Introduction

The family Culicidae is considered one of the most important insects affecting human health. ^{1 , 2} A wide range of arboviruses are transmitted by this family, including dengue virus, dengue hemorrhagic virus, and West Nile virus. ² Some mosquitoes of the Culicidae family are extremely active in terms of blood-feeding in urban environments. ³ The role of these species in the harassment caused by the bite, along with the transmission of pathogens, has doubled the medical importance of these species. ^{1 - 3} West Nile virus has attracted attention due to its rapid spread throughout much of the world. This virus was first detected in 1937 in Uganda in a 37-year-old woman. Currently, it is known as one of the most common arbovirus diseases that cause severe complications. ^{4 , 5} Since 1970, when this disease was first reported in Iran, many studies have been conducted throughout the country. In general, some different species of the genera Aedes and Culex have been reported as the main vectors of the West Nile virus from different regions of the world. ⁸ Potential vectors of the West Nile virus are distributed throughout most of Iran from north to south. ² The presence of Ae. caspius, along with Cx. pipiens, in Shiraz can indicate the possibility of a complete disease transmission cycle.

Aedes Caspius causes significant disturbance and harassment and is sometimes able to transmit pathogenic agents to humans in tropical latitudes. For example, we can mention the endemicity of the West Nile virus in the USA and the spread of the Zika virus in Italy by this mosquito. In most regions of Europe, sympatric dispersal between Ae. caspius and Ae. dorsalis has been reported. ¹⁰ West Nile virus (WNV), Tahyan virus, and Francisella tularensis bacteria, the causative agent of tularemia, have been isolated from the natural population of this species. ¹¹ Probably, Ae. caspius plays an important role in the spread of tularemia, the transmission of the Tahyan virus, and the rabbit myxoma virus. ^{12 , 13}

Culex mosquitoes constitute a wide range of mosquitoes involved in the transmission of arboviral diseases, including West Nile virus, Sindbis virus, equine encephalitis, St. Louis, Oro-pouch fever, avian malaria, lymphatic filariasis, and Rift Valley fever. ¹⁴ The genus Culex is one of the most important groups of the Cx. pipiens complex, which consists of six members: Cx. quinquifasciatus, Cx. pallens, Cx. australicus, Cx. globocoxitus, Cx. Molestus, and Cx. pipiens. ¹⁵

Diseases transmitted by arthropods impose a great health burden on the inhabitants of tropical and subtropical regions. Many efforts have been made over the years to fight these diseases. However, the prevalence and geographical distribution of these diseases are expanding, and the available methods to fight them are limited. Controlling mosquitoes with the endosymbiont bacterium Wolbachia is a new approach in this field. ¹⁶ Presently, in Australia, Brazil, Colombia, and Southeast Asia, the strategy of deploying mosquitoes infected with Wolbachia is being employed to control Dengue and chikungunya within their natural habitats. ¹⁷ This study aims to determine Wolbachia infection in two species of Ae. caspius and Cx. pipiens, in Shiraz City, Fars Province. The results of this study provide basic information for better control of arbovirus vectors and the possibility of using new environmentally friendly biological methods to control the transmission of dangerous arbovirus diseases such as dengue fever and West Nile in the country.

Methods

Study area

Shiraz is the capital of Fars province, situated at a latitude of 29.61 and longitude of 52.53, with an elevation of 1545 meters above the sea level. The total average annual precipitation has been reported to be 305.6 mm. Within the scope of this study, sampling sites were selected based on various factors, including the type of larval habitats, water availability, sunlight penetration, water flow, and water transparency. The selected sites with geographical coordinates are shown in Figure 1 and Table 1.

To determine the Wolbachia infection, we collected Culicidae samples from April to September 2022. Larvae were collected using the dipping method in selected locations of larval habitats. Subsequently, they were transported to the laboratory to determine their abundance and identity. Adult samples were caught during the day to determine the abundance and Wolbachia infection in each of the studied locations. To catch samples from methods such as Aspiration inside human dwellings, Landing catches on human baits, Animal baited trap, CO2-baited CDC light traps, Sticky trap, Total catch, and Shelter pits were used. ¹⁸ The samples collected were identified based on morphological identification keys and registered in standard forms. ^{19 , 20} Finally, they were kept in a freezer at minus 20 degrees Celsius until molecular investigations.

Figure 1. Sampling locations in the study area, Shiraz City, Fars Province, Iran. (Generated using ArcMap10.8)

Molecular study

Given that Cx. pipiens and Ae. caspius mosquitoes were the predominant species and significant vectors in the study area, they were subjected to investigation for Wolbachia infection. Based on the level of Wolbachia infection, 20% of Cx. pipiens mosquitoes and 50% of Ae. caspius mosquitoes were included in the study sample. DNA extraction from adult mosquitoes was done to determine Wolbachia infection using the Collins method²¹. The Wolbachia surface protein (WSP) genes were amplified by nested Polymerase Chain Reaction (PCR) to investigate the presence and genetic diversity of Wolbachia strains inside the body of Ae. caspius and Cx. pipiens mosquitoes. The specifications of primers and PCR thermal cycles are as follows. ^{22 , 23}

To perform the reaction in both PCR steps, we used Maxime PCR Premix Kit (i-Taq), Cat. No. 25025 manufactured by INtRON Company. In both steps, 2.5 microliters of forward and reverse primers were used, and 5 microliters of DNA were used in the first reaction, and 2.5 microliters of the PCR product of the first step were used in the second reaction. The PCR products were run on a 1% agarose gel, and then the bands were visualized under UV light. Each sequence obtained was compared with the sequences registered in GenBank using the NCBI database. MEGA6 and BioEdit software were used to investigate phylogenetic relationships. Finally, the sequences were recorded in GenBank (Tables 2 and 3).

Results

In this study, a total of 1404 mosquito samples, including 697 adults and 707 larvae, were collected from the study area in Shiraz City, Fars Province, Iran. In this study, eight species of four Culicidae genera were identified, including Cx. pipiens, Cx. theileri, Cx. sinaiticus, Cx. quinquefasciatus, Ae. caspius, Culiseta longiareolata, Anopheles Hyrcanus, and An. dethali. The results show that the most species caught was Cx. pipiens, and the least species caught was An. hyrcanus (Table 4).

In this study, we screened 110 Ae. caspius and 110 Cx. pipiens species for Wolbachia infection by PCR assays with wsp gene primers. From 110 adult Cx. pipiens screened, 75 (68%) Cx. pipiens mosquitoes were infected with Wolbachia. The molecular results showed that the level of Wolbachia infection in the female population of Cx. pipiens was between 60-100 percent; in the male population, it was between 35-70 percent (Table 5). Wolbachia sequences were obtained from infected individuals in each of the infected Cx. pipiens species in this study (sequences were registered in the GenBank; accession numbers OR478431, OR478432, OR478433, OR478434, OR478435). The results show that there is no Wolbachia infection in the population of Ae. caspius (Figure 2 and Table 6). In addition, the wsp gene phylogeny analysis indicated Cx. pipiens specimens were infected with strains of B Wolbachia supergroup (Figure 3).

Figure 2. Electrophoresis of the second stage of Nested-Polymerase Chain Reaction (PCR) for a part of Wolbachia surface protein (wsp) gene of 501 bp length in different populations of Aedes caspius and Culex pipiens collected from different parts of Shiraz City, Fars Province (M marker 100bp, 1: Drosophila melanogaster (positive control), 2-4: Culex pipiens populations, 5-6: Aedes caspius populations, 7: negative control)

Figure 3. Phylogenic analysis based on the Wolbachia surface protein gene gene. Sample sequences of Culex Pipiens collected in Shiraz City, Fars Province in Iran. Wolbachia sequences in the samples were done to show the degree of similarity (99-100%). Supergroup B was identified in this research, and Supergroup C was used as an outgroup.

Discussion

Aedes Caspius is found in most regions of the world and transmits many arbovirus diseases to humans. ²⁴ This species has been reported from most parts of Iran, such as West Azarbaijan and East Azarbaijan, Central, Semnan, Khorasan, Fars, Kurdistan, Chahar Mahal and Bakhtiari, Kohgiluyeh and Boyer Ahmad, Isfahan, and Hormozgan. ^{2 , 5} In the present study, Ae. caspius and Cx. pipiens were caught from all sampling sites in Shiraz City. Therefore, the results of the present study are similar to the results of previous studies in terms of the distribution of Ae. caspius. Culex Pipiens is widely distributed throughout the world. ²⁴ These species were collected from all the places under study, so in terms of distribution, the results of the present study are similar to those of previous research. ²⁴

This study showed that Ae. caspius are not infected with Wolbachia bacteria, while Cx. pipiens are highly infected with the Wolbachia bacterium (Tables 5 and 6). Indeed, the level of Wolbachia infection in Cx. pipiens mosquitoes can vary across different locations, and environmental factors such as temperature, vegetation, and humidity may play a role in influencing this variation. Studies have suggested that higher temperatures could potentially reduce the presence of Wolbachia within the host mosquitoes. This relationship between environmental conditions and Wolbachia infection highlights the complexity of vector biology and the importance of considering ecological factors in understanding vector-borne diseases and their control. ²⁵ In addition, the rates of infection with Wolbachia bacteria were between 60-100% among female populations and between 35-70% in males. The findings of this study are in the same line with previous research indicating that female mosquitoes tend to exhibit higher levels of Wolbachia infection compared to male mosquitoes. This observation is consistent with the fact that Wolbachia bacteria are often vertically transmitted from mother to offspring through the eggs, resulting in higher infection rates in female mosquitoes. ²⁶ Understanding the sex-specific patterns of Wolbachia infection in mosquitoes is important for elucidating the biology of both the bacteria and their mosquito hosts, as well as for informing strategies for vector control and disease management. Previous studies show that the rate of Wolbachia infection in different populations of Cx. pipiens is between 80-90%; also, a study conducted in California reported an infection rate between 93-100%, which indicates that the present study aligns with the aforementioned studies. ²⁷ The identification of Wolbachia strains in our study as belonging to supergroup B and their presence in Cx. pipiens mosquitoes are consistent with the findings reported in previous studies. Supergroup B is one of the major clades of Wolbachia bacteria, and its association with Cx. pipiens mosquitoes has been documented in various geographic regions. This consistency across studies suggests a stable relationship between Cx. pipiens mosquitoes and Wolbachia supergroup B strains. ^{28 - 30} A wide variety of Wolbachia strains including strains from supergroups A and B have been identified in Aedes albopictus and various Drosophila species. ^{22 , 31 , 32} Generally, mosquitoes are commonly infected by Wolbachia strains from supergroups A and B. Therefore, previous studies confirm the results of our study. ³³

In a study in Italy, Wolbachia infection was reported in Ae. cinereus, Ae. punctor, and Ae. detritus, but there were no reported infections in Ae. caspius and Ae. vexans. ³⁴ During this study, there was no infection with Wolbachia in Ae. caspius. Therefore, the results of the present study are completely consistent with those of the previous study. In a study in Portugal, researchers detected Wolbachia in Cx. Pipiens but did not find any infection with Wolbachia in Ae. caspius, Ae. Detritus, and An. maculipennis. ³⁵ Therefore, the present study confirms the results of the previous study. The higher prevalence of Wolbachia infection in Culex mosquitoes compared to Aedes mosquitoes is influenced by ecological, evolutionary, and biological factors. Culex mosquitoes, found in urban areas, have a higher infection rate due to prolonged co-evolution with Wolbachia and strong reproductive interactions. In contrast, Aedes mosquitoes inhabit different ecological niches with lower infection rates, possibly due to limited evolutionary history with Wolbachia and distinct biological traits. ³⁶

The results of a study that was conducted to determine Wolbachia infection in the population of Culicidae mosquitoes in Egypt showed that in Cx. pusillus, Cx. pipiens, Cx. antennatus, An. Pharoensis, and Ae. caspius, which were infected with Wucheraria bancroftii, Wolbachia infection was also reported from these species. ³⁷ Based on previous studies, it seems that when Anopheles pharoensis and Ae. caspius mosquitoes were not infected with Wucheraria bancrofti, bacterial infection was not reported in these species. However, when they were infected with Wucheraria bancrofti, bacterial infection was observed. From this observation, it is hypothesized that Wucheraria bancrofti, the filarial nematode, likely contains Wolbachia bacteria. This is consistent with previous findings where certain species of filarial nematodes, including Wucheraria bancrofti, have been shown to harbor endosymbiotic Wolbachia bacteria. ³⁷ Some studies conducted in Europe showed that Cx. pipiens, Cx, Ae. Punctor, and Coquillettidia richiardii are infected with Wolbachia. ³⁸ It appears that the present study findings align with previous research, indicating that Wolbachia infection is predominantly reported in the Cx. pipiens complex. This trend could potentially be attributed to the compatibility of Wolbachia bacteria with the Cx. pipiens complex. ^{5 , 39} While infection with Wolbachia was not reported in Ae. caspius in our study, Cx. pipiens showed a high rate of Wolbachia infection. This observation supports and corroborates the results of previous studies, which have also highlighted the prevalence of Wolbachia infection within the Cx. pipiens complex. This consistency across studies further strengthens our understanding of Wolbachia distribution among mosquito species. It underscores the importance of continued research to elucidate the factors driving Wolbachia prevalence and its implications for mosquito biology, vector competence, and disease transmission dynamics.

The results of our study and previous research suggest that Wolbachia infection varies among different species of Culicidae mosquitoes. This highlights the species-specific nature of Wolbachia infection in mosquitoes. Such factors as the mosquito's biology, ecology, and evolutionary history may influence its susceptibility to Wolbachia infection. Understanding these species-specific patterns of Wolbachia infection is crucial for developing targeted vector control strategies and leveraging Wolbachia-based interventions for disease control.

Conclusion

The findings of the current study revealed that the Wolbachia infection detected in Cx. pipiens belonged to supergroup B. Given the absence of Wolbachia infection in Ae. caspius observed in our study, there's an opportunity to artificially infect this species with Wolbachia. This can be achieved through laboratory-based methods, such as microinjection of Wolbachia-infected mosquito embryos or introduction of Wolbachia-infected adult mosquitoes into the wild.Ethics approval and consent to participate

This study was approved by the ethical committee of Shiraz University of Medical Sciences (IR.SUMS.SCHEANUT.REC.1400.102).

Acknowledgment

The authors would like to thank the Department of Biology and Control of Disease Vectors, School of Health, Shiraz University of Medical Sciences, Shiraz, Iran, for providing the mosquito laboratory facilities.

Author contributions

Kourosh Azizi and Saideh Yousefi analyzed and interpreted the data and drafted the manuscript. Narjes Moezi and Mozaffar Vahedi performed laboratory experiments. Reza Sadeghi and Saeed Shahabi reviewed and edited the manuscript. Azim Paksa conceived and designed the study and reviewed the manuscript. All authors read and approved the final manuscript.

Funding

This study was funded by Shiraz University of Medical Sciences, Shiraz, Iran, under grand NO. 24931.

Conflicts of Interest

None declared.

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