G. m. morsitans individuals (Table 1), were examinedfor the presence of T. congolense. Out of 19G. p. gambiensis flies, 9 (47.4 %) displayed T.congolense in the proboscis, indicating that theparasite had reached the maturation stage (Table 1).The other 10 T. congolense-infected G. p. gambiensisflies (52.6 %) did not bear any T. congolense in theproboscis and were thus displaying an immatureinfection (Table 1). With respect to G. m. morsitans,

out of 12 individuals, 9 (75%) showed T. congolense inthe proboscis whereas 3 (25%) were characteristic of animmature infection with no parasite in the proboscis(Table 1). Although T. congolense could be detected inthe probosces from both species, the distribution ofS. glossinidius was found to be drastically different,depending on the species. S. glossinidius was found inthe proboscis of all the G. p. gambiensis individualsanalyzed regardless of the status, mature or immature,of the T. congolense infection (Table 1; Fig. 2).Surprisingly, S. glossinidius was never detected in anyproboscis from G. m. morsitans (Table 1; Fig. 2).Running the same PCR detection on an increasingamount of DNA yielded the same negative result with

Table 1. Distribution of Sodalis glossinidius in midguts and probosces from Glossina morsitans morsitans and Glossina palpalis gambiensis

Presence of Sodalis glossinidius in %(PCR detection using specific GPO1 F/GPO1 R primers)

Midgut Proboscis

T. congolense T. congolense

Species Group of vectorial competence Infected Noninfected Mature infections Immature infections

G. p. gambiensisa Low 100 (19c) 100 (46d) 100 (9e) 100 (10f)G. m. morsitansb High 100 (12c) 100 (55d) 0 (9e) 0 (3f)

aMidguts and probosces of 65 individuals were dissected 48 days postinfection.b Midguts and probosces of 67 individuals were dissected 48 days postinfection.cNumber of dissected midguts with positive T. congolense savannah type detection.dNumber of dissected midguts with negative T. congolense savannah type detection.eNumber of dissected probosces with positive T. congolense savannah type detection.fNumber of dissected probosces with negative T. congolense savannah type detection.e,fProbosces were obtained from the G. p. gambiensis (19) and G. m. morsitans (12) individuals displaying T. congolense savannah type in theirmidgut.

Fig. 1. Detection of S. glossinidius from T. congolense-infected andnoninfected midguts. DNA isolated from midgut of flies was subjectedto polymerase chain reaction amplification using Sodalis-specific pri-mer set, GPO1 F/GPO1 R. Lanes 2–11: amplification on DNA ex-tracted from five T. congolense-infected midgut of G. m. morsitansflies (2–6) and from five noninfected midgut of G. m. morsitans flies(7–11). Lanes 12–21: amplification on DNA extracted from five T.congolense-infected midgut of G. p. gambiensis flies (12–16) and fromfive noninfected midgut of G. p. gambiensis flies (17–21). Lane 22:negative control. Lanes 1 and 23: molecular size markers.

A. Geiger et al.: Sodalis glossinidius and Vectorial Competence of Glossina 37

respect to detection of S. glossinidius in G. m. morsitansprobosces. G. p. gambiensis individuals found negativefor the presence of T. congolense in the midgut showedthe presence of S. glossinidius in the proboscis (data notshown). However, S. glossinidius was not present in theproboscis of the G. m. morsitans flies with noT. congolense-infected midgut.

Control PCR reactions were conducted on midgutsand probosces from healthy insectary tsetse flies of bothG. p. gambiensis and G. m. morsitans. The same resultsas for artificially infected flies were obtained. S-endo-symbionts were present in midguts from both fly speciesand in probosces from G. p. gambiensis. S. glossinidiuswas absent in probosces from G. m. morsitans.

Discussion

The objective of this work was to determine whether thepresence of S. glossinidius in organs critical for estab-lishment, maturation, and transmission of T. congolensesavannah type, i.e., midgut and proboscis, could becorrelated to the vectorial competence of Glossina.Previous studies have suggested that the susceptibility oftsetse flies for trypanosome transmission was dependenton the presence of S. glossinidius in midgut epithelialcells [35, 37]. S. glossinidius was considered to allowthe installation of the parasite in the insect midgutthrough the production of N-acetyl glucosamine (NAG)resulting from the hydrolysis of pupae chitin by aSodalis-produced endochitinase. NAG was reported toinhibit a tsetse midgut lectin lethal for the procyclicforms of the trypanosome. The presence of S. glossini-dius would thus allow the trypanosome to establish in

the midgut [35, 37]. One could therefore expect thepresence of S. glossinidius to be directly related to theability of the trypanosome to infect the insect vector.

Results reported here indicate that such a directcorrelation is not observed. S. glossinidius was detectedin the midgut of all parasite-infected and noninfectedindividuals of G. p. gambiensis and G. m. morsitans. Asimilar observation was reported on Glossina morsitanscentralis, in which, after T. congolense artificial infec-tion, Rickettsia-like organisms (RLO) were observed inall infected and noninfected midguts [18]. The authorsconcluded on the absence of any relationship between thepresence of RLO in the midgut and either the suscepti-bility of a laboratory-bred Glossina morsitans centralisto T. congolense infection, or the ability of T. congolenseinfection to undergo full cyclical development in thevector. Although all tsetse flies were found to carryWigglesworthia, considered an obligatory endosymbiont,microscopic observations revealed marked differenceswith respect to levels of S. glossinidius populationsin midgut tissues of different Glossina species [28].Furthermore, the symbiont was not present in everyindividual analyzed [28]. In other experiments, Welburnand Maudlin [34] established a quantitative relationshipbetween the number of RLO present in the midgut andsusceptibility to trypanosome infection.

Nevertheless, the persistent presence of S. glos-sinidius in either T. congolense-infected and nonin-fected midguts does not mean the symbiont is notinvolved in vectorial competence. S. glossinidiusmight be involved in the establishment of infection,but the interaction mechanisms are most likely morecomplex than previously thought. Other factors mightbe involved beyond the simple direct inhibition of aninsect lectin. Such factors could act in subsequent keysteps after lectin inhibition or facilitate the inhibitionof the lectin by S. glossinidius. The persistent pres-ence of S. glossinidius in midguts is well in line withthe involvement of other factors or steps in T. con-golense savannah-type establishment. The symbiontmight therefore play a necessary, but not sufficient,role in this process. Furthermore, the genetic diversityof S. glossinidius has not been investigated yet andone might well consider the possible existence ofpopulations of S. glossinidius differing in their abilityto modulate/mediate vectorial competence. However,one can also consider that the presence of S. glos-sinidius is a mere coincidence masking the actualmechanisms. The data reported here do not allow aconclusion on these various hypotheses, and furtherresearch is clearly needed to analyze functionalinteractions between the symbiont, the parasite, andthe vector. Further research is also required to inves-

Fig. 2. Detection of S. glossinidius from probosces of G. p. gambiensisand G. m. morsitans flies displaying mature or immature T. congolenseinfection. DNA isolated from probosces of flies was subjected topolymerase chain reaction amplification using Sodalis-specific primerset, GPO1 F/GPO1 R. Lanes 2–7: amplification on DNA extractedfrom the probosces of three G. m. morsitans flies with mature T.congolense infection (2–4) and of three G. m. morsitans flies withimmature T. congolense infection (5–7). Lanes 8–13: amplification onDNA extracted from the probosces of three G. p. gambiensis flies withmature T. congolense infection (8–10) and of three G. p. gambiensisflies with immature T. congolense infection (11–13). Lane 14: negativecontrol. Lanes 1 and 15: molecular size markers.

38 CURRENT MICROBIOLOGY Vol. 51 (2005)

tigate the genetic diversity of S. glossinidius and itspotential correlation with the vectorial competence ofGlossina.

The presence of mature T. congolense in probo-sces of G. m. morsitans individuals whereas S. glos-sinidius is absent also indicates that S. glossinidiusplays no role in the maturation steps of T. congolensesavannah type at least in G. m. morsitans. AlthoughS. glossinidius was absent in probosces from G. m.morsitans-infected individuals, this species was nev-ertheless fully capable of secreting and transmittinginfectious forms of T. congolense savannah type torabbits [23], thus indicating that the symbiont plays norole in the ability of the flies to secrete infectiousforms of T. congolense savannah type. The situation ismore complex with respect to G. p. gambiensis.

Our data are in agreement with previous reportsfrom Cheng and Aksoy [6] who, investigating the tis-sue tropism of S. glossinidius, only found the S-endosymbionts in the salivary glands of flies from thepalpalis group. The presence of S. glossinidius in thesalivary glands of G. palpalis was also reported fromultrastructural analyses [38]. Surprisingly, this verysimilar G. p. gambiensis population harboring matureT. congolense savannah type and S. glossinidius inprobosces was shown to be unable to secrete the par-asite [23], whereas it could secrete infectious forms ofTrypanosoma brucei gambiense [22]. This presence ofS. glossinidius in probosces of palpalis flies of lowvectorial competence, with respect to T. congolense,and their absence in probosces of morsitans flies ofhigh vectorial competence, further confirms that S.glossinidius seems to play no role in the maturationprocess of T. congolense savannah type.

This is also an indication that more complexmechanisms involving tripartite interactions might be atwork beyond the putative permissive role of S. glos-sinidius in the establishment of T. congolense savannahtype in the insect midgut. Investigations were reportedearlier on the possible role of components expressed inthe salivary glands [7, 26, 30]. Studies are under way tocharacterize proteins produced by S. glossinidius [1, 2,10].

Further studies are needed to understand therespective role of the vector, the parasite, and the sym-biont, and to describe the mechanisms involved. Popu-lations of T. congolense savannah type and Glossinaused in this work have been characterized from a geneticstandpoint, and investigating the genetic diversity ofS. glossinidius populations would provide a deeper in-sight on population interactions. Furthermore, geneti-cally well-known populations will also represent a goodbiological material for functional analyses of the

mechanisms of interaction. However, they may still bebiased with respect to wild-type populations, and futureexperiments using field-collected flies will certainlyhave to be designed to understand the relevance of theseresults for natural populations.

ACKNOWLEDGMENT

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

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