Identification of Wolbachia new strains from Aedes aegypti mosquitoes, the vector of dengue fever in Jeddah Province

Wolbachia are endosymbiotic bacteria found within many arthropods, including insects. A variety of benefits are provided by these bacteria to human and insect hosts, including protection from viruses and parasites and the ability to kill males. In this study, Wolbachia was identified in Aedes aegypti present in Jeddah, Saudi Arabia. A population of mosquitoes was collected from eight different areas, processed, and tested for Wolbachia using 16 S rRNA specific to Wolbachia bacteria and Wolbachia surface protein (wsp) under optimized PCR conditions. In five ecologically diverse sites to determine Wolbachia prevalence, we identified eleven diverse novel resident Wolbachia strains within Ae. Aegypti for the first time in Jeddah, Saudi Arabia. Future studies to evaluate the possible use of Wolbachia as a control agent in Aedes sp. in Saudi Arabia are necessary. Wolbachia prevalence rates and strain characterization through Sanger sequencing with multilocus sequence typing (MLST) and phylogenetic analysis revealed significant diversity. In developing biocontrol strategies, it is beneficial to consider the implications of resident Wolbachia strains. Supplementary Information The online version contains supplementary material available at 10.1186/s12866-023-03010-9.


Introduction
Mosquitoes belong to the order Diptera (Culicidae), and they are the most alarming insect threat to human health.Mosquitoes kill millions of people around the world by transmitting diseases.Mosquitoes and humans inhabit the same ecological area, clearly showing that there are more Aedes aegypti mosquitoes than any other insect group [1,2].The females of most species have piercing and sucking mouth parts, and it appears that for their Identification of Wolbachia new strains from Aedes aegypti mosquitoes, the vector of dengue fever in Jeddah Province E. Sharawi Somia 1* , Ihsan Ullah 1 , Hanan S. Alyahya 1 and Jazem A. Mahyoub 1,2* nontarget organisms, and health risks to humans and the environment [9,10].
Wolbachia are endosymbiotic bacteria that live within the cells of many arthropods, including insects such as mosquitoes [11].These bacteria provide a variety of benefits to their insect hosts, such as resistance to viruses and protection against parasitoids [12].These bacteria are thought to be the most common endosymbiont found in arthropods and are especially prevalent in insects [13].There are many different strains of Wolbachia, each of which is specific to a particular host species, and it is estimated that Aedes aegypti is infected with Wolbachia in up to 75% of wild populations [14].
The first evidence of Wolbachia infection in mosquito species was discovered in 1991 when researchers studying filarial nematodes in humans noticed that some patients had unusually high numbers of Wolbachia in their blood cells [15].They suspected that the bacterium was causing cytoplasmic incompatibility, where infected females produce eggs that lack sperm and therefore cannot develop into viable offspring [15].In 1999, scientists working on the yellow fever virus discovered that Wolbachia could also cause cytoplasmic male sterility (CMS) in mosquitoes [16].This meant that males carrying Wolbachia would not be able to reproduce because they lacked sperm [17].Additionally, Wolbachia pipientis (wPip) was the first strain discovered in the mosquito Culex pipiens.In addition, wAlb has been isolated from Aedes albopictus.In Australia, the Eliminate Dengue Programme approved the transfer of Wolbachia into mosquitoes to control dengue virus transmission.Many public health organizations use Wolbachia to control dengue virus and other arthropod-borne viruses.[14].With the increasing problem of pesticide resistance spreading in a number of cities and towns, including Jeddah, Makkah, Jizan and Al-Madinah [18], substantial efforts are being made to develop environmentally friendly strategies to reduce mosquito populations or limit their potential to transmit disease.
This study aims to identify different Wolbachia strains from laboratory and field strains of Ae. aegypti mosquitoes from Jeddah city and to determine the prevalence of Wolbachia strains in the area.

Collection of Insects Colony establishment (lab-strain)
A random group of Ae. aegypti population from the areas belonging to Al-Safa district was selected.The Jeddah Municipality, in cooperation with King Abdulaziz City for Science and Technology, had previously released large numbers of mosquitoes carrying the bacteria Wolbachia for the purpose of gathering adults of Ae. aegypti mosquitoes.The goal here was to ascertain the transmission of bacteria from parents to offspring by detecting the bacteria in the resulting offspring.The criteria used for sample size determination in the experiment were the district area, the number of cases of dengue fever, and the population density of the vector of dengue fever.The BG-Sentinel Mosquito Trap was used, and the Global Positioning System (GPS) coordinates of these sites were determined by projecting the coordinates of the sites covered by the study on the map of Jeddah Governorate using the Geographical Information System (GIS).Once per week, at least 12 h before sunset, traps were placed at each site and collected the next morning.The density of the released lab strains was approximately 500,000, according to the reports of the Jeddah Municipality.The contents of each trap were emptied into cages for rearing adult mosquitoes, which were cubic cages composed of metal frames of equal dimensions (30 × 30 × 30 cm).A special code number was given for each trap.To obtain eggs, mosquitoes were fed a blood meal using the Hemotek Membrane Feeding System.A small plastic cup with water was placed in the middle of the breeding cage after 4-6 days of blood feeding.Mosquitoes were reared in the laboratory at a temperature of 27 ± 1 °C, a relative humidity of 70 ± 5%, 14 h of light and 10 h of darkness until the exit of the adults (first generation).Adult males and females were taken 10 days after they left the pupal stage.Samples were then taken from each box, and the presence of Wolbachia bacteria in their tissues was determined using PCR separately for each sample.The positive mosquitoes were kept separate and propagated in the laboratory to obtain a laboratory strain carrying the bacteria.according to Table 1.

Collection of insects (field-strain)
To isolate bacteria from field strains, traps were distributed in several random areas according to Fig. 1; Table 2, and the insects belonging to the species Ae. aegypti were separated and preserved in 70% alcohol until the detection of bacteria, and each sample was labeled with a separate code number.

DNA extraction
A DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) was used to extract genomic DNA.The insects were first homogenized (bead homogenizer) and then digested overnight at 56 °C following the manufacturer's specifications.Total DNA was eluted into 200 µL and stored at − 40 °C.

Polymerase chain reaction (PCR)
The PCR technique was applied according to [19].The primer sequences were wsp 81 F for detecting Wolbachia infection using two molecular markers, wsp and 16 S rRNA (5′-TGGTCCAATAAGTGA TGAAGAAAC-3′), and wsp 691R (5′-AAAAATTAAA CGCTACTCCA-3′) for the wsp marker, and the 16 S Wolbachia-specific primers were Wolbachia_F (16S_ WspecF) (5′-CATACCTATTCGAA GGGATAG-3′) and Wolbachia_R (16S_WspecR) (5′-AGCTTCGAGT-GAAACCAATTC-3′).The wsp gene was amplified in a thermocycler.Each amplification was performed in 25 µl containing 1x GoTaq_Green Master Mix (Promega, USA), 2 µl of DNA template, and 1 µl each of forward and reverse primers (10 pmol).The amplification was performed by heating the sample at 95 °C for 5 min and 35 cycles of 94 °C for 30 s, 59 °C for 30 s and 72 °C for 45 s, followed by a final extension step of 72 °C for 10 min.In the final step, the temperature was set at 4 °C for an indefinite amount of time.The 16 S rRNA gene was amplified in 25 µl containing 1x GoTaq_Green Master Mix (Promega, USA), 2 µl of DNA template, and 1 µl each of forward and reverse primers (10 pmol).The amplification was performed by heating the sample at 95 °C for 5 min and 35 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 45 s, followed by a final extension step of 72 °C for 10 min.Then, we set it to 4 °C for an indefinite amount of time.All PCR amplification experiments included negative controls (water).Two microliters of DNA amplicon were assessed using 1.5% agarose gel electrophoreses at 100 V in an electrophoresis system for 25 min in TAE buffer (40 mM Tris-acetate, 1 mM EDTA, pH 8.0).A 100 bp DNA ladder (Promega, USA) was used as a marker.The PCR product of the wsp gene was approximately 610 bp, while that of the 16 S rRNA gene was approximately 438 bp.To validate the PCR amplification results, the positive sample was sequenced using Sanger sequencing at Macrogen, South Korea, using forward and reverse primers for each gene.

Resident strain density
Our study investigated whether Wolbachia bacteria are present in Aedes samples collected from different areas of Jeddah city.Twelve field samples were taken for bacterial isolation and species-level identification.The results showed that three samples were positive when examining the 16 S rRNA gene, while no infected samples were found when examining the wsp gene.In this study, we found that 13.63% of the Ae.aegypti individuals were infected with Wolbachia bacteria according to the 16 S rRNA gene test and (0.0%) according to the wsp gene (Fig. 2; Table 2).
After three generations of backcrossing between infected Ae. aegypti and noninfected Ae. aegypti under laboratory conditions, random samples were taken, and Wolbachia was detected in the tissues.In this study, twenty lab samples were taken for bacterial isolation and species-level identification.The results showed that 58.33% were positive when examining the 16 S rRNA gene, while 41% were positive when examining the wsp gene (Fig. 3; Table 2).

Wolbachia strain typing
The Wolbachia surface protein gene (wsp) is a molecular marker that is used to identify the Wolbachia bacteria in mosquitoes by its surface protein.In this study, the wsp gene, which encodes the surface protein of Wolbachia, was sequenced.It was demonstrated through the use of  Wolbachia-specific primers (wsp 81 F) that Ae. aegypti (n = 10) can be infected with Wolbachia.Wolbachia was detected using a specific primer, and the gene size was calculated at 600 bp (Fig. 4).
The presence of Wolbachia DNA was assessed in Ae. aegypti mosquitoes using 16 S rRNA.Wolbachia DNA was extracted and amplified through PCR using 16 S rRNA markers.The Wolbachia DNA was identified in samples, and the length of the DNA was 450 bp, which was appropriate for the 16 S rRNA gene length (Fig. 5).

Phylogenetic analysis of isolates based on 16 S rRNA
Phylogenetic analysis of Wolbachia was carried out based on 16 S rRNA sequences obtained from mosquitoes and other reference sequences in the NCBI through BLAST.Evolutionary distances were calculated using the maximum composite likelihood method, and bootstrap support values (1000 replicates) were used to construct the phylogenetic tree.According to the phylogeny based on 16 S rRNA sequences, all the bacterial strains found in Ae. aegypti were Wolbachia, which showed a high degree of similarity (> 98%) to 16 S rRNA Wolbachia sequences from endosymbionts and pipientis (Fig. 6).

Phylogenetic analysis of isolates based on the wsp sequence
The following results were obtained based on phylogenetic analyses of Wolbachia sequences from this study and reference sequences.Bootstrap tests (1000 replicates) indicate the percentage of trees in which a specific taxon clusters together next to a branch.Evolutionary distances were calculated by maximum composite likelihood.Bootstrap values strongly supported the relationship among the isolates and Wolbachia strains.Phylogenetic analyses showed that Ae. aegypti harboured Wolbachia strains (Fig. 7).In the current study, 11 new resident Wolbachia strains were recorded for the first time in Saudi Arabia and published in the NCBI database, according to Table (3).

Discussion
Aedes mosquitoes were tested for the presence of Wolbachia bacteria to determine whether they were harmful [20].There may still be some strains that remain undetected because of differences in tissue tropism [21].
When mosquitoes are infected with the Wolbachia bacterium, it can prevent diseases such as dengue fever and chikungunya from spreading [22].Furthermore, Wolbachia bacteria have been shown to be toxic to other parasites, such as Plasmodium, indicating that they may also affect the transmission of other parasites [20].Increasing temperatures reduce the density of Wolbachia endosymbionts in Aedes bacteria [23].According to [24], low endosymbiont density in Ae. aegypti probably contributed to the low infection rate.In addition, studies from non-vector systems show that Wolbachia replication, dissemination, vertical transmission, fitness effects, and cytoplasmic incompatibility vary with temperature, and because of this wide range of thermal sensitivities, patterns of Wolbachia-induced transmission blocking might be strongly influenced by local environment, and unfortunately, there are no current studies showing the effect of temperature on Wolbachia [23].In metabarcoding, studies found few sequences in the midgut of Ae. aegypti, indicating a low density of the endosymbiont [25].Several Wolbachia strains have been identified that presumably influence both the normal rate of human disease transmission and the manipulation of transmission rates by Wolbachia-infected mosquitoes as well as the transmission rate itself [25].Wolbachia strains are known to protect viruses in arboviral hosts.[20] claimed the Wolbachia they identified in Ae. bromeliae could reduce the transmission of arboviral diseases.Fig. 7 The wsp gene of Wolbachia was amplified through PCR using wsp-specific primers, and the sequences were searched against the NCBI nucleotide database for similar matches.Neighbour joining was used to construct a phylogenetic tree using bootstrap support values based on 1000 replicates According to our phylogenetic analysis in the present study, the Wolbachia strains found in our sample of Ae. aegypti mosquitoes belong to Wolbachia endosymbionts and Wolbachia pipientis, respectively.Eleven residents of Wolbachia strains were identified and published in the NCBI database as a first record.Mosquito species with medical significance display these types of characteristics, i.e., An.Gambiae and Ae.albopictus.It remains to be confirmed whether some Wolbachia strains cause pathogenic effects in Ae. aegypti mosquitoes [15,25].A number of these strains have been found in dipterans, particularly mosquitoes, and have been shown to cause cytoplasmic incompatibility, male killing, and feminization.Ae. aegypti mosquitoes carry Wolbachia strains that cause these phenotypic effects, but it is unclear whether they are responsible for them [21].
To explain the clustering of mosquito-infecting strains, several explanations can be offered.There is a possibility that Wolbachia was acquired by horizontal transmission from a previously infected ancestral species or that Wolbachia was cospecified with a previous host.Compared to other taxa, horizontal transmission has been shown to occur between close relatives most commonly in horizontal transmission [26].

Fig. 3
Fig. 3 Positive and negative rates in Ae. aegypti using two Wolbachia genes (16 S rRNA and wsp) in a lab strain

Fig. 2 Fig. 1
Fig. 2 Positive and negative rates in Ae. aegypti using two Wolbachia genes (16 S rRNA and wsp) in a field strain

Fig. 5 Fig. 4
Fig. 5 Gel electrophoresis results of the Wolbachia strain identification PCR assays for Ae.aegypti using the 16 S rRNA gene, M: Marker, 1 negative control sample from 2:7 Fig. 4 Gel electrophoresis results of the Wolbachia strain identification PCR assays for Ae.aegypti using the wsp gene, M: Marker, 1 negative control, sample from 2: 7

Fig. 6
Fig.6DNA sequence of Wolbachia was amplified using 16 S rRNA-specific primers through PCR.The sequences were searched against the NCBI nucleotide database, and the most similar match was downloaded.The neighbour-joining method was used to build the phylogenetic tree, and 1000 bootstrap values were used

Table 1
Distribution of the adult Ae. aegypti sampling sites (n = 22)

Table 2
A detailed summary of the results of Wolbachia in Ae. aegypti mosquitoes using the 16 S and wsp genes

Table 3
Novel resident Wolbachia strains using the 16 S and wsp genes