DOI: 10.5061/dryad.hmgqnk9x1
title: Data from: Molecular detection and phylogenetic characterization of <em>Wolbachia</em> strains in <em>Glossina</em> species from Nigerian conservation areas

description: Tsetse flies (Glossina spp.), the vectors of human and animal African
 trypanosomiasis, continue to exert a profound toll on public health and
 livestock productivity in Nigeria. This study investigates the prevalence,
 seasonal dynamics, and genetic diversity of Wolbachia endosymbionts in
 7,632 wild-caught tsetse flies sampled from four ecologically distinct
 conservation sites: Yankari Game Reserve, Kainji Lake National Park,
 Kagarko Forest, and Ijah Gwari Forest. Molecular screening based on wsp
 gene sequencing detected Wolbachia in 1,771 flies, with infection rates
 rising significantly during the wet season (e.g., G. morsitans
 submorsitans: 75.4% vs. 39.8% in dry season; p &lt; 0.001). Female
 flies showed consistently higher infection prevalence, reinforcing the
 role of vertical transmission. Phylogenetic reconstruction revealed nine
 Wolbachia strains spanning supergroups A and B, including a putatively
 unique regional variant (wsp9) restricted to northern Nigeria. Bacterial
 load exhibited a strong age-dependent pattern (r = 0.912, p &lt;
 0.001), and elevated GC content (~63%) suggested possible adaptation to
 savanna thermal conditions. These findings highlight the ecological
 flexibility of Wolbachia within natural tsetse populations and point to
 its potential application in vector control—particularly through
 mechanisms like cytoplasmic incompatibility. By combining molecular
 detection, ecological data, and evolutionary analysis, this study lays the
 groundwork for tailored, climate-sensitive Wolbachia-based strategies to
 reduce tsetse populations and support trypanosomiasis control in Nigeria.
description: <strong>Study Area</strong> The
 study examined four distinct conservation areas in Nigeria, focusing on
 different savanna and forest ecosystems. The Yankari Game Reserve in
 Bauchi State, Nigeria, features Sudan savanna vegetation and riparian
 forests. Kainji Lake National Park in Niger State, Nigeria, features a
 Guinea savanna ecosystem with gallery forests. Kagarko Forest in Kaduna
 State, Nigeria, has a derived savanna habitat with dense riverine
 thickets. Ijah Gwari Forest in Niger State, Nigeria, has a
 rainforest-savanna mosaic (Abubakar <em>et al.,</em> 2016;
 Isaac <em>et al.,</em> 2016).
 <strong>Sample Size Justification</strong>
 Sample sizes were determined based on prior estimates of tsetse
 population densities in Nigerian conservation areas (Shaida <em>et
 al.,</em> 2018), ensuring ≥80% statistical power to detect a minimum
 10% difference in <em>Wolbachia</em> prevalence between
 seasons at a significance level of α = 0.05.
 <strong>Sample Collection</strong>
 We collected samples monthly from April 2017 to July 2019,
 encompassing both dry and wet seasons a total of 7,632 flies were
 collected using Biconical traps, as described by Challier and Laveissière
 (1973), traps were baited with acetone and cow urine and deployed at
 50-meter intervals along transects in shaded microhabitats. Species
 identification was conducted using morphological keys developed by Potts
 (FAO, 2018), allowing for discrimination among <em>G. m.
 submorsitans</em>, <em>G. p. palpalis</em>, and
 <em>G. tachinoides</em>. Flies were dissected under sterile
 conditions, and the midgut, salivary glands, and reproductive tissues were
 preserved in 70% ethanol for subsequent DNA extraction (Weber <em>et
 al.,</em> 2019). <strong>Tissue Selection
 and DNA Extraction</strong>
 <em>Wolbachia</em> was targeted in reproductive
 tissues, midguts, and salivary glands due to their established or
 suspected roles in vertical transmission and systemic colonization in
 tsetse (Balmand <em>et al.,</em> 2013). Genomic DNA was
 extracted using the AccuPrep Genomic DNA Extraction Kit (Bioneer, Korea),
 based on manufacturer's instructions. Approximately 10 mg of
 reproductive tissues, midgut or salivary gland tissue was lysed in 200 µL
 G-Buffer with 20 µL Proteinase K at 56°C for one hour. The lysate was
 combined with 200 µL of Binding Buffer and transferred to AccuPrep DNA
 extraction columns. After two successive washes with 500 µL of W-Buffer,
 DNA was eluted in 50 µL of Elution Buffer (10 mM Tris-HCl, pH 8.5). DNA
 concentration and purity were assessed using a NanoDrop spectrophotometer
 (Thermo Fisher Scientific), and samples with A260/A280 ratios below 1.7
 were re-purified (Bioneer, 2018). <strong>PCR
 Amplification and Controls</strong>
 <em>AccuPower</em> PreMix PCR master mix from Bioneer
 was used based on manufactures instructions, two gene targets were used to
 detect and type <em>Wolbachia</em> strains: the
 <em>wsp</em> gene (<em>Wolbachia</em> surface
 protein), a primary marker for strain typing, and the 16S rRNA gene as a
 confirmatory marker. The primer sets used were 81F/691R for
 <em>wsp</em>. Primer pairs included wsp-specific 81F
 (5′-TGGTCCAATAAGTGATGAAGAAAC-3′) and 691R (5′-AAAAATTAAACGCTACTCCA-3′)
 (Zhou <em>et al.,</em> 1998) and along with 16S rRNA-targeting
 wsp F (5′-CATACCTATTCGAAGGGATAG-3′) and wsp R
 (5′-AGCTTCGAGTGAAACCAATTC-3′). PCR cycling conditions included an initial
 denaturation at 95°C for 5 minutes, followed by 35 cycles of denaturation
 at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at
 72°C for 1 minute, with a final elongation step at 72°C for 10 minutes.
 Positive controls included DNA from
 <em>Wolbachia</em>-infected <em>Drosophila</em>,
 and nuclease-free water was used as the negative controll (Bioneer, 2018,
 <em>Weber et al.,</em> 2019).
 <strong>Negative Controls</strong><br> Each PCR
 batch included a no-template control (nuclease-free water) and a positive
 control (<em>Wolbachia</em>-infected
 <em>Drosophila</em> DNA) to monitor for contamination and
 ensure amplification efficiency. <strong>Gel
 Electrophoresis and Sequencing and Phylogenetic
 Analysis</strong> Amplified PCR products were
 resolved on 1.5% agarose gels stained with ethidium bromide and visualized
 under UV illumination. Positive amplicons were purified using the QIAquick
 PCR Purification Kit (Qiagen) and sent to Macrogen Inc. (South Korea) for
 bidirectional Sanger sequencing (Sikkema‐Raddatz, 2013). Phylogenetic
 Analysis Sequence alignment and phylogenetic reconstruction were performed
 using MEGA-X software (Kumar <em>et al.,</em> 2018).
 Alignments were generated using the MUSCLE algorithm (Edgar, 2004) with
 default parameters. Phylogenetic trees were constructed using the Maximum
 Likelihood method under the GTR+G+I model with 1,000 bootstrap replicates.
 Reference sequences included known <em>Wolbachia</em> strains
 from <em>Glossina</em> spp. (GenBank accessions: KP715092,
 JX273258) and outgroup sequences from <em>Rickettsia</em>
 spp.
description: # Data from: Molecular detection and phylogenetic characterization of
 *Wolbachia* strains in *Glossina* species from Nigerian conservation areas
 Dataset DOI: [10.5061/dryad.hmgqnk9x1](10.5061/dryad.hmgqnk9x1) ##
 Description of the data and file structure ### Files and variables ####
 File: Dryad_data.xlsx **Description:** `Site`: Name of the conservation
 area (e.g., Yankari Game Reserve) ##### Variables: * `Season`: Sampling
 season — either “Dry” or “Wet” * `Screened`: Number of tsetse flies tested
 for Wolbachia * `Positive`: Number of flies that tested positive for
 Wolbachia * `Prevalence (%)`: Percentage of infected flies = (Positive /
 Screened) × 100 * `Chi-square`: Chi-square test statistic for seasonal
 comparison * `P-value`: Statistical significance of seasonal variation *
 `Species`: Scientific name of the tsetse species * *Glossina morsitans
 submorsitans* * *Glossina tachinoides* * *Glossina palpalis palpalis* *
 `Season`: “Dry” or “Wet” * `Screened`: Number of flies tested *
 `Positive`: Number of infected flies * `Prevalence (%)`: Infection rate *
 `Chi-square`: Test statistic comparing seasonal prevalence * `P-value`:
 Significance level