# Culex (Diptera: Culicidae) Mosquitoes in Jazan Region, Saudi Arabia, and Their Molecular Identification

Authors: Elsiddig Noureldin; Ommer Dafalla; Abdualziz Hakami; Mohammed Jubran; Ahmed Alzhrani; Muhannad Mujally; Othman Shajari; Ali Khardali; Zaki Eisa
Journal: International Journal of Zoology
 (2021)

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## Abstract

Morphological characteristics have been the gold standard method to identify mosquito species. However, morphological identification has many limitations including lack of expertise and damaging of external characters due to improper specimen handling. Therefore, we used the polymerase chain reaction technique (PCR) as an integrated tool to identifyCulex mosquito species to establish a more precise and reliable identification system related to their spatial distribution in Jazan region. We identified Culex mosquito species and subspecies using taxonomic keys, and then we used the polymerase chain reaction technique (PCR) as an integrated tool to confirm and refine the list of Culex mosquito species in the region. Phylogenetic trees were constructed for the identified species, and their distinctive clustering was compared with their reference’s species in the GenBank. We identified 7026 adult Culex mosquitoes belonging to 4 species. Culex tritaeniorhynchus was the predominant species (45%), followed by Cx. quinquefasciatus (32%), then Culex sitiens (20%), and Cx. pipiens (3%). The most infested areas by Culex in the region were Gizan and Sabya. The PCR achieved 100% success in identifying the four Culex mosquito species. We also report the molecular identification of Cx. quinquefasciatus and Cx. pipiens species for the first time in Jazan region while the molecular identification of Cx. tritaeniorhynchus and Cx. sitiens was reported for the first time in Jazan region and the whole Saudi Arabia. This study utilized for the first time PCR to identify Culex mosquito species in Jazan region. The PCR is a complementary and integrated taxonomy-based identification tool for mosquito species. This integration has the capacity to promote and enhance vector surveillance and control programs, as well as defining the genetic diversity of species in the region.


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## Body

## 1. Introduction

Mosquito-borne diseases, i.e., malaria [1, 2], filariasis [3], Rift Valley fever [4, 5], West Nile virus [6], dengue, and others [7, 8], pose potential public health threats in Saudi Arabia.Several authors have investigated the distribution of mosquito species in Saudi Arabia [1–16].Most of these studies reported the predominance ofCulex mosquitoes over other species. Within Culex species reported by these studies, Culex quinquefasciatus, Culex tritaeniorhynchus, and Cx. pipiens were the most dominant Culex mosquitoes in Saudi Arabia and Jazan region.The abundance and distribution of mosquito fauna are influenced by many factors including, but not limited to, host availability, climatic conditions, especially rain and temperature, human mobility and activities, and land cover [17]. These factors have a potential impact on the vectorial capacity of mosquitoes for disease transmission.The accurate identification of mosquito vector species and knowledge of their biology, ecology, and geographical distribution are considered important factors for surveillance and control of vectors and mosquito-borne diseases [18].Morphological identification is the gold standard and the conventional method to identify mosquito species depending on their external characters [19].However, morphological identification is highly time consuming and requires experienced taxonomists. Furthermore, improper handling of specimen damage is often occurred to some important characteristics such as bristles and scales leads to incomplete identification. Additionally, the similarity of shared morphological features by species complexes’ members makes identification based on taxonomic keys alone a difficult task [20].Polymerase chain reaction (PCR) is becoming a popular technique for mosquitoes identification based on their DNA sequencing and the fact that every species has its own genetic identity [19].Molecular techniques to differentiate betweenCx. pipiens complex or/and other Culex species, or to differentiate between the Cx. pipiens complex biotypes, are based on gel electrophoretic analysis of certain DNA fragments amplified by PCR [21]. PCR assay identifies Cx. pipiens, Cx. quinquefasciatus, and their hybrids based on the nucleotide sequence differences in the acetylcholinesterase gene Ace.2 [22].The aim of this study was to determine fauna of adultCulex mosquitoes (Diptera: Culicidae) and to produce precise and refine records of their species and their distribution in Jazan region based on morphological and genomic (molecular) identification. To the best of our knowledge, this is the first study in Jazan region to use molecular characteristics to identify Culex species and their genetic diversity.


## 2. Materials and Methods

### 2.1. Study Area

Jazan area is about 22,000 km2 and with 1.6 million population, lies between 16°–12 and 18°–25, latitude north, and located in the subtropical zone, southwestern of Saudi Arabia, with a coastal boundary of 250 km along the Red Sea and a 120 km border with the Republic of Yemen (Figure 1). This region includes over 3000 villages scattered along the area and about 100 islands located in the Red Sea, including the Farasan islands. It is surrounded by the Red Sea from the west and by Arabic Republic of Yemen from the south and east and Asir region from the north ([23]; GASTAT 2017: https://www.stats.gov.sa/en/5655).Figure 1
Boundaries of the different study areas in Jazan Region, southwest of Saudi Arabia.


### 2.2. Mosquito Collection

CDC miniature light traps were used for the adult mosquitos’ collection from different parts of Jazan region from February 2018 to December 2019 (Table1). Ten light traps were installed once per month in each of the houses, animals' shelters, wild vegetation, near wadies, sewerage plants, dams, and ponds from 1800 to 0600 hr.Table 1
Culex species in Jazan region.
AreaCx. tritaeniorhynchusCx. quinquefasciatusCx. pipiensCx. sitiensTotalGizan7509041733332160Al-shegairy243102024369Ahad Almasarha0002929Sabya (Qaem jaaferi)817004501267Sabya (Wadi sabya)3700340Samtah023570242Damad5300204257Baish40620825114753Aboareesh11931000429Al-tawal13006578Al-madhaya229000229Al-darb6700067Al-ariddah (Batin asar)489302143Haroob3610064Al-shegaig8400084Haroob (Wadi wasea)1500015Faifa9610097Al-gofl61321384190Al-dhabia0540256Al-horath18006179Al-sehi010900109Al-aeidabi161501142Al-aeidabi (Wadi qessi)2500934Aiban8180026Belgazzi1210300115Total3144226621813987026%4532320For outdoor collections, a 2 kilogram block of dry ice (CO2) was wrapped in a Hessian bag above the trap. To minimize the mortality of the collected mosquitoes due to desiccation, damp cotton pads were kept in the collection cups. Collected mosquitoes were brought to the insectary of the Saudi Center for Disease Prevention and Control in Gizan city for morphological and genetic identification.


### 2.3. Mosquito Morphological Identification

FemaleCulex pipens and Culex quinquefasciatus mosquitoes were morphologically identified and differentiated by using wing measurements of intersection of costa, subcosta, and bifurcation of R2 + 3 veins, as described by some taxonomic pictorial keys [24–26].For the Females ofCulex tritaeniorhynchus and Culex sitiens, relevant pictorial keys were used [24, 27, 28].


### 2.4. Molecular Identification ofCulex Species

Previously identifiedCulex mosquitoes of individual species by morphometric methods were selected according to their location of capture.DNA was extracted from the stored homogenate using GeneJET Genomic DNA Purification Kit (Thermo Scientific) following the manufacturer’s recommendations: For every individual mosquito, legs were homogenized in a mortar and pestle (mini borosilicate glass chamber length 60 mm/pestle diameter 9.0 mm, 3.0 Ml, Fisherbrand) in 150μL of digestion solution and then the homogenate was transferred to 1.5 tubes. Twenty microliters of Proteinase K Solution was added and mixed thoroughly by vortexing to obtain a uniform suspension and incubated in the thermo shaker for 30 minutes at 56°C, 1300 rpm. Twenty microliters of RNase A Solution which was added and mixed by vortexing and incubated for 10 min at room temperature, then 200 μL of lysis solution was added and mixed thoroughly by vortexing for 15 s until a homogeneous mixture is obtained. After adding 400 μL of 50% ethanol and mixed by vortexing the prepared lysate was transferred to a GeneJET Genomic DNA Purification Column inserted in a collection tube and centrifuged for 1 min at 8000 rpm. The GeneJET Genomic DNA Purification Column was placed into a new 2 mL collection tube and 500 μL of wash buffer 1 was added and centrifuged for 1 min at 8000 RPM, then the column was placed into a new collection tube and 500 μL of wash buffer 2 was added and centrifuged for 3 min at 13,000 rpm. DNA Purification Column was transferred to a sterile 1.5 mL microcentrifuge tube, and 200 μL of elution buffer was added to the center of the column membrane to elute genomic DNA. Incubated for 2 min at room temperature and centrifuged for 1 min at 8000 rpm.The molecular identification ofCulex species was performed by polymerase chain reaction technique (PCR) following the described procedure and using the primers provided in (Table 2.) [22, 29].Table 2
Primers used for molecular identification ofCulex species.
Consensus CPI6 RGCGGGTACCATGCTTAAATTTAGGGGTACulex pipiens complex PQIO FCCTATGTCCGCGTATACTA 698 bpCx. restuans R6 FCCAAACACCGGTACCCAA 506 bpCx. salinarius S20 FTGAGAATACATACCACTGCT 175 bpCx. nigripalpus N90I FATACCCATGCGAAAGCATAC 404 bpCx. nigripalpus NRI080 RGTACCGCGACCACACGACTTCx. quinquefasciatus FCQGGT CGT ATC AAC AAA GTC CA 500 bpCx. quinquefasciatus RCQATG GAT ACG GAC TAC ACT GGCOI (LCO1490)GGTCAACAAATCATAAAGATATTGG 740 bpCOI (HC02198)TAAACTTCAGGGTGACCAAAAAATCAThe PCR was carried out in a total volume of 50μL using Dream Tag Green Master Mix (Thermo Scientific) containing 25 μL master mix ready to use, 1 μL (10 pmol) of each reverse and species-specific forward primers, 5 μL of DNA template, and 18 μL nuclease-free water. The mix was subjected to initial denaturation at 94°C for 3 minutes, 30 cycles of denaturation (94°C, 60 s), primer annealing (51°C), primer extension (72°C, 60 s), and final extension for 5 minutes. In each run, negative and positive controls were included. The PCR products amplifications were analyzed by gel electrophoresis (1.5 agarose in Tris-Acetate EDTA buffer) staining with ethidium bromide. The visualization was carried out using Gel Doc XR Imaging System (Bio-Rad).Universal primers [30], LCO1490 and HCO2198 (Table 2), were used for the PCR amplification of a 710-bp fragment of the mitochondrial cytochrome oxidase subunit I gene (COI) for the samples not identified by the species-specific primers follow the same previous PCR procedure.


### 2.5. Sequencing

Purification and standard sequencing for PCR products were performed by Macrogen Company (Seoul, Korea). Sequencing reactions were performed in an ABI PRISM® 3730XL Analyzer (96 capillary type) using an ABI PRISM® BigDyeTM Terminator Cycle Sequencing Kit with AmpliTaq® DNA polymerase (FS enzyme) (Applied Biosystems), following the protocols supplied by the manufacturer. Single-pass sequencing was performed on each template using LCO1490 primer. Gel elution was performed using MG Gel Extraction SV (MD007) kit (MGmed), following the protocols supplied by the manufacturer.The sequences were searched for sequence similarity through Basic local Alignment Search Tool (BLAST) (http://www.ncbi.nlm.nih.gov/BLAST/) [31] and compared to reference sequences of Culex species detected in BLAST and downloaded from GenBank (http://www.ncbi.nlm.nih.gov/genbank/).Evolutionary relationship of taxa tree was constructed for each of theCulex species using the Molecular Evolutionary Genetic Analysis (MEGA 5 software).


## 2.1. Study Area

Jazan area is about 22,000 km2 and with 1.6 million population, lies between 16°–12 and 18°–25, latitude north, and located in the subtropical zone, southwestern of Saudi Arabia, with a coastal boundary of 250 km along the Red Sea and a 120 km border with the Republic of Yemen (Figure 1). This region includes over 3000 villages scattered along the area and about 100 islands located in the Red Sea, including the Farasan islands. It is surrounded by the Red Sea from the west and by Arabic Republic of Yemen from the south and east and Asir region from the north ([23]; GASTAT 2017: https://www.stats.gov.sa/en/5655).Figure 1
Boundaries of the different study areas in Jazan Region, southwest of Saudi Arabia.


## 2.2. Mosquito Collection

CDC miniature light traps were used for the adult mosquitos’ collection from different parts of Jazan region from February 2018 to December 2019 (Table1). Ten light traps were installed once per month in each of the houses, animals' shelters, wild vegetation, near wadies, sewerage plants, dams, and ponds from 1800 to 0600 hr.Table 1
Culex species in Jazan region.
AreaCx. tritaeniorhynchusCx. quinquefasciatusCx. pipiensCx. sitiensTotalGizan7509041733332160Al-shegairy243102024369Ahad Almasarha0002929Sabya (Qaem jaaferi)817004501267Sabya (Wadi sabya)3700340Samtah023570242Damad5300204257Baish40620825114753Aboareesh11931000429Al-tawal13006578Al-madhaya229000229Al-darb6700067Al-ariddah (Batin asar)489302143Haroob3610064Al-shegaig8400084Haroob (Wadi wasea)1500015Faifa9610097Al-gofl61321384190Al-dhabia0540256Al-horath18006179Al-sehi010900109Al-aeidabi161501142Al-aeidabi (Wadi qessi)2500934Aiban8180026Belgazzi1210300115Total3144226621813987026%4532320For outdoor collections, a 2 kilogram block of dry ice (CO2) was wrapped in a Hessian bag above the trap. To minimize the mortality of the collected mosquitoes due to desiccation, damp cotton pads were kept in the collection cups. Collected mosquitoes were brought to the insectary of the Saudi Center for Disease Prevention and Control in Gizan city for morphological and genetic identification.


## 2.3. Mosquito Morphological Identification

FemaleCulex pipens and Culex quinquefasciatus mosquitoes were morphologically identified and differentiated by using wing measurements of intersection of costa, subcosta, and bifurcation of R2 + 3 veins, as described by some taxonomic pictorial keys [24–26].For the Females ofCulex tritaeniorhynchus and Culex sitiens, relevant pictorial keys were used [24, 27, 28].


## 2.4. Molecular Identification ofCulex Species

Previously identifiedCulex mosquitoes of individual species by morphometric methods were selected according to their location of capture.DNA was extracted from the stored homogenate using GeneJET Genomic DNA Purification Kit (Thermo Scientific) following the manufacturer’s recommendations: For every individual mosquito, legs were homogenized in a mortar and pestle (mini borosilicate glass chamber length 60 mm/pestle diameter 9.0 mm, 3.0 Ml, Fisherbrand) in 150μL of digestion solution and then the homogenate was transferred to 1.5 tubes. Twenty microliters of Proteinase K Solution was added and mixed thoroughly by vortexing to obtain a uniform suspension and incubated in the thermo shaker for 30 minutes at 56°C, 1300 rpm. Twenty microliters of RNase A Solution which was added and mixed by vortexing and incubated for 10 min at room temperature, then 200 μL of lysis solution was added and mixed thoroughly by vortexing for 15 s until a homogeneous mixture is obtained. After adding 400 μL of 50% ethanol and mixed by vortexing the prepared lysate was transferred to a GeneJET Genomic DNA Purification Column inserted in a collection tube and centrifuged for 1 min at 8000 rpm. The GeneJET Genomic DNA Purification Column was placed into a new 2 mL collection tube and 500 μL of wash buffer 1 was added and centrifuged for 1 min at 8000 RPM, then the column was placed into a new collection tube and 500 μL of wash buffer 2 was added and centrifuged for 3 min at 13,000 rpm. DNA Purification Column was transferred to a sterile 1.5 mL microcentrifuge tube, and 200 μL of elution buffer was added to the center of the column membrane to elute genomic DNA. Incubated for 2 min at room temperature and centrifuged for 1 min at 8000 rpm.The molecular identification ofCulex species was performed by polymerase chain reaction technique (PCR) following the described procedure and using the primers provided in (Table 2.) [22, 29].Table 2
Primers used for molecular identification ofCulex species.
Consensus CPI6 RGCGGGTACCATGCTTAAATTTAGGGGTACulex pipiens complex PQIO FCCTATGTCCGCGTATACTA 698 bpCx. restuans R6 FCCAAACACCGGTACCCAA 506 bpCx. salinarius S20 FTGAGAATACATACCACTGCT 175 bpCx. nigripalpus N90I FATACCCATGCGAAAGCATAC 404 bpCx. nigripalpus NRI080 RGTACCGCGACCACACGACTTCx. quinquefasciatus FCQGGT CGT ATC AAC AAA GTC CA 500 bpCx. quinquefasciatus RCQATG GAT ACG GAC TAC ACT GGCOI (LCO1490)GGTCAACAAATCATAAAGATATTGG 740 bpCOI (HC02198)TAAACTTCAGGGTGACCAAAAAATCAThe PCR was carried out in a total volume of 50μL using Dream Tag Green Master Mix (Thermo Scientific) containing 25 μL master mix ready to use, 1 μL (10 pmol) of each reverse and species-specific forward primers, 5 μL of DNA template, and 18 μL nuclease-free water. The mix was subjected to initial denaturation at 94°C for 3 minutes, 30 cycles of denaturation (94°C, 60 s), primer annealing (51°C), primer extension (72°C, 60 s), and final extension for 5 minutes. In each run, negative and positive controls were included. The PCR products amplifications were analyzed by gel electrophoresis (1.5 agarose in Tris-Acetate EDTA buffer) staining with ethidium bromide. The visualization was carried out using Gel Doc XR Imaging System (Bio-Rad).Universal primers [30], LCO1490 and HCO2198 (Table 2), were used for the PCR amplification of a 710-bp fragment of the mitochondrial cytochrome oxidase subunit I gene (COI) for the samples not identified by the species-specific primers follow the same previous PCR procedure.


## 2.5. Sequencing

Purification and standard sequencing for PCR products were performed by Macrogen Company (Seoul, Korea). Sequencing reactions were performed in an ABI PRISM® 3730XL Analyzer (96 capillary type) using an ABI PRISM® BigDyeTM Terminator Cycle Sequencing Kit with AmpliTaq® DNA polymerase (FS enzyme) (Applied Biosystems), following the protocols supplied by the manufacturer. Single-pass sequencing was performed on each template using LCO1490 primer. Gel elution was performed using MG Gel Extraction SV (MD007) kit (MGmed), following the protocols supplied by the manufacturer.The sequences were searched for sequence similarity through Basic local Alignment Search Tool (BLAST) (http://www.ncbi.nlm.nih.gov/BLAST/) [31] and compared to reference sequences of Culex species detected in BLAST and downloaded from GenBank (http://www.ncbi.nlm.nih.gov/genbank/).Evolutionary relationship of taxa tree was constructed for each of theCulex species using the Molecular Evolutionary Genetic Analysis (MEGA 5 software).


## 3. Results and Discussion

Results of the identification and counts ofCulex species mosquitoes are shown in Table 1. Seven thousand twenty-six adult Culex mosquitoes were collected from 25 different areas of Jazan region during 2018 and 2019 (Figure 1).Clearly, the number of mosquito specimens was relatively low compared to the period of study (around 2 years). This is may be attributed to the fact that effective and extensive vector control activities were taken place during the last 5 years in the region to combat malaria, dengue, and rift valley, the most prevalent vector-borne diseases in the region.In this study, fourCulex species were present in the region, namely; Culex tritaeniorhynchus, Cx. quinquefasciatus, Cx. sitiens, and Cx. pipiens. Their respective percentages of occurrence were 45%, 32%, 20%, and 3%.Culex tritaeniorhynchus was the most abundant species encountered in 23 areas out of 25, followed by Cx. quinquefasciatus and Cx. sitiens which occurred in 14 areas, and Cx. pipiens in 4 areas.These results are in accordance with the findings of Al Ahmad et al. [15] who indicated that Culex tritaeniorhynchus is a common widespread species in Saudi Arabia and has been recorded from Jazan and other 14 provinces out of 15. Similar results were also reported by Alsheikh et al. [32], who found Cx. tritaeniorhynchus to be the most predominant species in the Red Sea coastal plain of Jazan region, followed by Cx. quinquefasciatus. The findings are also in line with those of Al Ahmad et al.and Bakr et al. [14, 33] who reported the same four Culex species from Jazan region. Culex tritaeniorhynchus, Cx. quinquefasciatus, and Cx. sitiens have also been reported from different parts of Jazan region with the predominance of Culex tritaeniorhynchus over other Culex species [34].In the present study, the most infested areas byCulex mosquitoes in Jazan region were Gizan (2160 specimens), Sabya (1267), Baish (753), and Aboareesh (429) (Table 1). This may be due to the nature of those areas, which are highly populated and relatively flat and permit for the formation of small stagnant water collections following rainfall and/or water pipe leakage in their urban areas [35]. The prevalence of the Culex species in those areas could be attributed also to their wide suitability to different breeding sites and variable extreme climatic factors prevailing there [11].Three of theCulex species reported in the present study are considered important disease vectors, namely Culex tritaeniorhynchus, Cx. quinquefasciatus, and Cx. pipiens.Characterizations of dominant mosquito vectors include well adaptation to a wide range of climatic conditions and habitats, high anthropophilic propensity, and variable adult resting behaviour [36].Mosquito fauna and their distribution in Saudi Arabia had been widely investigated by many authors. For example, forty-nine mosquito species belonging to seven genera from 1956 to 2017 (18 Anophelines and 31 Culicines) have been listed [37].Out of the 31 Culicines, 19 species were belonging to the genusCulex Linnaeus (1758): one species under the subgenus Barraudius Edwards, 1921, Cx. (Barraudius) pusillus; one species under subgenus Culiciomyia Theobald, 1907, Cx. (Culiciomyia) nebulosus; one species under subgenus Oculeomyia Theobald, 1907, Cx. (Oculeomyia) bitaeniorhynchus; two species under subgenus Maillotia Theobald, 1907, Cx. (Maillotia) arbieeni and Cx. (Maillotia) salisburiensis; and fourteen species under subgenus Culex Linnaeus, 1758: Cx. (Culex) decens, Cx. (Culex) duttoni, Cx. (Culex) laticinctus, Cx. (Culex) mattinglyi, Cx. (Culex) mimeticus, Cx. (Culex) perexiguus, Cx. (Culex) pipiens, Cx. (Culex) quinquefasciatus, Cx. (Culex) simpsoni, Cx. (Culex) sinaiticus, Cx. (Culex) sitiens, Cx. (Culex) theileri, Cx. (Culex) tritaeniorhynchus, and Cx. (Culex) univittatus.Likewise, 7 species ofCulex had also been identified in Jazan region: (1) Culex pipiens from Gizan City, Alariddah, Baish, Ahad Almasarha, Samtah, Eldarab, Haroob, Bani Malik, and Farasan Islands; (2) Culex quinquefasciatus from Gizan City, Alariddah, Baish, Ahad Almasarha and Farasan Islands; (3) Culex tritaeniorhynchus from Gizan City, Alariddah, Baish, Ahad almasarha, Samtah, Eldarab, Haroob, and Bani Malik; (4) Culex sitiens from Gizan City, Alariddah, Baish, Ahad almasarha, Samtah, Wadi Jazan, and Farasan Islands; (5) Cx. Sinaiticus; (6) Cx. decens; (7) Cx. bitaeniorhynchus [33].On the other hand, another 10 species have been identified from different areas of Jazan region includingCx. tritaeniorhynchus, Cx. laticinctus, Cx. pipiens, Cx. quinquefasciatus, Cx. sinaiticus, Cx. simpsoni, Cx. torrentium, Cx. sitiens, Cx. univittatus, and Cx. tigripes [14].Similarly, seven species were recorded from different parts of Jazan region includingCulex tritaeniorhynchus, Cx. quinquefasciatus, Cx. Sinaiticus, Cx. sitiens, Culex duttoni, Cx. Arbieeni, and Cx. mimeticus [34].Cx. (Culex) perexiguus Theobald, 1903, and Cx. pusillus (Macquart) have been reported from the region [24, 38].Consequently, 18 Culex species so far have been reported from Jazan region, namely:Culex tritaeniorhynchus, Cx. laticinctus, Cx. pipiens, Cx. quinquefasciatus, Cx. sinaiticus, Cx. univittatus, Cx. (Culex) perexiguus, Cx. pusillus, Cx. simpsoni, Cx. torrentium, Cx. sitiens, Cx. decens, Cx. bitaeniorhynchus, Cx. theileri, Cx. tigripes, Cx. duttoni, Cx. arbieeni, and Cx. mimeticus [37].Culex mosquitoes are proven or suspected vectors of West Nile, and Rift Valley fever viruses, microfilariae, and a variety of encephalitis [39, 40].It has been stated thatCx. quinquefasciatus occurs more in the south of the Arabian Peninsula with isolated occurring of Cx. pipiens in the southwestern mountains [41].Due to the close similarity ofCx. pipiens and Cx. quinquefasciatus, many of their occurrences and morphological identifications in Saudi Arabia are problematic, especially females [37].Considerable discrepancies in the larval and adult stages are exhibited between the two species, and it is difficult to distinguish them morphologically. Additionally, researchers working withCulex in Saudi Arabia have rarely, if not ever, used male genitalia diagnostic features to confirm identification of the two species [37].This was the reason why we adopted the molecular technique, which is known to be highly specific and sensitive for the identification ofCulex species complex and their hybrids [42, 43]. In addition, new methods of genomic (molecular) identification of species, subspecies, races, biotypes, and forms are more reliable and precise than older morphological identification methods of species complexes [44].


### 3.1.Culex quinquefasciatus (Figure 2)

Larvae ofCx. quinquefasciatus breed in water bodies with a high degree of organic pollution and close to human habitats. Females are nocturnal and enter houses and bite the man in preference to other mammals [45].Figure 2
Wing ofCulex quinquefasciatus showing the intersection of the vein subcosta with costa before the level of furcation of R1+2.Cx. quinquefasciatus was found breeding all year round with peaks during winter in Taif, Jeddah, and Tabuk of Saudi Arabia [46]. In Jazan region, this species was collected from rocky pits and streams. It was found together with Anopheles arabiensis and Cx. tritaeniorhynchus at the ratio of 1 : 4: 1 : 3 [34].The species is a primary vector ofWuchereria bancrofti, vector of avian malaria, western equine encephalomyelitis, and St. Louis encephalitis. It was implicated as a vector of dog heartworm [45, 47].Cx. quinquefasciatus and Cx. pipiens, which are the main vectors of bancroftian filariasis, Wuchereria bancrofti worldwide including the Middle East countries, have been reported from the south-western districts of Saudi Arabia [6].


### 3.2.Culex pipiens (Figure 3)

Larvae ofCulex pipiens breed in various places ranging from highly polluted sewages and cesspits to clear pools and containers. It breeds in stagnant water in shaded or unshaded habitats. Females bite the man outdoors and indoors [41].Figure 3
Wing ofCulex pipiens showing the intersection of the vein subcosta with costa at the level of furcation of R1+2.Cx. pipiens larvae were reported all year round in Taif, Jeddah, and Tabuk. The peak was at autumn while high density was in summer when temperature is high [46].Cx. pipiens is a primary vector of periodic Bancroftian filariasis. It has been naturally infected with West Nile and Sindbis viruses in Israel, while it was infected with Rift Valley Fever virus and West Nile virus in Egypt [41].W. bancrofti has been identified within foreign workers from five South-East Asian countries in Abha, southwest of the Saudi Arabia. Cx. pipiens may act as a potential vector of Bancroftian filariasis in Saudi Arabia [48].Culex pipiens complex has occurred in different climatic and habitat zones, particularly temperate and temperate-humid regions of the world [44].It is always difficult and problematic to differentiate morphologically between adults ofCx. quinquefasciatus and Culex pipiens especially females. Nonetheless, taxonomists used to differentiate between the two species by wing venations where the intersection of the vein subcosta with costa is before the level of furcation of R1+2 in the case of Cx. quinquefasciatus (Figure 2), while this intersection is at or beyond the level of furcation of R1+2 in the case of Cx. pipiens (Figure 3) [24]. However, this is readily achieved by molecular characteristics.Molecular identification of bothCx. pipiens (Figures 4 and 5) and Cx. quinquefasciatus (Figures 6 and 7) confirmed their initial morphological identification. Phylogenetic trees were constructed for Cx. pipiens and Cx. quinquefasciatus, and their distinctive clustering was compared with their reference’s species in the GenBank as shown in Figures 5 and 7. It is worthy to mention that the molecular characteristics of Cx. pipiens when compared with the records of the gene bank showed its similarity to those of Egypt, Kenya (100%) and very close to those of Iran (99.85%) (Table 3). While the molecular identification revealed one species of Cx. quinquefasciatus that is similar to the Turkish species (100%), and one subspecies of Cx. pipiens quinquefasciatus (Q primers 500 bp) is similar to the Sri Lankan subspecies (100%) (Table 3).Figure 4
PCR amplified 700 bp DNA bands using consensus CPI6 R andCx. pipiens complex PQIO F primers. Lane 1: 100 bp ladder, lanes 2–15 tested samples.Figure 5
Evolutionary relationship of taxa ofCulex pipiens identified in Jazan region using MEGA 5.Figure 6
PCR amplified 500 bp DNA bands usingCx. quinquefasciatus specific primers. Lane 1: 100 bp ladder, lane 2: positive control, lane 3: negative control, lane 4: negative sample, lanes 5–15 positive samples.Figure 7
Evolutionary relationship of taxa ofCulex quinquefasciatus identified in Jazan region using MEGA 5.Table 3
Similarity ofCulex species in Jazan Region related to gene bank references.
Culex speciesGene bank accession no.Similarity (%)CountryCulex tritaeniorhynchusKJ012245.1100USAAB738247.1100JapanMF179221100ChinaCulex pipiensMT199095100EgyptMK300250100KenyaJQ95837199.85IranCulex quinquefasciatusMK713993.1100TurkeyCulex pipiens quinquefasciatus (Q primers 500 bp)AF089002.1100Sri LankaCulex sitiensMN552296100GuineaMK300241100KenyaMF179212100ChinaIn Baljurashi Province of Saudi Arabia, the results of molecular identification ofCx. pipiens using PCR technique revealed that the Cx. pipiens cf-3 strain was the accurate definition after initial morphological identification of the species [49]. Likewise, the molecular characteristics of Cx. quinquefasciatus samples from Yanbu Province of Saudi Arabia indicated that they were very close to those found in Pakistan, Brazil, and India (GenBank references; KF406862.1, MK575480.1, MH538709.1, and MH538707.1.) [50].


### 3.3.Culex tritaeniorhynchus (Figure 8(b))

The main morphological features found inCx. tritaeniorhynchus are the presence of a median pale ring in the proboscis that extends proximally to the ventral surface of the proboscis (Figure 9(b)), and the fore and midfemora are entirely black (Figure 10(b)), whereas the furcation of R2+3 is proximal to the furcation of M1+2 (Figure 11(b)).Figure 8
Culex sitiens (a) and Culextritaeniorhynchus (b).
(a)(b)Figure 9
Proboscis ofCulex sitiens (a) and Culextritaeniorhynchus (b).
(a)(b)Figure 10
Fore and midfemora ofCx. sitiens (speckled) (a) and (entirely black) Cx.tritaeniorhynchus (b).
(a)(b)Figure 11
Furcation ofR2+3 of Cx. sitiens (distal to furcation of M1+2) (a) and (proximal to furcation of M1+2) Cx.tritaeniorhynchus (b).
(a)(b)Larvae ofCx tritaeinorhynchus are found in many habitats that are sunlit and contain vegetation like the temporary, semi-permanent, and permanent ground water habitats, low-salinity tidal marshes, streams, ground pools, and swamps. Females feed primarily on pigs and cattle, but in their absence will feed on men [27]. It has been reported that larvae of Cx. tritaeniorhynchus preferred aquatic habitats with wet muddy substrate and low total dissolved salts (TDS). Those areas are found in eastern Sarwat Mountain range near the cities in Ahd almasarha, Eleidabi, Sabya, Al-Ariddah, and Abuareesh [51].Cx. tritaeniorhynchus was found in all types of breeding sites in the Red Sea coastal plain of Jazan region (man-made pools, especially turbid ones, rain pools, dams, rock pools, and domestic water tanks). It was also noticed to share breeding sites with Anopheles arabiensis, the main malaria vector in the region [23].Cx. tritaeinorhynchus is a primary vector of Japanese B encephalitis in the oriental region [27] and Rift valley virus in Jazan region of Saudi Arabia with a biting preference for humans and sheep [4]. In the present study, the molecular identification of Cx. tritaeniorhynchus using the universal primer COI (Figure 12), along with the constructed phylogenetic trees and their distinctive clustering, was compared with their reference’s species in the GenBank (Figure 13) and showed that it is quite similar to the species of USA, Japan, and China (100% similarity, Table 3).Figure 12
PCR amplified 745 bp DNA bands using COI primers to identifyCulex tritaeniorhynchus and Culex sitiens. Lane 1: 100 bp ladder, lane 2: positive control, lanes 3–7: tested samples, lane 8: negative control.Figure 13
Evolutionary relationship of taxa ofCulex tritaeniorhynchus identified in Jazan region using MEGA 5.


### 3.4.Culex sitiens (Figure 8(a))

The main morphological features of the proboscis indicate that the median pale ring is present, but not extending proximally to the ventral surface as inCx. tritaeinorhynchus (Figure 9(a)); the fore and midfemora are speckled (Figure 10(a)). On the other hand, the furcation of R2+3 is distal to the furcation of M1+2 (Figure 11(a)).Bionomics ofCx. sitiens showed various breeding habitats including fresh ground water, salt, brackish, and artificial containers in coastal, urban, and suburban areas [41].It can also be found in almost every stagnant brackish waters with full sunlight [52]. Females feed primarily on pigs and birds, but will also bite men. It is an implicated vector of Japanese B encephalitis [41].In Jeddah, Saudi Arabia, the density ofCx. sitiens was directly related to temperature. Its peak of density is in summer, and it is highly abundant in spring, while it breeds throughout the year [46].Cx. sitiens molecular identification in the present study using the universal primer COI (Figure 12), along with the constructed phylogenetic trees and their distinctive clustering, when compared with their reference’s species in the GenBank (Figure 14) revealed that it is similar to the African species of Guinea and Kenya (100%) and China (100%) (Table 3).Figure 14
Evolutionary relationship of taxa ofCulex sitiens identified in Jazan region using MEGA 5.All mosquito species identified morphologically in this study have also been molecularly identified, assuring absolute compatibility between taxonomic and molecular identification, confirming that PCR is a complementary tool for the identification of mosquito species.


## 3.1.Culex quinquefasciatus (Figure 2)

Larvae ofCx. quinquefasciatus breed in water bodies with a high degree of organic pollution and close to human habitats. Females are nocturnal and enter houses and bite the man in preference to other mammals [45].Figure 2
Wing ofCulex quinquefasciatus showing the intersection of the vein subcosta with costa before the level of furcation of R1+2.Cx. quinquefasciatus was found breeding all year round with peaks during winter in Taif, Jeddah, and Tabuk of Saudi Arabia [46]. In Jazan region, this species was collected from rocky pits and streams. It was found together with Anopheles arabiensis and Cx. tritaeniorhynchus at the ratio of 1 : 4: 1 : 3 [34].The species is a primary vector ofWuchereria bancrofti, vector of avian malaria, western equine encephalomyelitis, and St. Louis encephalitis. It was implicated as a vector of dog heartworm [45, 47].Cx. quinquefasciatus and Cx. pipiens, which are the main vectors of bancroftian filariasis, Wuchereria bancrofti worldwide including the Middle East countries, have been reported from the south-western districts of Saudi Arabia [6].


## 3.2.Culex pipiens (Figure 3)

Larvae ofCulex pipiens breed in various places ranging from highly polluted sewages and cesspits to clear pools and containers. It breeds in stagnant water in shaded or unshaded habitats. Females bite the man outdoors and indoors [41].Figure 3
Wing ofCulex pipiens showing the intersection of the vein subcosta with costa at the level of furcation of R1+2.Cx. pipiens larvae were reported all year round in Taif, Jeddah, and Tabuk. The peak was at autumn while high density was in summer when temperature is high [46].Cx. pipiens is a primary vector of periodic Bancroftian filariasis. It has been naturally infected with West Nile and Sindbis viruses in Israel, while it was infected with Rift Valley Fever virus and West Nile virus in Egypt [41].W. bancrofti has been identified within foreign workers from five South-East Asian countries in Abha, southwest of the Saudi Arabia. Cx. pipiens may act as a potential vector of Bancroftian filariasis in Saudi Arabia [48].Culex pipiens complex has occurred in different climatic and habitat zones, particularly temperate and temperate-humid regions of the world [44].It is always difficult and problematic to differentiate morphologically between adults ofCx. quinquefasciatus and Culex pipiens especially females. Nonetheless, taxonomists used to differentiate between the two species by wing venations where the intersection of the vein subcosta with costa is before the level of furcation of R1+2 in the case of Cx. quinquefasciatus (Figure 2), while this intersection is at or beyond the level of furcation of R1+2 in the case of Cx. pipiens (Figure 3) [24]. However, this is readily achieved by molecular characteristics.Molecular identification of bothCx. pipiens (Figures 4 and 5) and Cx. quinquefasciatus (Figures 6 and 7) confirmed their initial morphological identification. Phylogenetic trees were constructed for Cx. pipiens and Cx. quinquefasciatus, and their distinctive clustering was compared with their reference’s species in the GenBank as shown in Figures 5 and 7. It is worthy to mention that the molecular characteristics of Cx. pipiens when compared with the records of the gene bank showed its similarity to those of Egypt, Kenya (100%) and very close to those of Iran (99.85%) (Table 3). While the molecular identification revealed one species of Cx. quinquefasciatus that is similar to the Turkish species (100%), and one subspecies of Cx. pipiens quinquefasciatus (Q primers 500 bp) is similar to the Sri Lankan subspecies (100%) (Table 3).Figure 4
PCR amplified 700 bp DNA bands using consensus CPI6 R andCx. pipiens complex PQIO F primers. Lane 1: 100 bp ladder, lanes 2–15 tested samples.Figure 5
Evolutionary relationship of taxa ofCulex pipiens identified in Jazan region using MEGA 5.Figure 6
PCR amplified 500 bp DNA bands usingCx. quinquefasciatus specific primers. Lane 1: 100 bp ladder, lane 2: positive control, lane 3: negative control, lane 4: negative sample, lanes 5–15 positive samples.Figure 7
Evolutionary relationship of taxa ofCulex quinquefasciatus identified in Jazan region using MEGA 5.Table 3
Similarity ofCulex species in Jazan Region related to gene bank references.
Culex speciesGene bank accession no.Similarity (%)CountryCulex tritaeniorhynchusKJ012245.1100USAAB738247.1100JapanMF179221100ChinaCulex pipiensMT199095100EgyptMK300250100KenyaJQ95837199.85IranCulex quinquefasciatusMK713993.1100TurkeyCulex pipiens quinquefasciatus (Q primers 500 bp)AF089002.1100Sri LankaCulex sitiensMN552296100GuineaMK300241100KenyaMF179212100ChinaIn Baljurashi Province of Saudi Arabia, the results of molecular identification ofCx. pipiens using PCR technique revealed that the Cx. pipiens cf-3 strain was the accurate definition after initial morphological identification of the species [49]. Likewise, the molecular characteristics of Cx. quinquefasciatus samples from Yanbu Province of Saudi Arabia indicated that they were very close to those found in Pakistan, Brazil, and India (GenBank references; KF406862.1, MK575480.1, MH538709.1, and MH538707.1.) [50].


## 3.3.Culex tritaeniorhynchus (Figure 8(b))

The main morphological features found inCx. tritaeniorhynchus are the presence of a median pale ring in the proboscis that extends proximally to the ventral surface of the proboscis (Figure 9(b)), and the fore and midfemora are entirely black (Figure 10(b)), whereas the furcation of R2+3 is proximal to the furcation of M1+2 (Figure 11(b)).Figure 8
Culex sitiens (a) and Culextritaeniorhynchus (b).
(a)(b)Figure 9
Proboscis ofCulex sitiens (a) and Culextritaeniorhynchus (b).
(a)(b)Figure 10
Fore and midfemora ofCx. sitiens (speckled) (a) and (entirely black) Cx.tritaeniorhynchus (b).
(a)(b)Figure 11
Furcation ofR2+3 of Cx. sitiens (distal to furcation of M1+2) (a) and (proximal to furcation of M1+2) Cx.tritaeniorhynchus (b).
(a)(b)Larvae ofCx tritaeinorhynchus are found in many habitats that are sunlit and contain vegetation like the temporary, semi-permanent, and permanent ground water habitats, low-salinity tidal marshes, streams, ground pools, and swamps. Females feed primarily on pigs and cattle, but in their absence will feed on men [27]. It has been reported that larvae of Cx. tritaeniorhynchus preferred aquatic habitats with wet muddy substrate and low total dissolved salts (TDS). Those areas are found in eastern Sarwat Mountain range near the cities in Ahd almasarha, Eleidabi, Sabya, Al-Ariddah, and Abuareesh [51].Cx. tritaeniorhynchus was found in all types of breeding sites in the Red Sea coastal plain of Jazan region (man-made pools, especially turbid ones, rain pools, dams, rock pools, and domestic water tanks). It was also noticed to share breeding sites with Anopheles arabiensis, the main malaria vector in the region [23].Cx. tritaeinorhynchus is a primary vector of Japanese B encephalitis in the oriental region [27] and Rift valley virus in Jazan region of Saudi Arabia with a biting preference for humans and sheep [4]. In the present study, the molecular identification of Cx. tritaeniorhynchus using the universal primer COI (Figure 12), along with the constructed phylogenetic trees and their distinctive clustering, was compared with their reference’s species in the GenBank (Figure 13) and showed that it is quite similar to the species of USA, Japan, and China (100% similarity, Table 3).Figure 12
PCR amplified 745 bp DNA bands using COI primers to identifyCulex tritaeniorhynchus and Culex sitiens. Lane 1: 100 bp ladder, lane 2: positive control, lanes 3–7: tested samples, lane 8: negative control.Figure 13
Evolutionary relationship of taxa ofCulex tritaeniorhynchus identified in Jazan region using MEGA 5.


## 3.4.Culex sitiens (Figure 8(a))

The main morphological features of the proboscis indicate that the median pale ring is present, but not extending proximally to the ventral surface as inCx. tritaeinorhynchus (Figure 9(a)); the fore and midfemora are speckled (Figure 10(a)). On the other hand, the furcation of R2+3 is distal to the furcation of M1+2 (Figure 11(a)).Bionomics ofCx. sitiens showed various breeding habitats including fresh ground water, salt, brackish, and artificial containers in coastal, urban, and suburban areas [41].It can also be found in almost every stagnant brackish waters with full sunlight [52]. Females feed primarily on pigs and birds, but will also bite men. It is an implicated vector of Japanese B encephalitis [41].In Jeddah, Saudi Arabia, the density ofCx. sitiens was directly related to temperature. Its peak of density is in summer, and it is highly abundant in spring, while it breeds throughout the year [46].Cx. sitiens molecular identification in the present study using the universal primer COI (Figure 12), along with the constructed phylogenetic trees and their distinctive clustering, when compared with their reference’s species in the GenBank (Figure 14) revealed that it is similar to the African species of Guinea and Kenya (100%) and China (100%) (Table 3).Figure 14
Evolutionary relationship of taxa ofCulex sitiens identified in Jazan region using MEGA 5.All mosquito species identified morphologically in this study have also been molecularly identified, assuring absolute compatibility between taxonomic and molecular identification, confirming that PCR is a complementary tool for the identification of mosquito species.


## 4. Conclusion

In this study,Culex tritaeniorhynchus was the predominant species in Jazan region, followed by Cx. quinquefasciatus, then Cx. sitiens, and Cx. pipiens. The most infested areas by Culex mosquitoes in the region were Gizan and Sabya.The results showed the potentiality of integrating the morphological and molecular identification to refine the list ofCulex mosquito species and accurately document their geographical distribution in the Jazan region. This integration has the capacity to promote and enhance vector surveillance and control programmes, as well as defining the genetic diversity of species in the region.



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*Source: 5563916-2021-07-08.xml*