Parasitol Res DOI 10.1007/s00436-015-4394-2
ORIGINAL PAPER
Pediculicidal treatment using ethanol and Melia azedarach L João Ricardo Rutkauskis & Debora Jacomini & Livia Godinho Temponi & Maria Helena Sarragiotto & Edson Antonio Alves da Silva & Tereza Cristina Marino Jorge
Received: 28 January 2015 / Accepted: 19 February 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Pediculosis is an infestation of the scalp caused by Pediculus humanus capitis, known as lice, which affects thousands of people throughout the world. Disease control is achieved by topical insecticides, whose indiscriminate use has led to the emergence of resistant populations of lice. Melia azedarach L. (Meliaceae) is an Asian tree that is found in Brazil, where it is popularly known as cinnamon or santabárbara. This study aimed to evaluate a pediculicidal treatment, made from a hydroethanolic extract of M. azedarach, and to study the effect of extraction solvents (ethanol and water) on insect mortality. The chemical composition of crude extract was studied by gas chromatography, identifying 32 methyl esters of fatty acids, with esters of heneicosanoic, palmitic, and arachidic acids present in greatest abundance. The 1H and 13C nuclear magnetic resonance (NMR) spectra suggested the presence of flavonoids and terpenes. QuercetinJ. R. Rutkauskis : D. Jacomini : T. C. M. Jorge Center of Medical Sciences and Pharmaceutical, UNIOESTE, Cascavel, Brazil L. G. Temponi Center of Biological and Health Sciences, UNIOESTE, Cascavel, Brazil M. H. Sarragiotto Center of Exact Sciences, Department of Chemistry, Universidade Estadual de Maringá (UEM), Colombo Av. no. 5790, Maringá, PR, Brazil E. A. A. da Silva Center of Exact Sciences and Technology, UNIOESTE, Cascavel, Brazil T. C. M. Jorge (*) Laboratory of Pharmacognosy, Universidade Estadual do Oeste do Paraná (UNIOESTE), University Street no. 2069, University Campus, 85819-110 Cascavel, PR, Brazil e-mail: [email protected]
3-O-β- D -glucopyranoside (1) and quercetin-3-O-β- D glucopyranosyl-(1→6)-O-β-D-glucopyranoside (2) were isolated from the extract. The bioassay of pediculicidal activity shows that the M. azedarach extract had a pediculicidal activity, inducing the death of all lice faster than 1 % permethrin, a topical insecticide commonly used to control lice. Keywords Melia azedarach . Meliaceae . Pediculicidal activity
Introduction Pediculosis is an infestation of the scalp caused by Pediculus humanus capitis (Anoplura: Pediculidae) De Geer, popularly known as head lice (Ko and Elston 2004). The disease is a public health problem, with global prevalence estimated at 300 million cases per year (Mumcuoglu et al. 2009). Lice measure 1 to 3 mm and can live on average for 30 days. During this period, each female louse deposits 300 eggs, known as nits. After the nits hatch, they form nymphs, which turn into adult insects in about 10 days (Chosidow 2000). Lice are not able to jump or fly. Transmission occurs through direct contact with infected individuals or personal objects (Gratz 1997). This infestation has been increasing in the world and is often endemic in developing countries. In Brazil, studies indicate that the disease affects all age groups in poor communities, and the prevalence can reach 40 % of the general population (Heukelbach et al. 2008). Female children are often the most affected, and the number of cases varies with the time of year (Feldmeier 2012). The most common symptom of lice infestation is severe pruritus, but the infestation also causes excoriations on the skin, allergies, sleep loss, secondary infections, and inflammatory reactions caused by the bite and saliva
Parasitol Res
of the lice (Oh et al. 2010). In addition to these symptoms, pediculosis can also cause psychological problems as a result of embarrassment and low self-esteem in infested children, particularly aggravated by the exclusionary practices adopted in some schools (Lebwhol et al. 2007). The main form of pediculosis control occurs by applying topical insecticides such as pyrethroids (Asenov et al. 2010). Permethrin is a pyrethroid widely used in pediculicidal treatments, mainly because it has low toxicity in humans (Isman 2006). In the USA, spending on pediculicidal treatments in the year 2000 was approximately US$ 1 billion, in addition to losses arising from school days and work days lost by infested children and family. It is estimated that most pediculicidal treatments occur unnecessarily, through the indiscriminate use of pesticides (Hansen and O’Haver 2004). Treatments usually begin after detecting the presence of infestation in nearby people or colleagues in classrooms. This fact contributes to the development of insect resistance to products used to control lice, creating the need to develop new alternatives for the treatment of this disease (Marshall Clark et al. 2013). Some species of plants have been used for therapeutic purposes for at least 60,000 years and are the basis of traditional medicine (Fabricant and Farnswoth 2001). The species Melia azedarach L. (Meliaceae) is a native tree of China and India, but is now found all over the planet. In Brazil, it can be found in the south and southeast, where it is popularly called cinnamon or santa-bárbara (Lorenzi and Matos 2003). This plant is well-known in popular medicine and has been investigated scientifically, with various biological activities such as antioxidant, antimicrobial, antiprotozoal, antinematodal, anti-inflammatory, antipyretic, and antiulcerative (Zhao et al. 2010). However, the main characteristic of M. azedarach is its insecticidal activity, which is generally related to the presence of limonoids (Viegas 2003). Many studies using natural products have been conducted in attempts to develop new options to control lice. Some had interesting results, for example, the use of Vitex agnus castus to repel lice (Semmler et al. 2010) and the treatment with seed extract and a shampoo based on Azadirachta indica A. Juss (Meliaceae), a species closely related to M. azedarach (AbdelGhaffar et al. 2012; Heukelbach et al. 2006; Mehlhorn et al. 2011. Although the insecticidal properties of these plants are well-known, little research has specifically investigated the pediculicidal activity of M. azedarach. In this sense, the phytochemical study of this plant has shown promising results, indicating that these extracts are active on both the eggs and nits of P. humanus capitis (Carpinella et al. 2007; Jorge et al. 2009). Thus, motivated by the increase in P. humanus capitis infestations in populations resistant to commercially available insecticides and pediculicides and knowing the activities of M. azedarach, we carried out this study in order to evaluate the pediculicidal activity of treatment with a 50 %
hydroethanolic extract produced from the fresh leaves and branches of M. azedarach and to study the effects of the extraction solvent (ethanol or water) on insect mortality.
Experimental Plant material M. azedarach leaves and branches were collected in March 2013 from an exemplar located in the city of Cascavel (24° 59′ 21″ S and 53° 26′ 54″ W), southern Brazil. The plant was identified by Prof. Dr. Livia Godinho Temponi, by comparison with voucher no. 4703 deposited in the herbarium of the Biological Sciences and Health Center of the State University of West of Paraná (UNIOESTE).
Materials and equipment Sephadex LH-20 (GE Healthcare) was used for the isolation and purification of extracts. The thin-layer chromatographic analyses were performed on silica gel 60 G and 60 GF-254 (Vetec). The 1H and 13C NMR spectra were obtained on a Varian Mercury (300 MHz) or a Bruker (500 MHz) spectrometer in the Department of Chemistry, State University of Maringá (UEM). Deuterated solvents used were D 2 O, CD3OD, and CDCl3 (Tedia). The gas chromatography (GC) analyses were performed on Trace GC Ultra chromatograph (Thermo Scientific), at the Department of Pharmacy UEM, based on the pattern of a C4-C24 fatty acid methyl ester (FAME) mix (Sigma-Aldrich). A fused silica column (100 m long, 0.25 mm internal diameter, and 0.20 mm in the SE-30 stationary phase) was used, as well as permethrin lotion 1 % (Multilab) and a magnifying glass (Tecnival).
Plant extract Fresh leaves and branches of M. azedarach (600 g) were triturated and immersed in 2000 mL of a 50 % (v/v) hydroethanol solution for 24 h. After this period, the extractive mixture was filtered and 600 mL (15 mg mL−1) was stored in a freezer (−4 °C) for subsequent bioassays. The remaining filtrate was concentrated on a rotary evaporator at reduced pressure, and the residual water was removed by lyophilization. After removing the solvent, 59,981 g of the crude extract (CE) was obtained, which was fractionated by liquid-liquid extraction with solvents of variable polarity to give 0.527 g of hexane extract, 2.203 g of dichloromethane extract (CH2Cl2), 0.940 g of ethyl acetate extract (EtOAc), and 53.577 g of hydromethanolic extract (H2O:MeOH).
Parasitol Res
Chemical characterization of the plant extract
Pediculicidal activity test
Analysis of the hexane fraction by gas chromatography
Collection of parasites
The esterification of fatty acids in the hexane fraction was performed before the chromatographic analysis by adding 1 mL of 1 % NaOH in MeOH to 100 μg of this fraction, followed by heating for 15 min at 55 °C. Then, 2 mL of a solution of 5 % HCl in MeOH was added to the mixture, which was heated at 55 °C for 15 min. At the end of the reaction, 1 mL of deionized water was added. The FAME was extracted using hexane in a separatory funnel, and the fractions were pooled and concentrated on a rotary evaporator under reduced pressure. After esterification, FAME was analyzed on a GC chromatograph equipped with a flame ionization detector (FID) and a fused silica capillary column. The sample (2 μL) was added into the injector and detector at 235 °C. The temperature changed over time; the heating ramp started at 65 °C for 4 min and was programmed up to 185 °C at a rate of 16 °C min−1 and then kept constant for 12 min, followed by heating to 235 °C at a rate of 20 °C min−1 and then held constant for 14 min. The whole chromatographic run took 40 min. The gas flow was 1.4 mL min−1 for the carrier gas (He), 35 mL min−1 for the make-up gas (N2) and flame, 350 mL min−1 for synthetic air, and 35 mL min−1 for H2. The identification of components was done by comparison with the retention times of the signals obtained with FAME standards. The integration and calculation of the area of each peak of the chromatogram was performed using the ChromQuest program.
P. humanus capitis were collected from children aged 3– 12 years old, from public schools and daycare centers in the city of Cascavel; the children were not under treatment for pediculosis. The collection of insects was performed according to protocol no. 322307 of the Ethics Committee for Research on Human Beings of UNIOESTE. The presence of head lice in children was found by visual inspection, and insects were carefully removed from the scalp with the aid of a comb.
Study of the ethyl acetate and hydroethanol fractions of the plant extract The EtOAc fraction (287 mg) was subjected to a Sephadex LH-20 column (60 cm×1.5 cm) eluted with a gradient of MeOH:CH2Cl2 (100:0; 76:25; 50:50), which afforded 16 fractions. Fractions 4 and 5, after analysis by TLC, were combined and subjected to another Sephadex LH-20 column in MeOH, yielding 12 fractions. Fraction 7 (25 mg), after being again subjected to a Sephadex LH-20 column, resulted in the isolation of 8 mg of a light-green solid named compound 1. The H2O:MeOH fraction (475 mg) was subjected to a Sephadex LH-20 column (60 cm×1.5 cm) and eluted with a gradient of H2O:MeOH (100:0; 75:25; 50:50; 25:75; 0:100), which produced 30 fractions. Fraction 13 (5 mg) was suspended in MeOH, and the soluble portion was transferred to another flask. After evaporation of the solvent, 3 mg of a light-green solid compound named 2 was obtained. The crude extract and CH2Cl2, EtOAc, H2O:MeOH fractions as well as compounds 1 and 2 were characterized by 1H and 13C NMR analyses.
Bioassay The pediculicidal activity test was carried out at room temperature employing a suitable method proposed by the World Health Organization (WHO 1981). For the bioassay, five treatments were used: the 50 % hydroethanolic extract of M. azedarach (Ma), ethanol (EtOH), the 50 % (v/v) hydroethanol solution (EtOH:H2O), as well as the positive control, permethrin lotion 1 % (Per 1 %) (10 mg mL−1), and the negative control, distilled water. The lice were removed from the scalp and placed in a watch glass in groups of five insects. Each group of five lice was placed in a Petri dish lined previously with a thin layer of cotton wool soaked in 5 mL of one of the treatments. Mortality assessments were made using a magnifying glass (10-fold magnification) after the parasites were placed on the treated plates. Lice not showing movement were considered dead. In total, 100 lice were used, distributed in 20 Petri dishes, separated into five groups of four plates. Each of the five groups of four plates received one of the treatments. The order in which treatments were placed on each plate was set at random. Mortality readings were taken at 1, 3, 5, 7, 10, 15, 20, 25, 30, 45, 60, 90, 120, 150, and 180 min, as well as at 6, 9, 12 18 and 24 h. Readings were taken at all measurement times, even after the verified death of insects.
Statistical analysis The experiment employed a completely randomized design. The results are expressed as the percentage mortality of parasites. Statistical analysis was performed using the KruskalWallis test (p value
ORIGINAL PAPER
Pediculicidal treatment using ethanol and Melia azedarach L João Ricardo Rutkauskis & Debora Jacomini & Livia Godinho Temponi & Maria Helena Sarragiotto & Edson Antonio Alves da Silva & Tereza Cristina Marino Jorge
Received: 28 January 2015 / Accepted: 19 February 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Pediculosis is an infestation of the scalp caused by Pediculus humanus capitis, known as lice, which affects thousands of people throughout the world. Disease control is achieved by topical insecticides, whose indiscriminate use has led to the emergence of resistant populations of lice. Melia azedarach L. (Meliaceae) is an Asian tree that is found in Brazil, where it is popularly known as cinnamon or santabárbara. This study aimed to evaluate a pediculicidal treatment, made from a hydroethanolic extract of M. azedarach, and to study the effect of extraction solvents (ethanol and water) on insect mortality. The chemical composition of crude extract was studied by gas chromatography, identifying 32 methyl esters of fatty acids, with esters of heneicosanoic, palmitic, and arachidic acids present in greatest abundance. The 1H and 13C nuclear magnetic resonance (NMR) spectra suggested the presence of flavonoids and terpenes. QuercetinJ. R. Rutkauskis : D. Jacomini : T. C. M. Jorge Center of Medical Sciences and Pharmaceutical, UNIOESTE, Cascavel, Brazil L. G. Temponi Center of Biological and Health Sciences, UNIOESTE, Cascavel, Brazil M. H. Sarragiotto Center of Exact Sciences, Department of Chemistry, Universidade Estadual de Maringá (UEM), Colombo Av. no. 5790, Maringá, PR, Brazil E. A. A. da Silva Center of Exact Sciences and Technology, UNIOESTE, Cascavel, Brazil T. C. M. Jorge (*) Laboratory of Pharmacognosy, Universidade Estadual do Oeste do Paraná (UNIOESTE), University Street no. 2069, University Campus, 85819-110 Cascavel, PR, Brazil e-mail: [email protected]
3-O-β- D -glucopyranoside (1) and quercetin-3-O-β- D glucopyranosyl-(1→6)-O-β-D-glucopyranoside (2) were isolated from the extract. The bioassay of pediculicidal activity shows that the M. azedarach extract had a pediculicidal activity, inducing the death of all lice faster than 1 % permethrin, a topical insecticide commonly used to control lice. Keywords Melia azedarach . Meliaceae . Pediculicidal activity
Introduction Pediculosis is an infestation of the scalp caused by Pediculus humanus capitis (Anoplura: Pediculidae) De Geer, popularly known as head lice (Ko and Elston 2004). The disease is a public health problem, with global prevalence estimated at 300 million cases per year (Mumcuoglu et al. 2009). Lice measure 1 to 3 mm and can live on average for 30 days. During this period, each female louse deposits 300 eggs, known as nits. After the nits hatch, they form nymphs, which turn into adult insects in about 10 days (Chosidow 2000). Lice are not able to jump or fly. Transmission occurs through direct contact with infected individuals or personal objects (Gratz 1997). This infestation has been increasing in the world and is often endemic in developing countries. In Brazil, studies indicate that the disease affects all age groups in poor communities, and the prevalence can reach 40 % of the general population (Heukelbach et al. 2008). Female children are often the most affected, and the number of cases varies with the time of year (Feldmeier 2012). The most common symptom of lice infestation is severe pruritus, but the infestation also causes excoriations on the skin, allergies, sleep loss, secondary infections, and inflammatory reactions caused by the bite and saliva
Parasitol Res
of the lice (Oh et al. 2010). In addition to these symptoms, pediculosis can also cause psychological problems as a result of embarrassment and low self-esteem in infested children, particularly aggravated by the exclusionary practices adopted in some schools (Lebwhol et al. 2007). The main form of pediculosis control occurs by applying topical insecticides such as pyrethroids (Asenov et al. 2010). Permethrin is a pyrethroid widely used in pediculicidal treatments, mainly because it has low toxicity in humans (Isman 2006). In the USA, spending on pediculicidal treatments in the year 2000 was approximately US$ 1 billion, in addition to losses arising from school days and work days lost by infested children and family. It is estimated that most pediculicidal treatments occur unnecessarily, through the indiscriminate use of pesticides (Hansen and O’Haver 2004). Treatments usually begin after detecting the presence of infestation in nearby people or colleagues in classrooms. This fact contributes to the development of insect resistance to products used to control lice, creating the need to develop new alternatives for the treatment of this disease (Marshall Clark et al. 2013). Some species of plants have been used for therapeutic purposes for at least 60,000 years and are the basis of traditional medicine (Fabricant and Farnswoth 2001). The species Melia azedarach L. (Meliaceae) is a native tree of China and India, but is now found all over the planet. In Brazil, it can be found in the south and southeast, where it is popularly called cinnamon or santa-bárbara (Lorenzi and Matos 2003). This plant is well-known in popular medicine and has been investigated scientifically, with various biological activities such as antioxidant, antimicrobial, antiprotozoal, antinematodal, anti-inflammatory, antipyretic, and antiulcerative (Zhao et al. 2010). However, the main characteristic of M. azedarach is its insecticidal activity, which is generally related to the presence of limonoids (Viegas 2003). Many studies using natural products have been conducted in attempts to develop new options to control lice. Some had interesting results, for example, the use of Vitex agnus castus to repel lice (Semmler et al. 2010) and the treatment with seed extract and a shampoo based on Azadirachta indica A. Juss (Meliaceae), a species closely related to M. azedarach (AbdelGhaffar et al. 2012; Heukelbach et al. 2006; Mehlhorn et al. 2011. Although the insecticidal properties of these plants are well-known, little research has specifically investigated the pediculicidal activity of M. azedarach. In this sense, the phytochemical study of this plant has shown promising results, indicating that these extracts are active on both the eggs and nits of P. humanus capitis (Carpinella et al. 2007; Jorge et al. 2009). Thus, motivated by the increase in P. humanus capitis infestations in populations resistant to commercially available insecticides and pediculicides and knowing the activities of M. azedarach, we carried out this study in order to evaluate the pediculicidal activity of treatment with a 50 %
hydroethanolic extract produced from the fresh leaves and branches of M. azedarach and to study the effects of the extraction solvent (ethanol or water) on insect mortality.
Experimental Plant material M. azedarach leaves and branches were collected in March 2013 from an exemplar located in the city of Cascavel (24° 59′ 21″ S and 53° 26′ 54″ W), southern Brazil. The plant was identified by Prof. Dr. Livia Godinho Temponi, by comparison with voucher no. 4703 deposited in the herbarium of the Biological Sciences and Health Center of the State University of West of Paraná (UNIOESTE).
Materials and equipment Sephadex LH-20 (GE Healthcare) was used for the isolation and purification of extracts. The thin-layer chromatographic analyses were performed on silica gel 60 G and 60 GF-254 (Vetec). The 1H and 13C NMR spectra were obtained on a Varian Mercury (300 MHz) or a Bruker (500 MHz) spectrometer in the Department of Chemistry, State University of Maringá (UEM). Deuterated solvents used were D 2 O, CD3OD, and CDCl3 (Tedia). The gas chromatography (GC) analyses were performed on Trace GC Ultra chromatograph (Thermo Scientific), at the Department of Pharmacy UEM, based on the pattern of a C4-C24 fatty acid methyl ester (FAME) mix (Sigma-Aldrich). A fused silica column (100 m long, 0.25 mm internal diameter, and 0.20 mm in the SE-30 stationary phase) was used, as well as permethrin lotion 1 % (Multilab) and a magnifying glass (Tecnival).
Plant extract Fresh leaves and branches of M. azedarach (600 g) were triturated and immersed in 2000 mL of a 50 % (v/v) hydroethanol solution for 24 h. After this period, the extractive mixture was filtered and 600 mL (15 mg mL−1) was stored in a freezer (−4 °C) for subsequent bioassays. The remaining filtrate was concentrated on a rotary evaporator at reduced pressure, and the residual water was removed by lyophilization. After removing the solvent, 59,981 g of the crude extract (CE) was obtained, which was fractionated by liquid-liquid extraction with solvents of variable polarity to give 0.527 g of hexane extract, 2.203 g of dichloromethane extract (CH2Cl2), 0.940 g of ethyl acetate extract (EtOAc), and 53.577 g of hydromethanolic extract (H2O:MeOH).
Parasitol Res
Chemical characterization of the plant extract
Pediculicidal activity test
Analysis of the hexane fraction by gas chromatography
Collection of parasites
The esterification of fatty acids in the hexane fraction was performed before the chromatographic analysis by adding 1 mL of 1 % NaOH in MeOH to 100 μg of this fraction, followed by heating for 15 min at 55 °C. Then, 2 mL of a solution of 5 % HCl in MeOH was added to the mixture, which was heated at 55 °C for 15 min. At the end of the reaction, 1 mL of deionized water was added. The FAME was extracted using hexane in a separatory funnel, and the fractions were pooled and concentrated on a rotary evaporator under reduced pressure. After esterification, FAME was analyzed on a GC chromatograph equipped with a flame ionization detector (FID) and a fused silica capillary column. The sample (2 μL) was added into the injector and detector at 235 °C. The temperature changed over time; the heating ramp started at 65 °C for 4 min and was programmed up to 185 °C at a rate of 16 °C min−1 and then kept constant for 12 min, followed by heating to 235 °C at a rate of 20 °C min−1 and then held constant for 14 min. The whole chromatographic run took 40 min. The gas flow was 1.4 mL min−1 for the carrier gas (He), 35 mL min−1 for the make-up gas (N2) and flame, 350 mL min−1 for synthetic air, and 35 mL min−1 for H2. The identification of components was done by comparison with the retention times of the signals obtained with FAME standards. The integration and calculation of the area of each peak of the chromatogram was performed using the ChromQuest program.
P. humanus capitis were collected from children aged 3– 12 years old, from public schools and daycare centers in the city of Cascavel; the children were not under treatment for pediculosis. The collection of insects was performed according to protocol no. 322307 of the Ethics Committee for Research on Human Beings of UNIOESTE. The presence of head lice in children was found by visual inspection, and insects were carefully removed from the scalp with the aid of a comb.
Study of the ethyl acetate and hydroethanol fractions of the plant extract The EtOAc fraction (287 mg) was subjected to a Sephadex LH-20 column (60 cm×1.5 cm) eluted with a gradient of MeOH:CH2Cl2 (100:0; 76:25; 50:50), which afforded 16 fractions. Fractions 4 and 5, after analysis by TLC, were combined and subjected to another Sephadex LH-20 column in MeOH, yielding 12 fractions. Fraction 7 (25 mg), after being again subjected to a Sephadex LH-20 column, resulted in the isolation of 8 mg of a light-green solid named compound 1. The H2O:MeOH fraction (475 mg) was subjected to a Sephadex LH-20 column (60 cm×1.5 cm) and eluted with a gradient of H2O:MeOH (100:0; 75:25; 50:50; 25:75; 0:100), which produced 30 fractions. Fraction 13 (5 mg) was suspended in MeOH, and the soluble portion was transferred to another flask. After evaporation of the solvent, 3 mg of a light-green solid compound named 2 was obtained. The crude extract and CH2Cl2, EtOAc, H2O:MeOH fractions as well as compounds 1 and 2 were characterized by 1H and 13C NMR analyses.
Bioassay The pediculicidal activity test was carried out at room temperature employing a suitable method proposed by the World Health Organization (WHO 1981). For the bioassay, five treatments were used: the 50 % hydroethanolic extract of M. azedarach (Ma), ethanol (EtOH), the 50 % (v/v) hydroethanol solution (EtOH:H2O), as well as the positive control, permethrin lotion 1 % (Per 1 %) (10 mg mL−1), and the negative control, distilled water. The lice were removed from the scalp and placed in a watch glass in groups of five insects. Each group of five lice was placed in a Petri dish lined previously with a thin layer of cotton wool soaked in 5 mL of one of the treatments. Mortality assessments were made using a magnifying glass (10-fold magnification) after the parasites were placed on the treated plates. Lice not showing movement were considered dead. In total, 100 lice were used, distributed in 20 Petri dishes, separated into five groups of four plates. Each of the five groups of four plates received one of the treatments. The order in which treatments were placed on each plate was set at random. Mortality readings were taken at 1, 3, 5, 7, 10, 15, 20, 25, 30, 45, 60, 90, 120, 150, and 180 min, as well as at 6, 9, 12 18 and 24 h. Readings were taken at all measurement times, even after the verified death of insects.
Statistical analysis The experiment employed a completely randomized design. The results are expressed as the percentage mortality of parasites. Statistical analysis was performed using the KruskalWallis test (p value
