Ticks
Ornithodoros phacochoerus ticks were collected in Mozambique in 2021 and 2022, in the Coutada 9 Game Reserve from the district of Macossa (n = 29, GPS coordinates: -17.7681; 33.8348) and in the Gorongosa National Park from the district of Gorongosa (n = 29, GPS coordinates: -18.9775; 34.3521). They were shared under Material Transfer Agreement with the Mozambique Institute of Agricultural Research (IIAM) and in compliance with the Nagoya Protocol.
O. moubata, O. porcinus, O. maritimus, and O. erraticus ticks (n = 2 per species) came from colonies maintained at the CIRAD laboratory (Montpellier, France, member of the Vectopole Sud network) since 2008, 2012, 2015 and 2016 respectively.
The Ornithodoros moubata colony originated from the Neuchâtel strain initially collected in Tanzania and maintained at the University of Neuchâtel (Switzerland). The Ornithodoros porcinus ticks were initially sampled in Mahitsy (Madagascar) [12]. The Ornithodoros maritimus ticks were collected in the field on the island of Carteau in Camargue (France) [13]. Finally, the Ornithodoros erraticus ticks originated from the “Alentejo” strain collected in the field in Alentejo (Portugal) in 2013 and 2016 [14].
DNA extractions
Nymph and adult ticks were washed in a 1% bleach bath for 30 seconds, then rinsed for 1 minute in three consecutive baths of Milli-Q water to eliminate cuticular bacteria and avoid contamination for other downstream analyses such as ticks microbiota characterization [15]. Ticks were then cut and crushed individually using small scissors and pellet pestles. DNA was extracted from the crushed tick homogenate, using the standard protocol from the DNeasy® Blood & Tissue genomic DNA extraction kit (Qiagen, Hilden, Germany). DNA extracts were finally eluted in 200 µl of elution buffer and stored at -20°C until further use.
Tick genomes and elimination of repeated sequences
After sequencing COI (primers: forward 5’-AATTTACAGTTTATCGCCT-3’, reverse 5’-CATACAATAAAGCCTAATA-3’ and forward 5’-GGAACAATATATTTAATTTTTGG-3’, reverse 5’-ATCTATCCCTACTGTAAATATATG-3’ [16]) 12S rRNA gene (primers: forward 5’-AAACTAGGATTAGATACCCT − 3’, reverse 5’-AATGAGAGCGACGGGCGATGT-3’ [17]) and 16S rRNA gene (primers: forward 5’-CTGCTCAATGATTTTTTAAATTGCTGTGG-3’, reverse 5’- CCGGTCTGAACTCAGATCAAGT-3’ [18]), the ticks sampled in Mozambique were identified as Ornithodoros phacochoerus (Additional file 1: Dataset S1). Since no genome was available for this species, three genomic datasets from closely related species were used for microsatellite design [11]: one genome from Ornithodoros moubata (cell line) and two genomes from Ornithodoros porcinus (Kenya and Madagascar ticks). These genomic data were published by the Friedrich Loeffler Institute in Germany.
Tick genomes contain multiple sequence repeats, making microsatellite design a challenging task [19,20]. To optimize this design, we employed the method published by Shah et al. for the elimination of repeated sequences in complex genomes [21]. For this purpose, the three tick genomes were screened for repeated sequences using RepeatExplorer2 clustering on Galaxy version 2.3.8.1 [22]. For each genome, reads identified as singletons by RepeatExplorer2 were retained for microsatellite mining, while sequences in clusters were discarded [21].
Microsatellite design and selection
After elimination of repeated sequences, Palfinder [23] and Primer3 [24] from the Galaxy palfinder pipeline [25] were used to screen for microsatellite motifs and to design primer sequences for the potential markers. In total, 40,170 potential markers were obtained from the O. moubata genome, 18,689 from O. porcinus Kenya, and 33,006 from O. porcinus Madagascar. Sequences were then compared between the three genomes to keep only the potential markers that were common between at least two of the genomes. To be selected for further analyses, the markers also needed to be polymorphic between the two genomes or have a microsatellite pattern repeated at least eight times. In the end, 74 markers were kept from the comparisons between O. moubata and O. porcinus genomes, and 77 between O. porcinus Kenya and O. porcinus Madagascar genomes, for a total of 151 potential microsatellite markers (named from ms-1 to ms-151).
PCR test on O. phacochoerus and genotyping
All 151 potential markers were amplified by touchdown PCR using a 5’-end M13 extension (5’-CACGACGTTGTAAAACGAC-3’) on the forward primer and fluorescent M13 dye (FAM, VIC or NED) added to the PCR mix [26]. These first tests were performed on a batch of thirty samples from different populations of O. phacochoerus and two samples of O. porcinus as positive controls. Genotyping was performed at the GPTR laboratory (Great Regional Technical Platform of genotyping, AGAP Institut/CIRAD, Montpellier, France) with an ABI 3500xL Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Of the 151 markers tested, 24 were selected according to the following criteria: successful amplification for O. phacochoerus, polymorphic between at least two O. phacochoerus samples, PCR products size ranging from 60bp to 500bp (for full sequences, see Additional file 2: Dataset S2).
For the 24 selected markers, fluorescent-labeled forward primers (FAM, VIC, NED or PET) were designed. Touchdown PCRs were performed in six multiplexes of four markers each. PCRs and genotyping tests were performed to adjust the concentration of each primer in the multiplexes (Additional file 3: Table S1).
The amplification mix consisted in 2 µL of DNA template, 10 µL of 2x Type-it Microsatellite PCR Kit (Qiagen, Courtaboeuf, France), adjusted volume of fluorescence-labeled forward primer and reverse primer for each of the four markers amplified in the PCR (according to the concentration chosen in Additional file 3) in a final volume of 20 µL. The touchdown PCR program was set as follow: 95°C for 3 min, then 10 cycles of 95°C for 20 sec, 60°C -0.5°C/cycle, for 30 sec and 72°C for 1 min, then 30 cycles of 95°C for 20 sec, 55°C for 30 sec, and 72°C for 1 min, followed by a final extension step at 72°C for 7 min.
Formamide for denaturation and GeneScan-600 (LIZ) Size Standard Kit for ladder (Applied Biosystems, Foster City, CA, USA) were added to the PCR products before genotyping by capillary electrophoresis at the GPTR platform.
The 24 selected markers were tested on two populations of Ornithodoros phacochoerus ticks (Coutada 9 and Gorongosa) and on closely related (O. porcinus and O. moubata) and more distant (O. maritimus and O. erraticus) Ornithodoros species with two individuals for each species.
Allelic diversity and statistics
Genotypes were read using GeneMapper® v.6 software (Applied Biosystems, Waltham, MA, USA). Allele bins were set manually after a review of all samples. Allele scoring was performed automatically according to the bin set designed for the marker, then manually checked by two different experimenters. Alleles were named according to their length in base pairs.
The resulting dataset was converted to Fstat and Micro-Checker format using CREATE [27]. Linkage disequilibrium p-values were calculated using Fstat v 2.9.4. [28], then corrected with Benjamini and Yekutieli correction [29] on R version 4.2.3 (2023-03-15) [30]. Presence of null alleles, stuttering and short allele dominance were tested using Micro-Checker [31]. When possible, correction for stuttering was performed by pooling alleles with overlapping signals, then stuttering was re-evaluated [32]. Observed and expected heterozygosity, Fis and Fst were calculated using Fstat v 2.9.4. [28].
After genotyping, two loci were readily eliminated: ms-46 which was monomorphic for all O. phacochoerus samples and ms-66 for which several individuals presented more than two alleles suggesting that the marker was duplicated in O. phacochoerus genome. Moreover, loci ms-96 and ms-64 were difficult to read due to the low quality of the profiles (low intensity profiles in which peaks were difficult to distinguish from each other). Finally, locus ms-82 was the only locus with an absence of heterozygote profiles in all samples leading to its elimination. The results obtained from these five markers are presented but they were not selected in the proposed subset of markers, leading to a subset of 19 markers selected out of 24 markers tested (Table 1). When peaks were of low intensity in some of the samples, a threshold of peak intensity was set, below which the samples were not scored.