ABSTRACT
With their diverse species, mosquitoes are known to transmit the causal agents of diseases such as malaria, dengue, and yellow fever. Their high adaptability, attraction to humans, and variable adult behaviors make them a significant health concern. The focus on Aedes aegypti is significant for reducing vector-human contacts, monitoring insecticide resistance, and developing innovative vector management strategies. Given the scarcity of studies on Ae. aegypti in the western region of Saudi Arabia, this research is a significant step forward. The study aims to analyze the genetic variations and conduct a phylogenetic study of forty Ae. aegypti samples collected from Taif and Jeddah governorates of Saudi Arabia. The mitochondrial cytochrome c oxidase subunit I (COI) locus was targeted for genetic variance and phylogenetic analysis. Sequences of COI of Ae. aegypti isolates were submitted to the DNA Data Bank of Japan (DDBJ) and National Center for Biotechnology Information (NCBI) Genbank and compared with other global Aedes species isolates. The phylogenetic analysis shows that Ae. aegypti samples from Jeddah have identities ranging from 96.9% to 99.8%, closely related to the Peru (MN299016) and Cambodia (MN299014) isolates. Taif isolates have genetic similarities ranging from 97.5% to 99.8%, closely related to the Germany (KY022526) isolate. Sequence alignment and pairwise comparison show variation among the populations of Ae. aegypti from Taif and Jeddah regions (74.24–98.84%) with a genetic divergence distance of 0.008–0.12. In comparison, ranges slightly change with other Ae. aegypti (79.92–95.96%, 0.008–0.01) as well as Ae. albopictus populations (74.13–83.58%, 0.13–0.20) found in the Genbank database. According to our findings, the present study provides information for a local variation of Ae. aegypti in the western region of Saudi Arabia that could help in disease mapping and risk mitigation, thereby enhancing our ability to manage disease vectors effectively.
INTRODUCTION
In Saudi Arabia, there are 49 confirmed mosquito species, including 18 anapheline and 31 culicine species (Alahmed et al. 2019). According to the World Health Organization (WHO 2022), Aedes aegypti (L.) is the primary vector for dengue, yellow fever, Zika, and chikungunya. In Saudi Arabia, dengue fever cases have been reported in the country’s southwestern regions, including Jeddah, Makkah, Madinah, Jazan, and Sahil. Additionally, cases of chikungunya have been reported in Jeddah and Jazan. No cases of Zika have been reported to date (Fakeeh and Zaki 2003, Al-Azraqi et al. 2013, Hussain et al. 2013, Altassan et al. 2019, Hakami et al. 2021). Dengue virus represents the most widespread arthropod-borne virus, infecting around 390 million people annually (Bhatt et al. 2013). The incidence rate of dengue cases changes from country to country over time, including in Saudi Arabia (Ministry of Health 2021). Considerable efforts have been made to control these arboviruses, which have become widespread (Weaver and Barrett 2004, Diallo et al. 2018).
Knowledge about mosquito species and their distribution is crucial for diversity studies and disease management strategies. Understanding their habitats and the pathogens they carry is essential for planning targeted vector control measures to prevent disease transmission (Walker et al. 2007, Laurito and Almirón 2015). Only 10% of species have been identified worldwide due to a lack of taxonomic expertise (Besansky et al. 2003, Pennisi 2003). The mitochondrial cytochrome c oxidase subunit I gene (COI) has been used to quickly and reliably identify Ae. aegypti and their taxonomic characteristics (Mousson et al. 2005). Due to its conserved functional domains and variable regions, the COI gene is highly valuable for evolutionary studies (Al Thabiani 2023). Molecular approaches are preferred for species identification, genetic diversity, and molecular phylogeny due to their high precision. They do not require a specific gender or life stage of the mosquito to give accurate results (Shepard et al. 2006, Kumar et al. 2007, Pfeiler et al. 2013). Therefore, the present study was carried out to explore the genetic diversity of Ae. aegypti populations of Taif and Jeddah governorates of Saudi Arabia using the COI gene as a significant marker. The study aims to examine COI sequences from 20 Ae. aegypti from Taif and 20 other samples from Jeddah, finding the resemblance/diversity in their DNA sequences to those of the same species from other regions worldwide.
MATERIALS AND METHODS
Study area
Makkah Region falls within the Hejaz region, which comprises a range of elevations with a maximum height of approximately 2,700 m to the south and 1,450 m to the west. Makkah Region is divided into the capital of the region—the city of Makkah— and 11 other governorates, including Jeddah and Taif. Taif, located in the Makkah Region, western Saudi Arabia, is at an elevation of 1,800 m on the eastern slopes of the Al Sarawat Mountains, covering an area of 1,378 km2. Despite its hot desert climate, Taif experiences milder weather than Jeddah due to its geographic location and higher altitude. In winter, temperatures can drop to as low as 3°C and rise to a maximum of 18°C. During the summer, temperatures typically range from 22°C to a maximum of 25°C, with an average of 27.1°C. July is the warmest month of the year, while January experiences the lowest average temperatures, dropping to 13.9°C. Taif has less relative humidity (RH) variation throughout the year than Makkah and Jeddah, ranging from 29% in winter to 57% in summer. Taif’s topography leads to varying rainfall rates, with hills and mountains experiencing heavier and more frequent rainfall than lower areas within the same region. In addition, Jeddah, a low-altitude region, represents one of the important governorates of the Makkah region. Jeddah is the largest coastal city in the kingdom. Compared to Taif, Jeddah experiences higher average temperatures throughout the year and minimal rainfall. Unlike other Saudi Arabian cities, Jeddah has warm winters, but its summers are extremely hot and humid, ranging from 30°C to 43°C. The average annual RH in Jeddah is approximately 63%, and it undergoes extreme seasonal variation. For both regions, the prevailing winds are mainly from the west and northwest, with moderate speeds, but during seasonal transitions, they can reach up to 36 km/h, often causing sandstorms (United Nations Habitat [UNH] 2019a, 2019b).
Samples collection
Adult mosquitoes were collected outdoors near human settlements using blackhole traps, suspended about 2 m above the ground from sunset to sunrise. The blackhole trap is a black plastic device measuring 25 × 25 × 32 cm and weighing 1.2 kg. It has an electrical fan and two 4-watt ultraviolet fluorescent bulbs powered by an alternating current electrical system of 220–240 V. The fluorescent light serves as the default illumination in the trap (Jhaiaun et al. 2021). Mosquitoes were collected from the Taif (21.2841°N, 40.4248°E) and Jeddah (21.5292°N, 39.1611°E) governorates during the winter season from December 2023 to February 2024 (Fig. 1). The collected mosquitoes were transported in paper cups to the laboratory and were examined using a dissecting microscope, using the Diptera: Culicidae key of Rueda (2004). From the collected samples, 40 adult mosquitoes (20 from each governorate) morphologically identified as Ae. aegypti were incorporated into the present study. The samples were preserved in 70% ethanol for further molecular evaluation.
Map showing the locations of Aedes aegypti sampling from Taif (A) and Jeddah (B), Saudi Arabia. Samples were collected from December 2023 to February 2024.
Map showing the locations of Aedes aegypti sampling from Taif (A) and Jeddah (B), Saudi Arabia. Samples were collected from December 2023 to February 2024.
DNA extraction and PCR
The deoxyribonucleic acid (DNA) was extracted from 40 Ae. aegypti mosquito samples (20 samples each from Taif and Jeddah regions), as described by Abdella et al. (2018). Briefly, samples were homogenized and treated with proteinase K. Then, the DNA was precipitated by ethyl alcohol (97%) and purified, using silica gel spin columns. Finally, the DNA was solubilized by deionized water (50 μl) and stored at −4°C for later use.
The mitochondrial cytochrome oxidase subunit I (COI) gene region was targeted for amplification via polymerase chain reaction (PCR). The polymerase chain reaction mixture (20 μl) was set up to include: 1 μl (100 ng/μl) extracted DNA, 7 μl sterile water, 1 μl forward primer (LCO1490, 5′-GGTCAACAAATCATAAAGATATTGG-3′), 1 μl reverse primer (HC02198, 5′-TAAACTTCAGGGTGACCAAAAAATCA-3′), and then 10 μl 2x master mix (Promega, USA) were added in 0.2 ml PCR Eppendorf to amplify 730 bp fragment from COI (Vrijenhoek 1994). The amplification was done through 35 cycles using the following parameters: 95°C (60 sec), 40°C (60 sec), and 72°C (90 sec), followed by a final extension step at 72°C (7 min). The PCR amplifications were confirmed by using ethidium bromide-stained 1.5% agarose gel electrophoresis.
Sequencing and phylogenetic analysis
The amplified DNA fragments were sequenced using the Sanger method (Crossley et al. 2020). The sequence data underwent preprocessing, including quality checks and trimming, utilizing the Geneious software version 2024.0.5 (Kearse et al. 2012) designed for such molecular biology tasks. Forty isolates were submitted to the National Center for Biotechnology Information (NCBI) Genbank (https://submit.ncbi.nlm.nih.gov/) and the DNA Data Bank of Japan (DDBJ) (https://www.ddbj.nig.ac.jp/index-e.html) for accession numbers as mentioned in Table 1. Next, the sequences were aligned using MUSCLE, renowned for its accuracy in handling large sequence alignments. MUSCLE is a fast, new computer program designed to create multiple DNA, RNA, or protein sequences’ alignments for high-throughput applications. For data analysis, MUSCLE uses k-mer counting, log-expectation score, and tree-dependent restricted partitioning of the sequences (Edgar 2004).
Country of origin of Aedes sp. COI sequences, accession numbers, and descriptions according to the Genbank database.

Phylogenetic analyses were conducted using BEAUti (Bayesian Evolutionary Analysis Utility) and BEAST (Bayesian Evolutionary Analysis by Sampling Trees), part of a suite that implements Bayesian statistical approaches to construct evolutionary trees. Bayesian Evolutionary Analysis represents a major new molecular evolutionary software package version. BEAUti is a graphical user interface (GUI) application that generates BEAST XML files. Default parameters within BEAUti were used to define the nucleotide substitution model, clock model, and tree priors. The Markov Chain Monte Carlo (MCMC) algorithm in BEAST was then employed to infer the phylogenetic tree (Drummond et al. 2012). The resulting phylogeny was visualized using FigTree (Rambaut 2007), providing a user-friendly interface for displaying and interpreting phylogenetic trees.
The nucleotide sequences of the present isolates were aligned by COI loci of another Ae. aegypti and Ae. Albopictus with accession numbers, geographic location names, and description, according to NCBI Genbank database (Table 1). The alignment algorithm has three parameters concerning gap costs: gap open cost (10), gap extension cost (1), and end gap cost (as any other). After alignment, a pairwise comparison was done regarding the percentage of identity and the genetic divergence distance between isolates with Bonferroni correction (Vega-Rúa et al. 2020). The analysis was made by CLC Genomics Workbench Version 24.0.2 QIAGEN Aarhus A/S (digitalinsights.qiagen.com/). In addition, a blast between two or more sequences was done for specific isolates using basic local alignment search tool (BLAST), (https://blast.ncbi.nlm.nih.gov/) (Zhang et al. 2000).
RESULTS
Forty isolates of Ae. aegypti from Taif and Jeddah were successfully amplified and sequenced at the COI gene locus. Their sequences were submitted online with different accession numbers, as shown in Table 2.
As shown in Table 3, the genetic examination of Ae. aegypti specimens from Jeddah (JED) and Taif (TAF) showed a substantial genetic resemblance among various isolates, as determined by COI gene. The samples collected in Jeddah display a variation in identity ranging from 96.9% to 99.8%. Most of these samples strongly resemble the reference sequences MN299016 (from Peru) and MN299014 (Cambodia), representing distinct Ae. aegypti isolates. The similarity ratings of JED16, JED8, JED9, and JED17 are about 97%, indicating a minor variance within this group. JED14 and JED15 have the greatest similarity score of 99.8%, showing a significant correspondence to the established reference sequences.
The percentage of similarity between the Aedes aegypti of the present study and other global isolates based on nucleotide basic local alignment search tool, National Center for Biotechnology Information (nBLAST, NCBI).

The similarity of the Taif samples varies between 97.5% and 99.8%. The Taif group has a comparable pattern of significant genetic similarity to the reference sequences. TAF12 and TAF17 have the maximum similarity score of 99.8% with KY022526 (Germany) and OR413797 (Saudi Arabia, Jeddah), while TAF19 and TAF4 display the lowest similarity score of 97.5% with MN299014 (Cambodia) and MN299016 (Peru) (Table 3). This suggests a significant degree of preservation in the COI gene while indicating some genetic variations. The Jeddah and Taif groups possess many samples that align closely with the reference sequence OR413797, also found in Jeddah, Saudi Arabia.
Based on the given information about the phylogenetic tree, there are two primary groups that categorize the Ae. aegypti samples according to their genetic resemblance to reference sequences (Fig. 2). The initial prominent cluster, indicated in yellow, indicates a strong genetic relationship between the samples and the reference sequences linked to the yellow fever mosquito, notably KY022526.1 (from Germany) and MN299016.1 (from Peru). The second cluster (Fig. 2), shown in green, corresponds to the samples that match the reference sequence OR413797. This suggests the presence of other samples that share a significant amount of genetic similarity, which may imply the existence of a distinct lineage or a subgroup within the Ae. aegypti species.
Phylogenetic relationships among Aedes aegypti collected from Taif, Jeddah (black color), and the most near-reference isolates found in Genbank (red color). The isolates are categorized into two clusters: the yellow cluster that indicates a strong genetic relationship between the present samples with KY022526.1 (from Germany) and MN299016.1 (from Peru) isolates. The green cluster corresponds to the samples that match the reference sequence OR413797.
Phylogenetic relationships among Aedes aegypti collected from Taif, Jeddah (black color), and the most near-reference isolates found in Genbank (red color). The isolates are categorized into two clusters: the yellow cluster that indicates a strong genetic relationship between the present samples with KY022526.1 (from Germany) and MN299016.1 (from Peru) isolates. The green cluster corresponds to the samples that match the reference sequence OR413797.
The TAF13 sample (LC831985) is distinct from the other samples in the phylogenetic tree, making it an outlier or an outgroup (Fig. 2). This sample does not exhibit a close clustering pattern with the two primary reference sequences. The fact that it is an outgroup indicates that it likely has a distinct genetic composition compared to the other samples.
The nucleotide sequence alignment was done between Taif and Jeddah isolates (40 samples), Ae. aegypti (KY022526, OR413797, OR413797-1, MK300226, MN299014, MN299016, and MN298998), and Ae. albopictus (PP596091 from Hong Kong, PP825981 from Portugal, and PP893185 from India) (Fig. 3). It shows the polymorphic sites of different populations among COI loci. The alignment was then analyzed by pairwise comparison to show their related percentage of identity and genetic divergence distance Fig. 4. The percentage of identity ranges from 74.24% (PQ058640 (TAF3) and LC832165 (JED6)) to 98.84% (LC832161 (JED2) and LC82163 (JED4)) between the present study populations (Taif and Jeddah) with genetic divergence distance ranges from 0.008 to 0.12. However, in the percentage of identity between the Taif and Jeddah populations and other studies, Ae. aegypti ranges from 79.92% ((PQ058640 (TAF3) and MN298998 (Congo)) to 95.96% (LC832160 (JED1) and MN299014 (Cambodia)) with a genetic divergence distance of 0.008–0.01. Finally, the percentage of identity was lower between Ae. aegypti Taif and Jeddah populations and Ae. albopictus 74.13% (PQ058640 (TAF3) and PP596091 from Hong Kong) to 83.58% (PQ013114 (TAF17) and PP825981 from Portugal) with divergence distance 0.13–0.20. OR413797 and OR413797-1 (whole genome with length = 15354 bp, from Jeddah, Saudi Arabia) are highly similar to the present study populations; however, they show a lower percentage of identity and more genetic divergence distance due to a low query coverage (3%) with other samples (∼ 700 bp).
Alignment between Aedes aegypti of the present study, the most near-reference isolates were found in Genbank and other Ae. albopictus.
Alignment between Aedes aegypti of the present study, the most near-reference isolates were found in Genbank and other Ae. albopictus.
Pairwise comparison between studied isolates and other Ae. aegypti and Ae. albopictus isolates found in Genbank. The upper right half refers to percent identity between isolates, while the lower left half refers to the genetic divergence, ranging from red (maximum) to blue (minimum) colors.
Pairwise comparison between studied isolates and other Ae. aegypti and Ae. albopictus isolates found in Genbank. The upper right half refers to percent identity between isolates, while the lower left half refers to the genetic divergence, ranging from red (maximum) to blue (minimum) colors.
DISCUSSION
Aedes aegypti showed a widely distributed potential globally across tropical and subtropical regions, including Saudi Arabia, influenced by weather, climatic conditions, and human activities (Campbell et al. 2015, Kamal et al. 2018). Additionally, poor sanitary conditions and uncontrolled urbanization have contributed to the range of expansion of Ae. species (Öztürk and Akiner 2023). In recent decades, Ae. aegypti has been found in various regions of Asia, Africa, and the Americas, with its distribution predicted to increase by the year 2050 (Kraemer et al. 2015, Kamal et al. 2018). Aedes aegypti, a vector of different arboviruses, is widely distributed due to its insecticide resistance, which hinders control efforts (Spadar et al. 2024). Therefore, we focused on studying Ae. aegypti at the molecular level, especially the COI gene, which is the most used among many molecular markers (Samanta et al. 2023, Sharawi et al. 2024). In the current study, 40 samples of Ae. aegypti were gathered from the Taif and Jeddah governorates since few studies have focused on Ae. aegypti research in the western region of Saudi Arabia (Aziz et al. 2012, Alikhan et al. 2014, Khater et al. 2021, Al Zahrani et al. 2023). Therefore, we aim to address this gap by studying the phylogenetic analysis of Ae. aegypti isolates from those 2 regions, Jedda and Taif. Jeddah is a major industrial city on the Red Sea in Saudi Arabia, serving as a key entry point for millions of animals (trade) and pilgrims from countries that could be arboviral disease endemic. On the other hand, Taif is a popular tourist destination among Saudis because of its location in the mountains, beautiful scenery, and unique relaxed atmosphere, providing thousands of jobs to residents through the tourism industry (UNH 2019a, 2019b).
Table 3 shows a high identification percentage in the Jeddah and Taif samples with reference isolates that indicate a genetically stable area inside the mitochondrial COI gene among the mosquitoes examined. The findings indicate that the COI gene is well preserved in Ae. aegypti from these areas. In addition, Jeddah and Taif groups possess many samples that align closely with the reference sequence OR413797 (Jeddah, Saudi Arabia). This suggests that these isolates share a remarkably high level of genetic similarity with the Ae. aegypti isolate represented by this specific accession. This information is valuable for categorizing them taxonomically and possibly for monitoring steps to restrict the spread of disease vectors. Nevertheless, the few deviations identified may offer valuable insights into the genetic variability influencing disease transmission dynamics and strategies for managing vectors. Genetic fluctuations in global Ae. aegypti populations are linked to current climatic conditions, human social behavior, and viral infection immunity (Khater et al. 2021).
According to the present study, there is a variation among the populations of Ae. aegypti from the Taif and Jeddah regions (74.24–98.84%), between current isolates and other Ae. aegypti (79.92–95.96%), and against Ae. albopictus populations (74.13–83.58%) (Fig. 4). Recent studies indicate that, Ae. aegypti showed minimal population genetic structure across different climates, including urban areas (Naim et al. 2020, Alghamdi et al. 2021). This low genetic diversity, with low nucleotide variation and high haplotype diversity, complicates efforts to control these mosquito populations (Laurito and Almirón 2015). The origins of the present populations remain unclear; it is not known whether they come from a single invasion or multiple separate invasions. However, the near similarity (< 95%) between the present isolates and others found in the Genbank database could have a common ancestor, such as those from Kenya, Peru, and Cambodia. If multiple invasions occurred in the same area, this could lead to an increase in the species’ genetic diversity. Consequently, this genetic diversity may enhance the species’ tolerance to ecological factors, such as climate adaptation, and biological factors, like pesticide resistance (Gao et al. 2021). Estimating pairwise genetic distances from COI sequences provided a quantitative method for evaluating DNA barcoding (Al Zahrani et al. 2023). As shown in Fig. 4, closely related isolates have a shorter genetic distance (0.008) compared with that from the GenBank Ae. albopictus samples ranges from 0.13 to 0.20.
According to the phylogenetic tree, the proximity of the samples in each cluster separately (yellow/green highlight) suggests a significant degree of genetic preservation, which may imply a shared evolutionary background or even a comparable ecological habitat to those reference isolates. The clear distinction between the green and yellow clusters may indicate genetic divergence over time due to variables such as geographic isolation, adaptation to diverse habitats, or other evolutionary influences (Fig. 2). The isolate TAF13 (LC831985) appears as an outgroup from yellow and green clusters in the phylogenetic tree. This might be because it is more evolutionarily distant or acquired genetic material from other species or subpopulations through introgression. The distinctive genetic makeup of LC831985 necessitates further examination, as it has the potential to unveil significant revelations on the genetic variation and adaptability of Ae. aegypti. It is a common challenge to study mitochondrial loci, including COI, as the number of mitochondria per cell, the number of mitochondrial DNA copies on chromosomes, and the allelic diversity are unknown (Richly and Leister 2004, Black and Bernhardt 2009, Spadar et al. 2024). Finally, the phylogenetic analysis highlights the genetic connections and possible variations among the Ae. aegypti populations in Jeddah and Taif. Comprehending these genetic differences is important for vector control tactics, as it could impact the efficacy of treatments and may provide insights into the processes of disease transmission by these mosquitoes.
Accurate identification and mapping of Ae. aegypti is important for its role in vector control. The present study illustrates that the genetic variance and phylogenetic relation of Ae. aegypti samples from Taif and Jeddah are crucial for understanding Aedes cryptic species’ evolutionary processes in the western region of Saudi Arabia. Many isolates share a close genetic similarity with other reference isolates from Argentina, Cambodia, Germany, and Kenya. Further investigations concerning Aedes distribution, mapping, and phylogenetic analysis in other regions of Saudi Arabia are encouraged.
ACKNOWLEDGMENTS
The researchers acknowledge the Deanship of the Graduate Studies and Scientific Research, Taif University, for funding this study.