I ' . .
Acta Tropica 65 (1997) 175-180
Microsatellite markers for genetic population
studies in Glossina palpalis (Dìptera : Glossìnìdae)
P. Solano a , G. Duvallet b, V. Dumas cy D. Cuisance b, G. Cuny '**
Cirdes Bobo Dioulasso, Burkina Faso
CIRAD-EA4 VT campus de Buillurgitet, i2.lonlpellier, Frorice
Laboratoire d'Epidhiologie des A4uluaies à Vecteurs, Orstoril,
Departemerit Suilté 91 I avenite Agropolis, Aiforitpellier, France
Received 5 November 1996; received in re&ed'form 30 December 1996; accepted 10 February 1997
Little is known about tsetse intraspecific variability and its consequences on vectorial
capacity. Since isoenzyme analyses revealed little polymorphism, microsatellite markers have
been developed for Glossiria palpalis gar71biei7sis species. Three loci have been identified and
showed size polyinorphisms for insectarium samples. Moreover, amplifications were ob-
served in different species belonging to palpalis group. These molecular markers will be
useful to estimate gene flow within G. p . gari~bier~sis
extended to related species. O 1997 Elsevier Science B.V.
populations and analyses could be
Keyivords: Microsatellite; Glossina palpalis; Population genetics; Trypanosomosis; G.
jiiscipes; G. ~norsiiaris
Most species of the genus Clossiria play a potential vector role in the transmission
of African TrJpaiiosoniosis, which has considerable economic impact in subsaharan
* Corresponding author. Present address: Centre Orstoni, Departement Santé, LEMV, BP 5045,
34 032 Montpellier Cedex. Tel.: + 33 67416298; fax: + 33 67547800; e-mail: firstname.lastname@example.org
0001-706X/97/SI 7.00 Q 1997 Elsevier Science B.V. AI1 rights reserved.
Fonds Documentaire ORSTOM
P. Solano et al. /Acta Tropìca 65 (1997) 175- IS0
Africa (Janhke et al., 19SS). In West Africa, species of the palpalis group (subgenus
Neinorhiira) are involved in transmission of Animal trypanosomosis (nagana) and
Human trypanosomosis (sleeping sickness). Despite their iniportance little is known
about tsetse population genetics and their implications for the transmission of
trypanosomes: very little information is .available on the possible structuration of
tsetse populations which could lead them to express resistance to control measures,
by avoiding traps or treated animals for example. Intraspecific variation and related
differential vectorial capacity is suspected to occur in natural G. palpulis gaiiibieirsis
populations (Bauer et al., 1995; Solano et al., 1996). However, previous studies
using isozyme analyses were undertaken only for interspecies comparisons (Good-
ing et al., 1991) or for purpose of genetic assignment of loci (Gooding and Rolseth,
1995). Natural populations of tsetse flies of Burkina Faso showed little polymor-
phism using isozyme data on five loci (Gooding, 1991). Genetic studies were hence
of limited value because of the lack of accurate technologies.
Among insects, microsatellite loci have mostly been developed for social species
like ants (Gertsch et al., 1995), bees (Estoup et al., 1993, 1995), or wasps (Hughes
and Queller, 1993). In the field of medical or veterinary entomology, studies are still
rare, and microsatelllites have only been developped in Anopheles gainbicre (Zheng
et al., 1993, 1996; Lanzar0 et al., 1995) and Siinuliuii? d~111111110s21111
This paper reports on the isolation of microsatellite sequences in G. p. gai~ibiemis,
a riverine species widespread in West Africa and their potential use for population
genetics in this taxa and in related species.
2. Material and methods
DNA (20 pg) from 50 individual G. p. gaiiibieiuis, originating from the CIRAD/
ORSTOM insectarium (Montpellier, France), was digested to completion overnight
with HueIII. The 400- SOO bp fraction was recovered and ligated into the deplios-
phorylated EcoRV site of M 13 BM 20 (Boehringer-Mannheim). Ligation products
were used to transform E.coli XL1 Blue cells and 4500 recombinants clones were
lifted on Hybond-N + membranes. Hybridizations were carried out with (CA)n and
(GA)n probes, labelled with ~CTP-CX[~~P],
sham) according to manufacturer instructions. Positive clones were dot-blotted and
re-screened to ensure specificity. Eleven clones were kept after this secondary
screening. Eight of them were sequenced by the dideoxy-chain termination method,
using the Taq dye primer kit and an automatic sequencer (Applied Biosystems).
Teniplate DNA for PCR was prepared by incubating two legs of a fly in 5%
chelex for 1 li at 56"(2', then 30 min at 95°C. Amplification reactions were
performed in a Perkin Elmer thermal cycler, in a final volume of 50 pl containing
as final concentrations 1 x Appligene incubation buffer with 1.5 mM MgCl,, 200
/i M of each dNTP, 15 pmol of each primer and 0.5 U Appligene Taq Polymerase.
Samples were first denatured during 90 s at 92°C and then processed through 35
cycles consisting of 30 s at 92"C, 30 s at 50° C for loci 55.3 and 19.62 and 4S"C for
using rapid hybridization buffer (Anier-
' 7 4
P. Solario et al. /Acta Tropica 65 (1997) 17S- Iso
locus 69.22 and 1 min at 72°C. The last elongation step was lengthened to 10 min.
An amount of 15 pl of each amplified saiiiple was resolved on 12% non-denaturing
3 . Results
Of these eight clones sequenced, four were false positives and microsatellite
sequences isere successfully obtained for four clones. The presence of false positives
can be explained by an imperfect homology of sequences between the clones and
the microsatellite probes due to the low stringency of the hybridization washes.
Tivo clones had microsatellite sequences located too close to the cloning site to
allow primer selection; fortunately, as one of thein (6G- )
also, three pairs of primers could be designed.
A sequence was considered as a microsatellite if the number of repetitions of the
dinucleotide motif was six at a minimuni (Stallings et al., 1991). According to
Weber (1990), the three microsatellite loci were classified as ‘perfect’ for 69.22 and
19.62 ((TA),, and (GT),,, respectively) and ‘imperfect’ for 55.3 (GT),, GC(GTh.
Eight individual G. p. guiizbieizsis from the insectarium were individually tested by
PCR with the three primer pairs and PCR products were size-fractioned on 12%
acrylamide gels with appropriate markers. Results were as follows: locus 55.3
showed four alleles, locus 19.62 showed three and locus 69.22 showed two alleles
(Table I). Allele size was highly variable; for example, 20 bp separated the largest
and smallest alleles at locus 19.62 (Table 1).
The three primer pairs gave also a strong signal with wild G. p. gumbiensis from
Mali; the three individuals tested had some alleles in common with sonie of the
insectarium tsetse, for example allele 176 bp at locus 19.62, which appears as the
most common. Wild G. pnlpalis palpulis from Cameroun and a laboratory colony of
G. jtscipes jtscipes gave also scorable signals for the three primer pairs with
intra-colony variability at the three loci for G. p. palpalis, and at loci 55.3 aiid 19.62
for G. f: jitscipes (Table 2). Only primer pairs 19.62 amplified an appropriate sized
product froin DNA of G, tuchii?oides. No amplification signal could be obtained at
owned a (TA) repeat
Characteristics of the three microsatellite loci among a laboratory sample of eight G . p. gariibierisis
Locus Repeat sequence Allele sizes (bp) Primer sequence
18 1, 183, 187, 197
176, 178, 196
1 7 5
P. Solario et al. /Acta Tropica 65 (1997) 175-IS0
Size of the bands (when observed) in other tsetse taxa
Bands observed (bp)
G. p. palpalis
G. J jìisc@es
G. ni. sirbiiiorsi- G. III. iiiorsitaris
any locus with either G. niorsitans morsitans nor than with G. niorsitans subniorsi-
The (CA)n and (GA)n probes used in this work allowed three primer pairs which
showed size polymorphisms in a laboratory sample of G. p. gambiensis, to be
designed. The fact that one locus (69.22) consisted of a (TA) repeat whereas the
probes used (CA)n and (GA)n could be explained by the presence of a second GT
repeat too close to the cloning site to allow primer selection.
The tsetse individuals from different origins used in this study were just tested for
scorable amplifications. After this first step, heritability of the presumed alleles
should be demonstrated, then the microsatellite markers will be used to estimate
gene flows within species. G. p. gai?ibimsis is of particular interest since previous
work has shown great plasticity of behaviour in Burkina Faso (Challier, 1973;
Bauer et al., 1995; Solano et al., 1996) and great variability in transmitting
Trupaiiosoriia brucei gariibieizse (Elsen et al., 1994). At the moment, there is no
strong evidence that tsetse populations are structured, as is the case in A~zopkeles
gaiiibiae, for example (Lanzar0 et al., 1995), but this could be due to the lack of
studies of this type of tsetse.
All tsetse belong to one genus Glossi~la, that is divided into three subgenera,
Aiisteiiiiia (fusca group), Nemorhiiia (palpalis group) and Glossiiia (morsitaiis
group). G. 117. srrbniorsitaiis and G. 1 1 1 . nors sit airs are in the 17iorsitaiis group and
could not be amplified with any primer sets used in this study. In contrast, all three
loci were correctly amplified in both G. p. palpalis and G. f. fuscipes from the
palpalis group. Indeed these species were first classified as a single one, G. palpalis
(Van der Planck, 1949), and they showed great similarity at isoenzyme loci (Fig. 1).
Finally, G. tackiiioides belongs also to the palpalis group, but is known to be quite
different from G. palpalis, regarding ecological behaviour as well as genetic data
(Gooding et al., 1991). In this work, the primer specificity reflected well the
generally accepted phylogenetic relationships between the tsetse taxa (Fig. 1). In the
future, these genetic studies could be extended to related species because interspe-
cific conservation of flanking sequences will support use of these loci.
p. Solano el a l . /Acla Tropica 45 (1997) 175-IS0
G. oaloalis Daloalis
G. morsilans submorsitan
Mean genetic identity
Fig. I. Phenogram for nine taxa of tsetse flies, based upon loci for 12 enzymes (Gooding, 1981,
This work was supported by the CIRAD-EMVT and the centre ORSTOM
Montpellier, France, and will continue at the CrRDES Bobo Dioulasso, Burkina
Faso. The authors acknowledge Dr O. Dial1 (Laboratoire Central Vétérinaire,
Bamako, Mali) for the gift of the specimens from Mali.
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