Abstract:

INTRODUCTION:

We report the larvicidal activity of two formulations from Amanita muscariaagainst Culex quinquefasciatus, as well as the viability of the aqueous extract after storage.

METHODS

The larvicidal activity of aqueous extract and powder from A. muscaria, and the viability of the aqueous extract after storage, were evaluated.

RESULTS

The aqueous extract caused larval deaths, which varied from 16.4% to 88.4%. The efficiency of the powder varied from 29.2% to 82.8%. Storage did not interfere with the larvicidal efficiency of the aqueous extract of A. muscaria.

CONCLUSIONS

These results show the potential of A. muscariato control C. quinquefasciatus.

Keywords:
Bioinsecticide; Fly agaric; Filariasis vector

The mosquito Culex quinquefasciatusSay, 1823 (Diptera, Culicidae), is the main vector of the human parasitic roundworm, Wuchereria bancrofti(Cobbold, 1877) (Spirurida, Onchocercidae) on the American continent ¹1. Lozovei AL Culicidae (mosquitos). Marcondes CB, editor. Entomologia médica e veterinária. São Paulo: Atheneu; 2011. p. 107-174.. Reisen ²2. Reisen WK, Fang Y, Martinez VM. Avian host and mosquito (Diptera: Culicidae) vector competence determine the efficiency of West Nile and St. Louis encephalitis virus transmission. J Med Entomol 2005; 42:367-375.reported that this mosquito can also act as a vector of arboviruses, such as West Nile and St. Louis encephalitis.

This species is usually controlled by the use of synthetic chemical insecticides. The use of these products can cause problems, such as human and environmental contamination, as well as the development of resistance in insect populations ³3. Jones CM, Machin C, Mohammed K, Majambere S, Ali AS, Khatib BO et al. Insecticide resistance in Culex quinquefasciatusfrom Zanzibar: implications for vector control programmes. Parasit Vectors 2012; 5:1-9..

Plant-based products are the most recently researched alternatives, showing great results in mosquito control ⁴4. Gkinis G, Michaelakis A, Koliopoulos G, Ioannou E, Tzakou O, Roussis V. Evaluation of the repellent effects of Nepeta parnassicaextract, essential oil, and its major nepetalactone metabolite against mosquitoes. Parasitol Res 2014; 113:1127-1134.^{) (5}5. Ahbirami R, Zuharah WF, Thiagaletchumi M, Subramaniam S, Sundarasekar J. Larvicidal efficacy of different plant parts of railway creeper, Ipomoea cairicaextract against dengue vector mosquitoes, Aedes albopictus(Diptera: Culicidae) and Aedes aegypti(Diptera: Culicidae). J Insect Sci 2014; 14:1-6.. However, although there are few studies that investigate the use of fungi as a control measure, some fungal species have shown promising results against insects ⁶6. Mier N, Canete S, Klaebe A, Chavant L, Fournie D. Insecticidal properties of mushroom and toadstool carpophores. Phytochemistry 1996; 41:1293-1299.^{) (7}7. Bucker A, Bucker NCF, Souza AQLD, Gama AMD, Rodrigues-Filho E, Costa FMD, et al. Larvicidal effects of endophytic and basidiomycete fungus extracts on Aedesand Anopheleslarvae (Diptera, Culicidae). Rev Soc Bras Med Trop 2013; 46:411-419..

Among the fungal substances known to be toxic to insects, ibotenic acid and muscimol are both found in the ectomycorrhizal fungus Amanita muscaria(Linnaeus, 1758) (Agaricales, Amanitaceae) ⁸8. Michelot D, Melendez-Howell LM. Amanita muscaria: chemistry, biology, toxicology, and ethnomycology. Mycol Res 2003; 107:131-146.. This fungus is generally found in temperate areas, and in Brazil, has been reported from São Paulo to the southern part of the country in association with plantations of Pinusspp. or Eucalyptusspp. ⁹9. Wartchow F, Maia LC, Cavalcanti MAQ. Taxonomic studies of Amanita muscaria(L.) Lam (Amanitaceae, Agaricomycetes) and its infraspecific taxa in Brazil. Acta Bot Brasilica 2013; 27:31-39.. Although A. muscariapossesses substances that are toxic to insects, there are no studies regarding the impact of this fungal species against culicids. Thus, this work aims to report the toxicity of two formulations, an aqueous extract, and a powder, from A. muscariaagainst C. quinquefasciatuslarvae, as well as to evaluate the ability to store the aqueous extract of this fungal species.

The larvae of C. quinquefasciatusused in this study were obtained from a laboratory colony, and raised according to the methodology proposed by Gerber ¹⁰10. Gerberg EJ. Manual for mosquito rearing and experimental techniques. Am Mosq Control Assoc 1979; 5:1-124., differing only in the diet offered to the larvae. In this investigation, the larvae were fed with minced fish feed.

Amanita muscariaspecimens were collected in July in a grove of Pinus elliottiiEngelmann, 1880 (Pinales, Pinaceae) in Capão do Leão, Brazil (31°48′08.7″S, 52°24′51.2″W). Basidiomata were collected when their rings were completely opened, and were subsequently put in an incubator at 45°C for 96h. After dehydration, basidiomata were milled in an electric mill with a mesh of 0.5mm.

Crude extract was obtained from the fungal powder and added to sterile distilled water at a concentration of 1:10 (w/v). The resulting mixture was put in a shaker (120rpm) at 50°C for 48h. Subsequently, the product was centrifuged for 10 min at 3,000rpm and the supernatant was filtrated using a paper filter. A portion of the extract was used immediately and the remainder was either stored in refrigerator (3°C ± 2°C) or freezer (-20°C) for six months.

The experiment to detect larvicidal activity was performed following the standard protocol from the World Health Organization ¹¹11. World Health Organization. Instructions for determining the susceptibility or resistance of mosquito larvae to insecticides. Vector Biol Control 1981; 81:1-6, with some modifications. Insects were maintained in a climatic chamber (25°C ± 2°C, 70% ± 10% RH, and a photoperiod of 12:12h) for all experiments.

We evaluated the efficiency of Amanita muscariaagainst Culex quinquefasciatuslarvae using two formulations, an aqueous extract and a fungal powder. We used five concentrations of each formulation. To evaluate the aqueous extract, we diluted the crude extract in water to the following concentrations: 14.5ppm, 29.0ppm, 58.0ppm, 116.0ppm, and 232.0ppm. The preparation of the powder formulation was created by adding varied concentrations of powder directly to water and shaking gently for 1 minute. The final concentrations of the fungal powder formulations were 0.2mg/ml, 0.4mg/ml, 0.8mg/ml, 1.6mg/ml, and 3.2mg/ml. Each concentration was composed of five replicates, each with 50ml of liquid (extract or water) and 50 third-instar larvae of C. quinquefasciatus. For each formulation, we established a control group, composed in the same way as larvicidal treatment groups, differing only in that there was no Amanita muscariaadded .The groups were evaluated after 24h and larvae that did not show any motility after being lightly touched were considered dead.

After six months of chilling or freezing, the larvicidal activity of stored extracts were evaluated against C. quinquefasciatuslarvae using a final concentration of 232.0ppm. For each group, five replicates were created using 50ml of extract and 50 third-instar larvae. Two treatments served as control conditions: a negative control (water only plus larvae) and a positive control (aqueous extract that had not been stored plus larvae). The evaluation of larvicidal activity was performed as previously described.

Mortality was evaluated using an analysis of variance (ANOVA) (p = 0.05), in which means were compared using Tukey's test (p = 0.05). The median lethal concentration (LC ₅₀) and 90% lethal concentration (LC ₉₀) of each formulation were analyzed using probit analysis. All analyses were performed using SPSS version 22.0 for Windows (IBM, Armonk, NY, USA) ¹²12. IBM Corporation. IBM SPSS Statistics for Windows. Version 22.0. Armonk, NY: IBM Corp; 2013..

The aqueous extract of Amanita muscariacaused significant mortality in C. quinquefasciatuslarvae (F = 156.11; DF = 5; p < 0.001). The mean mortalities caused by extracts varied from 16.4% to 88.4%, while in control groups, the mean mortality was approximately 3.2% ( Figure 1).

Figure 1:
Mean mortality (%) of Culex quinquefasciatuslarvae subjected to different concentrations of aqueous extract from Amanita muscaria. Different letters represent significant difference between groups (Tukey's test; p = 0.05). Error bars represent the standard deviation of the mean.

The powder of Amanita muscariacaused significant mortality of C. quinquefasciatuslarvae (F = 101.01; DF = 5; p < 0.001). The mean mortalities caused by the powder preparation varied from 29.2% to 82.8%, whereas, in the control group, the mean mortality was approximately 3.2% ( Figure 2).

Figure 2:
Mean mortality (%) of Culex quinquefasciatuslarvae subjected to different concentrations of powder from Amanita muscaria. Different letters represent significant difference between groups (Tukey's test; p = 0.05). Error bars represent the standard deviation of the mean.

The mean lethal concentration (LC ₅₀) of the aqueous extract of A. muscariawas approximately 51.46ppm and the LC ₉₀was approximately 248.07ppm. The powder formulation resulted in an LC ₅₀of approximately 1.19mg/ml and an LC ₉₀of approximately 3.98mg/ml ( Table 1).

The mortalities of C. quinquefasciatuslarvae caused by different methods of storing the aqueous extract of A. muscariadid not show significant differences between groups (F = 0.9; DF = 2; p = 0.43), which varied from 83% to 86.8%. The mortalities observed were significantly higher than those observed for the negative control (F = 261.31; DF = 3; p < 0.001), which was approximately 2.8%.

Both aqueous and powder formulations from A. muscariadisplayed larvicidal activity against C. quinquefasciatuslarvae in a dose-dependent manner. The aqueous extract possessed toxicity classified between moderate and high according to the categories proposed by Komalamisra ¹³13. Komalamisra N, Trongtokit Y, Rongsriyam Y, Apiwathnasorn C. Screening for larvicidal activity in some Thai plants against four mosquito vector species. Southeast Asian J Trop Med Public Health 2005; 6:1412-1422..

The larvicidal efficiency of the aqueous extract of A. muscariaobserved in this study was higher than the efficiencies of different fungal formulations from Pycnoporussp. (Linnaeus, 1758) (Polyporales, Polyporaceae) and Pestalotiopsissp. Steyaert, 1949 (Xylariales, Amphisphaeriaceae) against the yellow fever mosquito, Aedes aegypti(Linnaeus, 1762) (Diptera, Culicidae), as observed by Bucker ⁷7. Bucker A, Bucker NCF, Souza AQLD, Gama AMD, Rodrigues-Filho E, Costa FMD, et al. Larvicidal effects of endophytic and basidiomycete fungus extracts on Aedesand Anopheleslarvae (Diptera, Culicidae). Rev Soc Bras Med Trop 2013; 46:411-419.. The observed differences could be related to species-specific differences or to the methods of extraction, as the author observed different impacts according to tested formulations. This illustrates the importance of evaluating other methods of extraction for A. muscariain order to maximize the efficiency of this fungus against C. quinquefasciatuslarvae. However, we consider the use of water as a solvent for extraction to be an advantageous alternative, as it is a low-cost method and does not result in environmental problems as do other solvents.

The extract of A. muscariahad a higher larvicidal efficiency against C. quinquefasciatusthan extracts from four other fungal species tested by Chelela ¹⁴14. Chelela BL, Chacha M, Matemu A. Larvicidal potential of wild mushroom extracts against Culex quinquefasciatusSay, Aedes aegyptiand Anopheles gambiaeGiles S.S. Am J Res Commun 2014; 2:105-114.. The highly toxic activity of A. muscariais likely a result of ibotenic acid and muscimol, toxic substances found in A. muscariathat are known to be responsible for causing death in flies ⁸8. Michelot D, Melendez-Howell LM. Amanita muscaria: chemistry, biology, toxicology, and ethnomycology. Mycol Res 2003; 107:131-146..

The toxicity of Amanita muscariapowder against Culex quinquefasciatuslarvae was different than the effect observed by Mier ⁶6. Mier N, Canete S, Klaebe A, Chavant L, Fournie D. Insecticidal properties of mushroom and toadstool carpophores. Phytochemistry 1996; 41:1293-1299.against DrosophilaFallen, 1823 (Diptera, Drosophilidae) larvae. According to this author, although A. muscariais not toxic to Drosophila, other species of the genus Amanitawere shown to be toxic to this dipteran, such as A. phalloides(LC ₁₀₀= 0.1mg/ml), A. citrina(LC ₁₀₀= 30mg/ml), and A. rubescens(LC ₁₀₀= 53mg/ml). In the present study, the LC ₉₀was approximately 3.98mg/ml in A. muscaria. Direct comparisons between the results of various investigations are difficult because of differences in the dipteran species tested, highlighting the importance of evaluating the fungal specificity against various targeted insect species.

Even after six months of chilling or freezing, the extracts maintained their larvicidal efficiency against C. quinquefasciatus. This result is important considering the time disparity between the appearance of A. muscariaand the period of abundance of C. quinquefasciatus. C. quinquefasciatususually reaches its highest numbers during the warmest period of the year ¹⁵15. Ribeiro PB, Costa PRP, Loeck AE, Vianna EES, Silveira Júnior P. Exigências térmicas de Culex quinquefasciatus(Diptera, Culicidae) em Pelotas, Rio Grande do Sul, Brasil. Iheringia Ser Zool 2004; 94:177-180., while A. muscariausually occurs during months that have lower mean temperatures (such as July and August) in Rio Grande do Sul, Brazil (E Bernardi: personal communication, 2015).

In conclusion, we obtained promising results with the two methods of application of A. muscariaas a larvicide, even following long-term storage of the toxic extract, illustrating that this mushroom can be used as a source of bioinsecticide compounds. Despite these results, other aspects of its use, such as the impact of this insecticide against non-targeted populations and its efficiency under field conditions, require further evaluation.

ACKNOWLEDGMENTS

We are thankful for the Universidade Federal de Pelotas which provided technical support for the development of this study.

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