Insecticidal Efficacy of Metarhizium anisopliae Derived Chemical Constituents against Disease-Vector Mosquitoes

Insecticides can cause significant harm to both terrestrial and aquatic environments. The new insecticides derived from microbial sources are a good option with no environmental consequences. Metarhizium anisopliae (mycelia) ethyl acetate extracts were tested on larvae, pupae, and adult of Anopheles stephensi (Liston, 1901), Aedes aegypti (Meigen, 1818), and Culex quinquefasciatus (Say, 1823), as well as non-target species Eudrilus eugeniae (Kinberg, 1867) and Artemia nauplii (Linnaeus, 1758) at 24 h post treatment under laboratory condition. In bioassays, Metarhizium anisopliae extracts had remarkable toxicity on all mosquito species with LC50 values, 29.631 in Ae. aegypti, 32.578 in An. stephensi and 48.003 in Cx. quinquefasciatus disease-causing mosquitoes, in A. nauplii shows (5.33–18.33 %) mortality were produced by the M. anisopliae derived crude extract. The LC50 and LC90 values were, 620.481; 6893.990 μg/mL. No behavioral changes were observed. A low lethal effect was observed in E. eugeniae treated with the fungi metabolites shows a 14.0 % mortality. The earthworm E. eugeniae mid-gut histology revealed that M. anisopliae extracts had no more harmful effects on the epidermis, circular muscle, setae, mitochondrion, and intestinal lumen tissues than chemical pesticides. By Liquid chromatography mass spectrometry (LC-MS) analysis, camphor (25.4 %), caprolactam (20.68 %), and monobutyl phthalate (19.0 %) were identified as significant components of M. anisopliae metabolites. Fourier transform infrared (FT-IR) spectral investigations revealed the presence of carboxylic acid, amides, and phenol groups, all of which could be involved in mosquito toxicity. The M. anisopliae derived chemical constituents are effective on targeted pests, pollution-free, target-specific, and are an alternative chemical insecticide.


Introduction
Mosquitoes are a major health problem because they transmit diseases such as malaria, dengue fever, yellow fever, and the Zika virus, which impact 700 million people each year and kill over one million people [1]. Adult mosquito control has been the primary approach for avoiding disease transmission, and it usually requires the use of synthetic pesticides and repellents, primarily organophosphates and pyrethroids [2]. Mosquitoes have evolved resistance to organophosphate and synthetic pyrethroids [3][4][5][6]. The chemical pesticides have been accumulated in our green ecosystem soil and waterbodies as well as food chains [5]. For insect pest management, entomopathogenic bacteria, fungi, and nematodes are considered effective microbial control methods for insect pests [7][8][9].
Entomopathogenic fungi produce secondary metabolites that could be used as a source for biopesticide development [10][11][12][13][14]. The entomopathogenic fungi Metarhizium anisopliae secondary metabolites, in particular, are known to be effective biopesticides for the control of Aedes aegypti (Meigen, 1818) mosquitoes [7,15], and other fungi such as Tolypocladium [13], Beauveria [9], Fusarium [10,11], and Lagenidium giganteum [14] have also been bioprospected as insect pests. Several biopesticides have been hampered by the fact that they are slow acting, taking anywhere from a few days to a week to exhibit action, which has hampered their commercialization [16]. Furthermore, their effects on non-target organisms are understudied [7].
The brine shrimp, Artemia sp. (Anostraca: Artemiidae), is a branchiopod crustacean that can tolerate salinities of up to 250 gL −1 /L [17]. Artemia are commonly employed for the evaluation of marine contamination by synthetic chemicals because of their high sensitivity to chemicals or other toxicants [17], and Artemia nauplii (Linnaeus, 1758), an important component of the aquatic ecosystem, are regarded as indicators for environmental toxicity [17,18]. Earthworms, therefore, are considered to be bio-indicators of terrestrial ecosystems and are frequently used as biomarkers for assessing the environmental toxicity of chemical contaminants [19,20]. In the present study, we investigated the toxicity of secondary metabolites extracted from Metarhizium anisopliae (Metschn, 1879) strains and their toxicity effect was evaluated against disease-vector mosquitoes Aedes Aegypti (Meigen, 1818), Anopheles Stephensi (Liston, 1901), and Culex quinquefasciatus (Say, 1823), as well as their toxicity against non-target organisms, such as earthworm Eudrilus eugeniae (Kinberg, 1867) and brine shrimp Artemia nauplii (Linnaeus, 1758).

Mass Culturing of M. anisopliae
Metarhizium anisopliae was cultured on Potato Dextrose Broth (PDB), as a medium for fungal growth. Sixteen 500 mL conical flasks, each containing 250 mL of PDB (dextrose 8 g, peptone 2 g and distilled water 250 mL), were autoclaved at 15 psi for 25 min. Chloramphenicol antibiotics (150 mg/mL) (Sigma-Aldrich Chemicals Private Limited, Bangalore) was added to the culture medium to prevent bacterial contamination. The cultures were allowed to grow for 20 days, and spore concentration was counted using a hemocytometer. A concentration of 1 × 10 7 spores/mL of M. anisopliae conidia were transferred to the culturing medium using an inoculation needle. The culture medium was maintained at the optimized culture conditions (pH 7.0, temperature 28 ± 5 • C) for 30 days.

Extraction of Secondary Metabolites
Fungus mycelial biomass was washed with distilled water after 20 days to eliminate culture medium components. Metarhizium anisopliae biomass was cold extracted with ethyl acetate to extract the biologically active chemical constituents under laboratory conditions. The ethyl acetate solvent was fully pooled with fungal biomass and left for 25 days. Then, the organic phase (light-yellow color) was separated after 25 days using a separating funnel, and the solvent was evaporated using a rotary evaporator at 45 • C.

Larval Collection and Maintenance
The Institute of Vector Control and Zoonoses at Hosur, Tamil Nadu, India. The mosquito egg masses per species were separately placed in a plastic tray (22 cm × 27 cm × 12 cm) (wonder, India) in dechlorinated tap water. The containers were transferred to room temperature with 28 ± 2 • C, 70-80% RH relative humidity and 12:12 (L:D) photoperiod and kept in it for 10-15 days. Each stage (larvae, pupae, and adult) of the mosquitos were taken for bioassay. During this process, mosquito larvae were fed with 0.5 g Tetra Bit (Pellet Fish Food) in each container, and adults were given a 10% sugar solution as a feeding source.

Non-Target Organisms
The Eudrilus eugeniae stock were maintained under laboratory condition at a room temperature of 27 ± 2 • C. Artemia nauplii larvae were kept in 1000 mL of saltwater with a salinity of 30 ppt in a culture medium with a pH range of (7)(8). An aspirator was used to provide oxygen.

Mosquitocidal Bioassays
The fungal metabolites larvicidal and pupicidal efficacy was assessed using the World Health Organization protocol [21]. Stock solutions of fungal extract were dissolved in Dimethylsulfoxide (DMSO) (Sigma-Aldrich, India) at a concentration of 10% w/v (10 µg of extracts in 100 mL of DMSO) and diluted to five different concentrations: 10, 15, 30, 50, and 75 µg/mL. Twenty-five 4th instar larvae and pupae were each transferred to 249 mL of tap water with 1 mL of different concentrations of fungal extract and replicated three times. Dead insects were counted 24 h. As a negative control, DMSO at a concentration of 10% w/v was used.
The adulticidal activity was evaluated following methods described by the Centers for Disease Control and Prevention [22]. Twenty-five newly emerged adults of A. aegypti, A. stephensi and C. quinquefasciatus were exposed to different concentrations (25, 50, 100, 150 and 200 µg/mL) of M. anisopliae secondary metabolites. Metabolites solutions were dispensed to the screw cap bottle of 80 mL, and for solvent evaporation, it was air dried over-night. In the control treatment, adult mosquitoes were exposed to DMSO (0.1%). Mortality was recorded post 24 h of treatment. A cotton ball soaked with a 10% glucose solution was used as a food source for mosquitoes. Three replicates for each concentration were performed (n = 450).

Non-Target Bioassays
The effects of fungal metabolites on earthworms E. eugeniae was tested in an artificial soil composed of 15% sphagnum peat, 25% kaolinite clay, and 77 % fine sand. To keep the pH at 5.9, a few drops of CaCO 3 were added. The water content was reduced to 30% of the dry weight. Fungi metabolites from M. anisopliae were put into the artificial soil at concentrations of 50 g/mL and 75 µg/mL. The 15 E. eugeniae larvae were then moved to a plastic container (375 mm × 300 mm × 75 mm) containing 1 kg of sterile artificial soil, which was then sealed with a plastic lid to keep the worms from escaping. Dead worms were counted 24-h after exposure. Monocrotophos was used as a positive control, while the negative control was free of fungal metabolites. Each treatment was replicated three times.
On brine shrimp A. nauplii, the toxicity of fungal secondary metabolites was determined as follows: Mature A. nauplii were collected with a hand pipette and utilized in toxicity tests on A. nauplii with different concentrations of M. anisopliae secondary metabolites (10,15,30,50, and 75 µg/mL). As a negative control, the DMSO solution was employed. After 24 h of treatment, the A. nauplii dead mortality was calculated. Each concentration was tested three times, with each replicate containing 25 mature A. nauplii.

Fourier Transformed Infrared Spectroscopy Analysis
FT-IR analysis was conducted for the identification of the functional groups presents in the crude fungal metabolites. Two mg of fungi metabolites were properly mixed in 75 mg KBr; KBr acts as a binding agent on cleaned micro mortar and pestle. The mixed component was made into KBr pellets formed at low pressure. The KBr pellets were taken for FT-IR analysis using a BRUKER FT-IR spectrometer. FT-IR spectra scanning range was from 500 to 4000 cm −1 .

Liquid Chromatography-Mass Spectrophotometer Analysis
The chemical components profiling of crude fungal extracts was completed through the use of a Bruker Daltonik Impact II ESI-Q-TOF system (Bremen, Germany), ready with a Bruker Daltonik Elute, Ultra High Performance Liquid Chromatography (UHPLC) system (Bremen, Germany), in each positive (M + H) and negative (M − H) electrospray ionisation modes. Chromatographic separation was carried out on a Bruker Daltonik (Bremen, Germany) C18 reversed segment column (2.1 mm, 1.8 m, 120) at 30 • C, with an autosampler temperature of 8 • C and a total run time of 20 min, using water/methanol (90:10%) as eluent with five mM ammonium formate and 0.1% formic acid. The crude extract was dissolved in 2.0 mL of DMSO, and the quantity was multiplied to 50 mL with acetonitrile prior to centrifugation at 4000 rpm for two min and injection. The composition of the samples became mounted with the aid of figuring out the m/z ratio when it comes to the retention length of the utilised standards.

Statistical Analysis
The mortality rate was corrected using Abbot formula [23]. The dead A. nauplii, mosquito larvae, pupae, and adults were counted separately 24 h after treatment, and LC 50 and LC 90 were estimated using probit analysis. The SPSS-16.00 programme [18] was used to conduct all of the analyses.

Non-Target Organisms
Entomopathogenic fungi M. anisopliae constituents showed a minimal effect on nontargeted A. nauplii. This study clearly shows (5.33-18.33 %) mortality were produced by the M. anisopliae derived crude extract (Table 2; Figure 2). The LC 50 and LC 90 values were 620.481; 6893.990 µg/mL (Table 2). No behavioral changes were observed during the treatment with fungal extracts.
A low lethal effect was observed in E. eugeniae treated with the fungi metabolites; 14.0% mortality were observed in those treated with M. anisopliae secondary metabolites at 30 days after treatments. The highest earthworm mortality was observed in Monocrotophos pesticide treatment that shows 87.33 % mortality. Furthermore, the chemical treatment epidermis, intestinal and body wall thickness was reduced by the chemical (Figure 3; Tables 3 and 4).

Discussion
Recently, there has been a great interest in the use of biologically derived pesticides as an alternative to synthetic chemicals [9,10]. Entomopathogenic fungi-derived toxins have several advantages over synthetic pesticides in that they kill mosquitos at different stages in both laboratory and environmental conditions, have lower toxic effects on non-target organisms, and remain stable for several months in extreme cold and hot conditions [9,10,14]. In this study, we evaluated the toxic effects of secondary metabolites isolated from M. anisopliae strains against larvae, pupae, and adults of the disease-vector mosquitoes Ae. aegypti, An. stephensi and Cx. quinquefasciatus, and we assessed their target specificity and environmental safety by testing the extracts against the aquatic and terrestrial non-target species A. naupli L. and E. eugeniae.
Fungal secondary metabolites showed clear toxicity against all the tested instars of the mosquitoes and much lower toxicity against the non-target organisms. In the present study, M. anisopliae crude metabolites showed high toxicity towards the larvae, pupae, and adults of A. aegypti, A. stephensi and C. quinquefasciatus mosquitoes at 24 h post treat-ment under laboratory conditions ( Figure 1; Table 1). In line with our results, previous studies on entomopathogenic fungal derived pesticides from several species of Metarhizium, Fusarium, Aspergillus, Trichoderma and Lecanicillium showed that they are effective against medical and agricultural insect pests [24]. Soni and Prakash [25] reported that Chrysosporium keratinophilum derived secondary metabolites have strong larvicidal activity against C. quinquefasciatus and A. stephensi mosquito larvae, while [26] reported that different fungal metabolites cause strong larvicidal activity against larvae of A. stephensi and C. quinquefasciatus. Similarly, Metarhiziumanisopliae, Aspergillus flavus, Fusarium oxysporum, Verticillium lecanii, Paecilomyces fumosoroseus, Beauveria bassiana, and Fusarium moniliforme and their toxins have been shown to produce remarkable mosquitocidal potential on larvae, pupae, and adult mosquitoes [9][10][11]27]. C. tropicum, C. clavisporus and F. oxysporum culture filtrates showed strong larvicidal activity against A. stephensi, A. aegypti and C. quinquefasciatus [10,11,22,28], and secondary metabolites of A. fumigatus showed strong larvicidal activity against larvae of A. aegypti [29].
On the contrary, in our study, we observed low toxicity of the fungal metabolites against non-target species such as A. nauplii and E. eugeniae (Tables 2-4; Figures 2 and 3). Similarly, [30] reported few swimming speed alterations in Artemia adults after their treatment by different toxins. A similar study about the effects of the fungi secondary metabolites from Penicillium daleae on Artemia, observed morphological changes in eye shape, eye color, and eye fading [31]. These results suggest that secondary metabolites from different fungi may produce lower levels of toxicity to non-target organisms. For this reason, the assessment of the lower effects of fungal secondary metabolites in aquatic and terrestrial ecosystems on non-target species is of prime importance. The chemical composition of the secondary metabolites extracted from the M. anisopliae entomopathogenic fungi analysed in this study is in accord with previous research by [9,10] and by [32], who observed similar kinds of chemical constituents (Figure 4; Table 5). Previously, [9,10] reported that B. bassiana and F. oxysporum derived crude metabolites had the same chemical constituents showing a strong larvicidal activity on A. aegypti, A. stephensi and C. quinquefasciatus larvae. In this study, FT-IR analyses showed the presence of phenols, biogenic amines, and carboxylic acids, which may be involved in the toxic effects on mosquitoes ( Figure 5; Table 6).
Similarly, previous studies showed that the metabolites of entomopathogenic fungi are constituted by components belonging to several chemical classes (phenols, alcohols, carboxylic acids, misc, aromatics, phosphoramide, and disulfides), which may be involved in the mosquitocidal effects [9][10][11]26,[33][34][35][36]. The strong mosquitocidal activity and the low toxic effect on non-target organisms exhibited by M. anisopliae indicate that, besides entomopathogenic fungal conidia, their metabolites may also have a significant role in efficient microbial-derived mosquito control tools that can be used in mosquito control programmes as effective, cheaper, biodegradable, target-specific alternatives to chemical insecticides. Further research into the single crude metabolite chemical constituents under laboratory and semi-field conditions may result in the development of effective M. anisopliae derived bio-pesticides.

Conclusions
The strong mosquitocidal activity and the low toxic effect on non-target organisms exhibited by M. anisopliae indicate that, besides entomopathogenic fungal conidia, their metabolites may also have a significant role in efficient microbial-derived mosquito control tools that can be used in mosquito control programmes as effective, cheaper, biodegradable, target-specific alternatives to chemical insecticides. Further research into the single crude metabolite chemical constituents under laboratory and semi-field conditions may result in the development of effective M. anisopliae derived biopesticides.