Larvicidal effects of endophytic and basidiomycete fungus extracts on Aedes and Anopheles larvae (Diptera, Culicidae)

Mosquito culture and management

Aedes aegypti Linnaeus 1762: eggs were obtained from colonies at the Laboratório de Malária e Dengue of Instituto Nacional de Pesquisas da Amazônia – INPA, and the mosquitos were kept in cages in the insectary. Mosquitoes were maintained for oviposition in the insectary at 26 ± 2°C with a photoperiod of 12:12 (L/D) and 80 – 90% relative humidity²⁵.

Anopheles nuneztovari Gabaldón 1940: collections of species from the genus Anopheles was conducted in the Manaus east area (Amazonas State, Brazil) near Farm Natan (03°04′10″S, 59°51′40″W). Catches were performed in cattle pens, and only feeding females were selected. Samples were obtained between the hours of 18 to 21h by manual capture. Mosquitoes were maintained for oviposition in the insectary, according to the criteria of Scarpassa and Tadei²⁶. Females and eggs were identified according to a dichotomous key for the Culicidae family, generated by Faran²⁷.

Fungus collections and extractions

Plant origin and endophytic fungi: crude extracts of mycelium and metabolic medium were donated by the Laboratório de Produtos Bioativos de Origem Microbiana (LPBOM/UFAM). The endophytic fungi were isolated from Murraya paniculata (L.) Jack, Rutaceae, an exotic plant collected in São Paulo State, Brazil. And the endophytic fungus Pestalotiopsis virgulata was isolated from Gustavia cf. elíptica (S.A.), the Mori fruit, which is a member of the Lecythidaceae family from the Brazilian Amazon, which was collected from Fazenda Experimental da Agronomia (UFAM) in October 2008 (02°38′58″S, 60°03′09″W) and was preserved at LPBOM/UFAM, using the methodology described by Castelanni²⁸. Crude extracts were used to perform the screening for larvicidal activity (data not shown).

Reactivation and culture of Pestalotiopsis virgulata: after preliminary test results to verify Ae. aegypti larva mortality (data not shown), the fungal stock and experimental cultures were grown at 26±2°C in stationary conditions in Erlenmeyer flasks, containing 300mL of PDA (potato dextrose agar) growth medium with glucose (10g/L) as a carbon source and L-asparagine (2.5g/L) as a nitrogen source. The stock culture was inoculated with a section from the agar culture, and the stock culture was grown until the mycelia had totally covered the surface. The 47 experimental flasks of liquid culture were inoculated with floating mycelium fragments (~4mm in diameter, aseptically cut from the stock culture) and were grown for 14 days.

Secondary metabolite extractions from Pe. virgulata: After 28 days, the mycelia from the liquid culture medium (LCM) of Pe. virgulata were isolated by filtration through Whatman no. 4 Chr filter paper. Mycelia were added to ethanol for macerating, and after 24h, the resulting mixture was filtered. This process was repeated twice. The mycelia extracts obtained were then concentrated in a rotary evaporator at reduced pressure. After observing the boiling point of each solvent, the extracts were weighed to obtain yield. The LCM was extracted in solvents with different polarity: ethyl acetate (EtOAc) and water (aqueous extract).

Secondary metabolite extractions from Pycnoporus sanguineus: Pycnoporus sanguineus specimens were collected in Reserva Forestal Adolpho Ducke (02°58′30″S, 59°56′01″W) of Instituto Nacional de Pesquisas da Amazônia – INPA (Manaus-AM, Brazil). The criteria described by Smânia et al.²⁹ were used for cultivating and obtaining crude extracts from Py. sanguineus, with some modifications. The above protocol was used to perform secondary metabolite extractions from Py. sanguineus extracts.

Larvicidal selective bioassay

These experiments were performed following the results of the first stage and were aimed to calculate the LC₅₀ of selected extracts against the third instar larvae of the Ae. aegypti mosquito species. The selective bioassay results were used as a biological model for subsequent application of the extracts at the bioassay dose, to obtain the lethal concentrations (LC₅₀ and LC₉₀) for larvae of Ae. aegypti and An. nuneztovari. The trials were performed at two concentrations (250 and 500ppm). Two selected extracts were targeted in these trials (data not shown), and these extracts showed larval mortality of more than 50% after 24h of exposure. The experiment was repeated three times. The percentage mortality was calculated using the mortality formula, and corrections were made when necessary using Abbott’s³⁰ formula.

Larvicidal bioassay dose

The larvicidal bioassay was performed according to the recommendations of the WHO³¹, with minor modifications. The following criteria were considered: the mortality in the control group should not exceed 10%, the confidence limit was set at 95%, and the bioassays were repeated on three alternate days. The bioassay dose experiments were performed using two selective bioassay extracts: GaFr3 2.3 Mycelium EtOAc (Pe. virgulata) and Pyc Mycelium EtOAc (Py. sanguineus) (data not shown). The larvicidal bioassay dose experiments were performed using five concentrations: 100, 200, 300, 400 and 500ppm. The negative control received only DMSO (dimethylsulfoxide) at the same concentrations, and the mortality rate of the control was not to exceed 10%.

Statistical analysis

The lethal concentrations (LC₅₀ and LC₉₀) were determined by a probit analysis using POLO-PC^®32 LeOra Software, Berkeley, CA, USA. The mortality rate was corrected using Abbott’s³⁰ formula.

Selective bioassay evaluation

The percentage mortality of Ae. aegypti following treatment with different concentrations of extracts from endophytic fungi and basidiomycetes fungi was analyzed in a selective bioassay evaluation (data not shown). The ethyl acetate mycelia (EAM) extract from Py. sanguineus was very effective against the third instar larvae of Ae. aegypti, promoting mortality of 67% of the larvae at a concentration of 250ppm and up to 98% of the larvae at a concentration of 500ppm. The EAM extract of the Pe. virgulata endophyte caused 81% mortality of the larvae at 250ppm and up to 100% of the larvae at 500ppm, thus showing greater effectiveness than the extract from the basidiomycete.

Dose evaluation to obtain the lethal concentrations (LC₅₀ – LC₉₀)

Aedes aegypti. As shown in Table 1, three of six fungal extracts that were assessed to determine their lethal concentrations against Ae. aegypti larvae showed toxicity values in which LC₅₀ < 500ppm. The results calculated using the Polo-PC^® program indicate that the data fit the probit model. The ethyl acetate mycelia extracts from Pe. virgulata and Py. sanguineus showed better mortality results against Ae. aegyptithird instar larvae than the LCM extracts (Table 1, Figures 1 and 2).

FIGURE 1 Graphical representations of the probit values of mortality against the third instar larvae of Aedes aegyptifollowing 24h of treatments with: A: ethyl acetate mycelia (EAM) extract and liquid culture medium (LCM) from Pestalotiopsis virgulata; and B: ethyl acetate mycelia (EAM) extract and liquid culture medium (LCM) from Pycnoporus sanguineus. Pyc: Pycnoporus; EtOAc: ethyl acetate. 

FIGURE 2 Graphical representations of the probit values following 24h of treatment with ethyl acetate mycelia (EAM) extract from Pestalotiopsis virgulata and Pycnoporus sanguineus against third instar larvae of Aedes aegypti (A) and Anopheles nuneztovari (B). Pest: Pestalotiopsis; Pyc: Pycnoporus. 

Figure 1 shows the regression lines for the larval mortality induced by 24h of treatment with the mycelia or LCM EtOAc extracts from Pycnoporus and Pestalotiopsis. We first compared the results from thePestalotiopsis extracts (Figure 1A). In this comparison between the LCM EtOAc-treated Ae. aegypti larvae and the EAM-treated Ae. aegypti larvae, the value of X² was significant. This finding led us to reject the hypothesis that the larvicidal effects of the two extracts would be equal (Table 2). Therefore, the effective doses were qualitatively equal but quantitatively different. The relative potency of the larvicidal effects of the Pestalotiopsis LCM EtOAc extract (0.128) were less than those of the Pestalotiopsis EAM extract (7.769) (Table 2).

We next compared the results of the Pycnoporus extracts against Ae. aegypti larvae. (Figure 1B). In the comparison between the mortality induced by the Pycnoporus EAM extract and that induced by the Pycnoporus LCM EtOAc extract, the hypothesis that the extracts would result in equal rates of mortality was rejected, while the hypothesis that the slopes of the mortality curves would be parallel was not rejected. These data suggest that the effective doses of these fungal extracts were qualitatively equal but quantitatively different. The greatest larvicidal effects were observed for the mycelia extract from P. sanguineus, which also had the highest relative potency (Table 2). Comparing the data from the mycelia fungus extracts from the two fungal species, we observed that the greatest potential larvicidal effects against Ae. aegypti were obtained for the genus Pestalotiopsis. We obtained an LC₅₀ value of 101.8ppm for the Pestalotiopsis EAM and 156.8ppm for Pycnoporus EAM (Table 1 and Figure 2A). As shown in Table 2, the relative potency (RP) values were also calculated, and the Pestalotiopsis EAM (1.634) was shown to be more effective than the Pycnoporus EAM (0.612).

Anopheles nuneztovari

As shown in Table 1, three of five fungal extracts that were assessed to determine the lethal concentration against An. nuneztovari larvae showed toxicity values in which LC₅₀ < 500ppm. The results presented using the Polo-PC^® program indicated that the data fit the probit model. As shown in Table 2, the relative potency (RP) values were calculated, and the Pestalotiopsis EAM extract (RP = 36.221) was more effective than the Pestalotiopsis Mycelium Aqueous Fr. extract (RP = 0.027). In addition, the Pestalotiopsis EAM extract (PR = 47.981) was more effective than the Pestalotiopsis LCM EtOAc extract (PR = 0.019). However, the Pestalotiopsis LCM EtOAc (PR = 1.318) extract was more effective than the Pestalotiopsis LCM Aqueous Fr. (PR = 0.758). The Pestalotiopsis LCM EtOAc was compared with the Pestalotiopsis LCM Aqueous Fr., and statistical analysis led us to reject the hypothesis that the larvicidal effects of the two extracts would be equal, while the hypothesis that the slopes of the mortality curves would be parallel was not rejected. These data indicate that the effective doses of these extracts were qualitatively equal but quantitatively different.

The larvicidal activity of the Pestalotiopsis EAM extract against An. nuneztovari larvae was compared with the larvicidal effects of the Pycnoporus EAM extract against the same larval species, and the LC₅₀ values were observed to be 16.3 and 87.2ppm, respectively, (Table 1 and Figure 2B) while the LC₉₀ values were observed to be 74.4 and 343.7ppm. Table 2 shows that the Pestalotiopsis EAM extract (PR = 4.770) was more effective than the Pycnoporus EAM extract (PR = 0.209). Statistical analysis led us to reject the hypothesis that the larvicidal effects of the two extracts would be equal, while the hypothesis that the slopes of the mortality curves would be parallel was not rejected. These data indicate that the effective doses were qualitatively equal but quantitatively different. Probit results indicated that the hypothesis that the larvicidal effects of the two extracts would be equal could be rejected and that the regression lines were parallel and were thus not significantly different.

Classification following the criteria of Komalamisra et al.³³, with minor modifications, classifies plant larvicidal activities as nontoxic when the LC₅₀ is greater than 750ppm, weakly effective when the LC₅₀ranges from 200 to 750ppm, moderate when the LC₅₀ is 50 – 100ppm and high when the LC₅₀ is less than 50ppm. Thus, the activity of the ethyl acetate extract from Pe. virgulata against Ae. aegypti was considered moderate, while the ethanol extract was considered effective, and the aqueous (alkaline hydroethanolic) extracts showed without/low activity. Among the extracts evaluated against Ae. aegyptilarvae (Table 3), three of the fractions were nontoxic, one showed weak activity, and the other two fractions exhibited moderate toxicity. Among the extracts evaluated against An. nuneztovari larvae (Table 3), two of the fractions were nontoxic, one had weak activity, one displayed moderate activity, and one displayed high activity. Against Ae. aegypti larvae, the EAM extracts from Pe. virgulata and Py. sanguineusshowed weak toxicity, while the aqueous extracts were, for the most part, nontoxic. In contrast, against An. nuneztovari larvae, the EAM extracts from Pe. virgulata and Py. sanguineus had moderate to high toxicity, while the aqueous extracts showed without/low activity.

General

There is growing interest in the use of natural insecticides to reduce the use of synthetic pesticides and avoid environmental damage. The use of larvicidal compounds involves the application of chemicals to habitats to kill pre-adult mosquitoes. This practice can reduce overall pesticide use in a control program, by reducing or eliminating the need for ground or aerial chemical applications to kill adult mosquitoes³⁴. The efficiency in killing larval instars of important vector species and the lack of effects on non-target organisms, as well as the biological stability of extracellular metabolites, make this practice a promising alternative to mycelium- and conidial-based larvicides⁹. These products could be considered fungal-based natural larvicides for vector control. Biological control of immature forms of Anopheles nuneztovari and Anopheles darlingi, which are species that are endemic to the Brazilian Amazon, under both laboratory and field conditions, has been well-documented in the literature³⁵,³⁶.

The purpose of a general screen for bioactivity is to isolate as many potentially active constituents from a species as possible. This goal is achieved using two solvents: water, the most polar solvent, with a polarity index (PI) of 10.2; and an intermediary solvent, such as ethyl acetate (PI = 4.4)³⁷. In this study, the separated fractions were assessed for their ability to control two major species of mosquito larvae (Ae. aegypti and An. nuneztovari). This experiment validated this approach and revealed the efficacies of the extracellular metabolites from Py. sanguineus and Pe. virgulata that were extracted with ethyl acetate.

Liquid culture medium (LCM)

The insecticidal activity assessment of an aqueous extract allows for rapid and easy exploration of many species and compounds that are effective for the control of mosquito larvae³⁸. In the first stage of this study, the aims were to evaluate ethyl acetate extracts from the mycelia and LCM and to evaluate the aqueous fractions of the mycelia and LCM. Thus, the larvicidal activities of the secondary metabolites and extracellular metabolites in the LCM from Pe. virgulata and Py. sanguineus were not confirmed, as our results showed weak/nontoxic activity. As shown in Table 3, we verified that, against Ae. aegypti larvae, only the LCM extract from Py. sanguineus showed weak toxicity (LC₅₀ > 200ppm) at 24h. Unfortunately, it was not possible to perform a bioassay using the Pycnoporus LCM against An. nuneztovari larvae, due to insufficient amounts of the extract.

Liquid culture medium extracts from Pe. virgulata showed an LC₅₀ > 500ppm in every assay; therefore, these extracts had weak/nontoxic activity against both vector species tested (Tables 1 and 3). However, this fungal species was relevant in the initial screening, as the aqueous extract caused larva mortality at high extract concentrations³⁸. This relevance was also evident using aqueous extracts from LCM, which did not have significant larvicidal effects against any of the tested larvae.

The results observed showed that ethyl acetate extracts from the studied fungus mycelia presented better larvicidal activity and that the ethyl acetate extract from the LCM did not induce satisfactory results. Fungi secrete secondary metabolites into their external environment to perform several activities, including obtaining food and providing defense against microorganisms¹²,²². These results suggest that the studied fungi did not produce metabolites with larvicidal activity when grown in liquid culture medium. It is also possible that these metabolites were not produced by these fungi due to other factors, such as the lack of the necessity to produce such compounds in the absence of competition from microbes in the LCM or the lack of some other compound that would induce a fungal response. Finally, it might not be possible to obtain these secondary metabolites from LCM using the methodology applied in this work. Vyas et al.¹³used a different extract protocol to obtain second metabolites, and they demonstrated that Lagenidium giganteum metabolites, when filtered through Whatman filter paper applied directly to third instar larvae of An. stephensi, Cx. quinquefasciatus and Ae. aegypti, had LC₅₀ and LC₉₀ values against An. stephensi of 7.21 and 24.29ppm, respectively and against Cx. quinquefasciatus of 4.09 and 12.12ppm. However, theAe. aegypti larval instars and all nontarget organisms were not found to be susceptible. Mohanty and Prakash¹¹ demonstrated that filtrate extracellular metabolites from Trichophyton ajelloi were efficacious against larvae of the mosquito species Cx. quinquefasciatus and An. stephensi. Further results of Mohanty and Prakash¹² indicated that an LC₅₀ of secondary metabolites against third instar An. stephensi of SDB-2.33mg/250mL, CB-1.28mg/250mL and against Cx. quinquefasciatus of SDB-1.08mg/250mL, CB-0.65mg/250mL was obtained. The purposes of these filtration processes were to decrease impurities in the filtrates and to enhance efficacy. This finding might suggest that further bioassays should utilize the successful techniques used by these authors¹¹−¹³, which might enable investigators to select more efficiently the active components present in the cellular mycelium of Pe. virgulata and Py. sanguineus.

However, An. nuneztovari larvae were susceptible to the aqueous extract fraction from the endophytic fungus Pe. virgulata (LC₅₀ 389.3 ppm; Table 1). Prakash et al.³⁹ utilized filtered metabolites of Fusarium oxysporum as test materials for larvicidal activity against Cx. quinquefasciatus and An. stephensi larvae, while the mycelium mass was discarded.

Ethyl acetate mycelium extract

The ethyl acetate mycelium (EAM) extracts from the entophytic fungus Pe. virgulata and from the basidiomycete fungus Py. sanguineus demonstrated larvicidal activity against both mosquito species tested. The EAM extract from Py. sanguineus (less polar), containing at least two major components (cinnabarin and a mixture of other phenoxazin-3-one substituted components), had higher larvicidal activity and presented greater potentially lethal results than the aqueous extracts (more polar). This finding indicates that the active larvicidal components of mycelia extracts of Pe. virgulata and Py. sanguineus must be low-polarity constituents. This observation agrees with the results of Aivazi and Vijay⁴⁰, who examined the ethyl acetate extract from oak gall (LC₅₀ 116.92ppm). The aqueous extract in this study (more polar) had less activity against the two mosquito species.

The highest larval mortality was induced by the EAM extract from Pe. virgulata and Py. sanguineus. The results of the LC₅₀ calculation of these two extracts demonstrated that these extracts were more effective against Ae. aegypti, with the EAM extract of Pestalotiopsis leading to an LC₅₀ of 101.8ppm (Table 1) and the EAM extract of Pycnoporus leading to an LC₅₀ of 156.8pm. Against An. nuneztovari, the EAM extract ofPestalotiopsis led to an LC₅₀ of 16.3 ppm (Table 1), and the EAM extract of Pycnoporus led to an LC₅₀ of 87.2ppm. Therefore, different sensitivities of mosquito species were observed in response to these extracts. Similar to these results, Bagavan et al.⁴¹ showed that the ethyl acetate extract from the Achyranthes aspera leaf had effective larvicidal activity against Ae. aegypti and Cx. quinquefasciatus of 18.2 and 27.2 ppm, respectively, due to the presence of saponin. The xanthone sterigmatocystin, isolated from the endophytic fungus Podospora sp. and the plant Laggera alata (Asteraceae), was reported⁴² to have a high level of potency against third instar larvae of Anopheles gambiae, with LC₅₀ and LC₉₀ values of 13.3 and 73.5ppm, respectively.

As shown in Table 1, many different reactions to mycelia extract appeared between the two tested mosquito species. The LC₅₀ values of the mycelia extract were very high against Ae. aegypti cases, compared to An. nuneztovari, verifying the increased susceptibility of An. nuneztovari compared to Ae. aegypti larvae. According to Amer and Hehlhorn³⁴, these variations are not abnormal. In their results, the LC₅₀ values of many oils were very high against An. stephensi, compared to the two other mosquito species tested, and except for few cases, Ae. aegypti was more sensitive than Cx. quinquefasciatus. Prakash et al.³⁹ showed that the extracellular metabolites from F. oxysporum were less effective against An. stephensi but were highly effective against Cx. quinquefasciatus larvae. This finding might have been due to the size of Culex, which has more surface area.

With the primary information available, studies can be undertaken to standardize extracts and to identify and isolate active components. Our results clearly show that Py. sanguineus extract, which contains cinnabarin, demonstrates high larval mortality. From the results, we can conclude that the two mosquito species’ larvae were susceptible to the compounds in fungus extracts. Such findings could be useful in promoting research aimed at the development of new mosquito control agents, based on bioactive chemical compounds from indigenous fungus sources as an alternative to chemical larvicides, to suppress malaria vector mosquito populations.

The evaluation of the role of fungus extracts in the larvicidal bioassay against Aedes aegypti and Anopheles nuneztovari has demonstrated promising larvicidal activity. Studies investigating new substances are important to advancing the availability of control alternatives against malaria. Therefore, we conclude that the ethyl acetate mycelia extracts from Pe. virgulata and Py. sanguineus were more effective than ethyl acetate aqueous extracts and liquid culture extracts. The mycelia extract from Pe. virgulata was more effective than the ethyl acetate mycelia extract from Py. sanguineus against Ae. aegypti, as well as against An. nuneztovari larvae. An. nuneztovari larvae were shown to be more susceptible to mycelia extracts from both fungi, in comparison to Ae. aegypti larvae.

The results reported here provide the possibility for further efficient investigation of the larvicidal properties of natural product extracts. The isolation and purification of endophytic fungus crude extracts are in progress.