Mortality, Biological, and Biochemical Response of Musca domestica (Diptera: Muscidae) to Selected Insecticides1
The concentration–mortality response of Musca domestica L. (Diptera: Muscidae) to nine insecticides, and the impacts of these insecticides on selected biological and biochemical parameters of the insect, were determined in laboratory assays. Adults displayed a concentration-dependent response for each insecticide. Median lethal concentration (LC50) values in baits were: acetamiprid (0.39 μg/ml), bifenthrin (0.22 μg/ml), chlorpyriphos (0.21 μg/ml), deltamethrin (0.41 μg/ml), emamectin benzoate (0.001 μg/ml), fipronil (0.002 μg/ml), imidacloprid (0.27 μg/ml), profenophos (0.63 μg/ml), and lufenuron (0.001 μg/ml). Based on 95% confidence intervals, fipronil proved to be the most lethal of the insecticides tested. LC10, LC30, and LC50 values of each of the insecticides were used to assess impacts on M. domestica longevity, fecundity, percentage eclosion, larval duration, percentage pupation, pupal weight, pupal duration, adult emergence, and sex ratio. In general, development parameters, with the exception of larval duration, were significantly (P > 0.05) altered in a concentration-dependent manner for each insecticide. Furthermore, enzymatic activity of total glutathione S-transferases, total esterases, acetylcholinesterase, and acid and alkaline phosphates was elevated at the LC10, LC30, and LC50 levels of the nine insecticides, which may contribute to development of resistance to these insecticides.Abstract
The house fly, Musca domestica L. (Diptera: Muscidae), is a highly mobile insect pest that is often associated with decomposing matter and can vector disease-causing microbes (Fasanella et al. 2010; Ugbogu et al. 2006). Management relies mainly on insecticides (Ahmed et al. 2004; Shi et al. 2011); however, nonjudicial use of insecticides has resulted in development of resistance to the insecticide (Butler et al. 2007; Kozaki et al. 2009; Memmi 2010) and to environmental contamination (Yadav 2010). Insecticides with novel modes of action are now employed due to their effectiveness and low mammalian toxicity (Korrat et al. 2012; Shi et al. 2011) but should be used wisely to avoid development of resistance (Khan et al. 2013).
In addition to the direct lethal effects of insecticides on the target insect, which is indicated by lethal concentration or lethal dose values (Piri et al. 2014), sublethal effects of exposure to insecticides may affect physiological, behavioral, and developmental factors that will impact the next generation of the insect (Desneux et al. 2007; Miao et al. 2014). Longevity, fecundity, fertility, and changes in enzymatic activity reflect physiological impacts while behavioral changes may result in altering feeding and oviposition (Fujiwara et al. 2002; Liu and Trumble 2005; Zalizniak and Nugegoda 2006). Previous studies have demonstrated stimulated reproductive potential of target pests at low concentrations of insecticides (Tang et al. 2015; Zhang et al. 2015), decreased adult fecundity and survival at low concentrations (Han et al. 2012; Rehan and Freed 2015a), and alteration of the function of glutathione S-transferases (GSTs), esterases (ESTs), and other metabolic enzymes (Mouches et al. 1986; Piri et al. 2014).
In insects, GSTs are involved in the defense of target insects against insecticides (Yu 2004) and induce resistance against insecticides by coalescing reduced glutathione to the insecticide, as observed with organophosphate and pyrethroid resistance in insect species (Fragoso et al. 2003; Wei et al. 2001). Increased esterase levels also have been reported to illicit resistance to different insecticide groups such as organophosphates, carbamates, and pyrethroids (Mouches et al. 1986; Peiris and Hemingway 1993). Acetylcholinesterase (AChE) plays a vital role in neurotransmission and its function is targeted by organophosphate and carbamate insecticides. AChE found not to respond to those insecticides is an important detoxification mechanism against insecticides in many insect species (Walsh et al. 2001; Weill et al. 2003). For identification of underlying resistance mechanisms, enzyme assay therefore is an easy and insightful method for identifying underlying resistance mechanisms. The positive correlation of insecticide resistance with detoxification enzyme activity underlines the need for quantification of these enzymes in monitoring resistance development for improved management of insect pests (Yaqoob et al. 2013).
The importance of studying sublethal effects of insecticides on target insects is critical to developing and using new insecticides, delaying development of resistance, and decreasing pest resurgence risk (Xu et al. 2016). Our objectives were to define the toxicity of acetamiprid, bifenthrin, chlorpyriphos, deltamethrin, emamectin benzoate, fipronil, imidacloprid, profenophos, and lufenuron against M. domestica and to determine the sublethal effects of these insecticides on selected developmental and biological parameters of the insect as well as the activity of selected enzymes (i.e., total glutathione S-transferases, total esterases, acetylcholinesterase, acid and alkaline phosphatases) following exposure.
Materials and Methods
Insects
Musca domestica adults were reared in the Laboratory of Insect Microbiology and Biotechnology, Department of Entomology, Bahauddin Zakariya University, Multan, Pakistan. The adults were maintained in rearing cages (30 × 30 × 30 cm) covered with mesh screen and equipped with a cloth sleeve at the front for handling rearing cage contents. The rearing conditions were 26 ± 2°C, 50 ± 5% relative humidity (RH), and a 12:12 (L:D) period (Farooq and Freed 2016). Adults were provided with sugar and powdered milk (3:1) and water ad libitum while wheat bran, rice meal, yeast, and dry milk powder (40:10:3:3:1, respectively) as a water-based paste was provided in cages as an egg-laying medium (Bell et al. 2010).
Concentration–mortality response
Nine commercial-grade formulated insecticides belonging to different mode of action groups (Table 1) were tested in these bioassays; all were being applied at local poultry farms for control of M. domestica. Concentrations, ranging from 0.25 to 2.0 ppm for each insecticide, were prepared by serial dilution. Each suspension was mixed with sugar, which was used as a food bait for adult M. domestica. Two hundred flies including controls were employed for each insecticide, which was replicated three times. Insects and baits were placed in plastic containers (15 × 6 × 6 cm) and maintained at the aforementioned conditions. Mortality was recorded at 24, 48, and 72 h after initiation of the test.
Sublethal effects
The LC10, LC30, and LC50 concentrations of each insecticide were used to determine the sublethal effects on longevity, fecundity, eclosion, pupal weight, and sex ratio. The bait method was again used, with each concentration of insecticide being mixed with sugar, and with four replicates per treatment and 40 insects per replicate. Adults 4–5 days old at a sex ratio of 1:1 were placed in the plastic containers and provided baits and egg-laying medium as previously described. Baits with no insecticide served as controls. Adult longevity was recorded for each sex as per Fletcher et al. (1990). The egg-laying medium was examined daily for eggs and, if present, the eggs were counted with the aid of a hand lens. Eggs remained in the medium until eclosion. Fecundity was calculated by dividing the total number of eggs oviposited in the medium by the number of females in the containers (Crystal 1964). Percentage eclosion was calculated by dividing the number of larvae hatched by the total number of eggs oviposited (Sanil and Shetty 2012). Neonates remained in the rearing medium and were examined daily to estimate larval duration as being the interval between initiation of the 1st instar until pupation (Elkattan et al. 2011). To calculate percentage pupation, numbers of pupae were counted and divided by the total number of larvae. Pupae were also weighed and placed in separate containers until adult emergence, at which point pupal duration and percent emergence could be calculated as per Khazanie (1979) while number of males and females were counted to calculate sex ratio.
Enzyme and protein activity
Musca domestica adults were exposed to the LC10, LC30, and LC50 concentrations of each insecticide, and subsequent survivors at 24, 48, and 72 h after exposure were homogenized in PBG (100 mM phosphate buffer, pH = 7.5, 20% glycerol) and centrifuged at 13684.3 g for 10 min at 4°C. The supernatant was stored at −20°C for further testing. Total GST activity was determined with 1-chloro-2,4dinitrobenzene (CDNB) as substrate (Kristensen 2005). The reaction rate was determined for 5 min at 30°C at 340 nm using kinetic and lag period of 2 min. The incubation mixture for a 1-ml quartz cuvette contained 30 μl sample, 950 μl phosphate buffer (PB), 10 μl of CNDB (100 mM), and 10 μl of GSH (100 mM) for each sample. Total EST activity was determined by hydrolysis rate of p-nitrophenylacetate (PNPA) (Joffe et al. 2012). The reaction rate was determined for 5 min at 405 nm using a kinetic and lag period of 1 min. Each sample 1-ml quartz cuvette contained 10 μl sample, 980 μl PB, and 10 μl of PNPA (100 mM). AChE activity was determined by ATCI and DTNB solution at 30°C (Kristensen et al. 2006). The incubation mixture contained a 15-μl sample, 950 μl PB, and was incubated for 2 min at 30°C. Later, 30 μl of DTNB (10 mM) and 5 μl of ATCI (10 mM) were added for color development. The optical density was measured every 30 s for 5 min at 412 nm. Acid and alkaline phosphatase activity was determined by the hydrolysis rate of p-nitrophenyl phosphate (Serebrov et al. 2006). For acid phosphatase, a 25-μl sample and 535 μl citrate PB (pH = 5.0) were incubated for 2 h at 30°C while alkaline phosphatase employed a 25-μl sample and 535 μl Tris HCl buffer (pH = 8.8) and was incubated for 2 h at 30°C. Later, 425 μl NaOH (0.05N) was added to each well for color development. The optical density was measured at 410 nm. Protein concentration of the samples was determined by the Bradford (1976) assay with bovine serum albumin as the standard at 595 nm.
Statistical analysis
Morality data were corrected using Abbott's formula (Abbott 1925), and the concentration–mortality response was analyzed by probit analysis (Finney 1971) using POLO-PC software (Polo-PC 1987) to determine LC10, LC30, and LC50 values with associated slopes and 95% confidence intervals. Data from biological parameter testing and enzymatic activity assays were analyzed using analytical software Statistix version 8.1 (McGraw-Hill 2008), and treatment means were compared using the honest significant difference (HSD) test at P = 0.05.
Results
Concentration–mortality responses
Lethal concentrations (LC) as determined by probit analysis with associated confidence intervals and regression line slopes for each insecticide are listed in Table 1. Based on comparison of LC50 values, fipronil proved be to the most toxic insecticide against M. domestica adults followed by emamectin benzoate and lufenuron. The LC10, LC30, and LC50 values were used in assessing the sublethal effects of the insecticides on M. domestica adults and their progeny.
Sublethal effects on longevity
Male longevity decreased as insecticide concentration increased (Table 2). In comparison to the controls, male longevity was significantly reduced by the LC50 concentration of acetamiprid (8.03 ± 0.09 d) and the LC50 concentration of emamectin benzoate (8.68 ± 0.32 d) (F = 2.81; df = 9, 18; P = 0.0008) (Table 2). A similar trend was observed with female longevity where the LC50 concentrations of fipronil and acetamiprid significantly reduced longevity to 8.25 ± 0.25 d and 8.66 ± 0.27 d, respectively (F = 5.51; df = 9, 18; P < 0.0001) (Table 2) .
Sublethal effects on fecundity and eclosion
In general, fecundity decreased with the increased concentration of insecticide. The fewest number of eggs was observed in the treatment with an emamectin benzoate LC50 concentration (116.50 ± 5.18) followed by the LC50 concentrations of lufenuron (125.00 ± 5.93) and acetamiprid (128.00 ± 5.35) (F = 5.09; df = 9, 18; P < 0.0001) (Table 2). A similar trend was observed with egg eclosion. The lowest percentage of eclosion was observed with the LC50 concentrations of fipronil (69.25 ± 1.00), emamectin benzoate (72.15 ± 1.63), and imidacloprid (74.50 ± 0.65) (F = 3.31; df = 9, 18; P = 0.0001) (Table 2).
Sublethal effects on larvae and pupae
Larval duration was not significantly affected by insecticide or sublethal insecticide concentration. However, the pupation percentage decreased with increasing concentration of insecticide. Imidacloprid, emamectin benzoate, and fipronil at their LC50 concentrations caused significant reductions in percentage of larvae successfully pupating (F = 2.27; df = 9, 18; P = 0.006) (Table 2). Pupal weight was also significantly reduced in treatments of the LC50 concentrations of fipronil, imidacloprid, and profenophos (F = 3.57; df = 9, 18; P < 0.0001) (Table 2). Insecticidal treatments also prolonged the duration of pupal period with the longest durations observed with the LC50 concentrations of lufenuron (8.91 ± 0.33 d), emamectin benzoate (8.63 ± 0.18 d), and profenophos (8.51 ± 0.19 d) (F = 4.40; df = 9, 18; P < 0.0001) (Table 2).
Sublethal effects on adult emergence and sex ratio
A significantly lower percentage of adults emerged in the LC50 (64.83 ± 1.77) and LC30 (67.58 ± 1.31) concentrations of fipronil (F = 1.92; df = 9, 18; P < 0.03) (Table 2). A significantly lower percentage of females emerged in the acetamiprid LC50 treatment (42.25 ± 2.29) (F = 1.83; df = 9, 18; P = 0.03) (Table 2).
Sublethal effects on enzymatic activity
GST activity was significantly increased after 72 h of exposure to the acetamiprid, bifenthrin, and imidacloprid LC50 concentrations as compared to other treatments (F = 3.39; df = 9, 18; P < 0.001) (Fig. 1A). Similar trends were observed for the LC30 and LC10 concentrations of acetamiprid and bifenthrin (Fig. 1B, C) while chlorpyriphos induced a much different GST activity after 24 h of exposure to the LC10 concentration (F = 5.54; df = 9, 18; P < 0.001) (Fig. 1C).
Citation: Journal of Entomological Science 53, 1; 10.18474/JES17-22.1
In comparison to other treatments, deltamethrin significantly increased EST activity in the LC50 concentration after 24 h (F = 2.39; df = 9, 18; P = 0.01) (Fig. 2A). Increased activity was also observed in treatments with the LC30 concentrations of deltamethrin at 24 h and bifenthrin and acetamiprid at 72 h (F = 2.02; df = 9, 18; P = 0.03) (Fig. 2B). At the LC10 concentration, bifenthrin, acetamiprid, and deltamethrin showed elevated EST activity after 72 h. Elevated EST activity was also observed with LC10 concentrations of fipronil and chlorpyriphos after 24 h of treatment (F = 4.47; df = 9, 18; P < 0.001) (Fig. 2C).
Citation: Journal of Entomological Science 53, 1; 10.18474/JES17-22.1
Acetamiprid, fipronil and bifenthrin at their LC50 concentrations significantly amplified AChE activity at 72 h (F = 2.46; df = 9, 18; P = 0.01) (Fig. 3A). Elevated enzymatic activities were also observed with fipronil and acetamiprid at 48 and 72 h (F = 2.09; df = 9, 18; P = 0.03) (Fig. 3B) while acetamiprid and bifenthrin at their LC10 concentrations elevated AChE activity at 72 h and deltamethrin, emamection benzoate, and fipronil elevated AChE activity at 48 and 72 h (F = 2.23; df = 9, 18; P = 0.01) (Fig. 3C).
Citation: Journal of Entomological Science 53, 1; 10.18474/JES17-22.1
The LC50 concentrations of acetamiprid and deltamethrin elevated acid phosphatase activity 48 and 24 h, respectively (F = 2.88; df = 9, 18; P < 0.001) (Fig. 4A) while the LC30 concentration of bifenthrin significantly increased enzymatic activity at 72 h (F = 1.13; df = 9, 18; P = 0.01) (Fig. 4B). Elevated activity was also observed with the LC10 concentration of acetamiprid at 72 and 48 h, bifenthrin at 72 h, deltamethrin and lufenuron at 48 h, and fipronil at 24 h (F = 3.54; df = 9, 18; P < 0.001) (Fig. 4C). Alkaline phosphatase activity was elevated by LC50 concentrations of fipronil at 48 h, profenophos at 24 and 48 h, and imidacloprid at 72 h (F = 2.69; df = 9, 18; P < 0.001) (Fig. 5A). Acetamiprid at the LC30 concentration significantly increased enzymatic activity (F = 3.70; df = 9, 18; P < 0.001) (Fig. 5B) while for LC10 the concentration of chlorpyriphos elevated activity at 24 h and acetamiprid at 72 h (F = 3.98; df = 9, 18; P < 0.001) (Fig. 5C).
Citation: Journal of Entomological Science 53, 1; 10.18474/JES17-22.1
Citation: Journal of Entomological Science 53, 1; 10.18474/JES17-22.1
Discussion
In the current study, LC10, LC30, and LC50 of nine different insecticides calculated by preliminary experimentation were assessed for their sublethal effects on biological and biochemical parameters of M. domestica. The sublethal effects of insecticides must be taken into account for their impact on the next generation of insect pests, as it explains the behavioral and physiological impacts which enable insects to survive after pesticide exposure (Desneux et al. 2007). In addition, a life table study was deemed as an inclusive method to evaluate the insecticide for its total effect on an insect population (Tuan et al. 2016).
In the current study, the insecticide concentrations significantly reduced the longevity of adults, especially in the case of emamectin benzoate and fipronil. Lee (2000) reviewed the sublethal effects of insecticides on longevity and fecundity of insect pests including Aedes aegypti (L.) (Diptera: Culicidae), Plutella xylostella (L.) (Lepidoptera: Plutellidae), and M. domestica. The results of the current research are in accordance to Hamilton and Schal (1990), who reported a shorter life span of Blattella germanica L. (Dictyoptera: Blattellidae) as a result of application of chlorpyrifos-methyl at LC10, LC20, and LC60 levels. In the current study, the shorter life span of female flies affected the fecundity in all treatments, which is in accordance with Ahmed and Wilkins (2001), who reported the reduction in the fecundity of insecticide-resistant strains of M. domestica. Furthermore, it may also be speculated that the insecticides affected ovaries of female flies, resulting in reduced egg laying (Perveen and Miyata 2000).
The hatching percentage was reduced at higher levels of insecticide in comparison to lower concentrations and the control, favoring the prior studies where the LC25 concentration of methoxyfenozide reduced the hatching percentage of Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae) (Enríquez et al. 2010), while the larval duration was not significantly reduced for all levels of insecticides. In addition, pupation percentage of M. domestica was reduced and significantly differed in comparison to the control. Similar results comparable to the current study were observed by Abouelghar et al. (2013) when sublethal concentrations of spinosad reduced the pupal percentage of Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae). The results regarding pupal weight are in accordance to Rehan and Freed (2015b), where spinosad significantly affected the pupal weight of the Spodoptera litura (F.) (Lepidoptera: Noctuidae). Pupal duration was prolonged, in accordance with Xu et al. (2016), who found that doses of cyantraniliprole resulted in prolonged pupal duration of Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae). In our study, maximum reduction in adult emergence from 94.25–62.83% was observed in comparison to the control. Similar results were observed by Miao et al. (2016), who showed a significant reduction in adult emergence of Megacopta cribraria (F.) (Hemiptera: Plataspidae) at below-lethal concentrations of imidacloprid. In addition, our results showed sex ratio to significantly differ among the treatments, which is in agreement with Sanil and Shetty (2012), who observed sex ratio changes with Anopheles stephensi (Liston) (Diptera: Culicidae) following treatments with temephos and propoxur at LC10, LC30, and LC50 concentrations.
In general, increased activity of detoxification enzymes indicates existence of a resistance mechanism in the insects in which the activity occurs. The important detoxification enzymes involved during degradation of toxic compounds include GSTs, ESTs, cytochrome P450 monooxygenases, acid phosphatases, and alkaline phosphatases (Yang et al. 2001). In our study, higher activities of detoxification enzymes were recorded with the assumption that increased levels of activity would result in resistance development. GST activity increased with exposure to acetamiprid, bifenthrin, and imidacloprid at the higher concentrations tested. The increase in GST activity may indicate its involvement in the detoxification process of acetamiprid, bifenthrin, and imidacloprid. Earlier studies recognized the GST system as a major mechanism involved in insecticide resistance in metabolizing several endogenous compounds (Flores et al. 2006; Gunasekaran et al. 2011; Yu 2004). In addition, noticeably increased levels of EST activities were recorded with deltamethrin, bifenthrin, and acetamiprid as compared to other treatments. However, no fixed trend was found for the increase in EST activity over time. In earlier studies, esterase-based resistance was reported for organophosphorus, carbamate, and pyrethroid insecticides (El-Latif and Subrahmanyam 2010; Field et al. 1988).
Our study demonstrates that AChE activity in M. domestica significantly increased after exposure to bifenthrin, acetamiprid, and fipronil. Regardless of the fact that AChE is not a target for bifenthrin, acetamiprid, and fipronil, the current study corroborates similar findings where AChE activity can be utilized as a biomarker for insecticide sensitivity (Jemec et al. 2007). Moreover, significantly increased AChE activity in M. cribraria by imidacloprid (LC40) (Miao et al. 2016) further supports the validity of our findings.
In addition, acid and alkaline phosphatases hydrolyze phospho-monoesters under acid or alkaline conditions. For acid phosphatases, the acetamiprid, bifenthrin, and deltamethrin showed significantly higher activities. Elevated levels of acid phosphatases were reported in Schistocerca gregaria (Forskal) (Orthoptera: Acrididae) by application of Ammi visnaga L. extracts (Ghoneim et al. 2014). Furthermore, chlorpyriphos, acetamiprid, bifenthrin, fipronil, and profenophos showed increased activities of alkaline phosphatases in comparison to other treatments. Similar results were reported by Emtithal and Thanaa (2012), where alkaline phosphatases may be the possible cause of detoxification of chlorpyriphos in Culex pipiens (L.) (Diptera: Culicidae).
Insect survival to sublethal levels of an insecticide results from increased selection pressure in favor of insecticide resistance based on physiological changes (i.e., increased gene copy number) in coding of a supplementary protective enzyme to aid breakdown of toxins into less toxic compounds (Daly et al. 1978). Moreover, pesticide adaption usually results in decreased relative fitness, and resistant insects have reduced reproductive potential and longevity (Stenersen 2004). The sublethal effects of LC10, LC30, and LC50 of insecticides in our study affected the normal developmental stages and longevity of M. domestica. In addition, increased activity of detoxification enzymes may aid in resistance development. Insecticide application may result in population decline by not only killing susceptible individuals but also by reducing reproductive potential in the surviving insects (Rao and Shetty 1992). Moreover, elevated enzyme activity at LC10, LC30, and LC50 of insecticides provides information for underlying resistance development in the M. domestica population. However, further research is needed for exploring the association of these findings in field conditions as involves insecticide selection and resistance management.
Total glutathione S-transferases (GST) activity of M. domestica at (A) LC50, (B) LC30, and (C) LC10 of different insecticides. Asterisk (*) shows significant difference between the enzyme activity at 24, 48, and 72 h (honest significant difference test, P ≤ 0.05).
Total esterase (EST) activity of M. domestica at (A) LC50, (B) LC30, and (C) LC10 of different insecticides. Asterisk (*) shows significant difference between the enzyme activity at 24, 48, and 72 h (honest significant difference test, P ≤ 0.05).
Acetylcholinesterase (AChE) activity of M. domestica at (A) LC50, (B) LC30, and (C) LC10 of different insecticides. Asterisk (*) shows significant difference between the enzyme activity at 24, 48, and 72 h (honest significant difference test, P ≤ 0.05).
Acid phosphatase activity of M. domestica at (A) LC50, (B) LC30, and (C) LC10 of different insecticides. Asterisk (*) shows significant difference between the enzyme activity at 24, 48, and 72 h (honest significant difference test, P ≤ 0.05).
Alkaline phosphatase activity of M. domestica at (A) LC50, (B) LC30, and (C) LC10 of different insecticides. Asterisk (*) shows significant difference between the enzyme activity at 24, 48 and 72 h (honest significant difference test, P ≤ 0.05).
Contributor Notes