Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Geiger, A. Articles by Frutos, R. Search for Related Content PubMed PubMed Citation Articles by Geiger, A. Articles by Frutos, R. Agricola Articles by Geiger, A. Articles by Frutos, R.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, December 2005, p. 8941-8943, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8941-8943.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

Two Tsetse Fly Species, Glossina palpalis gambiensis and Glossina morsitans morsitans, Carry Genetically Distinct Populations of the Secondary Symbiont Sodalis glossinidius

Anne Geiger,^* Gérard Cuny, and Roger Frutos

UMR 17, IRD-CIRAD, CIRAD TA 207/G, Campus International de Baillarguet, 34398 Montpellier Cedex 5, France

Received 16 February 2005/ Accepted 1 August 2005

Top Abstract Introduction References

 
Genetic diversity among Sodalis glossinidius populations was investigated using amplified fragment length polymorphism markers. Strains collected from Glossina palpalis gambiensis and Glossina morsitans morsitans flies group into separate clusters, being differentially structured. This differential structuring may reflect different host-related selection pressures and may be related to the different vector competences of Glossina spp.

Top Abstract Introduction References

 
Tsetse flies are vectors of African trypanosomes, the causative agents of sleeping sickness in humans and nagana, a tropical disease of cattle. African trypanosomiasis in humans is reemerging and has considerable impact on public health and economic development in sub-Saharan Africa (13, 35), whereas African trypanosomiasis in animals costs $4.5 billion per year.

To be transmitted, the parasite must first establish itself in the insect midgut and undergo a subsequent maturation process into the salivary gland or the mouthparts, depending on the species of trypanosome (28, 30). Factors involved in establishment are largely unknown, and only a small proportion of flies develop mature infection and transmit the disease (16). Variability in vector competence depends on the species of Glossina and trypanosomes. Glossina morsitans is a good vector of Trypanosoma congolense (10, 18, 29), whereas Glossina palpalis is a poor vector (12, 18, 22). Conversely, Glossina palpalis is the main vector of Trypanosoma brucei gambiense (11), the causative agent of African trypanosomiasis in humans, whereas Glossina morsitans is not (7, 15).

Tsetse flies harbor three different symbionts (3), among which Sodalis glossinidius (1, 4, 5) is considered to be involved in vector competence (14) and to favor the establishment of the parasite in the insect midgut (32, 34). This role is still discussed (14, 19, 27, 31, 33). To investigate whether vector competence could be related to genetic diversity, we conducted an amplified fragment length polymorphism (AFLP) analysis of S. glossinidius strains from two species of Glossina.

Hemolymph of 20 G. palpalis gambiensis and 19 G. morsitans morsitans female flies was individually collected in phosphate-buffered saline. The bacteria were separated from insect cells by differential centrifugation (6). DNA was extracted from these bacteria and from the reference strain, S. glossinidius type strain M1 (5), using the DNeasy tissue kit.

The identity of the bacteria, including strain M1, was assessed by amplification of a specific PCR fragment using primers GPO1 F and GPO1 R (4, 5, 20) and analysis of the 16S rRNA gene as previously described (9). For each fly, sequencing of different clones did not show any difference, suggesting that only one bacterial strain was present or was the main component of a population. Bacterial DNA was digested with EcoRI and MseI. Double-stranded oligonucleotide adaptors (Table 1) were ligated to the restriction fragments. Preamplification was performed with nonselective primers. Amplification was performed using the first PCR products as template and five selective primer combinations (I to V [Table 1]). PCR products labeled with different markers were separated on a two-dye, model 4200 LI-COR automated DNA sequencer. Infrared images were analyzed using the AFLP-Quantar program. Only clear and unambiguous bands ranging between 150 and 500 bp were considered. DNA from strain M1 was used as a control to avoid artifactual polymorphism. The presence/absence of fragments was scored in a binary matrix. A similarity matrix (Jaccard coefficient) was calculated, and an unweighted neighbor-joining tree (8, 26) was built using DARwin version 4.0 (25).

View this table: [in this window] [in a new window]

TABLE 1. Double-stranded oligonucleotide adaptor sequences and combinations of primers used for AFLP selective amplification

Five combinations of primers (Table 1) were used to perform AFLP analysis on 39 S. glossinidius strains, which generated a variable number of AFLP markers depending on the primer pair (Table 2 and 3). One hundred sixty-five markers were selected for both genetic distance calculation and cluster analysis. About 14.5% of the markers from G. palpalis gambiensis bacterial strains were polymorphic (Table 2), whereas polymorphism was found in only 6% of those from G. morsitans morsitans symbionts (Table 3).

View this table: [in this window] [in a new window]

TABLE 2. AFLP markers generated on S. glossinidius strains from G. palpalis gambiensis using five primer pair combinations

View this table: [in this window] [in a new window]

TABLE 3. AFLP markers generated on S. glossinidius strains from G. morsitans morsitans using five primer pair combinations

The dendrogram representing the cluster distribution of reference strain M1 and 39 S. glossinidius strains sampled from G. palpalis gambiensis and G. morsitans morsitans is shown in Fig. 1. Strains from G. palpalis gambiensis are distributed within three clusters (I, II, and III) associated with high bootstrap values (i.e., 75 to 100). Three other clusters (IV, V, and VI), with low bootstrap values, can be distinguished and correspond to strains from G. morsitans morsitans. Reference strain M1 branches separately. Populations of S. glossinidius isolated from G. palpalis gambiensis and G. morsitans morsitans are genetically distinct. Furthermore, populations of S. glossinidius from G. palpalis gambiensis are strongly structured in genetically distinct groups, whereas populations from G. morsitans morsitans are not stringently structured and display limited genetic diversity.

View larger version (12K): [in this window] [in a new window]

FIG. 1. 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 (1,000 replicates). Genetic similarities were calculated using the Jaccard coefficient based on the 29 polymorphic band positions observed for five AFLP primer pairs. The tree was constructed with DARwin version 4.0 (25). Numbers represent S. glossinidius strains as follows: 1 to 20, strains from G. palpalis gambiensis; 21 to 39, strains from G. morsitans morsitans; M1, S. glossinidius reference strain M1.

This variation in the structure of populations must be regarded in connection with the specific biology of both S. glossinidius and tsetse flies. Glossina flies reproduce by adenotrophic viviparity, and S. glossinidius is vertically transmitted to the intrauterine developing larva (2, 4). Exchange of genetic material between strains of S. glossinidius is thus very unlikely, and, furthermore, G. palpalis gambiensis and G. morsitans morsitans are geographically separated. Vertical transmission, sequence similarity of cloned PCR product, high bootstrap values for cluster I to III, and clear genetic structure of the S. glossinidius populations suggest that the bacterial strains analyzed in this work are most likely clonal. The difference in genetic diversity observed between S. glossinidius strains from G. palpalis gambiensis and G. morsitans morsitans might therefore reflect differential host-driven selective pressure of closely related microorganisms, in agreement with hypotheses on the origin and evolution of S. glossinidius (24).

Vector competence is a major difference between G. palpalis gambiensis and G. morsitans morsitans (7, 10, 12, 15, 17, 18, 21, 22, 23, 29) which relates directly to the suggested role of S. glossinidius on the inhibition of trypanocidal insect lectins through the production of N-acetylglucosamine (32, 34). S. glossinidius in G. palpalis gambiensis might have been selected to facilitate the establishment and transmission of the parasite, explaining the high bootstrap values and the structured population. On the other hand, the presence of genetically different populations of S. glossinidius in G. morsitans morsitans might also be related to its differing vector competence. However, further research is needed to clearly establish the correlation between a given genotype of S. glossinidius and vector competence. The demonstration of the existence of genetic diversity in S. glossinidius is a first step towards the characterization of natural populations and a better understanding of the tripartite Glossina-Sodalis-Trypanosoma interactions most likely involved in the transmission of this deadly reemerging disease.

 
We are grateful to D. Verhaegen for his help in data analysis. We are particularly grateful to B. Tchicaya and J. Janelle for maintenance and management of the tsetse colonies.

 
* Corresponding author. Mailing address: UMR 17, IRD-CIRAD, CIRAD TA 207/G, Campus International de Baillarguet, 34398 Montpellier Cedex 5, France. Phone: 33 4 67 59 39 25. Fax: 33 4 67 59 39 20. E-mail: Anne.Geiger{at}mpl.ird.fr.

Top Abstract Introduction References

 

1
1. Aksoy, S., A. A. Pourhosseini, and A. Chow. 1995. Mycetome endosymbionts of tsetse flies constitute a distinct lineage related to Enterobacteriaceae. Insect Mol. Biol. 4:15-22.[Medline]
2
2. Aksoy, S., X. Chen, and V. Hypsa. 1997. Phylogeny and potential transmission routes of midgut-associated endosymbionts of tsetse (Diptera: Glossinidae). Insect Mol. Biol. 6:183-190.[Medline]
3
3. Aksoy, S. 2000. Tsetse—a haven for microorganisms. Parasitol. Today 16:114-118.[CrossRef][Medline]
4
4. Cheng, Q., and S. Aksoy. 1999. Tissue tropism, transmission and expression of foreign genes in vivo in midgut symbionts of tsetse flies. Insect Mol. Biol. 8:125-132.[CrossRef][Medline]
5
5. Dale, C., and I. Maudlin. 1999. Sodalis gen. nov. and Sodalis glossinidius sp. nov., a microaerophilic secondary endosymbiont of the tsetse fly Glossina morsitans morsitans. Int. J. Syst. Bacteriol. 49:267-275.[Abstract/Free Full Text]
6
6. Dale, C., S. A. Young, D. T. Haydon, and S. C. Welburn. 2001. The insect endosymbiont Sodalis glossinidius utilizes a type III secretion system for cell invasion. Proc. Natl. Acad. Sci. USA 98:1883-1888.[Abstract/Free Full Text]
7
7. Dukes, P., A. Kaukas, K. M. Hudson, T. Asonganyi, and J. K. Gashumba. 1989. A new method for isolating Trypanosoma brucei gambiense from sleeping sickness patients. Trans. R. Soc. Trop. Med. Hyg. 83:636-639.[CrossRef][Medline]
8
8. Gascuel, O. 1997. Concerning the NJ algorithm and its unweighted version UNJ, p. 149-171. In B. Mirkin, F. R. McMorris, F. Roberts, and A. Rzhetsky (ed.), Mathematical hierarchies and biology, vol. 37. American Mathematical Society, Providence, R.I.
9
9. Geiger, A., S. Ravel, R. Frutos, and G. Cuny. Sodalis glossinidius (Enterobacteriaceae) and vectorial competence of Glossina palpalis gambiensis and Glossina morsitans morsitans for Trypanosoma congolense savannah type. Curr. Microbiol., in press.
10
10. Harley, J. M. B., and A. J. Wilson. 1968. Comparison between Glossina morsitans, G. pallidipes and G. fusca as vectors of trypanosomes of Trypanosoma congolense group: the proportions infected experimentally and the numbers of infective organisms extruded during feeding. Ann. Trop. Med. Parasitol. 62:178-187.[Medline]
11
11. Hoare, C. A. 1972. The trypanosomes of mammals, a zoological monograph. Blackwell Scientific Publications, Oxford, England.
12
12. Kazadi, J. M. 2000. Interactions between vector and trypanosome in determining the vectorial competence of tsetse flies. PhD dissertation. University of Liège, Liege, Belgium.
13
13. Louis, F. J., P. P. Simarro, and J. Jannin. 2003. Status of sleeping sickness in 2003. Med. Trop. 63:228-230.
14
14. Maudlin, I., and D. S. Ellis. 1985. Association between intracellular Rickettsia-like infections of midgut cells and susceptibility to trypanosome infection in Glossina spp. Z. Parasitenkd. 71:683-687.[CrossRef][Medline]
15
15. Maudlin, I., P. Dukes, A. G. Luckins, and K. M. Hudson. 1986. Extrachromosomal inheritance of susceptibility to trypanosome infection in tsetse flies. II. Susceptibility of selected lines of Glossina morsitans morsitans to different stocks and species of trypanosome. Ann. Trop. Med. Parasitol. 80:97-105.[Medline]
16
16. Maudlin, I., and S. C. Welburn. 1994. Maturation of trypanosome infections in tsetse. Exp. Parasitol. 79:202-205.[CrossRef][Medline]
17
17. Moloo, S. K., T. Asonganyi, and L. Jenni. 1986. Cyclical development of Trypanosoma brucei gambiense from cattle and goats in Glossina. Acta Trop. 43:407-408.[Medline]
18
18. Moloo, S. K., and S. B. Kutuza. 1988. Comparative study on the infection rates of different laboratory strains of Glossina species by Trypanosoma congolense. Med. Vet. Entomol. 2:253-257.[Medline]
19
19. Moloo, S. K., and M. K. Shaw. 1989. Rickettsial infections of midgut cells are not associated with susceptibility of Glossina morsitans centralis to Trypanosoma congolense infection. Acta Trop. 46:223-227.[Medline]
20
20. O'Neill, S. L., R. H. Gooding, and S. Aksoy. 1993. Phylogenetically distant symbiotic microorganisms reside in Glossina midgut and ovary tissues. Med. Vet. Entomol. 7:377-383.[Medline]
21
21. Ravel, S., P. Grébaut, D. Cuisance, and G. Cuny. 2003. Monitoring the developmental status of Trypanosoma brucei gambiense in the tsetse fly by means of PCR analysis of anal and saliva drops. Acta Trop. 88:161-165.[Medline]
22
22. Reifenberg, J. M., D. Cuisance, J. L. Frezil, G. Cuny, and G. Duvallet. 1997. Comparison of the susceptibility of different Glossina species to simple and mixed infections with Trypanosoma (Nannomonas) congolense savannah and riverine forest types. Med. Vet. Entomol. 11:246-252.[Medline]
23
23. Richner, D., R. Brun, and L. Jenni. 1988. Production of metacyclic forms by cyclical transmission of West African Trypanosoma (T.) brucei isolates from man and animals. Acta Trop. 45:309-319.[Medline]
24
24. Rio, R. V. M., C. Lefebvre, A. Heddi, and S. Aksoy. 2003. Comparative genomics of insect-symbiotic bacteria: influence of host environment on microbial genome composition. Appl. Environ. Microbiol. 69:6825-6832.[Abstract/Free Full Text]
25
25. Roumagnac, P., L. Gagnevin, L. Gardan, L. Sutra, C. Manceau, E. R. Dickstein, J. B. Jones, P. Rott, and O. Pruvost. 2004. Polyphasic characterization of xanthomonads isolated from onion, garlic and Welsh onion (Allium spp.) and their relatedness to different Xanthomonas species. Int. J. Syst. Evol. Microbiol. 54:15-24.[Abstract/Free Full Text]
26
26. Saitou, N., and M. Nei. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406-425.[Abstract]
27
27. Shaw, M. K., and S. K. Moloo. 1991. Comparative studies on Rickettsia-like organisms in the midgut epithelial cells of different Glossina species. Parasitology 102:193-199.
28
28. Van Den Abbeele, J., Y. Claes, D. van Bockstaele, D. Le Ray, and M. Coosemans. 1999. Trypanosoma brucei spp. development in the tsetse fly: characterization of the post-mesocyclic stages in the foregut and proboscis. Parasitology 118:469-478.
29
29. Van den Bossche, P. 2000. The development of a new strategy for the sustainable control of bovine trypanosomiasis in Southern Africa. Thesis, University of Pretoria, Pretoria, South Africa.
30
30. Vickerman, K., L. Tetley, A. Hendry, and C. M. Turner. 1988. Biology of African trypanosomes in the tsetse fly. Biol. Cell 64:109-119.[CrossRef][Medline]
31
31. Welburn, S. C., and I. Maudlin. 1991. Rickettsia-like organisms, puparial temperature and susceptibility to trypanosome infection in Glossina morsitans. Parasitology 102:201-206.
32
32. Welburn, S. C., K. Arnold, I. Maudlin, and G. W. Gooday. 1993. Rickettsia-like organisms and chitinase production in relation to transmission of trypanosomes by tsetse flies. Parasitology 107:141-145.
33
33. Welburn, S. C., I. Maudlin, and D. H. Molyneux. 1994. Midgut lectin activity and sugar specificity in teneral and fed tsetse. Med. Vet. Entomol. 8:81-87.[Medline]
34
34. Welburn, S. C., and I. Maudlin. 1999. Tsetse-trypanosome interactions: rites of passage. Parasitol. Today 15:399-403.[CrossRef][Medline]
35
35. World Health Organization. 2001. African trypanosomiasis or sleeping sickness, fact sheet no. 259. [Online.] http://www.who.int/mediacentre/factsheets/fs259/en/.

---

Applied and Environmental Microbiology, December 2005, p. 8941-8943, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8941-8943.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Geiger, A., Ravel, S., Mateille, T., Janelle, J., Patrel, D., Cuny, G., Frutos, R. (2007). Vector Competence of Glossina palpalis gambiensis for Trypanosoma brucei s.l. and Genetic Diversity of the Symbiont Sodalis glossinidius. Mol Biol Evol 24: 102-109 [Abstract] [Full Text]  
• Weiss, B. L., Mouchotte, R., Rio, R. V. M., Wu, Y.-n., Wu, Z., Heddi, A., Aksoy, S. (2006). Interspecific Transfer of Bacterial Endosymbionts between Tsetse Fly Species: Infection Establishment and Effect on Host Fitness. Appl. Environ. Microbiol. 72: 7013-7021 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Geiger, A. Articles by Frutos, R. Search for Related Content PubMed PubMed Citation Articles by Geiger, A. Articles by Frutos, R. Agricola Articles by Geiger, A. Articles by Frutos, R.