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

Abstract

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.

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).

TABLE 1.

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

Direction or no.	EcoRI primer	MseI primer
Adaptors
Forward	5′-CTCGTAGACTGC GTACC-3′	5′-GACGATGAGTCC TGAG-3′
Reverse	5′-AATTGGTACGCA GTCTAC-3′	5′-TACTCAGGACTC AT-3′
Combinations of primersc
I	EcoRI-AG*a	MseI-0
II	EcoRI-AG*a	MseI-C
III	EcoRI-C*b	MseI-0
IV	EcoRI-C*b	MseI-C
V	EcoRI-0	MseI-C*b

^aPrimer labeled with infrared dye IRD800.

^bPrimer labeled with infrared dye IRD700.

^cLetters after the restriction enzyme name represent the nucleotide base(s) added to each primer to select only a subset of the fragments using PCR amplification. A zero indicates that no base was added. *, primer labeled for detection.

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).

TABLE 2.

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

No.	Primer pairc	Total no. of markers	No. of polymorphic markers	% Polymorphic markers
I	EcoRI-AG*a/MseI-0	24	5	20.8
II	EcoRI-AG*a/MseI-C	15	3	20
III	EcoRI-C*b/MseI-0	55	7	12.7
IV	EcoRI-C*b/MseI-C	35	6	17.1
V	EcoRI-0/MseI-C*b	36	3	8.3
Total		165	24	14.5

^aPrimer labeled with infrared dye IRD800.

^bPrimer labeled with infrared dye IRD700.

^cLetters after the restriction enzyme name represent the nucleotide base(s) added to each primer to select only a subset of the fragments using PCR amplification. A zero indicates that no base was added. *, primer labeled for detection.

TABLE 3.

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

No.	Primer pairc	Total no. of markers	No. of polymorphic markers	% Polymorphic markers
I	EcoRI-AG*a/MseI-0	24	2	8.3
II	EcoRI-AG*a/MseI-C	15	0	0
III	EcoRI-C*b/MseI-0	55	6	10.9
IV	EcoRI-C*b/MseI-C	35	2	5.7
V	EcoRI-0/MseI-C*b	36	0	0
Total		165	10	6

^aPrimer labeled with infrared dye IRD800.

^bPrimer labeled with infrared dye IRD700.

^cLetters after the restriction enzyme name represent the nucleotide base(s) added to each primer to select only a subset of the fragments using PCR amplification. A zero indicates that no base was added. *, primer labeled for detection.

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.

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.