First record of a medically important vector, the Asian tiger mosquito Aedes albopictus (Skuse, 1895) (Diptera: Culicidae), using morphological and molecular data in northern Iran

Shahyad Azari-Hamidian

Research Center of Health and Environment, School of Health, Guilan University of Medical Sciences, Rasht, Iran [1]; Department of Medical Parasitology, Mycology and Entomology, School of Medicine, Guilan University of Medical Sciences, Rasht, Iran [2].

https://orcid.org/0000-0002-9370-9638

Behzad Norouzi, Hannaneh Maleki

Research Center of Health and Environment, School of Health, Guilan University of Medical Sciences, Rasht, Iran.

https://orcid.org/0000-0002-9788-5890

https://orcid.org/0000-0001-6952-7319

Seyed Mahmoud Rezvani, Morteza Pourgholami

Vice-Chancellorship of Health, Guilan University of Medical Sciences, Rasht, Iran.

https://orcid.org/0009-0006-2092-1909

https://orcid.org/0000-0002-1921-4784

Mohammad Ali Oshaghi

Department of Vector Biology and Control of Diseases, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran. 

https://orcid.org/0000-0003-3004-0364

ABSTRACT. Iranian mosquitoes (Diptera: Culicidae) include 73 species across eight genera. The fauna of mosquitoes in Guilan Province comprises 34 species classified into seven genera. A faunistic study of mosquitoes was conducted with emphasis on an aggressive biter mosquito reported by local people in Anzali and Rasht Cities of Guilan Province, northern Iran. Collections were made by hand catches using manual aspirators, light traps, BG lure traps and ovitraps during August–November 2023 in all 17 counties of the province. Species identification was carried out using morphological keys and molecular analysis of the barcode region of the cytochrome c oxidase subunit I (COI) gene. In total, 29 larvae and 896 adult specimens were collected which were not recognized as a species previously known to occur in the province. The aggressive biter mosquito was morphologically identified as Aedes albopictus (Skuse, 1895). The sequences of the barcode region of the COI gene of the species were generated for the first time in the country. This species was collected in 14 counties of the province. Thus, the mosquito fauna of Guilan Province increased to 35 species.

Keywords: Cytochrome c oxidase I, dengue fever, Guilan Province, invasive species, Stegomyia albopicta

Citation: Azari-Hamidian, S., Norouzi, B., Maleki, H., Rezvani, S.M., Pourgholami, M. & Oshaghi, M.A. (2024) First record of a medically important vector, the Asian tiger mosquito Aedes albopictus (Skuse, 1895) (Diptera: Culicidae), using morphological and molecular data in northern Iran. Journal of Insect Biodiversity and Systematics, 10 (4), 953–963.

INTRODUCTION 

The biting nuisance and their role in the transmission of different pathogens make mosquitoes (Diptera: Culicidae) the most important arthropods in public health entomology (Wilkerson et al., 2021). The family includes two subfamilies, Anophelinae and Culicinae, 41 genera and a total of 3,726 species (Harbach, 2024). Six known invasive Aedes species including A. aegypti (Linnaeus, 1762), A. albopictus (Skuse, 1895), A. atropalpus (Coquillett, 1902), A. japonicus (Theobald, 1901), A. koreicus (Edwards, 1917) and A. notoscriptus (Skuse, 1889) (Azari-Hamidian, 2023). To date, Iranian mosquitoes consist of 73 species representing eight genera (Azari-Hamidian et al., 2019, 2024). The fauna of mosquitoes in Guilan Province, northern Iran, includes 34 species representing seven genera (Azari-Hamidian & Norouzi, 2018; Azari-Hamidian et al., 2024).

Aedes albopictus is cosmopolitan, although originally confined to the Oriental Region, and occurs in Armenia, Oman, Pakistan and Türkiye neighbouring Iran (Azari-Hamidian et al., 2019; Wilkerson et al., 2021). This species is a known aggressive biter and invasive mosquito and is of great medical and veterinary importance as a competent vector of at least 22 arboviruses including chikungunya, dengue, West Nile and Zika viruses, all of which, except for Zika virus, have been recorded in Iran (Gratz, 2004; Azari-Hamidian & Harbach, 2023). Also, it is a known vector of some avian plasmodia such as Plasmodium fallax, P. galliceum and P. lophurae and filarial nematodes such as the dog heart worm Dirofilaria immitis and D. repens (Gratz, 2004; Wilkerson et al., 2021). There is one record of A. albopictus in Sistan and Baluchistan Province of southeastern Iran (Doosti et al., 2016). However, after that, this species has not been collected in the province and other areas of southern Iran in other surveys (Keshavarzi et al., 2017; Soltani et al., 2017; Nejati et al., 2020, 2024; Jaberhashemi et al., 2022). Thus, it seems that the species has not been established in the region.

Consistency between morphological identification, as the gold standard, and identification using the barcode region of the cytochrome c oxidase subunit I (COI) gene were reported from 69% to 98% and the COI barcode region can be successfully used to identify mosquito species (Ruiz-Lopez et al., 2012; Talaga et al., 2017; Laurito et al., 2022; Wu et al., 2022). Inconsistency between morphological identification and barcoding might be due to misidentification, synonymy, evidence of species complex, some degree of hybridization, incomplete lineage sorting between species and evidence of the effect of the geographical scale of sampling (Laurito et al., 2022). Although there are a few reports on the COI barcode region for some species of the genera Aedes, Anopheles and Culex in Iran (Azari-Hamidian et al., 2009, 2010; Karimian et al., 2014; Koosha et al., 2017; Nejati et al., 2024); however, there is no information about the COI barcode region for many other mosquito species of the country.

The aim of this study was a faunistic investigation of mosquitoes using morphological and molecular data with emphasis on an aggressive biter mosquito reported by local people in Anzali and Rasht Cities of Guilan Province.

MATERIAL AND METHODS

Sampling and morphological identification. Initial hand catch collections were made by manual aspirators in the reported localities, Anzali and Rasht Cities, during August 2023, followed by extensive collections using manual aspirators, light traps, BG lure traps and ovitraps up to November 2023 in all 17 counties of Guilan Province (Fig. 1). The specimens were transported to the Laboratory of Medical and Veterinary Entomology of the School of Health, Guilan University of Medical Sciences. Species identification was carried out utilizing morphological identification keys (Huang, 2004; Azari-Hamidian & Harbach, 2009; Becker et al., 2020). Adult voucher specimens are deposited in the Museum of Medical and Veterinary Entomology, the School of Health, Guilan University of Medical Sciences.

DNA barcoding. Molecular analysis was carried out using the sequences of the barcode region of the COI gene of mitochondrial DNA (mtDNA). The polymerase chain reaction (PCR) mixes and thermocycler parameters were those described by Folmer et al. (1994). The barcode region of the COI gene was amplified using the LCO (5′-GGT CAA CAA ATC ATA AAG ATA TTGG-3′) and HCO (5′ -TTA AAC TTC AGG GTG ACC AAA AAA TCA-3′) primers (Folmer et al. 1994). The product was 709 bp (658 bp without primers) for all mosquitoes. The sequences were generated in both directions and edited and aligned using MEGA v. 7 (Kumar et al., 2016).

Figure 1. Map of Iran highlighting the location of Guilan Province in northern Iran where the specimens of Aedes albopictus (Skuse, 1895) were collected in 2023.

Pairwise sequence divergence and the maximum likelihood tree, using 1,000 replicates, based on the Kimura’s two-parameter (K2P) model were obtained using MEGA v. 7 (Kimura, 1980; Kumar et al., 2016). Similarity with sequences in GenBank was assessed using BLAST (http://blast.ncbi.nlm.nih.gov). The sequences which were generated with the aforementioned primers and 100% coverage were chosen from GenBank for comparison. Template DNA from this study is retained in the Molecular Laboratory of the School of Health, Guilan University of Medical Sciences.

RESULTS

Taxonomic hierarchy

Class Insecta Linnaeus, 1785

Order Diptera Linnaeus, 1758

Suborder Nematocera Duméril, 1805

Infraorder Culicomorpha Hennig, 1948

Family Culicidae Meigen, 1818

Genus Aedes Meigen, 1818

Subgenus Stegomyia Theobald, 1901

Aedes albopictus (Skuse, 1895)

Material examined. In total, 29 larvae, were reared from the eggs, and 1529 adult specimens were collected. Among them, 29 larvae and 896 adult specimens were caught which were not recognized as a species previously known to occur in the province. They were captured using manual aspirators (886 specimens), BG lure traps (10 specimens) and ovitraps during August–November 2023. The aggressive biter mosquito species was morphologically identified as A. albopictus (Figs 2 and 3). This species was collected in 14 counties of the province, out of 17 (Fig. 1).

Figure 2. Female of Aedes albopictus (Skuse, 1895), captured in Guilan Province, northern Iran in 2023.
A. General habitus; B. Proboscis; C. Thorax, dorsal view; D. Abdomen, dorsal view; E. Head; F. Thorax, lateral view.

Figure 3. Larva and male genitalia of Aedes albopictus (Skuse, 1895), captured in Guilan Province, northern Iran in 2023. A. Head and antenna; B. Seta 1-A; C. Pecten; D. basal tubercules of 9–12-M; E. Annal papillae and auricle (absent); F. Comb; G. Spine of the gonostylus; H. Gonostylus; I. Mesal surface of the gonocoxite.

Other species, which were collected with A. albopictus by hand catches, were: A. caspius (Pallas, 1771) s.l. (4 females), A. cinereus Meigen, 1818 (94 females), A. vexans (Meigen, 1830) (312 females), Coquillettidia richiardii (Ficalbi, 1889) (7 females), Culex pipiens Linnaeus, 1758 (116 females), C. tritaeniorhynchus Giles, 1901 (82 females) and Uranotaenia unguiculata Edwards, 1913 (2 females). Also, C. pipiens (12 females) and C. tritaeniorhynchus (4 females) were collected with A. albopictus using BG lure traps. Also, 29 larvae were reared from the eggs, collected by egg traps, and were morphologically identified as A. albopictus (Fig. 3). Moreover, four microscope slides of the male genitalia verified the morphological identification of females and larvae (Fig. 3). No specimen of the species was captured by means of light traps.

DNA barcoding. The sequences of the COI barcode region were obtained from two specimens of
A. albopictus collected from Ghalamgoudeh, Anzali, 37°27'39.6"N, 49°27'43.2"E, 6.VIII.2023 (GenBank accession number: PP564390) and Astara City, 38°25'26.4"N, 48°52'12"E, 19.X.2023 (GenBank accession number: PP564391). The nucleotide sequences were 709 bp (658 bp without primers) as expected. The COI sequences of specimens from Anzali and Astara shared 100% coverage and identity with each other, one haplotype, and identical with the sequences in GenBank of specimens of A. albopictus from China (Shanghai, KX266726.1), Italy (JX679374.1), South Korea (MW829500) and Türkiye (MK714006.1). Eight selected sequences with 100% coverage were compared from China (MT890465.1, MZ007511.1, OR237214.1), Congo (MN299017.1), Germany (JQ388786.1), India (KJ410335.1), Malaysia (KY817524.1) and Thailand (OK413073.1) (similarity range 99.54–99.85%) (Fig. 4, Table 1). Variable bases of the COI barcode sequences obtained from the Iranian specimens and overlapping sequences from GenBank were presented in Table 1. Translation of nucleotide sequence to amino acids resulted in an alignment of 219 amino acids, eight (3.6%) of which were variable (Table 1).

Figure 4. Consensus tree of cytochrome c oxidase I (COI) barcode sequences of Aedes albopictus (Skuse, 1895) collected in Anzali and Astara Cities, Guilan Province of northern Iran, 2023, using maximum likelihood method based on the Kimura’s two-parameter (K2P) model.

Table 1. Variable bases of the cytochrome c oxidase I (COI) barcode sequences generated from the specimens of Aedes albopictus (Skuse, 1895) found in Anzali and Astara Cities, Guilan Province of northern Iran, 2023, with overlapping sequences from GenBank. Dots stand for identical bases as the Iranian specimens.

DISCUSSION

Based on the present record, the occurrence of A. albopictus in Iran is verified and it is found for the first time in Guilan Province, adding this invasive species to the mosquito fauna of the province. Henceforth, the current mosquito fauna of Guilan Province includes 35 species. Also, the sequences of the COI barcode region were obtained from the specimens of this species for the first time in the country. In the previous report of this species in southeastern Iran, different parts of the COI region (including 460 base pairs) were amplified using different primers (Doosti et al., 2016; GenBank accession number: KU351083). Thus, it was not possible to compare the sequences generated in the present work with the previous sequence from Iran. In addition to the nucleotide sequences from Asia and Europe which displayed 100% coverage and similarity to the present investigation sequences (Fig. 4, Table 1), there are numerous sequences from Africa, Asia and Europe with 100% coverage and more than 99% similarity in GenBank. Eight selected sequences were used to compare (Fig. 4, Table 1). All those selected sequences displayed less than one percent difference in nucleotides (range 0.15–0.46%). Thus, the variation of sequences generated in the present investigation surely is within intra-specific variation (Ruiz-Lopez et al., 2012; Hernández-Triana et al., 2019; Madeira et al., 2021; Chaiphongpachara et al., 2022; Wu et al., 2022).

In view of the recent finding of the Asian tiger mosquito in northern Iran, it is necessary to engage the national surveillance program for monitoring its population and interventions for its control. Studies of the bionomics, host preferences and resistance situation of the species to different insecticides in the region are recommended.

AUTHOR′S CONTRIBUTION

The authors confirm their contribution to the paper as follows: S. Azari-Hamidian: conceptualization, methodology, investigation, data curation, writing original draft, resources, supervision; B. Norouzi: investigation, data curation, writing original draft; H. Maleki: investigation, data curation, writing original draft; S.M. Rezvani: investigation, data curation, writing original draft, resources; M. Pourgholami: investigation, data curation, writing original draft; M.A. Oshaghi: methodology, investigation, data curation, writing original draft. All authors have read and agreed to publish this manuscript.

FUNDING

This research was partially supported by the Research Vice-Chancellorship of Guilan University of Medical Sciences (Approval ID: IR.GUMS.REC.1400.537).

AVAILABILITY OF DATA AND MATERIAL

Sequence data that support the findings of this study have been deposited in the GenBank (GenBank accession numbers: PP564390, PP564391). The specimens mentioned in this article are deposited in the Museum of Medical and Veterinary Entomology, the School of Health, Guilan University of Medical Sciences. Template DNA from this study is retained in the Molecular Laboratory of the School of Health, Guilan University of Medical Sciences. All other data are provided within the manuscript.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

This study only included arthropod material, and all required ethical guidelines for the treatment and use of animals were strictly adhered to in accordance with international, national, and institutional regulations. No human participants were involved in any studies conducted by the authors for this article.

CONSENT FOR PUBLICATION

Not applicable.

CONFLICT OF INTERESTS

The authors declare that there is no conflict of interest regarding the publication of this paper.

ACKNOWLEDGMENTS

The specimens were collected during the National Aedes Surveillance Program. The staff of the Health Vice-Chancellorship of Guilan University of Medical Sciences are acknowledged for their cooperation in the field. The knowledge and fast reports of local people in Anzali and Rasht Cities of Guilan Province, which caused fast reactions for collecting and reporting the species, are very appreciated.

REFERENCES

Azari-Hamidian, S. (2023) The invasive Aedes mosquitoes (Diptera: Culicidae) and their medical and veterinary importance: a mini review. Caspian Journal of Health Research, 8 (4), 241–246.

https://doi.org/10.32598/CJHR.8.4.473.2

Azari-Hamidian, S. & Norouzi, B. (2018) A checklist of mosquitoes (Diptera: Culicidae) of Guilan Province and their medical and veterinary importance. Caspian Journal of Health Research, 3 (3), 91–96.

https://doi.org/10.29252/cjhr.3.3.91

Azari-Hamidian, S. & Harbach R.E. (2009) Keys to the adult females and fourth-instar larvae of the mosquitoes of Iran (Diptera: Culicidae). Zootaxa, 2078, 1–33. https://doi.org/10.11646/zootaxa.2078.1.1

Azari-Hamidian, S. & Harbach, R.E. (2023) Arthropod-borne and arthropod-related viruses in Iran and neighboring countries. Parazitologiya, 57 (5), 356–440. https://doi.org/10.31857/S0031184723050010

Azari-Hamidian, S., Yaghoobi-Ershadi, M.R., Javadian, E., Abai, M.R., Mobedi, I., Linton, Y.M. & Harbach R.E. (2009) Distribution and ecology of mosquitoes in a focus of dirofilariasis in northwestern Iran, with the first finding of filarial larvae in naturally infected local mosquitoes. Medical and Veterinary Entomology, 23 (2), 111–121. https://doi.org/10.1111/j.1365-2915.2009.00802.x

Azari-Hamidian, S., Linton, Y.-M., Abai, M.R., Ladonni, H., Oshaghi, M.A., Hanafi-Bojd, A.A., Moosa-Kazemi, S.H., Shabkhiz, H., Pakari, A. & Harbach, R.E. (2010) Mosquito (Diptera: Culicidae) fauna of the Iranian islands in the Persian Gulf. Journal of Natural History, 44, 913–925.

https://doi.org/10.1080/00222930903437358

Azari-Hamidian, S., Norouzi, B. & Harbach, R.E. (2019) A detailed review of the mosquitoes (Diptera: Culicidae) of Iran and their medical and veterinary importance. Acta Tropica, 194, 106–122.

https://doi.org/10.1016/j.actatropica.2019.03.019

Azari-Hamidian, S., Norouzi, B., Maleki, H., Rezvani, S.M., Pourgholami, M. & Oshaghi, M.A. (2024) First record of Aedes (Aedes) cinereus (Diptera: Culicidae) in Iran. Zoology in the Middle East, 70 (2), 136–142.

https://doi.org/10.1080/09397140.2024.2359168

Becker, N., Petrić, D., Zgomba, M., Boase, C., Madon, M., Dahl, C. & Kaiser, A. (2020) Mosquitoes: Identification, Ecology and Control. 3rd Edition. Springer Verlag, Nature Switzerland AG, Cham, Switzerland. 570 p.

https://doi.org/10.1007/978-3-030-11623-1

Chaiphongpachara, T., Changbunjong, T., Laojun, S., Nutepsu, T., Suwandittakul, N., Kuntawong, K., Sumruayphol, S. & Ruangsittichai, J. (2022) Mitochondrial DNA barcoding of mosquito species (Diptera: Culicidae) in Thailand. PLoS ONE, 17 (9), e0275090. https://doi.org/10.1371/journal.pone.0275090

Doosti, S., Yaghoobi-Ershadi, M.R., Schaffner, F., Moosa-Kazemi, S.H., Akbarzadeh, K., Gooya, M.M., Vatandoost, H., Shirzadi, M.R. & Mosta-Favi, E. (2016) Mosquito surveillance and the first record of the invasive mosquito species Aedes (Stegomyia) albopictus (Skuse) (Diptera: Culicidae) in southern Iran. Iranian Journal of Public Health, 45, 1064–1073.

Folmer, O., Black, M., Hoeh, W., Lutz, R. & Vrijenhoek, R. (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology, 3, 294–299.

Gratz, N.G. (2004) Critical review of the vector status of Aedes albopictus. Medical and Veterinary Entomology, 18 (3), 215–227. https://doi.org/10.1111/j.0269-283X.2004.00513.x

Harbach, R.E. (2024) Mosquito Taxonomic Inventory. Available from: www.mosquito-taxonomic-inventory.info [Accessed 15 March 2024]

Hernández-Triana, L.M., Brugman, V.A., Nikolova, N.I., Ruiz-Arrondo, I., Barrero, E., Thorne, T., de Marco, M.F., Krüger, A., Lumley, S., Johnson, N. & Fooks, A.R. (2019) DNA barcoding of British mosquitoes (Diptera, Culicidae) to support species identification, discovery of cryptic genetic diversity and monitoring invasive species. ZooKeys, 832, 57–76. https://doi.org/10.3897/zookeys.832.32257

Huang, Y.-M. (2004) The subgenus Stegomyia of Aedes in the Afrotropical Region with keys to the species (Diptera; Culicidae). Zootaxa, 700, 1–120. https://doi.org/10.11646/zootaxa.700.1.1

Jaberhashemi, S.-A., Azari-Hamidian, S., Soltani, A., Azizi, K., Dorzaban, H., Norouzi, M. & Daghighi, E. (2022) The fauna, diversity, and bionomics of Culicinae (Diptera: Culicidae) in Hormozgan Province, southern Iran. Journal of Medical Entomology, 59 (3), 987–996. https://doi.org/10.1093/jme/tjac003

Karimian, F., Oshaghi, M.A., Sedaghat, M.M., Waterhouse, R.M., Vatandoost, H., Hanafi-Bojd, A.A., Ravasan, N.M. & Chavshin, A.R. (2014) Phylogenetic analysis of the Oriental-Palearctic-Afrotropical members of Anopheles (Culicidae: Diptera) based on nuclear rDNA and mitochondrial DNA characteristics. Japanese Journal of Infectious Diseases, 67 (5), 361–367. https://doi.org/10.7883/yoken.67.361

Keshavarzi, D., Soltani, Z., Ebrahimi, M., Soltani, A., Nutifafa, G.G., Soltani, F., Faramarzi, H., Amraee, K. & Hassanzadeh, A. (2017) Monthly prevalence and diversity of mosquitoes (Diptera: Culicidae) in Fars Province, southern Iran. Asian Pacific Journal of Tropical Disease, 7, 112–120.

https://doi.org/10.12980/apjtd.7.2017D6-369

Kimura, M. (1980) A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution, 16, 111–120.

https://doi.org/10.1007/BF01731581

Koosha, M., Oshaghi, M.A., Sedaghat, M.M., Vatandoost, H., Azari-Hamidian, S., Abai, M.R., Hanafi-Bojd, A.A. & Mohtarami, F. (2017) Sequence analysis of mtDNA COI barcode region revealed three haplotypes within Culex pipiens assemblage. Experimental Parasitology, 181, 102–110.

https://doi.org/10.1016/j.exppara.2017.08.003

Kumar, S., Stecher, G. & Tamura, K. (2016) MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Molecular Biol and Evolution, 33, 1870–1874. https://doi.org/10.1093/molbev/msw054

Laurito, M., Ayala, A.M., Arias-Builes, D.L. & Almirón, W.R. (2022) Improving the DNA barcode library of mosquito species with new identifications and discoveries in north-central Argentina. Journal of Medical Entomology, 59 (1), 173–183. https://doi.org/10.1093/jme/tjab160

Madeira, S., Duarte, A., Boinas, F. & Osório, H.C. (2021) A DNA barcode reference library of Portuguese mosquitoes. Zoonoses and Public Health, 68, 926–936. https://doi.org/10.1111/zph.12885

Nejati, J., Zaim, M., Vatandoost, H., Moosa-Kazemi, S.H., Bueno-Marí, R., Azari-Hamidian, S., Sedaghat, M.M., Hanafi-Bojd, A.A., Yaghoobi-Ershadi, M.R., Okati-Aliabad, H., Collantes, F. & Hoffmann, A.A. (2020) Employing different traps for collection of mosquitoes and detection of dengue, Chikungunya and Zika vector, Aedes albopictus, in borderline of Iran and Pakistan. Journal of Arthropod-Borne Diseases, 14, 376–390. https://doi.org/10.18502/jad.v14i4.5275

Nejati, J., Azari-Hamidian, S., Oshaghi, M.A., Vatandoost, H., White, V.L., Moosa-Kazemi, S.H., Bueno-Marí, R., Hanafi-Bojd, A.A., Endersby-Harshman, N.M., Axford, J.K., Karimian, F., Koosha, M., Choubdar, N. & Hoffmann, A.A. (2024) The monsoon-associated equine South African pointy mosquito ‘Aedes caballus’; the first comprehensive record from southeastern Iran with a description of ecological, morphological, and molecular aspects. PLoS ONE, 19 (5), e0298412. https://doi.org/10.1371/journal.pone.0298412

Ruiz-Lopez, F., Wilkerson, R.C., Conn, J.E., McKeon, S.N., Levin, D.M., Qui­ñones, M.L., Póvoa, M.M. & Linton, Y.-M. (2012) DNA barcoding reveals both known and novel taxa in the Albitarsis group (Anopheles: Nys­sorhynchus) of Neotropical malaria vectors. Parasites and Vectors, 5, 44. https://doi.org/10.1186/1756-3305-5-44

Soltani, Z., Keshavarzi, D., Ebrahimi, M., Soltani, A., Moemenbellah-Fard, M., Soltani, F., Faramarzi, H., Amraee, K. & Elyasigomari, A. (2017) The fauna and active season of mosquitoes in west of Fars Province, southwest of Iran. Archives of Razi Institute, 72, 203–208.

Talaga, S., Leroy, C., Guidez, A., Dusfour, I., Girod, R., Dejean, A. & Murienne, J. (2017) DNA reference libraries of French Guianese mosquitoes for barcoding and metabarcoding. PLoS ONE, 12 (6), e0176993.

https://doi.org/10.1371/journal.pone.0176993

Wilkerson, R.C., Linton, Y.-M. & Strickman, D.A, (2021) Mosquitoes of the world. Volumes 1 and 2. Johns Hopkins University Press, Baltomore, 1308 p. https://doi.org/10.1186/s13071-021-04848-6

Wu, J., Li, D., Boyd, B., Balan, R.K., George, S., Peacock, L. & Pal, C. (2022) Comparative performance of a multi-locus barcoding approach to enhance taxonomic resolution of New Zealand mosquitoes (Diptera: Culicidae). Austral Entomology, 62 (1), 77–95. https://doi.org/10.1111/aen.12630