Synergistic Toxicity of Plant Essential Oils Combined with Pyrethroid Insecticides against Blow Flies and the House Fly

Blow flies (Diptera: Calliphoridae) and the house fly (Diptera: Muscidae) are filth flies of medical importance, and control of their population is needed. As insecticide applications have resulted in fly resistance, and the exploration of plant essential oils (EOs) has increased against filth flies, this study assessed the combination of EOs with pyrethoids to enhance toxic efficacy. The EOs of five effective plants were screened initially against the house fly (Musca domestica L.). Their chemical constituent was performed using gas chromatography-mass spectrometry (GC-MS) analysis. The main components of Boesenbergia rotunda (Zingiberaceae) rhizome, Curcuma longa (Zingiberaceae) rhizome, Citrus hystrix (Rutaceae) fruit peel, Ocimum gratissimum (Lamiaceae) seed, and Zanthoxylum limonella (Rutaceae) fruit were δ-3-caren (35.25%), β-turmerone (51.68%), β-pinene (26.56%), p-cumic aldehyde (58.21%), and dipentene (60.22%), respectively. The screening test revealed that the three most effective plant EOs were from B. rotunda, C. longa and O. gratissimum, which were selected for the combination with two pyrethroid insecticides (permethrin and deltamethrin), in order to enhance their synergistic efficacy against the blow flies, Chrysomya megacephala Fabricius, Chrysomya rufifacies Macquart, and Lucilia cuprina Wiedemann, and the house fly. Synergistic action was presented in almost all of the flies tested with permenthrin/deltamethrin/EOs mixtures. It was interesting that the combination of deltamethrin with three EOs showed a synergistic effect on all of the tested flies. However, an antagonistic effect was observed in C. megacephala and M. domestica treated with permethrin-B. rotunda and C. megacephala treated with permethrin-O. gratissimum. The LD50 of insecticides decreased when combined with plant EOs. This alternative strategy will be helpful in developing a formula for effective fly control management.

insecticide [26]. Therefore, the aim of this study was to determine the synergistic effect of EOs with pyrethroid insecticides (permethrin and deltamethrin) against C. megacephala, C. rufifacies, L. cuprina and M. domestica. The combinations are expected to provide a promising alternative strategy for reducing the use of insecticides, which might be more effective than individual insecticides and plant extracts.

Rearing of Flies
The fly colonies of C. megacephala, C. rufifacies, L. cuprina and M. domestica were obtained from laboratory strains maintained for at least 10 years at the Department of Parasitology, Faculty of Medicine, Chiang Mai University, Thailand. They were reared in cages (30 × 30 × 30 cm) providing water and granulated sugar as food. Then, pork liver was put into the cage as a protein source and oviposition site. After female laid their eggs, approximately 100-200 first instars were transferred to transparent plastic boxes (12 × 15 × 6 cm) and fed with pork liver until the third instar reached prepupa and had no more need for feeding. The lid of each box was replaced with a fine silkscreen cloth sealed with adhesive paper tape for ventilation and prevention of larvae crawling out. The boxes were kept at natural temperatures and relative humidity until adult emergence [27].

Preparation of Plant EOs
Five species of plants, including finger root, Boesenbergia rotunda (root); kaffir lime, C. hystrix (fruit); turmeric, Curcuma longa (root); caraway, Ocimum gratissimum (seed) and szetchwan pepper, Zanthoxylum limonella (seed) (Figure 1) were purchased from a herb stores in Chiang Mai province, Thailand, and identified by a plant taxonomist at the Department of Pharmaceutical Sciences, Faculty of Pharmacy, Chiang Mai University, Thailand. Voucher specimens were deposited at the Department of Parasitology, Faculty of Medicine, Chiang Mai University.
Insects 2019, 10, x 3 of 16 EO and insecticide [26]. Therefore, the aim of this study was to determine the synergistic effect of EOs with pyrethroid insecticides (permethrin and deltamethrin) against C. megacephala, C. rufifacies, L. cuprina and M. domestica. The combinations are expected to provide a promising alternative strategy for reducing the use of insecticides, which might be more effective than individual insecticides and plant extracts.

Rearing of Flies
The fly colonies of C. megacephala, C. rufifacies, L. cuprina and M. domestica were obtained from laboratory strains maintained for at least 10 years at the Department of Parasitology, Faculty of Medicine, Chiang Mai University, Thailand. They were reared in cages (30 × 30 × 30 cm) providing water and granulated sugar as food. Then, pork liver was put into the cage as a protein source and oviposition site. After female laid their eggs, approximately 100-200 first instars were transferred to transparent plastic boxes (12 × 15 × 6 cm) and fed with pork liver until the third instar reached prepupa and had no more need for feeding. The lid of each box was replaced with a fine silkscreen cloth sealed with adhesive paper tape for ventilation and prevention of larvae crawling out. The boxes were kept at natural temperatures and relative humidity until adult emergence [27].

Preparation of Plant EOs
Five species of plants, including finger root, Boesenbergia rotunda (root); kaffir lime, C. hystrix (fruit); turmeric, Curcuma longa (root); caraway, Ocimum gratissimum (seed) and szetchwan pepper, Zanthoxylum limonella (seed) (Figure 1) were purchased from a herb stores in Chiang Mai province, Thailand, and identified by a plant taxonomist at the Department of Pharmaceutical Sciences, Faculty of Pharmacy, Chiang Mai University, Thailand. Voucher specimens were deposited at the Department of Parasitology, Faculty of Medicine, Chiang Mai University. Each plant was removed manually, shade dried in ambient temperature for 5-10 days, and ground to a coarse powder. Then, the powder was distilled following the protocol of Champakaew et al. [28]. About 250-300 g of ground plant powder was placed in an extraction column connected to a distillation flask containing ~1600 mL of distilled water and 10-15 glass beads. The distilled water was boiled to 100 °C in an immersion heater to produce steam that passed through the plant materials. The distillated mixture of essential oil and water was collected and allowed to settle into 2 layers over 3-5 days with the EO on top of the water. The water was released slowly, so that only the Each plant was removed manually, shade dried in ambient temperature for 5-10 days, and ground to a coarse powder. Then, the powder was distilled following the protocol of Champakaew et al. [28]. About 250-300 g of ground plant powder was placed in an extraction column connected to a distillation flask containing~1600 mL of distilled water and 10-15 glass beads. The distilled water was boiled to 100 • C in an immersion heater to produce steam that passed through the plant materials. The distillated mixture of essential oil and water was collected and allowed to settle into 2 layers over 3-5 days with the EO on top of the water. The water was released slowly, so that only the EO remained. The EO was dried over anhydrous sodium sulfate (Na 2 SO 4 ) for 24 h and kept at 4 • C until used.

Gas Chromatographic-Mass Spectrometry Analysis
EOs were analyzed by gas chromatography-mass spectrometry (GC-MS) using a Hewlett-Packard GC-MS system (Model 7890). The analysis was equipped with a split-splitless injector and HP5 mass-selective detector (MSD) (30 m × 0.25 mm ID × 0.25 µm film thickness), coupled with a mass spectrometer selective detector 5975. The column temperature was increased from 50 • C to 250 • C at a rate of 10 • C/min; inlet, 250 • C splitless; injection volume, 0.5 µL; helium as the carrier gas at 1.0 mL/min; injection in split mode (250:1); total run time, 24 min. The MSD 5975 Network (EI) worked with scan parameters, 30-550 amu.; MS Quadrupole, 150 • C and MS Source, 230 • C. This analysis was performed at the Science and Technology Service Center, Chiang Mai University (STSC-CMU).

Preliminary Screening of EOs
Screening for adulticidal toxicity of the five EOs was conducted against adult M. domestica. Each EO was diluted in acetone to obtain five concentrations; and acetone alone was used as a control. Each dose was given to 30 female flies (3-5 days old). The topical application method [29] was performed by applying substances (1 µL/fly) with an autopipette (Sartorius ® , Gottingen, Germany) on the pronotum of CO 2 anesthetized flies. Each experiment was conducted in triplicate, and fly mortality following treatment was checked after 24 h. The lethal dose (LD) was calculated by using Finney's probit analysis [30] for analyzing the mortality response. The three most effective EOs were selected for combining with insecticides.

Adulticidal Bioassay of EOs
Based on preliminary adulticidal screening results, the three most effective EOs were selected for adulticidal toxicity against C. megacephala, C. rufifacies and L. cuprina. The experiment was performed by topical application, with the procedure being the same as that in the preliminary screening of EOs. Each experiment was conducted in triplicate and fly mortality following treatment was checked after 24 h. The LD was calculated using Finney's probit analysis [30].

Adulticidal Bioassay of Insecticides
Technical grade insecticides (permethin and deltamethrin) were used for synergistic study. Permethrin (98.1% analytical standard, Pestanal ® , Seelze, Germany) was diluted in 10 mL of acetone to obtain the stock solution of 500 ppm; while deltamethrin (99.5% analytical standard, Dr. Ehrenstorfer ® , Augsburg, Germany) was dissolved in 10 mL of acetone to prepare stock solution of 500 ppm. Thirty females (3-5 days old) of each fly species were treated with both insecticides. Two hundred µl of each substance was diluted in acetone to a stock solution of five concentrations. Acetone alone was used as a control. Bioassay was carried out as described in Section 2.4. Fly mortality following treatment was checked after 24 h. The LD for insecticides was calculated using Finney's probit analysis [30] for analyzing the mortality response.

Adulticidal Bioassays of Binary Mixtures of EOs with Insecticides
Five variable descending concentrations were prepared for each mixture. These tests were carried out to determine the synergistic/antagonistic action resulted from vary descending at 5 concentrations of insecticides (permethrin/deltamethrin) with EO at its LD 25 value (LD 25 values were estimated mathematically for the expected lowest mortality dose of active compounds). The variation of permethrin and deltamethrin (min-max) concentrations for combined with three EOs (LD 25 ) were shown in Table 1. Then, 30 female flies/replicates were tested using topical application. The control group was treated with acetone only. Experiments were conducted in triplicate. Fly mortality following treatment was checked after 24 h. The synergistic factor (SF) for the mixed formulation was computed after calculating LD 50 for each combination.
The SF [31] for the combined formulation was calculated after calculating the LD 50 for each combination. SF = LD 50 of active substance alone LD 50 of active substance with a synergist SF > 1, indicates synergism; SF < 1, indicates antagonism. Table 1. Variation of permethrin and deltamethrin concentrations for combining with three EOs (LD 25 ).

Statistical Analysis
Regarding the toxicity assays of individual substances (EOs/deltamethrin/permethrin) and the combination of insecticides with EOs, the mortality data were analyzed using LdP line Software (Ehab Mostafa Bakr, Dokki, Cairo, Egypt) to calculate the LD. The Chi-square test was used in each bioassay for the assessment of significance and the measurement of the difference between the test samples. Statistically significant results were considered at p < 0.05.

Physical and Chemical Compositions of EOs
The organoleptic properties and physical characteristics of EOs were different in name, part used, odor, color, density and yield, as shown in Table 2. The percentage of EO yields, measured as yield of EO to dry weight, ranged from 0.57% (v/w) for C. longa to 6.00% (v/w) for C. hystrix. The GC-MS analysis for the EOs is displayed in Table 3. Twenty compounds in the EO from B. rotunda were identified, accounting for 96.77% of the whole oil. The three highest components were δ-3-caren (35.25%), followed by alcanfor (28.08%) and methyl cinnamate (15.10%). The EO of C. longa contained 17 identified compounds, representing 74.73% of the EO obtained. The 3 major components were β-turmerone (51.68%), followed by α-curcumene (7.59%) and β-sesquiphellandrene (4.74%). Twenty-eight compounds were identified from the EO of C. hystrix, accounting for 93.28% of the whole oil, with β-pinene (26.56%) being the principal constituent, followed by α-limonene (25.94%), and sabinene (14.01%). As for O. gratissimum, the EO analysis presented 18 compounds, accounting for 92.84% of the whole oil, with p-cumic aldehyde (58.21%) as the main chemical compound, followed by p-cymene (10.65%) and phenylacetylcarbinol (8.46%). With regard to Z. limonella, 32 compounds were identified, which represented 100% of the whole oil. The main chemical constituents were dipentene (60.22%), followed by sabinene (13.07%) and O-cymol (4.72%).

Adulticidal Activity of EOs
C. longa had the most effective toxicity in the adulticidal screening experiment on M. domestica, with the LD 50 being 77.01 µg/fly, followed by O. gratissimum (83.11 µg/fly), B. rotunda (103.59 µg/fly), C. hystrix (106.87 µg/fly) and Z. limonella (225.50 µg/fly) (Table 4). Therefore, the three most effective EOs-C. longa, B. rotunda and O. gratissimum-were selected for adulticidal activity against the three blow fly species, C. megacephala, C. rufifacies and L. cuprina. LD 25 = lethal dose that killed 25% of the exposed adult flies; LD 50 = lethal dose that killed 50% of the exposed adult flies; LD 99 = lethal dose that killed 99% of the exposed adult flies; UCL: Upper confidence limit; LCL: Lower confidence limit; χ 2 = chi-square; Conc. = concentration; df = degrees of freedom; SE = standard error. The unit of LD is µg/fly. Table 5 shows the results of adulticidal toxicity of the three most effective EOs against the three blow flies. The EO of C. longa was the most effective, with an LD 50 of 59.83 µg/fly, 94.52 µg/fly and 129.73 µg/fly against L. cuprina, C. megacephala and C. rufifacies, respectively. The adulticidal LD 50 of O. gratissimum oil was 68.50 µg/fly, 110.41 µg/fly, and 166.29 µg/fly against L. cuprina, C. megacephala and C. rufifacies, respectively. Regarding B. rotunda oil, the LD 50 was 124.64 µg/fly, 207.32 µg/fly, and 249.73 µg/fly against L. cuprina, C. megacephala and C. rufifacies, respectively (Table 5). No mortality was observed in the control flies.  25 = lethal dose that killed 25% of the exposed adult flies; LD 50 = lethal dose that killed 50% of the exposed adult flies; LD 99 = lethal dose that killed 99% of the exposed adult flies; EO: Essential oil; UCL: Upper confidence limit; LCL: Lower confidence limit; χ 2 = chi-square; df = degrees of freedom; SE = standard error. The unit of LD is µg/fly.

Discussion
Combined toxicity of insecticides with biopesticides has been investigated increasingly in order to reduce the use of insecticides, overcome insecticide resistance, enhance toxicity and more environmental safety. Examples were given in the combination of insecticides with fungicides applied to the honey bee, Apis mellifera [32,33]. This study evaluated the toxicity of pyrethorid insecticides (deltamethrin and permethrin) combined with promising plant EOs against four filth fly species (C. megacephala, C. rufifacies, L. cuprina and M. domestica).
Plants display the phenomenon of allelopathy, whereby they produce or release bioactive compounds that inhibit other organisms. More than 100,000 allelochemical groups are known, e.g., alkaloids, amines, amino acids, cyanogenic compounds, glucosides, glucosinolates, non-proteins, organic acids, peptides phenolics, polyacetylenes, quinones, and terpenoids [34]. Each EO selected herein had a distinct principle component, being δ-3-caren for B. rotunda, β-turmerone for C. longa and p-cumic aldehyde for O. gratissimum. Monoterpene was the compound of these oils and classified as a terpene type [35]. Rice and Coats [36] reported insecticidal activity of several monoterpenoids against M. domestica, the red flour beetle (Tribolium castaneum), and the Southern corn rootworm (Diabrotica undecimpunctata howardi). Molecules of these terpenes can penetrate into the cuticle of insects and interact with the target site; however, some of their portions may be metabolized and neutralized by insect defense mechanisms. Eventually, the amount of terpene molecules (toxicants) reaching the target site determines the toxicity of the substance [37]. Pavela [38] investigated the insecticidal activity of 34 EOs from a range of plants and found that the most effective, with their main components, were Mentha pulegium (pugelone 83.3%), Origanum compactum (carvacrol 58.3% and thymol 12.6%), and Pogostemon cablin (sesquiterpenes 95.3%), which expressed toxicity against M. domestica. The LD 50 of C. longa, B. rotunda, and O. gratissimum EO was lower in this study than permethin/deltamethrin administered alone. This was not surprising, because EOs are composed of both active and inactive compounds, while synthetic insecticides are a single purified active compound [39]. Furthermore, the efficacy of EOs depends on the geographical origin of plants and environmental conditions, which significantly influences the biological properties of EOs [40]. Regarding the adulticidal activity of insecticides, deltamethrin exhibited higher toxicity than permethrin, which is represented by lower LD 50 values in C. megacephala, C. rufifacies and L. cuprina. This result was in contrast to investigations in Chiang Mai during 2015, when permethrin (LD 50 = 0.0028 µg/fly) indicated more susceptibility than deltamethrin (LD 50 = 0.0461 µg/fly) in C. megacephala [7]. The result of the analysis for M. domestica in this study indicated that permethrin exhibited more toxicity than deltamethrin. This agreed with a previous study that showed M. domestica exhibiting more susceptibility to permethrin (LD 50 = 0.0049 µg/fly) than deltamethrin (LD 50 = 0.1058 µg/fly) [7]. Based on permethrin per se, both C. megacephala and M. domestica displayed a slight increase in insecticide resistance from 2015, showing~18-fold and 5-fold increased resistance, respectively [7]. Management of fly populations using chemical control with insecticide could lead to developing insecticide resistance. Meanwhile, biological control with plant EOs could not yield the efficacy achieved by insecticides, but this approach is environmentally safe. The combination in this study of deltametrin with each EO (B. rotunda, C. longa and O. gratissimum) and permethrin-C. longa mixtures showed a synergistic effect in all of the fly species, whereas an antagonistic effect was seen in C. megacephala and M. domestica treated with permethrin-B. rotunda, and in only C. megacephala with permethrin-O. gratissimum treatment. However, the synergistic action of binary mixtures of two insecticides is rarely known [41]. The possible reason for the synergistic efficacy could be the susceptibility of insect, development of resistance, and the different chemical compositions [42]. It is probable that the observed synergism was the result of laboratory flies overwhelmed by the mixture of insecticide combined with EOs that disrupt different target sites. Pyrethroid type I (permethrin) causes presynaptic neuron repetitive discharge in insects, and pyrethroid type II (deltamethrin) causes a tonic release of transmitters which is indicative to membrane depolarization [43], whereas EO causes deterring oviposition, inhibiting growth, repelling, and mimicking juvenile hormones [8,9]. Tong and Bloomquist [39] revealed that all of the 14 EOs, including oils of camphor (Cinnamomum camphora L.), thyme (Thymus serpyllum), Amyris (Amyris balsamifera L.), lemon (Citrus limon L.), cedarwood (Juniperus virginiana), frankincense (Boswellia carteri L.), dill (Anethum graveolens L.), myrtle (Myrtus communis L.), juniper (Juniperus communis L.), black pepper (Piper nigrum L.), verbena (Lippia citriodora L.), Helichrysum (Helichrysum italicum G.), sesame (Sesamum indicum L.), and sandalwood (Santalum album L.), combined with permethrin, had an antagonistic effect against Ae. aegypti larvae after 24 h exposure. Due to the high hydrophobicity and affinity of permethrin with EOs, the authors hypothesized that the molecules of permethrin were dispersed into these EOs and might reduce permethrin bioavailability. Faraone et al. [44] reported that the EO of L. angustifolia and Thymus vulgaris synergized the activity of imidacloprid against the green peach aphid, Myzus persicae, whereas these EOs antagonized another insecticide (spirotetramat). They suggested that the insecticide mode of action or chemical/physical properties are a crucial determinate of whether or not synergism or antagonism of insecticide-EO mixtures would occur. However, the idea of increasing opportunities for the use of EOs in pest management and reducing inputs of synthetic insecticides might be possible. One study reported the synergism of Solanum xanthocarpum with cypermethrin against the mosquito, Anopheles stephensi, with a 1:1 ratio of LD 50 between the EO and insecticide [45]. In addition, Silva et al. [24] showed the same toxicity of Ocimum basilicum-deltamethrin mixtures, which were relative to deltamethrin alone against the fall armyworm, Spodoptera frugiperda, and this combination reduced~80% LD 50 of the deltamethrin used. Combinations between the ethanolic extracts of Cichorium intybus, Azadirachta indica, Conyza aegyptiaca, Piper nigrum, Salix safsaf and Sonchus oleraceus with insecticides (chlorpyrifos, deltamethrin, flufenoxuoron and methomyl) increased the potential effect and reduced the amount of insecticides used against the larvae and adults of the mosquito, Anopheles pharoensis [46]. Maurya et al. [47] revealed the larvicidal activity of Ocimum basilicum-imidacloprid mixtures (ratio 1:1) against An. stephensi, with decreasing LD 50 of the insecticide used, and this formulation was safe for the aquatic mosquito predator, Anisops bouvieri, and other non-target aquatic cyclops. Such information revealed that the combination of plant EOs with insecticides increases the toxic effect against insect vectors/pests. In future, more mixtures of plant EOs with insecticides (e.g., C. longa + O. gratissimum + deltamethrin) merit investigation for enhancing synergistic efficacy for filth flies.
After being administered EO of C. hystrix, morphological aberration of C. megacephala, C. rufifacies, L. cuprina, and M. domestica larvae exhibited deformed midgut and hindgut, vacuolated fat cells, a decreased number of nuclei in the fat cells, and degenerative nuclei [16]. Only the adulticidal toxicity of C. longa, B. rotunda and O. gratissimum EOs combined with insecticides was assessed in this study. More bionomic investigations merit further study to determine the post-exposure of flies; for example, the development of immature stages, adult longevity and survival pattern, reproductive potential, and morphological abnormality.

Conclusions
This study assessed the synergistic action of adulticidal toxicity of plant EOs combined with permethrin/deltamethrin against C. megacephala, C. rufifacies, L. cuprina, and M. domestica. Although this study found the antagonistic effect in some cases, the most synergistic effect was found in the combination of Permethrin-C. longa against L. cuprina, with the highest SF of 10.35 and Deltamethrin-C. longa against C. rufifacies, with the highest SF of 7.57. These EOs presented a promising role as synergists to improve the efficacy of permethrin and deltamethrin. The mode of binary mixture application (e.g., repeated exposure with different dosages) merits further investigation for facilitating fully-competent fly management.