Transmission of malaria, filariasis, Japanese encephalitis, dengue fever, and other arbovirus diseases by mosquitoes has turned mosquitoes into the most important group of arthropods in medicine and health . In Iran, mosquitoes are vectors of two protozoal, two bacterial, four filarial, and seven arboviral diseases [2, 3]. There are 70 species and 8 (or 12) genera of Iranian mosquitoes depending on the classification of the tribe Aedini . Anopheles mosquito species are responsible for the transmission of malaria, but the majority of mosquito species from the genera of Culex and Aedes are responsible for the transmission of arboviruses to humans . Aedes aegypti, a highly anthropophilic species, Ae. albopictus the highly invasive mosquito and Culex species are important targets for the prevention of arboviruses including dengue virus, chikungunya virus, yellow fever virus, Zika virus, Japanese encephalitis virus and West Nile virus [5, 6].
Globally, there were an estimated 229 million malaria cases in 2019 in 87 malaria endemic countries. The disease is a major endemic infectious disease in Iran, especially in the south and southeastern provinces including; the Sistan-Baluchestan, Hormozgan, and Kerman [7–12]. Most of the cases in the central counties of Iran were imported from other countries (90.4%), mainly from Afghanistan (56.5%) and Pakistan (16.3%). Plasmodium vivax was the causative agent of 93.75% of cases, followed by P. falciparum (6.25%). Above-15-year-old group contained the most malaria reported cases (66.7%) .
Anopheles species is responsible for the transmission of malaria. So far, seven malaria vectors have been recognized and reported in Iran, including: Anopheles stephensi, An. culicifacies, An. dthali, An. fluviatilis, An. superpictus, An. maculipennis and An. sacharovi . The first five of these vectors can be found in the southeast of the country, together with the majority of malaria cases. Also, An. pulcherrimus has been considered as a potential malaria vector in this area based on immunological parasite detection (two-site immunoradiometric assay (IRMA)) .
Anopheles maculipennis s.l. distributed in Eurasia and North America and comprises nine Palearctic members [16, 17]. Some research indicated the occurrence of this malaria vector in central, the Caspian coast in the north, and North West of Iran [18–20].
Anopheles superpictus s.l. is distributed in Europe, Asia, and North Africa [21–25]. This species is one of the seven species of malaria vectors and reported in the Iranian Plateau, the slopes of the Alborz Mountains and southern Zagros, as well as the coastal plains of the Caspian Sea and the Persian Gulf in both malaria- endemic and non-endemic areas [8, 22]. Oshaghi et al in 2008 reported three genotypes X, Y, and Z in Iran. Interestingly, while the sympatric Y and Z genotypes appear to be exclusive to the populations from the southeastern part of the country, genotype X is geographically separated, and present in the North, the West, the South and the Central territories .
A malaria eradication programme was initiated in Iran in 1951 and changed to malaria control in 1985 due to the challenges and restrictions . Iran has been in an elimination stage since 2010. In 2017, the total number of recorded cases was 89, and incidence cases decreased from 0.01/1000 cases in 2017 to zero in 2019 [14, 26].
The goal of all control methods is to reduce the size of vector populations. There is a risk of insecticide resistance and off-target effects on other arthropod species in chemical control . Biological control is biodegradable and ecologically friendly . Entomopathogenic fungi were first used on An. gambiae with a fungus from the genus of Coelomomyces . Azari- Hamidian and Abaei reported Coelomomyces sp. from the larvae of An. culicifacies s.l. in Sistan and Baluchestan Province, Southeast Iran, where 5.8% of larvae were infected with the fungus . The use of pathogenic insect fungi against mosquito larvae has been reported in many studies and fungi is proven to be an effective way of killing mosquito larvae. Unlike other biological control agents, pathogenic insect fungi can infect mosquitoes directly by penetrating the cuticle . Some insect pathogenic fungi have been used effectively in recent years to control vector mosquitoes and have a wide range of species diversity. This group of pathogens is found among all phyla of fungi. The Ascomycota is the largest group of fungi. This group is extremely ecologically diverse, just like the pathogenesis pathogen of plants, animals, and humans. Pathogenic insect ascomycetes include a large group of fungi that attack a wide range of insects and are the most common insect pathogens . The entomopathogenic ascomycete fungi including Metarhizium anisopliae, and Beauveria bassiana have been reported as insecticides . The use of B. bassiana for control of Ae. aegypti  and Lagenidium giganteum in California targeted to control Cx. tarsalis  reduced the survival rate, blood-feeding, fecundity, and disease transmission power of targeted mosquitoes. Two species, Metarhizium anisopliae, and M. brunneum, are pathogenic to a wide range of mosquitoes in the genera of Aedes, and Culex [36, 37]. In many studies, spores and secondary metabolites of insect pathogenic fungi have been reported as biocontrol agents against mosquitoes [38–41]. The fungal hyphae produces endotoxins and penetrates through the larval. These toxins cause larval damage and toxicity in the hemocoel and larval mosquito guts [42, 43]. Metabolites of B. bassiana caused changes in the body and tissues of treated Cx. pipiens larvae, especially in the cuticle and midgut .
This study aimed to isolate and identify entomopathogenic fungi associated with mosquito larvae in Kashan County, Central Iran, and their infection, effects on mosquito larvae, and the investigation of new ways of biological control for disease vector mosquitoes.