Aedes aegypti mosquitoes are the main vectors of arboviruses such as those that cause dengue, chikungunya, and Zika illnesses (Consoli and Oliveira, 1994; de Oliveira Barbosa Bitencourt et al., 2021). These diseases have occupied a prominent position in public health in several countries of the Americas, including Brazil where the occurrence of all these arboviruses has been recorded simultaneously since 2015 (Saúde and Saúde, 2016).

In 2014, chikungunya fever was first recorded in Brazil and spread rapidly throughout the country (Araújo et al., 2020). A short time later, Zika virus was detected in northeastern Brazil in 2015 (Possas et al., 2017). In 2016, the country reached a peak of cases, with more than 215 thousand estimated cases of Zika (Saúde and Saúde, 2018), which resulted in thousands of cases of neonatal microcephaly (Zanotto and Leite, 2018). Dengue is characterized as one of the main arboviruses with worldwide outbreaks occurring in the Americas, Africa, the Middle East, Asia, and the Pacific Islands. About 3.9 billion people in 129 countries are at risk of infection by the dengue virus, a notable increase from previous decades, partially explained by the improvement of records and recognition of the disease burden by governments (WHO, 2020). According to the Pan American Health Organization, the highest number of dengue cases ever reported globally was in 2019. Brazil alone reported about 2.2 million cases in 2019, representing 70% of the total recorded in the Americas (PAHO, 2020).

Since specific antiviral drugs and effective vaccines against these arboviruses are not available, measures to curb the transmission of these diseases remain focused on vector control, mostly through the elimination of breeding sites and the use of chemical insecticides (Zara et al., 2016). However, the frequent use of chemical insecticides is toxic to the environment and has resulted in the selection of insecticide-resistant mosquito populations (Seetharaman et al., 2018; Araújo et al., 2020). It is therefore urgently necessary to explore new approaches to control these vectors.

Fungal secondary metabolites constitute a rich source of bioactive molecules (Daniel et al., 2017), potentially useful for mosquito control. More specifically, fungi isolated from aquatic habitats are a rich and unexplored source of new natural products. In order to adapt and survive in the aquatic environment, fungi accumulate unique bioactive secondary metabolites, not found in terrestrial environments (Bhakuni and Rawat, 2006; Imhoff, 2016).

The Amazon rainforest contains ∼ 25% of the world’s terrestrial biodiversity (Malhi et al., 2008), including microorganisms potentially useful for A. aegypti control programs. In this work, we explored the larvicidal potential of the fungi isolated from the aquatic environments of the Amazon region. Our results suggest the possibility of utilizing fungi-derived extracts and/or their metabolites as part of integrated vector management programs.

In this study, 36 isolates belonging to 23 genera of fungi were analyzed regarding their ability to produce mosquito larvicidal compounds. Extracts from isolated strain were obtained from both mycelium and culture liquid medium fractions of the cultures. The growth time of the fungi until no glucose was detected in the medium ranged from 17 to 85 days. The pH of the cultures ranged from 6 to 7.5 in comparison to the pH of 5.5 in the control (non-inoculated medium). The extracts obtained from the liquid medium presented yielded between 82 and 724 mg, after extraction and drying procedures. Mycelium extracts from fungi strains 1132 and 1126 yielded 256 and 5872 mg, respectively, being the lowest and the highest obtained values (Table 1).

Seven mycelium extracts originating from fungi belonging to six genera (Aspergillus, Cladosporium, Fusarium, Diaporthe, Talaromyces, and Trichoderma) caused larval mortality from 3.3 to 43.3%, and none presented mortality equal to or greater than 50% up to 72 h of exposure (Supplementary Table 1). Larvicidal activity equal to or greater than 50% was observed in 15 of the 36 extracts of liquid medium; six liquid medium extracts belonging to five genera (Albifimbria, Aspergillus, Diaporthe, Emmia, and Sorocladium) killed 100% of the larvae within 72 h.

Eight extracts (from strains 1126, 1132, 1133, 1160, 1203, 1232, 1242, and 1266) showed larvicidal activity equal to or greater than 50% at 24 h, four (1244, 1246, 1248, and 1280) resulted in 50% mortality only at 48 h and three extracts (1184, 1240, and 1283) caused 50% mortality only at 72 h of exposure. Six extracts caused 100% larval mortality, three (1160, 1203, and 1242) in less than 24 h, two (1126 and 1266) at 48 h and one (1232) at 72 h (Figure 1).

Extracts with larvicidal activity equal to or greater than 90% (Albifimbria lateralis 1160, Aspergillus sp. 1126, Cladosporium sp. 1132, D. ueckerae 1203 and 1242, Emmia sp. 1232, Sorocladium sp. 1266, and Trichoderma atroviride 1133) were further studied and LC₅₀ and LC₉₀ values determined (Figure 1 and Supplementary Table 1).

Overall, the liquid medium extracts from Al. lateralis 1160 and D. ueckerae 1203 showed the best results, with highest mortality rates and lowest LC values. The extract of the strain 1160 (Al. lateralis) presented the lowest LC₅₀ (0.268 μg/ml) at 24 h. The extract of the 1203 strain (D. ueckerae) had the lowest LC₉₀ (2.928 μg/ml) at 24 h. Furthermore, D. ueckerae 1203 extracts had the lowest LC₅₀ (0.108 μg/ml) and LC₉₀ (0.894 μg/ml) at 48 h, and at 72 h with an LC₅₀ of 0.062 μg/ml and an LC₉₀ of 0.476 μg/ml (Table 2).

The public health importance of Ae. aegypti in tropical regions has attracted the attention of local authorities and the World Health Organization due to the wide geographical distribution and severity of diseases transmitted by these mosquitoes in the last decades, especially dengue, chikungunya, and Zika (Rodrigues-Alves et al., 2020). As such, there is a growing interest in new insecticides and larvicides capable of controlling this vector. Naturally derived insecticides have been pursued as potentially less toxic alternatives, aiming at reducing environmental pollution and preventing the selection of mosquitoes resistant to chemical insecticides (Al-Mekhlafi, 2017; Araújo et al., 2020).

For the control of Ae. aegypti, insecticides are frequently applied directly in natural bodies of water and/or artificial containers, usually located closely or kept inside human households. Therefore, the use of natural, potentially less-toxic, insecticides is desirable from both environmental and social perspectives. This work investigated for the first time extracts of fungi isolated from aquatic habitats of the Amazon region in order to identify fungal lineages that can produce larvicidal bioactive metabolites against Ae. aegypti.

Following the protocols described here, none of the mycelium extracts showed larvicidal activity resulting in mortality rates above 50% within 72 h. However, 15 extracts from the liquid culture medium resulted in more than 50% mortality. Six killed 100% of the larvae within 72 h and three of these were lethal in less than 24 h, resembling the positive control Temephos. These results demonstrate that some of the isolated fungal strains secrete metabolites with larvicidal activity against Ae. aegypti.

The fungi tested in this study were subjected to the similar cultivation conditions. However, cultivation times varied for each fungal strain. To avoid differences in nutrient availability, we used total glucose consumption in the culture medium as determinant of the cultivation time length. Fungi growth styles and physical, chemical and biological factors, among others, influence development time and the production of bioactive metabolites (Kavanagh, 2011; Costa and Nahas, 2012). Species-specific traits explain the difference in cultivation time and biological activities among the isolated fungi studied in our work.

Mosquito larvicidal activities of the mycelial extracts and the liquid culture medium extracts, have been described for other fungi such as Stereum sp. (JO5289) (Chirchir et al., 2013), Beauveria bassiana (UNI 40) (Daniel et al., 2017), Trametes sp. (Waweru et al., 2017), Pestalotiopsis virgulata and Pycnoporus sanguineus (Bücker et al., 2013). However, the LC₅₀ values revealed in our work are lower than those previously published, such as Metarhizium anisopliae (LC₅₀ = 59.83 μg/ml, Vivekanandhan et al., 2020), B. bassiana (LC₅₀ = 1.230 μg/ml, Daniel et al., 2017), and Aspergillus terreus (LC₅₀ = 80.407 μg/ml, Ragavendran and Natarajan, 2015), indicating the potency of the metabolites obtained from the strains tested in our study.

It is worth noting that to date there have been no reports of biological activities against insect species of metabolites produced by fungal strains of the species Al. lateralis, D. ueckerae, and Emmia sp. For the first time, lineages of these species of fungi with larvicidal activity against Ae. aegypti have been identified.

The genus Albifimbria consists of four species, i.e., Al. lateralis, Albifimbria terrestris, Albifimbria verrucaria, and Albifimbria viridis, which are usually found in soil, leaves, fruits, and in the air (Lombard et al., 2016). The species Albifimbria lateralis (Lombard et al., 2016) has been recently described and needs better investigation regarding the production of secondary metabolites, though our investigation indicates promising applications of this fungus species in vector control. Metabolites produced by Al. verrucaria exhibit antimicrobial activities (Zou et al., 2011) and bioherbicidal activities (Walker and Tilley, 1997) and are considered to be a potential biocontrol agent against the fungus Botrytis cinerea in grapes (Li et al., 2019).

The species D. ueckerae was described by Udayanga et al. (2015). Its occurrence in Brazil was identified by Soares et al. (2018) who isolated this species of fungus from Costus spiralis (Jacq.) Roscoe (Costaceae), a plant native to the Amazon region used in traditional medicine. Fungal species of the genus Diaporthe are known to be a rich source of secondary metabolites (Chepkirui and Stadler, 2017).

Currently, 106 compounds derived from Diaporthe exhibiting biological activities, such as cytotoxic, antifungal, antibacterial, antiviral, antioxidant, anti-inflammatory, phytotoxic, antiparasitic, and herbicidic activities, have been studied (Ash et al., 2010; Meepagala et al., 2018; Xu et al., 2021). Two cyclohexeneoxidediones, phyllostine acetate (1) and phyllostine (2), from the fungus Diaporthe miriciae, showed insecticidal activity against Plutella xylostella larvae (Ratnaweera et al., 2020). Meepagala et al. (2018) isolated a compound from the liquid medium extract of Diaporthe eres, identified as 3,4-dihydro-8-hydroxy-3,5-dimethylisocoumarin (1), which has larvicidal activity against Ae. aegypti.

In addition, other fungi from different genera such as Beauveria, Fusarium, Metarhizium, Neosartorya, and Paecilomyces, also produce compounds with insecticidal activity such as beauvericin, gliotoxin, enniatin, oosporein, destruxins, cytochalasins, etc (Vyas et al., 2007; Masi et al., 2017; Berestetskiy and Hu, 2021).

This study is the first to evaluate aquatic fungi strains from the Amazon for their ability of producing mosquito larvicidal metabolites. Our findings open opportunities for the development of new larvicides that may be used as mosquito control agents. Crude fungal extracts, such as those studied here, are a complex mixture of different classes of molecules. The process of fractionation and purification of raw extracts guided by bioactivity (Chirchir et al., 2013) is necessary for the isolation and characterization of the chemical compounds responsible for the larvicidal activities observed in our work. Further studies are needed to characterize the active larvicidal metabolites produced by these fungi and define their mechanisms of action.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

MO, WT, ADS, and AQS designed the study. MO, ADS, and AQS performed the production of fungal extracts, analyzed the results, and wrote the manuscript. MO and RK reared mosquitoes and carried out bioassays. MO, GS, OM, OT, ADS, and AQS supervised and finalized the manuscript. All authors read and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.