Visceral leishmaniasis is not endemic in West Africa, but prevalence of canine leishmaniasis and seroprevalence of Leishmania infantum infection in humans are high in the Mont Rolland community (Thiès region, Senegal). Previous studies in this area showed that Sergentomyia schwetzi could be the potential vector of Le. infantum. To precisely describe the biology and population structure of this potential vector, we identified eight novel microsatellite loci to characterize Se. schwetzi populations. We tested these loci in Se. schwetzi populations from five locations at Mont Rolland (Thiès, Senegal). All the loci were polymorphic, with a mean of 17.25 alleles (observed heterozygosity: 0.455). We did not detect any evidence of scoring errors due to stuttering and large allele dropout. Moreover, several of these loci were also amplified in six other sand fly species (Sergentomyia magna, Sergentomyia dubia, Sergentomyia minuta, Phlebotomus duboscqi, Phlebotomus perniciosus, and Phlebotomus ariasi). These preliminary results demonstrate the utility of these microsatellite markers for Se. schwetzi (and for the other sand fly species) population genetic studies.
Leishmaniasis is a neglected disease that affects 0.9–1.6 million people worldwide, and causes 26,000–65,000 deaths each year. Over a billion people live in endemic areas at risk of infection (World Health Organization 2016). This disease can present different clinical forms, mostly in function of the infecting Leishmania species (Banuls et al. 2011). Only the visceral form is lethal if untreated, and represents 0.2–0.4 million cases each year. Since 2014, an increase of visceral leishmaniasis cases and deaths and of its geographical expansion has been observed (Ibarra-Meneses et al. 2020).
Leishmaniasis is caused by a protozoan parasite belonging to the Leishmania genus, transmitted through the bite of infected female sand flies (Abonnenc 1972). Sand flies are relatively small (2–3 mm) insects belonging to the Phlebotominae subfamily within the Psychodidae family (Tesh 1988). Both sexes feed on sweet juices, but only females take bloodmeals that are needed for egg maturation (Abonnenc 1972). Sand flies are distributed in tropical and subtropical areas, arid and semiarid areas, and temperate zones worldwide (Killick-Kendrick 1999). On the approximately 800 sand fly species recorded, two main genera are present in the Old World: Phlebotomus (Diptera: Psychodidae) (94 species) and Sergentomyia (258 species) (Kato et al. 2010). Species belonging to Phlebotomus genus are described as proven vectors of Leishmania in the Old World, although several studies also suggest the involvement of Sergentomyia species as Leishmania vectors (Kanjanopas et al. 2013, Jaouadi et al. 2015, Senghor et al. 2016).
Fig. 1.
Map of the study area. Description of the Mont-Rolland community (Thiès region, Senegal). Identification of the villages: Fouloum (Fm), Guidieur (G), Khaye Diagal (KaD), Keur Lat Diop (KLD), Ndiaye Bopp (NB), Pallo Diale (PD), Thiaye (T), and Twin Djassa (TD). The number of Sergentomyia schwetzi individuals collected (in brackets) and the soil characteristics are indicated. Adapted from Senghor et al. (2011) and Cassan et al. (2016).
This work is specifically focused on Se. schwetzi Adler, Theodor et Parrot 1929. Even if identification keys were established (Abonnenc 1972, Ba 1999), few articles were published on the systematic classification of Sergentomyia genus (Davidson 1990, Randrianambinintsoa et al. 2014) since until a few years ago they were not considered as potential vectors.
Visceral leishmaniasis is not endemic in West Africa. However, the prevalence of infections by Leishmania infantum, a species that causes visceral leishmaniasis in dogs, and its seroprevalence in humans are high in the Mont Rolland community (Thiès region, Senegal) (Faye et al. 2010, Faye et al. 2011). Entomological studies in this area showed that unsuspected sand fly species could be the Le. infantum vectors: Sergentomyia dubia and Sergentomyia schwetzi (Senghor et al. 2011, Senghor et al. 2016). However, no information is available on the population structure of these potential vectors. Yet, a better knowledge of these vectors is needed to precisely understand Leishmania transmission. Although few full sand fly genome sequences are available, the microsatellite marker-based method still remains one of the best approaches to study their population structure (Prudhomme et al. 2020).
Therefore, the aim of this work was to identify microsatellite markers to study Se. schwetzi population structure. We describe preliminary results based on 35 Se. schwetzi samples collected in the Mont Rolland community and using eight new polymorphic microsatellite loci. Moreover, we tested the transferability of these microsatellite markers to six other sand fly species (Sergentomyia magna, Se. dubia, Sergentomyia minuta, Phlebotomus duboscqi, Phlebotomus perniciosus, and Phlebotomus ariasi).
Material and Methods
Sand Fly Collection and Identification
In December 2012, sand flies were captured in the rural community of Mont Rolland, in the Thiès region (West Senegal), using sticky traps (20 × 20 cm white paper strips covered with castor oil), and CDC miniature light traps (John W. Hock Co., FL) (Alten et al. 2015, Ayhan et al. 2017). Se. schwetzi specimens were collected at Pallo Diale, Fouloum, Twin Djassa, Ndiaye Bopp and Thiaye. Se. magna, Se. dubia, and Ph. duboscqi specimens were collected at Fouloum, Guidieur, Keur Lat Diop, Khaye Diagal and Ndiaye Bopp (Fig. 1; Table 1, and Supp Table 1 (tjac109_suppl_supplementary_table_s1.xlsx) [online only]). Se. minuta, Ph. Perniciosus, and Ph. ariasi specimens were captured at Roquedur (Gard, France) between July 2011 and 2013 (Table 1, and Supp Table 1 (tjac109_suppl_supplementary_table_s1.xlsx) [online only]).
Samples were prepared, dissected, mounted, and identified as previously described in Prudhomme et al. (2020).
Selected Specimens
Fifty Se. schwetzi individuals were pooled and used to designed and synthesized the microsatellite markers. Then, 66 sand fly specimens were used to test these markers: 35 Se. schwetzi (Senegal), 5 Se. magna (Senegal), 5 Se. dubia (Senegal), 5 Se. minuta (Senegal), 5 Ph. duboscqi (Senegal), 6 Ph. perniciosus (France), and 5 Ph. ariasi (France) (Table 1 and Supp Table 1 (tjac109_suppl_supplementary_table_s1.xlsx) [online only]).
DNA Extraction, Isolation of Microsatellite Loci, and Identification
DNA was extracted from the thorax, wings, legs, and abdomen of each sand fly specimen using the Cetyl Trimethyl Ammonium Bromide (CTAB) method, as previously described in Prudhomme et al. (2015). Se. schwetzi specimens were used, by a private company (StarSEQ, Mainz, Germany), to isolate, clone, and sequence microsatellite markers using an Illumina MiSeq system (Prudhomme et al. 2015). Among the 84 locus pairs designed and synthesized, 8 were successfully amplified (Table 2).
DNA Amplification and Genotyping
All sand fly DNA samples were individually amplified. Each 25 µL reaction mix included 10 pmol of forward (labeled with a fluorochrome, FAM, ATT0565, or HEX) and reverse primers (Table 2), 2 µL of DNA template, 5 pmol of dNTP mix, 5 µL of 5X buffer, 2 µL of MgCl2, and 2 units of Taq polymerase (Promega). DNA was amplified using the following program: initial denaturation step at 95°C for 3 min, then 35 cycles at 95°C for 30 s, the annealing temperature of each locus (Table 1) for 30 s, extension at 72°C for 1 min, and a final extension step at 72°C for 5 min.
Genotyping was performed on a 3500xL Genetic Analyzer (Applied Biosystems) automated sequencer with 1 µL of PCR product added to a standard loading mix: 0.2 µL of internal standard-size GENESCAN 500LIZ and 15 µL of formamide (HiDi) (both Applied Biosystems). The GENEMAPPER 4.0 (Applied Biosystems) software was used to read and analyze the profiles. As the PCR products (loci) were of different sizes and labeled with different fluorochromes (Table 2), multiplex amplification was possible.
Table 1.
Description of the sand fly specimens used in this work
Table 2.
Details of the eight microsatellite markers selected: name, primer sequences, structure of the repeat array, annealing temperature (Ta), labeling dye, multiplexing group, allele number (Na), allele size range, observed heterozygosity (Ho), mean genetic diversity (Hs), expected heterozygosity (He), allelic richness (AR) and GenBank accession number
Data Analysis
Raw data were formatted for CREATE (Coombs et al. 2008) that allows their conversion for the different software programs used. Linkage disequilibrium (LD) between each locus pair was measured with the G-based test (10,000 permutations) (Goudet et al. 1996, Prudhomme et al. 2020).
FSTAT, version 2.9.4 (Goudet 1995) was used to compute the observed heterozygosity (Ho), expected heterozygosity (He), mean genetic diversity (Hs), allelic richness (AR), and inbreeding coefficient (FIS). Deviations from the Hardy–Weinberg equilibrium were measured using GENEPOP, version 4.2.2 (Rousset 2008). The individual estimations of Ho, Hs, He, and fixation index (FST) in the populations from the different locations, as well as the Cavalli-Sforza and Edwards genetic distances (Cavalli-Sforza and Edwards 1967) between populations were calculated using GENETIX 4.05 (Belkhir et al. 2009). These distances were used to build a Neighbor Joining (NJ) tree using Mega V6 (Tamura et al. 2013). Scoring errors due to stuttering, large allele dropout, and homozygote excess were computed with MicroChecker 2.2.3 (Van Oosterhout et al. 2004).
Results and Discussion
Selected Microsatellite Markers
Among the 84 microsatellite loci found (available on request), eight successfully amplified loci were selected on the basis of the criteria described in Prudhomme et al. (2015). These dinucleotide microsatellite loci are described in Table 2 (sequences available in GenBank). Their polymorphism in Se. schwetzi was tested using the 35 specimens from Senegal.
Genetics Analysis
In the Se. schwetzi samples from the five localities of Mont Rolland (Senegal), all eight loci were polymorphic (mean: 17.25 alleles per loci; range 9–22) (Table 3). The mean Ho, Hs, and He were 0.455 ± 0.187, 0.849 ± 0.103, and 0.888 ± 0.092, respectively. No deviation from the Hardy–Weinberg equilibrium was observed, and no LD was detected between loci. No evidence of scoring errors due to stuttering and large allele dropout was found.
Table 3.
Estimation of the allele number (Na), number of dinucleotide repeats (allele size range), observed heterozygosity (Ho), mean genetic diversity (Hs), expected heterozygosity (He), inbreeding coefficient (FIS), and allelic richness (AR) for the five Se. schwetzi populations (Pop, population; PD, Pallo Diale; Fm, Fouloum; TD, Twin Djassa; T, Thiaye; and NB, Ndiaye Bopp)
The allelic richness per locus and sampling localities varied from 1 to 1.945 (Table 3). The fixation index (FST) revealed a low genetic differentiation between Pallo Diale and Fouloum (FST = 0.0091), Pallo Diale and Twin Djassa (FST = –0.0018), Pallo Diale and Ndiaye Bopp (FST = 0.02460), Fouloum and Twin Djassa (FST = –0.00790), Fouloum and Ndiaye Bopp (FST = 0.00189), and Twin Djassa and Ndiaye Bopp (FST = –0.01626). The FST was moderate between Thiaye and Pallo Diale (FST = 0.10489), Thiaye and Fouloum (FST = 0.07588), and Thiaye and Twin Djassa (FST = 0.12828). The FST was higher between Ndiaye Bopp and Thiaye (FST = 0.18741). However, none of these FST values was significant, possibly due to the small number of individuals collected at each locality. In agreement with the low FST value, no strong structuring was observed between sampling localities on the NJ phenogram (Fig. 2). Additional molecular studies will be performed with more samples to determine Se. schwetzi genetic structure at different geographic scales. These analyses will bring genetic information on Se. schwetzi vector versus nonvector population. Nevertheless, these preliminary results suggest that these eight microsatellite loci are suitable for Se. schwetzi genetic study.
Fig. 2.
Neighbor-joining phenogram based on the Cavalli-Sforza chord measure, for the five Sergentomyia schwetzi populations collected at Fouloum (SS_Fm), Ndiaye Bopp (SS_NB), Pallo Diale (SS_PD), Thiaye (SS_T), and Twin Djassa (SS_TD). Sergentomyia dubia (Se. dubia) was used as outgroup.
Table 4.
Transferability of the eight microsatellite loci selected to other sand fly species (n = number of tested individuals)
Transferability Test to Other Species
Some loci could be amplified also in Se. magna (SSC5, SSC6, SSC7), Se. dubia (SSC3, SSC5, and SSC8), Se. minuta (all loci), Ph. duboscqi (SSC3, SSC5, SSC7, and SSC8), Ph. perniciosus (SSC1, SSC2, SSC3, SSC4, SSC6, and SSC7), and Ph. ariasi (SSC2 and SSC3) DNA samples. The number of alleles per locus for these loci are listed in Table 4, and Supp Table 1 (tjac109_suppl_supplementary_table_s1.xlsx) (online only). Therefore, these microsatellite loci can be used for genetic, phylogeographic, and evolutionary analyses of different species within the Phlebotomus and Sergentomyia genera.
Conclusions
Microsatellites are useful for assessing genetic diversity, specimen identification, phylogenetic studies, and as a tool to measure inbreeding and differences among populations. As microsatellites have a rapid evolution rate, they are particularly valuable to understand the relationships among closely related species (Okumu et al. 2017). These preliminary results demonstrate the utility of these microsatellite markers for Se. schwetzi (and for other sand fly species) population genetic studies.
Acknowledgments
We wish to thank the GenSeq technical facilities of ISEM, Institut des Sciences de l'Evolution de Montpellier and Labex Centre Méditerranéen de l'Environnement et de la Biodiversité where some of the sample preparations were performed. We also thank Université Cheikh Anta DIOP (UCAD), Institut de Recherche pour le Développement (IRD), Centre national de la recherche scientifique (CNRS) and Research Infrastructures for the control of vector-borne diseases (INFRAVEC2) for financial support and the community of Mont Rolland for their welcome. We also thank Elisabetta Andermarcher for assistance in preparing and editing the manuscript.
Author Contributions
J.P., B.F., M.W.S, A.A.N, and A.L.B. designed the study, T.M. and S.H. performed the molecular characterization. J.P. and T.M. analyzed the data. J.P., T.M., S.H., C.T., C.C., M.W.S, A.A.N, B.F. and A-L.B. contributed to field sampling, sand fly species identification or sample preparation. J.P. and A-L.B. wrote the manuscript with support from T.M., S.H., C.T., M.W.S., A.A.N., B.F. and C.C. All authors have read and approved the actual version of the manuscript.
Data Availability
All resources used in this article are provided in the Supp Table S1 (tjac109_suppl_supplementary_table_s1.xlsx) [online only] and all the analyses are detailed allowing the assessment or verification of the manuscript's findings.
