Microsatellite markers for genetic population studies in Glossina palpalis gambiensis (Diptera: Glossinidae).
ABSTRACT Little is known about intraspecific variability in tsetse flies and its consequences for vectorial capacity. Microsatellite markers have been developed for Glossina palpalis gambiensis. Three loci have been identified and showed size polymorphisms for insectarium samples. G. palpalis gambiensis from Burkina Faso were also subjected to PCR to investigate then genetic variability. Amplifications were observed in different species belonging to the palpalis group. These molecular markers will be useful to estimate gene flow within G. palpalis gambiensis populations and analysis could be extended to related species.
- SourceAvailable from: Elvina Viennet[Show abstract] [Hide abstract]
ABSTRACT: The emergence and massive spread of bluetongue in Western Europe during 2006-2008 had disastrous consequences for sheep and cattle production and confirmed the ability of Palaearctic Culicoides (Diptera: Ceratopogonidae) to transmit the virus. Some aspects of Culicoides ecology, especially host-seeking and feeding behaviors, remain insufficiently described due to the difficulty of collecting them directly on a bait animal, the most reliable method to evaluate biting rates.Our aim was to compare typical animal-baited traps (drop trap and direct aspiration) to both a new sticky cover trap and a UV-light/suction trap (the most commonly used method to collect Culicoides). Collections were made from 1.45 hours before sunset to 1.45 hours after sunset in June/July 2009 at an experimental sheep farm (INRA, Nouzilly, Western France), with 3 replicates of a 4 sites×4 traps randomized Latin square using one sheep per site. Collected Culicoides individuals were sorted morphologically to species, sex and physiological stages for females. Sibling species were identified using a molecular assay. A total of 534 Culicoides belonging to 17 species was collected. Abundance was maximal in the drop trap (232 females and 4 males from 10 species) whereas the diversity was the highest in the UV-light/suction trap (136 females and 5 males from 15 species). Significant between-trap differences abundance and parity rates were observed. Only the direct aspiration collected exclusively host-seeking females, despite a concern that human manipulation may influence estimation of the biting rate. The sticky cover trap assessed accurately the biting rate of abundant species even if it might act as an interception trap. The drop trap collected the highest abundance of Culicoides and may have caught individuals not attracted by sheep but by its structure. Finally, abundances obtained using the UV-light/suction trap did not estimate accurately Culicoides biting rate.Parasites & Vectors 06/2011; 4:119. · 3.25 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Glossina palpalis palpalis (Diptera: Glossinidae) is widespread in west Africa, and is the main vector of sleeping sickness in Cameroon as well as in the Bas Congo Province of the Democratic Republic of Congo. However, little is known on the structure of its populations. We investigated G. p. palpalis population genetic structure in five sleeping sickness foci (four in Cameroon, one in Democratic Republic of Congo) using eight microsatellite DNA markers. A strong isolation by distance explains most of the population structure observed in our sampling sites of Cameroon and DRC. The populations here are composed of panmictic subpopulations occupying fairly wide zones with a very strong isolation by distance. Effective population sizes are probably between 20 and 300 individuals and if we assume densities between 120 and 2000 individuals per km2, dispersal distance between reproducing adults and their parents extends between 60 and 300 meters. This first investigation of population genetic structure of G. p. palpalis in Central Africa has evidenced random mating subpopulations over fairly large areas and is thus at variance with that found in West African populations of G. p. palpalis. This study brings new information on the isolation by distance at a macrogeographic scale which in turn brings useful information on how to organise regional tsetse control. Future investigations should be directed at temporal sampling to have more accurate measures of demographic parameters in order to help vector control decision.Parasites & Vectors 07/2011; 4:140. · 3.25 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Feeding success of free-living hematophagous insects depends on their ability to be active when hosts are available and to reach places where hosts are accessible. When the hematophagous insect is a vector of pathogens, determining the components of host-seeking behavior is of primary interest for the assessment of transmission risk. Our aim was to describe endo/exophagy and circadian host-seeking activity of Palaearctic Culicoides species, which are major biting pests and arbovirus vectors, using drop traps and suction traps baited with four sheep, as bluetongue virus hosts. Collections were carried out in the field, a largely-open stable and an enclosed stable during six collection periods of 24 hours in April/May, in late June and in September/October 2010 in western France. A total of 986 Culicoides belonging to 13 species, mainly C. brunnicans and C. obsoletus, was collected on animal baits. Culicoides brunnicans was clearly exophagic, whereas C. obsoletus was able to enter stables. Culicoides brunnicans exhibited a bimodal pattern of host-seeking activity with peaks just after sunrise and sunset. Culicoides obsoletus was active before sunset in spring and autumn and after sunset in summer, thus illustrating influence of other parameters than light, especially temperature. Description of host-seeking behaviors allowed us to discuss control strategies for transmission of Culicoides-borne pathogens, such as bluetongue virus. However, practical vector-control recommendations are difficult to provide because of the variation in the degree of endophagy and time of host-seeking activity.PLoS ONE 01/2012; 7(10):e48120. · 3.53 Impact Factor
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: email@example.com
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.
Bauer, B., Amsler-Delafosse, S . , Clausen, P. et al. (1995) Successful application of deltamethrin pour on
to cattle in a campaign against tsetse flies (Glossirin spp) in the pastoral zone of Samorogouan,
Burkina-Faso. Trop. Med. Parasitol. 46, 183-189.
Challier, A. (1 973)' Ecologie de Glossiria palpalis gariibierisis en savane d'Afrique Occidentale. %em.
ORSTOM, Paris, 64, 274 pp.
Elsen, P., Roelants, P. and De Lile (1994) Cytogenetic and isozymic comparisons of two laboratory lines
of Glossina pnlpalis gariibierisis. Ann. Trop. Med. Parasitol. 88(S), S I 1-522.
Estoup, A., Solignac, M., Harry, M. and Cornuet, J.M. (1993) Characterization of (GT)n and (CT)n
microsatellites in two insect species: Apis n?e/li/i.ra atid Boiitbw 'IPrresJris. Nucleic Acids Res. 21 (6),
1427- 143 I.
Estoup, A., Scholl, A., Pouvreau, A. and Solignac, M. (1995) Monoandry and polyandry in bumble bees
as evidenced by highly variable microsatellites. Mol. Ecol. 4, 89-93.
Gertsch, P., Pamilo, P. and Varvio, S.L. (1995) Microsatellites reveal high genetic diversity within
colonies of Camporiotics ants. Mol. Ecol. 4, 257-260.
Gooding, R.H. (1981) Genetic polymorphism in three species of tsetse flies in Upper Volta. Acta Trop.
Gooding, R.H., Moloo, S.K. and Rolseth, B.M. (1991) Genetic variation in Glossina breoipalpis, G.
Longipeririis and G. pallidipes and the phenetic relationships of Glossina species. Med. Vet. Entomol.
Gooding, R.H. and Rolseth, B.M. (I 995) Genetics of Clossiria palpolis palpalis: designation of linkage
groups and the mapping of eight biochemical and visible marker genes. Genome 38, 833-837.
P. Solmio el al. /Acta Tropica 65 (1997) 175- 160
Hughes, C.R. and Queller, D.C. (1993) Detection of highly polymorphic microsatellite Ioci in a species
with little allozyme polymorphism. Mol. Ecol. 2, 131-137.
Janhke, H.E., Tacher, G., Keil P et al. (1988) Livestock production in tropical Africa, \!#¡th special
reference to the tsetse-affected zone. In: Livestock Production in Tsetse Affected Areas of Africa.
Proc. Meeting 23-27/1 I/S8, Nairobi, Kenya, ILCA-ILRAD.
Lanzaro, G.C., Zheng, L. and Toure, Y.T. (1995) Microsatellite DNA and isozyme variability ¡II a West
African population of Anopheles Gariibiae. Insect Mol. Bio]. 4(2), 105-1 12.
Solano, P., Reifenberg, J.M. and Atnsler-Delafosse, S. (1996) Trypanosome characterization by PCR in
Glossina pulpalis gumbierisis and G. facltimides from Burkina Faso. Med. Vet. Entomol. IO. 354-35s.
Stallings, R.L., Ford, A.F and Nelson, D. (1991) Evolution and distribution of (GT)n repetitive
sequences in mammalian genomes. Genomics IO, 807-815.
Van der Planck, F.L. (1949) The classification of Glossirin palpalis, including the descriptions of new
subspecies and hybrids. Proc. R. Entomol. Soc. London (B)18, 69-77.
Weber, J.L. (1990) Informativeness of human (dC-dA)n- (dG-dT)n polymorphisms. Genoniics 7,
Zheng, L., Collins, F.H., Kumar, V. and Kafatos, F.C. (1993) A detailed genetic map for the S
chromosome of the malaria vector, Auoplieles gariibiae. Science 26(1), 605-608.
Zheng, L., Benedict, hl.Q., Cornell, A.J., Collins, F.H. and Kafatos, F.C. (1996) An integrated genetic
map the African human malaria vector mosquito, Anopkeles gambiue. Genetics 143(2), 9-11 -952.