Chinnasamy Ragavendrana,
Nawal Kishore Dubeyb and
Devarajan Natarajan*a
aNatural Drug Research Laboratory, Department of Biotechnology, School of Biosciences, Periyar University, Salem – 636 011, Tamil Nadu, India. E-mail: mdnataraj@rediffmail.com; natarajpu@gmail.com
bCentre of Advanced Study in Botany, Banaras Hindu University, Varanasi-221005, India
First published on 13th January 2017
Mosquitoes are the carriers of severe and well-known illnesses such as malaria, arboviral encephalitis, dengue, chikungunya and yellow fever, which cause significant morbidity and mortality in humans and domestic animals around the world. Entomopathogenic fungal metabolites act as a mosquito control agent and are potential alternatives to chemical control because they can be innovative and more selective than chemical insecticides. The main aim of the present study was to perform experiments on the larvicidal and pupicidal effects of the entomopathogenic fungus Beauveria bassiana (isolated from infected grasshopper) against the first to fourth instar larvae of Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti. The larval and pupal mortality were observed after 24 h of exposure. The efficacy of an ethyl acetate mycelium extract at all the tested concentrations (50, 100, 150, 200, 250 and 300 μg mL−1) exhibited better activity against the 1st to 4th instar larvae of An. stephensi (LC50 = 42.82, 39.45, 25.72, and 32.66; LC90 = 254.67, 367.11, 182.27, and 199.20 μg mL−1), Cx. quinquefasciatus (LC50 = 72.38, 68.11, 27.06, and 35.495; LC90 = 481.68, 254.69, 129.83, and 146.24 μg mL−1) and Ae. aegypti (LC50 = 62.50, 52.89, 58.60, and 47.12; LC90 = 314.82, 236.18, 247.53, and 278.52 μg mL−1), respectively. The pupicidal activity of the fungal mycelium extracts was tested against An. stephensi, Cx. quinquefasciatus and Ae. Aegypti, where the ethyl acetate extracts had different LC50 values (LC50 = 40.66, 54.06, 44.26, and LC90 = 184.02, 225.61, and 263.02 μg mL−1). Based on Fourier transform infrared spectroscopy (FTIR) analysis and gas chromatography-mass spectrometry (GC-MS) analyses, the ethyl acetate mycelium extract contained six major chemical compounds identified as 9,12-octadecadienoic acid (ZZ)– (63.16%), n-hexadecanoic acid (21.28%), octadecanoic acid, phenyl methyl ester (10.45%), dehydroegosterol 3,5-dinitrobenzoate (1.86%), squalene (1.66%) and bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)prophyl]maleate (1.56%). The n-hexadecanoic acid standard was found to be better larvicidal against An. stephensi, Cx. quinquefasciatus, followed by Ae. aegypti. The HPLC analysis of the ethyl acetate mycelium extract was compared with that of the n-hexadecanoic acid standard and it was found to show a similar chromatographic peak (at a retention time of 3.383 and 3.378 min). The outcome of the present study identifies the bioactive compounds obtained from B. bassiana that can be used as effective and alternate larvicidal and pupicidal agents against the An. stephensi Cx. quinquefasciatus and Ae. aegypti mosquito vectors.
Culex mosquitoes are painful and persistent biters and are responsible for filariasis. Lymphatic filariasis is a neglected tropical disease. Lymphatic filariasis is commonly known as elephantiasis and infection occurs when filarial parasites are transmitted to humans through mosquitoes.3 When a mosquito with infective stage larvae bites a person, the parasites are deposited on the person's skin from where they enter into the body. The larvae then migrate to the lymphatic vessels where they develop into adult worms in the lymphatic system. Worldwide, more than 1.3 billion people from 72 countries are threatened by lymphatic filariasis, commonly known as elephantiasis.4 Chikungunya is a viral tropical disease transmitted by Aedes mosquitoes. The disease is prevalent in Africa, Asia, the islands in the Caribbean, India and Pacific oceans. Typical symptoms are an acute illness with fever, skin rash and incapacitating joint pain that can last for weeks.5 The latter distinguishes chikungunya virus from dengue, which otherwise shares the same vectors, symptoms and geographical distribution. There is no cure or commercial vaccine for the disease. Most patients recover fully; however, in some cases, joint pain may persist for several months or even years. As with dengue, the only method to reduce the transmission of the chikungunya virus is to control vector mosquitoes and protect against mosquito bites. Yellow fever is an acute viral hemorrhagic disease transmitted by Aedes mosquitoes. The “yellow” in the name refers to the jaundice that affects some patients. There are an estimated 200000 cases of yellow fever, which cause 30
000 deaths worldwide per year. The virus that causes yellow fever is endemic in densely populated countries, viz., Tropical Africa and Latin America. Small numbers of imported cases occur in countries free of yellow fever.6
The common control agents for mosquito larvicides are mainly dependent on chemical methods using synthetic insecticides that are likely to include, organophosphates such as temephos, fenthion, phytochemicals and insect growth regulators such as diflubenzuron, and methoprene.7 However, most of these synthetics have adverse effects on the environment. Due to their residual nature there are reports on the development of pesticide resistance in mosquitoes8 rendering them ineffective for further applications. These problems encourage the search for safer and better alternative bioactive larvicidal agents. Although various biocontrol measures are in vogue, to date, their effective control of larval mosquitoes has not been practically highlighted. Microbial control is recommended as an alternative way, and microbial based larvicides are employed for minimizing the mosquito population, which provides an effective, environmentally friendly and sociable approach to bring the mosquito population to the lowest level.9,10
Beauveria bassiana (Clavicipitaceae) is a soil borne fungus that feeds on insects and can be used effectively to control thrips, aphids, whitefly, caterpillars, beetles, and subterranean insects like ants and termites. B. bassiana is non-toxic to mammals, birds and plants, and its use is not expected to have any deleterious effects on human health or the environment.11 Conidia of B. bassiana has been reported to be effective in killing mosquito larvae when applied as conidia dust in the breeding sites. Besides infecting larvae, the fungus has also proven to be virulent to adult mosquitoes.12 B. bassiana is applied to the target pest as a spore, which is the reproductive and dispersal structure of the fungus. Once the spores have made contact with the insect exoskeleton, they grow hyphae (long, branching vegetative appendages) that secrete enzymes, which in turn dissolve the cuticle (outermost layer of the skeleton). These fungal hyphae grow into the insect, feed on its body tissue, produce toxins, and reproduce. It takes up to seven days for the insect to die. During favorable (moist) conditions (92% humidity or greater), B. bassiana will “bloom” and release more spores into the environment to repeat the cycle on other pest insects.13 The species of Beauveria has been reported to produce secondary metabolites, including bassianin, bassiacridin, beauvericin, bassianolide, beauverolides, tenellin and oosporein.14–16 It also produces proteases, chitinases and lipases, which can degrade the insect cuticle.17 In this regard, the entomopathogenic fungi, viz., Aspergillus flavus, A. parasiticus, Penicillium falicum, Fusarium vasinfectum and Trichoderma viride and soil bacteria, Bacillus thuringiensis and B. sphaericus have been reported to be effective against Cx. quinquefasciatus.18 Hence, the present study was focused on the insecticidal potential of Beauveria bassiana mycelial extracts against target mosquitoes.
The larvicidal activity of mycelium ethyl acetate and methanol extracts obtained from B. bassiana was investigated. The ethyl acetate mycelium extract had a promising larvicidal activity (Table 1) against the 1st to 4th instar larvae (after 24 h of exposure period) on An. stephensi (LC50 = 42.82, 39.45, 25.72, and 32.66; LC90 = 254.67, 367.11, 182.27 and 199.20 μg mL−1) Cx. quinquefasciatus (LC50 = 72.38, 68.11, 27.06, and 35.495; LC90 = 481.68, 254.69, 129.83, and 146.24 μg mL−1) and Ae. aegypti (LC50 = 62.50, 52.89, 58.60, and 47.12; LC90 = 314.82, 236.18, 247.53, and 278.52 μg mL−1). The methanol mycelium extract (Table 2) showed considerable mortality against the vector mosquitoes i.e. An. stephensi, which had the better LC50 and LC90 values (LC50 = 65.22, 68.96, 67.64 and 52.95; LC90 = 317.77, 431.59, 345.35 and 687.70 μg mL−1) followed by Cx. quinquefasciatus (LC50 = 98.56, 80.85, 61.72 and 41.16; LC90 = 678.66, 399.97, 336.85 and 470.47 μg mL−1) and Ae. aegypti (LC50 = 64.94, 72.61, 61.90 and 57.65; LC90 = 961.97, 901.21, 439.32 and 916.04 μg mL−1). At a concentration of less than 50 μg mL−1 from B. bassiana, the mortality rates were slower, but the larvae became very slow-moving when compared with the control. The sub-lethal effects on the first and second larval instars were correlated with the minimum survival of the third and fourth instar larvae. The third and fourth instars larvae were also susceptible in the bioassay at the lowest lethal concentration. The dose dependent assay results showed that maximum (100%) mortality was obtained at a higher concentration (300 μg mL−1) against the different stages of instar larvae of the An. stephensi, Cx. quinquefasciatus and Ae. aegypti mosquitoes. At a higher concentration of extracts, the mortality rate was exhibited within 5 h of exposure. More than 50% mortality was observed within the first 10 h. The control showed a nil mortality in the concurrent assay. The χ2 value was significant at the P < 0.05 level.
Mosquito species | Larvae stage | Concentration (μg mL−1) | Percentageb mortality ± SE | LC50 (LCL–UCL) (μg mL−1) | LC90 (LCL–UCL) (μg mL−1) | χ2 (df = 3) |
---|---|---|---|---|---|---|
a Control (deionized water) – nil mortality. LC50 – lethal concentration that kills 50% of the exposed larvae, LC90 – lethal concentration that kills 90% of the exposed larvae, LCL = lower confidence limit, UCL = upper confidence limit, df degree of freedom, * χ2 – chi-square values are significant at P < 0.05 levels.b The mean value of five replicates (±SE). | ||||||
An. stephensi | I | 50 | 62.66 ± 2.5 | 42.826 (22.661–59.994) | 254.679 (196.072–400.697) | 14.266 |
100 | 68.00 ± 1.0 | |||||
150 | 71.66 ± 2.0 | |||||
200 | 81.66 ± 1.5 | |||||
250 | 93.66 ± 5.1 | |||||
300 | 99.33 ± 1.1 | |||||
II | 50 | 63.33 ± 3.5 | 39.459 (15.560–60.018) | 367.114 (253.777–811.269) | 14.442 | |
100 | 66.66 ± 1.5 | |||||
150 | 68.66 ± 1.5 | |||||
200 | 74.66 ± 2.0 | |||||
250 | 87.33 ± 2.0 | |||||
300 | 98.33 ± 2.0 | |||||
III | 50 | 71.00 ± 1.0 | 25.727 (8.271–42.558) | 182.275 (140.331–278.069) | 9.289 | |
100 | 79.00 ± 5.5 | |||||
150 | 83.33 ± 3.0 | |||||
200 | 86.33 ± 3.0 | |||||
250 | 93.66 ± 5.0 | |||||
300 | 100 ± 0.0 | |||||
IV | 50 | 67.66 ± 3.2 | 32.664 (14.232–49.187) | 199.206 (155.735–297.324) | 8.545 | |
100 | 73.00 ± 2.0 | |||||
150 | 84.00 ± 1.0 | |||||
200 | 85.33 ± 1.5 | |||||
250 | 93.00 ± 3.6 | |||||
300 | 100 ± 0.0 | |||||
Cx. quinquefasciatus | I | 50 | 48.33 ± 1.5 | 72.385 (47.687–92.674) | 481.686 (334.801–960.417) | 17.270 |
100 | 54.00 ± 2.0 | |||||
150 | 62.33 ± 1.5 | |||||
200 | 65.00 ± 1.7 | |||||
250 | 77.33 ± 1.1 | |||||
300 | 98.33 ± 0.5 | |||||
II | 50 | 48.33 ± 3.2 | 68.117 (51.556–82.429) | 254.698 (208.256–343.894) | 13.911 | |
100 | 52.00 ± 2.6 | |||||
150 | 73.33 ± 3.0 | |||||
200 | 83.00 ± 3.0 | |||||
250 | 90.00 ± 3.6 | |||||
300 | 100 ± 0.0 | |||||
III | 50 | 74.66 ± 1.5 | 27.063 (11.301–41.487) | 129.836 (103.262–172.038) | 8.658* | |
100 | 81.66 ± 1.5 | |||||
150 | 85.33 ± 2.0 | |||||
200 | 95.33 ± 2.5 | |||||
250 | 98.66 ± 1.5 | |||||
300 | 100 ± 0.0 | |||||
IV | 50 | 70.33 ± 1.5 | 35.495 (19.588–49.247) | 146.249 (119.821–190.880) | 15.145 | |
100 | 75.00 ± 2.0 | |||||
150 | 82.33 ± 3.2 | |||||
200 | 95.00 ± 5.5 | |||||
250 | 99.66 ± 0.5 | |||||
300 | 100 ± 0.0 | |||||
Ae. aegypti | I | 50 | 53.00 ± 1.0 | 62.506 (42.337–79.404) | 314.823 (242.389–487.932) | 14.334 |
100 | 55.33 ± 2.0 | |||||
150 | 66.66 ± 1.5 | |||||
200 | 80.00 ± 2.0 | |||||
250 | 85.66 ± 2.5 | |||||
300 | 99.33 ± 1.1 | |||||
II | 50 | 56.00 ± 3.0 | 52.896 (34.846–68.158) | 236.183 (189.696–332.423) | 13.939 | |
100 | 65.00 ± 3.0 | |||||
150 | 73.66 ± 4.1 | |||||
200 | 82.33 ± 5.8 | |||||
250 | 93.66 ± 4.0 | |||||
300 | 100 ± 0.0 | |||||
III | 50 | 52.00 ± 2.0 | 58.603 (40.851–73.647) | 247.535 (199.550–345.351) | 16.537 | |
100 | 65.33 ± 1.5 | |||||
150 | 68.33 ± 2.0 | |||||
200 | 81.00 ± 7.0 | |||||
250 | 94.00 ± 4.5 | |||||
300 | 100 ± 0.0 | |||||
IV | 50 | 57.00 ± 1.0 | 47.125 (26.419–64.574) | 278.528 (212.833–445.541) | 15.999 | |
100 | 69.00 ± 1.3 | |||||
150 | 74.00 ± 1.0 | |||||
200 | 78.66 ± 0.5 | |||||
250 | 87.66 ± 1.5 | |||||
300 | 100 ± 0.0 |
Mosquito species | Larvae stage | Concentration (μg mL−1) | Percentageb mortality ± SE | LC50 (LCL–UCL) (μg mL−1) | LC90 (LCL–UCL) (μg mL−1) | χ2 (df = 3) |
---|---|---|---|---|---|---|
a Control (deionized water) – nil mortality. LC50 – lethal concentration that kills 50% of the exposed larvae, LC90 – lethal concentration that kills 90% of the exposed larvae, LCL = lower confidence limit, UCL = upper confidence limit, df degree of freedom, χ2 – chi-square values are significant at P < 0.05 levels.b The mean value of five replicates (±SE). | ||||||
An. stephensi | I | 50 | 44.33 ± 1.5 | 65.224 (45.224–82.072) | 317.772 (246.041–484.853) | 5.135 |
100 | 64.33 ± 3.0 | |||||
150 | 69.00 ± 1.0 | |||||
200 | 79.00 ± 1.0 | |||||
250 | 83.33 ± 4.1 | |||||
300 | 96.00 ± 5.2 | |||||
II | 50 | 43.33 ± 2.8 | 68.964 (45.281–88.493) | 431.598 (308.932–799.046) | 7.405 | |
100 | 64.00 ± 2.0 | |||||
150 | 66.00 ± 0.5 | |||||
200 | 68.33 ± 1.5 | |||||
250 | 81.00 ± 1.0 | |||||
300 | 92.66 ± 2.5 | |||||
III | 50 | 49.00 ± 4.3 | 67.647 (46.999–85.017) | 345.357 (262.909–547.261) | 8.495 | |
100 | 52.66 ± 4.1 | |||||
150 | 68.00 ± 1.0 | |||||
200 | 73.00 ± 1.0 | |||||
250 | 85.00 ± 2.0 | |||||
300 | 92.33 ± 6.6 | |||||
IV | 50 | 51.66 ± 2.5 | 52.954 (21.812–77.823) | 687.709 (398.781–2673.123) | 3.488 | |
100 | 62.66 ± 2.5 | |||||
150 | 67.66 ± 0.5 | |||||
200 | 71.66 ± 2.0 | |||||
250 | 75.33 ± 1.1 | |||||
300 | 88.33 ± 1.5 | |||||
Cx. quinquefasciatus | I | 50 | 40.00 ± 7.2 | 98.565 (72.255–121.752) | 678.665 (441.025–1565.144) | 16.361 |
100 | 45.00 ± 2.0 | |||||
150 | 54.33 ± 1.5 | |||||
200 | 60.00 ± 3.6 | |||||
250 | 67.00 ± 2.6 | |||||
300 | 94.00 ± 6.9 | |||||
II | 50 | 45.00 ± 5.5 | 80.851 (59.967–98.793) | 399.970 (300.347–648.342) | 7.986 | |
100 | 45.6 ± 3.0 | |||||
150 | 66.00 ± 1.0 | |||||
200 | 73.33 ± 1.5 | |||||
250 | 81.33 ± 1.5 | |||||
300 | 92.33 ± 6.8 | |||||
III | 50 | 48.33 ± 3.2 | 61.721 (40.554–79.435) | 336.852 (255.152–542.614) | 1.783 | |
100 | 62.66 ± 2.5 | |||||
150 | 69.00 ± 1.0 | |||||
200 | 84.33 ± 1.5 | |||||
250 | 88.33 ± 1.5 | |||||
300 | 88.33 ± 1.5 | |||||
IV | 50 | 59.66 ± 4.7 | 41.165 (14.889–63.584) | 470.474 (302.367–1328.572) | 3.475 | |
100 | 66.66 ± 2.0 | |||||
150 | 72.33 ± 2.5 | |||||
200 | 74.33 ± 1.5 | |||||
250 | 83.66 ± 4.0 | |||||
300 | 90.00 ± 1.0 | |||||
Ae. aegypti | I | 50 | 50.66 ± 4.1 | 64.944 (29.362–92.251) | 961.973 (501.659–5352.134) | 4.495 |
100 | 51.66 ± 1.5 | |||||
150 | 60.33 ± 1.5 | |||||
200 | 67.33 ± 2.5 | |||||
250 | 72.00 ± 1.7 | |||||
300 | 85.00 ± 4.5 | |||||
II | 50 | 43.33 ± 0.5 | 72.613 (38.530–99.148) | 901.215 (494.937–3877.842) | 0.352 | |
100 | 55.00 ± 1.0 | |||||
150 | 62.66 ± 4.9 | |||||
200 | 72.00 ± 2.6 | |||||
250 | 74.33 ± 1.5 | |||||
300 | 76.33 ± 2.8 | |||||
III | 50 | 46.33 ± 6.6 | 61.909 (37.187–82.136) | 439.325 (307.906–869.960) | 2.492 | |
100 | 63.00 ± 2.0 | |||||
150 | 66.66 ± 3.5 | |||||
200 | 71.00 ± 1.0 | |||||
250 | 84.00 ± 1.0 | |||||
300 | 85.00 ± 1.7 | |||||
IV | 50 | 51.00 ± 3.6 | 57.651 (22.651–84.807) | 916.043 (478.320–5338.628) | 0.937 | |
100 | 55.00 ± 2.6 | |||||
150 | 67.60 ± 2.0 | |||||
200 | 71.00 ± 1.0 | |||||
250 | 76.66 ± 3.0 | |||||
300 | 79.66 ± 4.7 |
The results of the pupal mortality of mosquitoes (Table 3) were tested with six different concentrations (50 to 300 μg mL−1) of the fungus extracts. The fungal ethyl acetate mycelium extracts show better results against An. stephensi (LC50 = 40.66; LC90 = 184.02 μg mL−1) followed by Cx. quinquefasciatus (LC50 = 54.06; LC90 = 225.61 μg mL−1) and Ae. aegypti (LC50 = 44.26; LC90 = 263.02 μg mL−1) (Fig. 1), whereas the methanol extract revealed moderate pupicidal effects against An. stephensi (LC50 = 51.92; LC90 = 1196 μg mL−1), Cx. quinquefasciatus (LC50 = 69.29; LC90 = 862.25 μg mL−1) and Ae. aegypti (LC50 = 76.34; 1178.15 μg mL−1), (Table 4). At the concentrations of 300 μg mL−1 for the B. bassiana ethyl acetate constituents, about 90% of the mortality was observed within 18 h for An. stephensi and Cx. quinquefasciatus, followed by Ae. Aegypti, and a 100% pupal mortality was observed at the higher concentration of the extracts. The pupal toxicity revealed a dose-dependent mortality in the treatment against the An. stephensi, Cx. quinquefasciatus and Ae. aegypti. Based on the results, the ethyl acetate extract obtained from the fungal species was found to be an excellent pupicidal agent against the targeted mosquitoes An. stephensi, Cx. quinquefasciatus and Ae. aegypti.
Mosquito species | Concentration (μg mL−1) | Percentageb mortality ± SE | LC50 (LCL–UCL) (μg mL−1) | LC90 (LCL–UCL) (μg mL−1) | χ2 (df = 3) |
---|---|---|---|---|---|
a Control (deionized water) – nil mortality. LC50 – lethal concentration that kills 50% of the exposed larvae, LC90 – lethal concentration that kills 90% of the exposed larvae, LCL = lower confidence limit, UCL = upper confidence limit, df degree of freedom, * χ2 – chi-square values are significant at P < 0.05 levels.b The mean value of five replicates (±SE). | |||||
An. stephensi | 50 | 64.66 ± 1.0 | 40.661 (23.465–55.408) | 184.022 (149.315–250.834) | 14.510 |
100 | 73.66 ± 1.1 | ||||
150 | 74.33 ± 2.5 | ||||
200 | 91.33 ± 1.5 | ||||
250 | 97.33 ± 1.5 | ||||
300 | 100 ± 0.0 | ||||
Cx. quinquefasciatus | 50 | 54.33 ± 1.5 | 54.064 (36.734–68.769) | 225.619 (183.306–309.150) | 10.558 |
100 | 66.66 ± 1.5 | ||||
150 | 73.00 ± 1.0 | ||||
200 | 85.00 ± 1.0 | ||||
250 | 93.66 ± 3.2 | ||||
300 | 100 ± 0.0 | ||||
Ae. aegypti | 50 | 62.00 ± 2.6 | 44.263 (23.883–61.530) | 263.002 (201.843–417.120) | 14.921 |
100 | 67.00 ± 1.0 | ||||
150 | 71.00 ± 2.6 | ||||
200 | 81.33 ± 4.1 | ||||
250 | 91.66 ± 3.7 | ||||
300 | 100 ± 0.0 |
![]() | ||
Fig. 1 The pupicidal efficacy of the ethyl acetate extracts of B. bassiana against Cx. quinquefasciatus after 24 h of exposure: (a) control pupa, (b) pupa treated at a concentration of 300 μg mL−1. |
Mosquito species | Concentration (μg mL−1) | Percentageb mortality ± SE | LC50 (LCL–UCL) (μg mL−1) | LC90 (LCL–UCL) (μg mL−1) | χ2 (df = 3) |
---|---|---|---|---|---|
a Control (deionized water) – nil mortality. LC50 – lethal concentration that kills 50% of the exposed larvae, LC90 – lethal concentration that kills 90% of the exposed larvae, LCL = lower confidence limit, UCL = upper confidence limit, df degree of freedom, χ2 – chi-square values are significant at P < 0.05 levels.b The mean value of five replicates (±SE). | |||||
An. stephensi | 50 | 51.00 ± 3.6 | 51.925 (14.109–81.604) | 1196.224 (541.648–15![]() |
1.285 |
100 | 61.33 ± 1.5 | ||||
150 | 65.00 ± 1.0 | ||||
200 | 68.33 ± 1.5 | ||||
250 | 71.33 ± 2.0 | ||||
300 | 81.33 ± 1.5 | ||||
Cx. quinquefasciatus | 50 | 48.66 ± 3.7 | 69.299 (35.648–95.455) | 862.253 (477.816–3641.619) | 1.827 |
100 | 54.33 ± 1.5 | ||||
150 | 62.00 ± 2.0 | ||||
200 | 68.33 ± 1.5 | ||||
250 | 77.66 ± 2.5 | ||||
300 | 80.33 ± 1.5 | ||||
Ae. aegypti | 50 | 48.66 ± 3.2 | 76.346 (38.351–105.396) | 1178.151 (578.043–7953.579) | 4.314 |
100 | 51.33 ± 2.5 | ||||
150 | 58.33 ± 1.5 | ||||
200 | 62.00 ± 4.3 | ||||
250 | 70.66 ± 2.0 | ||||
300 | 82.66 ± 2.5 |
In addition, the toxicity of the n-hexadecanoic acid standard was tested against An. stephensi, Cx. quinquefasciatus and Ae. aegypti. The LC50 values of n-hexadecanoic acid against the first, second, third and fourth instar larvae of An. stephensi (LC50 = 50.22, 58.72, 2.27 and 38.61; LC90 = 105.09, 148.19, 15.910 and 81.98) and Cx. quinquefasciatus (LC50 = 10.64, 23.23, 12.75 and 0.72; 39.82, 55.53, 38.47 and 5.18) followed by Ae. aegypti (LC50 = 5.53, 12.46, 8.13 and 9.41; 21.25, 33.75, 30.57 and 27.36 μg mL−1) were recorded from present investigation. Similar observations were made for the pupicidal activity against An. stephensi, Cx. quinquefasciatus and Ae. aegypti; the LC50 and LC90 values were represented as follows: 8.66, 0.69, 3.05; 28.86, 4.38 and 11.43 μg mL−1, respectively. n-Hexadecanoic acid was found to show effective insecticidal activity against An. stephensi and Cx. quinquefasciatus, followed by Ae. aegypti.
Simultaneously, the Acremonium mycelium ethyl acetate extract showed larvicidal effects after 24 h of exposure. Considerable mortality was evident after the treatment of Acremonium for 1–4th instar larvae of three important mosquitoes. The LC50 and LC90 values of the first, second, third and fourth instars of An. stephensi (LC50 = 11.38, 8.18, 8.56 and 5.30; LC90 = 22.42, 17.19, 17.23 and 11.84 μg mL−1); Cx. quinquefasciatus (LC50 = 10.11, 13.35, 4.01 and 8.06; LC90 = 20.23, 25.13, 9.83 and 17.83 μg mL−1) and Ae. aegypti (LC50 = 8.50, 9.58, 15.26 and 10.35; LC90 = 18.02, 20.00, 28.88 and 21.51 μg mL−1) and the LC50 and LC90 values of the pupae (LC50 = 5.48, 9.60 and 3.99; LC90 = 14.46, 20.56 and 11.10 μg mL−1) were obtained from the present study.
FTIR spectroscopy was used to identify the functional groups of the active compounds based on the peak value in the infra-red region. FTIR analysis of the ethyl acetate mycelium extract showed the presence of prominent bands due to the O–H group of hydrogen-bonded alcohols or phenols (3420.94), C–H aromatics (3002.58), C–H alkanes (2916.88), –C
C– nitriles (2122.99), –C
C– alkanes (1654.84), C–H alkanes (1436.22), C–C aromatics (1409.89), C–O carboxylic acids (1315.64), C–N aliphatic amines (1021.42),
C–H alkenes (953.59), N–H primary amines (901.85) and C
O ketones (706.10) cm−1 (Fig. 2 and Table 5).
Observed wavenumber (cm−1) | Functional group | Bonding pattern |
---|---|---|
3420.94 | O–H stretch alcohols or phenols | Strong, broad |
3002.58 | ![]() |
Sharp |
2916.88 | C–H alkanes | Medium |
2122.99 | –C![]() |
|
1654.84 | –C![]() |
Medium |
1436.22 | C–H bend alkanes | Medium |
1409.89 | C–C stretch aromatics | Medium |
1315.64 | C–O stretch alcohols, carboxylic acids, esters, ethers | Sharp |
1021.42 | C–N stretch aliphatic amines | Medium |
953.59 | ![]() |
Sharp |
901.85 | N–H wagging primary amines | Strong, broad |
706.10 | C![]() |
Sharp |
The GC-MS results obtained from the ethyl acetate extract of B. bassiana indicated the presence of six major compounds viz. 9,12-octadecadienoic acid (ZZ)– (63.16%), n-hexadecanoic acid (21.28%), octadecanoic acid, phenyl methyl ester (10.45%), dehydroegosterol 3,5-dinitrobenzoate (1.86%), squalene (1.66%), and bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)prophyl]maleate (1.56%) (Fig. 3 and Table 6). Hence, the isolated bioactive compounds obtained from the B. bassiana derived products, with proven potential as an insecticide, can play an important role in the interruption of the transmission of mosquito-borne diseases. The larvicidal and pupicidal activity of the ethyl acetate extract may be due to the presence of major bioinsecticide constituents such as 9,12-octadecadienoic acid (ZZ)– and n-hexadecanoic acid.
![]() | ||
Fig. 3 The insecticidal compounds identified in the ethyl acetate mycelium extracts obtained from B. bassiana. |
Rt | Area | Area% | Molecular weight/formula | Compound name | Biological activity | References |
---|---|---|---|---|---|---|
a Components identified based on computer matching of the mass peaks with the NIST-2008 Library. | ||||||
17.519 | 80![]() ![]() |
21.286 | 256, C16H32O2 | n-Hexadecanoic acid | Nematicide, pesticide | Ragavendran and Natarajan 2015,60 Rajeswari et al. 2012,73 Zahir Hussain et al. 2010 (ref. 74) |
19.120 | 240![]() ![]() |
63.160 | 280, C18H32O2 | 9,12-Octadecadienoic acid (ZZ)– | Larvicide | Velu et al. 2014 (ref. 75) |
24.032 | 6![]() ![]() |
1.663 | 410, C3CH50 | Squalene | Pesticide, antioxidant and antitumor | Rajeswari et al. 2012,73 WHO 1997 ref. 76 |
25.253 | 7![]() ![]() |
1.865 | 588, C35H44O6N2 | Dehydroegosterol 3,5-dinitrobenzoate | Not known | Nil |
26.098 | 39![]() ![]() |
10.458 | 374, C25H42O2 | Octadecanoic acid, phenyl methyl ester | Hypocholesterolemic and nematicide | Dr Duke's Phytochemical and Ethnobotanical Database77 |
30.390 | 5![]() ![]() |
1.566 | 608, C38H56O6 | Bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)prophyl]maleate | Not known | Nil |
HPLC analysis of the ethyl acetate mycelium extract of B. bassiana and the n-hexadecanoic acid standard showed a similar chromatographic peak (at the retention time 3.383 and 3.378 min) (Fig. 4a and b).
![]() | ||
Fig. 4 (a) The HPLC chromatogram of the n-hexadecanoic acid standard and (b) the HPLC chromatogram of the ethyl acetate mycelium extract obtained from B. bassiana. |
The present study exhibited that the bioactive metabolites of B. bassiana have larvicidal and pupicidal activity against Anopheles, Culex and Aedes mosquitoes. These metabolites may destroy the cuticle layer of the larvae and pupae, which leads to the death of the larvae and pupa. A similar study has been reported by Ababutain et al.,42 which identified Streptomyces sp. having better mosquitocidal properties. The use of fungus and their products are virulent and are a promising alternative insecticidal control agent.43 The efficacy of the insecticidal activity of B. bassiana products against the larvae of An. stephensi, Cx. quinquefasciatus and Ae. aegypti larvae showed that the LC50 and LC90 values for Cx. quinquefasciatus and Ae. aegypti were higher than An. stephensi. The LC50 values for the 1st to 4th instar larvae values were observed to be as follows: 65.22, 68.96, 67.64 and 52.95; LC90 = 317.77, 431.59, 345.35 and 687.70 μg mL−1, respectively. In the present study, after the treatment of the various larval stages of An. stephensi, Cx. quinquefasciatus and Ae. aegypti with the B. bassiana mycelia extracts at different concentrations, 100% mortality was observed based on the dose-dependent manner. Recently, Kovendan et al.,44 studied B. thuringiensis var. israelensis against the larvae of Cx. quinquefasciatus at different concentrations. The LC50 and LC90 values were reported as follows: the LC50 value of I instar was 9.332%, II instar was 9.832%, III instar was 10.212%, and IV instar was 10.622%, whereas the LC90 value of I instar was 15.225%, II instar was 15.508%, III instar was 15.887% and IV instar was 15.986%. Similar studies have been carried out by several researchers using bacteria Bacillus thuringiensis,45,46 Bacillus sphaericus47 and fungus Trichoderma viride48 and Actinobacteria,49 entomopathogenic fungi Metarhizium,50 Trichophyton,41 Tolypocladium,51 Chrysosporium52 and Lagenidium53 were reported as potential insecticidal agents.
The outcome of present study proved that mycelium extract of B. bassiana had a broad spectrum larval mortality against An. stephensi, Cx. quinquefasciatus and Ae. aegypti and the values were found to be as follows: for An. stephensi, LC50 = 65.22, 68.96, 67.64, and 52.95; LC90 = 317.77, 431.59, 345.35 and 687.70 μg mL−1; for Cx. quinquefasciatus, LC50 = 98.56, 80.85, 61.72, and 41.16; LC90 = 678.66, 399.97, 336.85 and 470.47 μg mL−1 and for Ae. aegypti, LC50 = 64.94, 72.61, 61.90 and 57.65; LC90 = 961.97, 901.21, 439.32 and 916.04 μg mL−1. Similarly, Vijayan and Balaraman54 isolated 94 actinomycetes from marine soil samples collected at different locations, out of which 35 samples exhibited improved larvicidal activity against Cx. quinquefasciatus, An. stephensi and Ae. aegypti with LC50 values in the range of 1–3 μL mL−1.
The larval and pupal mortality of Cx. quinquefasciatus after 24 h of treatment with the n-hexadecanoic acid standard showed the highest larvicidal (LC50 = 2.27 and LC90 = 15.91 μg mL−1) and pupal toxicity (LC50 = 0.69 and LC90 = 4.38 μg mL−1) than An. stephensi and Ae. aegypti. Similarly, Rahuman et al.55 reported a bioassay-guided fractionation of the acetone extract of Feronia limonia, which was shown as a potent mosquito larvicide, identified as n-hexadecanoic acid and found to be effective against fourth instar larvae of Ae. aegypti, Cx. quinquefasciatus and An. stephensi. Similarly, Sivakumar et al.31 found the larvicidal and repellent activity of pure tetradecanoic acid against Ae. aegypti and Cx. quinquefasciatus. The LC50 values were 14.08 and 25.10 μg mL−1. More recently, Srinivasan et al.56 reported the larvicidal potential of isolated thujone against the 4th instar larvae of Ae. aegypti (LC50 = 4.23 mg L−1) and An. stephensi (LC50 = 3.30 mg L−1). Fungal secondary metabolites have play an important roles in pathogenesis and the larvicidal activity, which can help in controlling mosquito populations and reduce the spread of vector borne diseases. Acremonium ethyl acetate metabolites were found to be more effective against Ae. aegypti and Cx. quinquefasciatus, followed by An. stephensi larvae. Furthermore, the pathogenicity of Acremonium sp. was also reported to possess good parasitic properties.57 Similarly, Stanly Pradeep et al.58 proved that F. oxysporum metabolites are more effective against An. stephensi than Cx. quinquefasciatus larvae.
The FTIR results indicated that the ethyl acetate mycelium extract showed the presence of chemical bands due to O–H group hydrogen-bonded alcohols or phenols (3420.94), C–H aromatics (3002.58), C–H alkanes (2916.88), –C
C– nitriles (2122.99), –C
C– alkanes (1654.84), C–C aromatics (1409.89), C–O carboxylic acids or alcohols (1315.64), C–N aliphatic amines (1021.42), N–H primary amines (901.85) and C
O ketones (706.10) cm−1. Similar functional groups were obtained by Nagajyothi et al.59 The GC-MS analysis results revealed that the larvicidal and pupicidal activity of mycelium ethyl acetate extracts from B. bassiana were exhibited due to six major compounds, namely 9,12-octadecadienoic acid (ZZ)– (63.16%), n-hexadecanoic acid (21.28%), octadecanoic acid, phenyl methyl ester (10.45%), dehydroegosterol 3,5-dinitrobenzoate (1.86%), squalene (1.66%), bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)prophyl]maleate (1.56%). Earlier, Ragavendran and Natarajan60 reported that the Aspergillus terreus ethyl acetate extract contains six bioactive compounds and its constituents showed better larvicidal and the pupicidal effects on selected mosquito vectors, namely An. stephensi (LC50 = 97.410, 102.551, 29.802 and 8.907; LC90 = 767.957, 552.546, 535.474 and 195.677 μg mL−1), Cx. quinquefasciatus (LC50 = 89.584, 74.689, 68.265 and 67.40; LC90 = 449.091, 337.355, 518.793 and 237.347 μg mL−1) and Ae. aegypti (LC50 = 83.541, 84.418, 80.407 and 95.926; LC90 = 515.464, 443.167, 387.910 and 473.998 μg mL−1). Pupicidal activity was also reported against An. stephensi (LC50 = 25.228; LC90 = 140.487 μg mL−1), Cx. quinquefasciatus (LC50 = 54.525; LC90 = 145.366 μg mL−1) and Ae. aegypti (LC50 = 10.536; LC90 = 63.762 μg mL−1). Squalene is considered as an important substance for practical and clinical use with huge potential in the nutraceutical and pharmaceutical industries.61 Similarly, Thimiri et al.62 reported that the Streptomyces sp. produced the isolated compound (2S,5R,6R)-2-hydroxy-3,5,6-trimethyloctan-4-one observed against the larvae of R. microplus (LC50 = 88.74 ppm; r2 = 0.865), An. subpictus (LC50 = 162.59 ppm; r2 = 0.817) and Cx. quinquefasciatus (LC50 = 120.15 ppm; r2 = 0.782). Kumar Saurav et al.63 reported that Streptomyces VITSVK5 sp. yielded the bioactive/isolated compound 5-(2,4-dimethylbenzyl) pyrrolidin-2-one, which had larvicidal activity against the larvae of R. microplus (LC50 = 210.39 ppm, r2 = 0.873), An. stephensi (LC50 = 169.38 ppm, r2 = 0.840) and Cx. tritaeniorhynchus (LC50 = 198.75 ppm, r2 = 0.887). Previously, some researchers have reported the insecticidal activity of isolated compounds obtained from the species of Streptomyces, namely tetranectin,64 avermectins,65 faeriefungin66 and macrotetrolides.67
The HPLC analysis of the ethyl acetate mycelium extract was compared with the n-hexadecanoic acid standard and they showed a similar chromatographic peak (at a retention time of 3.383 and 3.378 min). The HPLC results were in agreement with the earlier reports of Ragavendran and Natarajan,60 and Manilal et al.68 who obtained (15.31 and 42%) n-hexadeconoic acid using different extracts. Previously, several researchers have isolated n-hexadecanoic acid from different plants and microbes i.e. Vitex altissima, V. negundo and V. trifolia,69 Aspergillus fumigatus,70 A. versicolor71 and Pestalotiopsis sp.72 The use of fungus based products would be cheaper, target-specific, self-sustained and highly toxic to mosquitoes, even at low doses.
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