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 < 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 < 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.

Study Area

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 et al., 2016; Isaac et al., 2016).

Sample Size Justification

Sample sizes were determined based on prior estimates of tsetse population densities in Nigerian conservation areas (Shaida et al., 2018), ensuring ≥80% statistical power to detect a minimum 10% difference in Wolbachia prevalence between seasons at a significance level of α = 0.05.

Sample Collection

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 G. m. submorsitans, G. p. palpalis, and G. tachinoides. Flies were dissected under sterile conditions, and the midgut, salivary glands, and reproductive tissues were preserved in 70% ethanol for subsequent DNA extraction (Weber et al., 2019).

Tissue Selection and DNA Extraction

Wolbachia 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 et al., 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).

PCR Amplification and Controls

AccuPower PreMix PCR master mix from Bioneer was used based on manufactures instructions, two gene targets were used to detect and type Wolbachia strains: the wsp gene (Wolbachia 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 wsp. Primer pairs included wsp-specific 81F (5′-TGGTCCAATAAGTGATGAAGAAAC-3′) and 691R (5′-AAAAATTAAACGCTACTCCA-3′) (Zhou et al., 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 Wolbachia-infected Drosophila, and nuclease-free water was used as the negative controll (Bioneer, 2018, Weber et al., 2019).

Negative Controls
Each PCR batch included a no-template control (nuclease-free water) and a positive control (Wolbachia-infected Drosophila DNA) to monitor for contamination and ensure amplification efficiency.

Gel Electrophoresis and Sequencing and Phylogenetic Analysis

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 et al., 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 Wolbachia strains from Glossina spp. (GenBank accessions: KP715092, JX273258) and outgroup sequences from Rickettsia spp.