Larvicidal efficiency of the fungus Amanita muscaria ( Agaricales , Amanitaceae ) against Musca domestica ( Diptera , Muscidae )

This work reports the larvicidal action of two formulations of Amanita muscaria against Musca domestica. Two methods of extraction were tested: an aqueous extract from dried, powdered basidiomes (DPB); and an extract from fresh basidiomes liquefied in water (FLB). The mortality caused by the DPB extract varied from 14.67% to 100%. The efficiency of the FLB extract varied from 10.67% to 89.33%. The mean lethal concentration (LC50) of the DPB extract was approximately 1,931.02 ppm, whereas the LC50 for the FLB extract was about 30%. The extracted substances from these methods did not interfere with the development period of immatures and did not influence pupal weight. These results show the potential of A. muscaria extracts for controlling M. domestica.


Musca domestica
Due to its vectorial ability and large populations, this species often needs to be controlled.Control is usually done with synthetic chemical insecticides, which cause many problems, such as environmental contamination, human and domestic animal poisoning, and the appearance of resistance in populations (ACEVEDO et al., 2009;KOZAKI et al., 2009;WANG et al., 2012).
As an alternative to synthetic insecticides, natural products have been studied (CABRAL et al., 2008).The most important ones are obtained from plants and, although poorly understood, some fungal species show insecticidal activity (MIER et al., 1996).Amanita muscaria (Linnaeus, 1758) (Agaricales, Amanitaceae) is popularly known as fly agaric because some traditional communities use solutions of this fungus to control flies (CORDIER, 1876;MOYEN, 1888apud MIER et al., 1996).Insecticidal properties of Amanita muscaria are mainly associated with the action of ibotenic acid and muscimol (TAKEMOTO et al., 1964).
A l t h o u g h t h e u s e o f t h i s f u n g u s b y traditional communities is known (MICHELOT; MELENDEZ-HOWELL, 2003), there are no reports of its use against M. domestica immatures.Thus, this study aimed to evaluate the efficiency of aqueous extracts of A. muscaria against M. domestica immatures.
Adults of M. domestica were collected in Pelotas, Rio Grande do Sul, Brazil (31°48'S, 52°25'W), identified with the help of a dichotomous key (CARVALHO et al., 2002), and maintained in cages (30 x 30 x 30 cm).The M. domestica specimens were maintained according to Ribeiro et al. (2000) for rearing Ophyra aenescens (Diptera, Muscidae), differing only in the diet of the larvae, which in this work was composed of meat meal and sawdust (proportion 2:1).Throughout the experimental period, stock and test colonies were maintained in a climatic chamber (25 ± 2°C, RH 70 ± 10%, 12L: 12D).
For the first test (DPB extract), as soon as the basidiomes were collected they were placed in an incubator at 45°C for 96 h, and then milled with an electric mill with 0.5 mm mesh.The crude extract was obtained from the fungal powder added to distilled sterile water in the proportion of 1:10 (w/v).The resulting mixture was placed in a shaking incubator at 120 rpm and 25°C for 48 h.Then, the product was centrifuged for 10 min at 3,000 rpm and the supernatant was filtered using filter paper.
For this test, we prepared six concentrations: 0 ppm (control group), 928 ppm, 1,856 ppm, 2,784 ppm, 3,712 ppm and 4,640 ppm.We used 75 first-instar larvae divided in three replicates of 25 larvae for each concentration, totaling 450 individuals.Replicates were formed with 25 g of diet (composed of meat meal and sawdust in the proportion of 2:1) moistened with 25 mL of extract or distilled sterile water (control group).The container with the diet and larvae was placed in a larger container with moistened sawdust so the post-feeding larvae could abandon the substrate and pupariate in the sawdust.The larger container was closed with voile.
We observed the treatments daily and the postfeeding larvae found outside of the substrate were placed in containers with slightly moistened sawdust.We weighed the pupae after 48 hours and watched them until adult emergence.

Larvicidal efficiency of fly agaric against the house fly
The second test (FLB extract) followed the same procedure as the first test, differing only in the solution that was used to moisten the diet that, in this test, was obtained from the liquefaction of fresh basidiomes in water.
We prepared six concentrations with the fresh basidiomes and distilled sterile water (w/v): 0% (control group), 10%, 20%, 30%, 40%, and 50%.The mixture was filtered with filter paper, the solid portion was discarded, and the liquid was used to moisten the diet where the larvae were placed.
For both tests, total mortality (larval and pupal), development period, and pupal weight were evaluated using an analysis of variance (ANOVA) and means compared by Tukey's test (p < 0.05).LC 50 and LC 90 of each test were estimated with a Probit analysis.All analyses were performed using the software SPSS for Windows, version 22.0 (SPSS INC., 2013).
The mean mortality varied from 44.00% to 100%, whereas the control group showed a mean mortality of 10.67% (Table 1).
The FLB extract also caused significant mortality (F = 178.87;DF = 5; p < 0.001).The mean mortality varied from 25.33% to 89.33%, whereas the control group showed a mean mortality of about 6.67% (Table 1).For the first concentration of both extracts, we did not observe an insecticidal effect.
The mean lethal concentration (LC 50 ) of the DPB extract was 1,931.02ppm and the LC 90 was 3,174.30ppm.For the FLB extract, the LC 50 was 29.77% and the LC 90 was 49.42% (Table 2).
Our study shows, for the first time, that aqueous formulations of A. muscaria exhibit larvicidal activity against M. domestica.Due to resistance of this insect species to synthetic chemical insecticides, similar studies had already been described as important in the 1960s (PETZSCH, 1960); however, little was known beyond reports from traditional communities.
We observed that the larvicidal effect was dosedependent, as reported by Cárcamo et al. (2016) for Culex quinquefasciatus (Diptera, Culicidae) larvae subjected to the same fungal species.However, the efficiency that we observed was lower than observed by these authors.They reported an LC 50 of about 50 ppm, and in the present study we estimated a concentration of 1,931 ppm to kill the same percentage of larvae.These differences are probably associated with specific features of these species, since the method of extraction was the same for both experiments.
The larvicidal effect of A. muscaria was not observed by Mier et al. (1996) for Drosophila melanogaster (Diptera, Drosophilidae) larvae.For this fly, other Amanita species were considered toxic: A. phalloides, A. citrina and A. rubescens.Larvicidal action can be associated with different factors, such as the method used to obtain the active ingredient, since the way fungal compounds are extracted influences larvicidal activity (THONGWAT et al., 2015).
The two tested formulations killed immatures of M. domestica, and the FLB extract was easier to produce.Nevertheless, it can only be produced in colder seasons when fresh basidiomes of A. muscaria grow in Rio Grande do Sul, Brazil (BERNARDI, E.: personal communication, 2015).On the other hand, the DPB extract can be stored up to six months without losing efficiency (CÁRCAMO et al., 2016).
The inability of these two extracts to alter the development period and pupal weight is probably due to the mode of action of the toxic compounds.The main compound found in A. muscaria is muscimol (MICHELOT; MELENDEZ-HOWELL, 2003), which is a potent agonist of GABA A (FRØLUND et al., 2002) that acts as a neurotoxic insecticide.
We obtained promising results with two methods of application of A. muscaria as a larvicidal insecticide.In addition, this is the first scientific report of the use of aqueous extracts as an insecticide against M. domestica larvae, which is an important species in urban and animal production environments.Nonetheless, other aspects of its use, such as the impact of this insecticide against Larvicidal efficiency of fly agaric against the house fly non-target populations and its efficiency under field conditions, must be thoroughly evaluated.
* In the columns, values followed by different letters represent a significant difference between mean mortalities of each concentration (Tukey's test -p < 0.05).

TABLE 2 :
Lethal concentrations of Amanita muscaria aqueous extracts (from dried, powdered basidiomes -DPB, and fresh basidiomes liquefied in water -FLB) required to cause the death of 50% and 90% of Musca domestica immatures.

TABLE 3 :
Development period (larva to adult) (days) and pupal weight (mg) of Musca domestica originating from larvae subjected to different concentrations of aqueous extracts of Amanita muscaria: from dried, powdered basidiomes (DPB), and from fresh basidiomes liquefied in water (FLB).Groups that were not evaluated because they did not have the minimum number of individuals. *