Ecotoxicology and Environmental Safety 100 (2014) 1–6
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Biocontrol potential of essential oil monoterpenes against housefly, Musca domestica (Diptera: Muscidae) Peeyush Kumar, Sapna Mishra, Anushree Malik n, Santosh Satya Applied Microbiology Laboratory, Centre for Rural Development & Technology, Indian Institute of Technology Delhi, New Delhi 110016, India
art ic l e i nf o
a b s t r a c t
Article history: Received 17 September 2013 Received in revised form 11 November 2013 Accepted 18 November 2013
Housefly (Musca domestica L.), one of the most common insects in human settlements, has been associated as vectors for various food-borne pathogens, causing food spoilage and disease transmission. The control of housefly was attempted using plant monoterpenes; menthone, menthol, menthyl acetate, limonene, citral and 1,8-cineole, against different life stages of housefly. Bioefficacy against housefly adults revealed highest repellent activity by menthol (95.6 percent) and menthone (83.3 percent). Against housefly larvae, menthol with an LC90 of 0.02 ml/cm2 in contact toxicity assay and menthone with a LC90 value of 5.4 ml/L in fumigation assay were found to be most effective control agent. With respect to pupicidal activity, superior performance was shown by menthol, citral and 1,8-cineole in contact toxicity assay and citral and 1,8-cineole in fumigation assay. Limonene was found to be the poorest performer in all the assays. Overall, highest efficacy observed for menthol and menthone in various bioassays was in agreement with the results of essential oil activity obtained previously. Significant activity of monoterpenes against various life stages of housefly demonstrates their potential as excellent insecticides with prospects of monoterpenes being developed into eco-friendly and acceptable products for housefly control. & 2013 Elsevier Inc. All rights reserved.
Keywords: Musca domestica Essential oil Monoterpenes Bioefficacy Repellency
1. Introduction The housefly (Musca domestica L.), one of the most common insects in human settlements, has been associated as vectors for various food borne pathogens. Houseflies breed in rotting vegetable matter or animal feces, from where they acquire and transmit pathogenic organisms to foodstuffs, causing food spoilage and disease transmission. On a conservative estimate, houseflies are associated with vectoring of more than 100 etiological agents of bacterial, protozoan, and viral diseases (Fotedar, 2001; Kumar et al., 2012a). Conventional control of housefly is based on the use of chemical insecticides. However, these chemicals are not only detrimental to animal and human health but also cause various types of soil and water pollution. Besides, gradually insects tend to develop resistance against these insecticides leading to pest resurgence (Kumar et al., 2011, 2012a. Additionally, these insecticides are toxic to a large spectrum of animal species, thus, also killing non-target organisms. Efforts are thus being made world over to replace these synthetic chemicals with alternatives which are safer and do not cause any toxicological effects on the environment.
n
Corresponding author. Fax: þ 91 11 26591121. E-mail address: [email protected] (A. Malik).
0147-6513/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2013.11.013
In our search for housefly control agents compatible with human surroundings and food materials, essential oils and terpenoids could be a good alternative. Terpenoids are major components of plant essential oils. Terpenoids mostly constitute of monoterpenes and sesquiterpenes and are supposedly responsible for pesticidal activity of essential oils. The insecticidal activity of monoterpenes has been reported against various agricultural and household pests with a potential effect as contact toxicant, fumigant, repellent and antifeedant (Lee et al., 2003; Abdelgaleil et al., 2009; Nathan et al., 2008). Rice and Coats (1994) investigated a number of monoterpenes and their derivatives in different bioassays against adult housefly. Similarly, Palacios et al. (2009a) reported potent fumigant activity of various monoterpenes against adult housefly while their repellency activity was demonstrated by Roitberg and Isman (1992). The above literature studies establish pesticidal potential of monoterpenes against housefly. However, most of these studies have been conducted against adult housefly, which at a given time form only 15 percent of the total housefly population (Novartis, 2013). In comparison, the investigations on use of monoterpenes for control of other stages of housefly remain neglected. Moreover, most of the studies in literature reports have utilized fumigation assay for housefly control. Although breeding sites of housefly (larvae or pupae) may create a microhabitat, ensuring fumigation activity of monoterpenes; fumigation assay against housefly adults may be an impractical approach, as in real life situation housefly adults rarely remain confined within a space to allow fumigation
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P. Kumar et al. / Ecotoxicology and Environmental Safety 100 (2014) 1–6
action of essential oils or monoterpenes to take place. In view of the above facts, the present study was conducted to evaluate the activity of six monoterpenes; menthone, menthol, menthyl acetate, limonene, citral and 1,8-cineole, against different stages of housefly. The activity was assessed through repellency (against adults) and contact toxicity and fumigation (larvae and pupae). Monoterpenes, menthone, menthol and menthyl acetate were chosen as they formed the major components of Mentha x piperita (peppermint) essential oil, which was found to be highly effective against housefly in earlier studies (Kumar et al., 2011, 2012b. Limonene is principal constituent of Citrus sinensis (orange) essential oil, which was reported to be most potent against housefly adults (Palacios et al., 2009a). Citral and 1,8-cineole are major components of Cymbopogon citratus (lemongrass) and Eucalyptus globulus (blue gum), respectively. The insecticidal activities of both the essential oils against housefly have been reported in earlier studies (Kumar et al., 2011, 2012c, 2013.
2. Materials and Methods
2.5. Fumigation assay Fumigation assay was performed in a 1 L conical flask (Kumar et al., 2013). Cotton swabs impregnated with different concentrations of monoterpenes (1, 3, 5, 7, 10 ml/L of air) were attached to the cork of the flask, while larvae/pupae were placed at the bottom surface of conical flask. Control was treated with acetone alone. Each treatment incorporated three replicates. During the assay, flasks were kept at the temperature of 28 7 2 1C and RH of 65 percent. Larvicidal assay was done with twenty lab reared larvae (2nd instars) placed in conical flask along with larval diet. Any change in treated larvae or mortality was observed till 48 h. Pupicidal assay was carried out with twenty pupae (2–3 days old). Observations for adult emergence in treated pupae were made till six days.
2.6. Statistical analysis The repellency data of essential oils was abbot corrected by accounting the number of flies repelled in the control (Abbott, 1925). The values for LC50, LC90, chi-square and 95 percent-confidence intervals for each regression coefficient were calculated by using probit analysis (Finney, 1971). A two-way analysis of variance (ANOVA) was used to analyze the effect of varying doses and exposure periods on larval mortality. All statistical analysis including preparation of dendogram was performed using the statistical software SPSS version 17.0 (SPSS, 2008).
2.1. Materials The monoterpenes (menthone, menthol, menthyl acetate, limonene, citral and 1,8-cineole) were purchased from Sigma Aldrich, India.
2.2. Housefly Adult houseflies collected from field by sweep net method, were reared in cylindrical boxes containing a diet of groundnut oil cake and wheat bran (1:3), according to the method described earlier (Kumar et al., 2011). Hatched larvae were transferred individually to cylindrical vials (28 12 mm2) containing a larval diet (constituents: 2 g groundnut oil cake, 5 g wheat bran, 2 g milk powder, 1 g honey mixed with 10 ml of water) which was changed daily until larvae reached the pupal stage. For repellency bioassay, field flies were used while larvicidal and pupicidal bioassay were done with larvae and pupae obtained from rearing of field flies. 2.3. Repellency assay The repellent effect of different monoterpenes was evaluated against field collected adult houseflies, irrespective of their sex. Repellency assay was performed in a repellency chamber described earlier (Kumar et al., 2011). Outer chamber of the repellency chamber was connected to the inner chamber through an opening (dia., 60 mm). Adult flies (50) were placed in the outer chamber along with petri plate (dia., 90 mm) containing filter paper impregnated with monoterpene solutions ( E 0.16 μl/cm2) diluted in acetone. Control was treated with 0.5 ml of acetone. Treated and control discs were air-dried for 5 min to evaporate the solvent completely, before experiment initiation. Houseflies moving from outer to inner chamber were counted as being repelled. Three replicates of each monoterpene treatment were performed.
2.4. Contact toxicity assay
3. Results 3.1. Repellency assay The repellency activity against housefly adults varied significantly among different monoterpenes (F¼93.9; df ¼ 5; Po 0.001). Also, significant variation between monoterpenes was observed for different time of observation (F¼56.7; df ¼7; Po 0.001). Repellency assay with different monoterpenes is represented in Fig. 1. Menthol was found to be most superior repellent (95.6 percent), while limonene was a poor performer with only 38 percent of overall repellency after 2 h of observation. The figure clearly showed almost similar trend in percentage repellency with monoterpenes of menthone (83.3 percent) and citral (76.0 percent) and menthyl acetate (67.3 percent) and 1,8-cineole (64.0 percent). Dendogram based on the repellency results for different monoterpenes showed a cluster formed by monoterpenes (menthone, citral, menthyl acetate and 1,8-cineole), indicating close repellency relationship between them (Suppl. Fig. 1). The higher repellency activity of menthol was also reflected by its RT50, the time for repellency of 50 percent of adult flies, which was 16.28 min (Suppl. Table 1). Menthone and citral with RT50 of 21.39 and 33.54 min, respectively, showed adequate housefly repellency activity, as well as followed the repellency activity trend reflected by percentage repellency of adult housefly using monoterpenes.
The contact toxicity assay was performed in a Petri plate (dia., 90 mm) according to the method described by Kumar et al. (2013). Different concentrations of monoterpenes (0.016, 0.047, 0.079, 0.11 and 0.16 μl/cm2) were prepared by mixing their variable volumes with 0.5 ml of acetone and applied on the filter paper using a micropipette. Control Petri plate was treated with acetone alone. Treated filter paper was air dried for 5 min, before introduction of larvae/pupae to Petri plates. Three replicates of each oil treatment were performed. Petri plates were kept at the temperature of 28 7 2 1C and relative humidity (RH) of 65 percent. For each larvicidal bioassay, twenty larvae (2nd instars) were kept on filter paper along with larval diet and the treated larvae were observed for any change in appearance and mobility for next four days. Assay on housefly pupae was performed with twenty pupae (2–3 days old). Any adult emergence in the control and treated pupae were observed till six days. Activity of oil on housefly pupae was calculated (Kumar et al., 2011) as the percentage reduction in adult emergence or inhibition rate. Percentage inhibition rate (PIR) was calculated as: %IR or PIR ¼
Cn T n 100 Cn
where Cn is the number of newly emerged insects in the untreated (control) Petri plates and Tn is the number of insects in the treated Petri plates.
Fig. 1. Repellency efficacy (%) of different monoterpenes (0.16 ml/cm2) against adult housefly, Musca domestica, in a repellency chamber experiment.
P. Kumar et al. / Ecotoxicology and Environmental Safety 100 (2014) 1–6
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Table 1 Lethal concentrations of essential oil monoterpenes against housefly larvae in contact toxicity assay. Monoterpenes
Day 1
Day 2
Day 3
LC50 (LCI–UCI)a
LC90 (LCI–UCI)a
Chi-square (χ2)
LC50 (LCI–UCI)a
LC90 (LCI–UCI)a
Chi-square (χ2)
LC50 (LCI–UCI)a
LC90 (LCI–UCI)a
Chi-square (χ2)
Menthol
–b
0.785
–
0.020 (–)
2.49
–
–
–
Menthyl acetate
0.038 (0.0–0.067)c 0.023 (0.0–0.066) 0.068 (0–0.133) 0.033 (–) 0.111 (0.058–1.385)
0.051 (0.018–0.106) 0.179 (0.133–0.350) 0.144 (0.095–1.154) 0.295 (0.188–2.677) 0.194 (–) 0.356 (0.217–12.181)
0.684
0.011 (0.0–0.035)
0.096 (0.073–0.151)
1.433
–
0.663
0.115 (–)
3.037
0.200 (0.135–0.946)
0.915
0.004 (0.0–0.025) –
0.079 (0.55–0.141) 0.064
0.120 (–) 0.151 (–)
1.211 0.631
– 0.0 (0.0–0.018)
Menthone Limonene Citral Cineole
a b c
3.304 0.493 1.276 0.178
0.001 (0.0–0.048) – 0.012 (–)
0.109 (0.081–0.197) 0.033 (–) 0.043 (0.028–0.088)
1.173 1.514 2.101 0.850
LC50 and LC90, values in μl/cm2; LCI, lower limit of 95% confidence interval; UCI, upper limit of 95% confidence interval. –, the values could not be obtained during statistical analysis. 0.0, negative value obtained through statistical analysis.
3.2. Larvicidal assay through contact toxicity The percent mortality of housefly larvae with different monoterpenes was highly significant at different concentration (F¼76.66; df¼ 4, 89; Po0.0001) and time (F¼30.26; df¼17, 89; Po0.0001). Significant results were also obtained for interaction between concentration and time (F¼38.28; df¼ 68, 89; Po0.0001). Activity of different monoterpenes in contact toxicity assay is represented in terms of lethal concentrations (Table 1). The monoterpene concentrations required to kill housefly larvae varied with time of observation. For day 2, the values of lethal concentration, LC90 was lowest for menthol (LC90, 0.02 ml/cm2), followed by citral (LC90, 0.092 ml/cm2), menthyl acetate (LC90, 0.096 ml/cm2), menthone (LC90, 0.115 ml/cm2) and 1,8-cineole (LC90, 0.151 ml/cm2). Limonene with a LC90 value of 0.2 ml/cm2 was observed to be least effective. The statistical evaluation of insecticidal activity revealed similarity in activity for menthone and citral and menthyl acetate and limonene (Suppl. Fig. 2). 3.3. Larvicidal assay through fumigation Housefly larvicidal activity through fumigation toxicity varied significantly between different monoterpenes (Po 0.01). Significant results were also obtained for the percent mortality of housefly larvae for different concentrations of monoterpenes (F¼ 102.76; df ¼ 4, 179; Po 0.0001), exposure period (F¼126.67; df ¼35, 179; P o0.0001) and interaction between concentration and time (F¼ 42.17; df ¼140, 179; P o0.0001). Menthol was found to be the most effective fumigant with an LC90 value of 5.4 ml/L, at 32 h (Table 2). At 32 h, values of LC90 for different monoterpenes varied between 5.4 and 18.53 ml/L. Observation of relationship between monoterpenes based on their fumigation toxicity resulted in the formation of two distinct groups; those with higher activity (menthol, citral, 1,8-cineole, menthyl acetate) and another with comparatively lower larvicidal activity (menthone, limonene) (Suppl. Fig. 3).
indicate PIR values at lower concentrations. The box plot indicates absolute inhibition in adult emergence (PIR-100 percent) with menthol, citral and 1,8-cineole, at their highest concentration. Value of PIR at different tested concentrations varied between 47.3 and 100 percent for menthol, while for menthyl acetate and menthone, it was 36.8–73.7 percent and 42.1–94.7 percent, respectively. For limonene, PIR varied between 36.8 and 89.5 percent. Citral and 1,8-cineole were observed to have higher PIR values even at lower concentration, implying suppression of adult emergence at very low concentrations. Although, menthol and menthone showed comparatively low values for PIR at lower concentrations, pupae treated with these monoterpenes showed high incidence of incomplete emergence and malformation in emergence of adult flies. Moreover, the emerged flies showed overall stunted growth. 3.5. Pupicidal assay through fumigation In the fumigation assay against housefly pupae, significant results were observed for growth inhibition of housefly pupae for different monoterpenes (F¼20.98; df ¼5, 29; P o0.0001), and at different concentrations of monoterpenes (F¼14.97; df ¼ 4, 29; Po 0.0001). From the box plot, the highest effectivity was observed for 1,8-cineole with PIR values varying between 90 and 100 percent (Fig. 3). Asterisk in the box plot represents 90 percent PIR observed at lowest concentration of 1,8-cineole. The activity of 1,8-cineole was followed closely by citral (88.9–100 percent). Menthol (73.7–100 percent), menthone (68.4–100 percent), menthyl acetate (57.9–89.5 percent) and limonene (36.8–89.5 percent) also showed appreciable pupicidal activity. Malformation, reduced size and stunted growth was observed in emerged adult flies obtained from pupae treated with menthol, menthone and menthyl acetate.
4. Discussion 3.4. Pupicidal assay through contact toxicity Pupicidal assay through contact toxicity showed significant variation in growth inhibition for housefly pupae with different monoterpenes (F¼27.31; df ¼ 5, 29; Po0.0001), and with variation in concentration of monoterpenes (F¼29.19; df ¼4, 29; P o0.0001). Values of PIR for monoterpenes treated housefly pupae are represented through a box plot (Fig. 2). Horizontal lines on the box and stem of the box indicate PIR values for a particular concentration. Further, the lower horizontal lines in a vertical bar
The present study has shown higher activity of menthol in almost all the assays against housefly. The result is supported by the study of Rice and Coats (1994), who investigated a number of monoterpenes and their derivatives in fumigation assay against housefly adults and reported higher activity of menthol (LC50, 3.6 mg/cm3) than menthone (LC50,13.7 mg/cm3). The higher fumigation efficacy of menthol was also reflected in the study of Lee et al. (2003), which reported 100 percent mortality of adult housefly at 50 mg/cm3. Samarasekera et al. (2008) found superior repellent
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P. Kumar et al. / Ecotoxicology and Environmental Safety 100 (2014) 1–6
Table 2 Lethal concentrations of essential oil monoterpenes against housefly larvae in fumigation assay. Statistical values
Time (h)
Monoterpenes Menthol
Menthyl acetate
Menthone
Limonene
Citral
Cineole
LC50 (LCI–UCI)a LC90 (LCI–UCI)a Chi-square (χ2)
8
12.69 (–)b 17.26 (–) 0.614
12.65 (–) 18.02 (–) 2.226
12.69 (–) 17.27 (–) 0.62
– – –
12.69 (–) 17.27 (–) 0.620
12.69 (–) 17.27 (–) 0.614
LC50 (LCI–UCI) LC90 (LCI–UCI) Chi-square (χ2)
16
10.03 (–) 24.51 (–) 0.156
14.19 (–) 21.28 (–) 0.102
12.28 (–) 24.47 (–) 0.921
11.72 (9.00–40.05) 17.46 (12.45–81.02) 0.870
9.93 (–) 22.29 (–) 0.126
10.60 (7.94–29.49) 17.69 (12.33–66.59) 0.834
LC50 (LCI–UCI) LC90 (LCI–UCI) Chi-square (χ2)
24
0.39 (–) 14.76 (–) 0.282
8.67 (–) 20.38 (–) 0.722
2.93 (–) 16.77 (–) 0.281
9.30 (6.18–322.14) 19.85 (12.42–1211.15) 0.686
0.99 (–) 15.43 (–) 0.110
2.93 (–) 16.77 (–) 0.282
LC50 (LCI–UCI) LC90 (LCI–UCI) Chi-square (χ2)
32
0.43 (0.0–2.81)c 7.11 (4.84–23.19) 1.258
0.65 (0.0–2.47) 5.40 (3.68–13.24) 1.339
5.19 (0.0–25.91) 16.09 (10.24–965.04) 0.029
7.08 (–) 18.53 (–) 0.767
1.08 (0.0–2.96) 6.44 (4.55–14.41) 0.305
1.45 (–) 14.19 (–) 0.336
LC50 (LCI–UCI) LC90 (LCI–UCI) Chi-square (χ2)
40
– 4.21 (2.17–20.28) 0.533
– – –
2.13 (0.0–4.60) 10.61 (7.28–43.65) 0.587
5.19 (0.0–25.91) 16.09 (10.24–965.24) 0.029
0.24 (0.0–2.21) 5.09 (3.30–13.99) 1.497
0.26 (0.0–258) 6.50 (4.37–19.58) 0.430
LC50 (LCI–UCI) LC90 (LCI–UCI) Chi-square (χ2)
48
– – –
– 10.79 (–) 0.236
– – –
1.99 (–) 14.78 (–) 0.153
– 3.31 (–) 0.772
– 2.38 (–) 0.367
a b c
LC50 and LC90, values in μl/L; LCI, lower limit of 95% confidence interval; UCI, upper limit of 95% confidence interval. –, the values could not be obtained during statistical analysis. 0.0, negative value obtained through statistical analysis.
Fig. 2. Box plot representing values of percentage inhibition rate (PIR) against housefly pupae for different monoterpenes in a contact toxicity assay [♯Horizontal lines on the box and stem of the box indicates PIR values for particular concentrations; €the lower horizontal lines in a vertical bar indicates PIR values at lower concentrations].
activity of menthol against Anopheles tessellatus (KD50, 0.54 mg/ml) and Culex quinquefasciatus (KD50, 0.5 mg/ml) compared to menthone against An. tessellatus (KD50, 4.31 mg/ml) and menthyl acetate against Cx. quinquefasciatus (KD50, 1.27 mg/ml). Similarly, Pavela (2011) reported significant effect of thymol (LD50, 30 μg/ml), carvacrol (LD50, 36 μg/ml), and other phenols against larvae of Cx. quinquefasciatus. In their study, higher bio-efficiency of phenol over other terpenoids was affirmed, ascertaining higher efficacy of menthol obtained in the present study. However, the higher activity of menthol shown in this study was contradicted by the study of Palacios et al. (2009b), which reported menthol as poor fumigant against housefly adults (LC50 4100 mg/dm3) than menthone (LC50, 8.6 mg/dm3) and 1,8-cineole (LC50, 3.3 mg/dm3). The variation in results for above studies highlights the effect of age and strain
Fig. 3. Box plot representing values of percentage inhibition rate (PIR) against housefly pupae for different monoterpenes in a fumigation toxicity assay [♯Horizontal lines on the box and stem of the box indicate PIR values for a particular concentrations; €the lower horizontal lines in a vertical bar indicate PIR values at lower concentrations; *100% PIR observed at four upper concentrations, represented by bar, asterisk represents 90% PIR observed at lowest concentration of 1,8cineole].
variation of target insects and disparity in bioassay methodology on efficacy of control agents. The present study showed various incidences of incomplete emergence of adult flies from pupal case as well as stunted growth for emerged flies with application of menthol and menthone in pupicidal assay. Similar observations were reported with sub-lethal doses of essential oils for control of various other insets (Hummelbrunner and Isman, 2001; Pavela, 2007). Hummelbrunner and Isman (2001) reported growth inhibition, weight loss and high agitation in insects' larvae at sub-lethal doses of essential oils. The effect of sub-lethal doses on housefly adults showed decreased longevity, increased natality and higher mortality in F1 larvae and adults (Pavela, 2007). The above discussion suggests that sub-lethal dose of
P. Kumar et al. / Ecotoxicology and Environmental Safety 100 (2014) 1–6
essential oils or monoterpenes might manifest overall biocontrol by affecting insects' behavior. Similarly, Kabir et al. (2013) concluded that growth-disrupting and neurobehavioral toxicity effects of plant control agents may be a valuable approach in integrated vector management. 1,8-cineole has been reported to have an appreciable activity against housefly adults in fumigation [100 percent mortality at 50 mg/cm3 (Lee et al., 2003) and LC50 of 3.3 mg/dm3 (Palacios et al., 2009a)] and topical toxicity assay [LC50, 240 mg/fly (Lee et al., 1997)]. However, in contrast to the present study, the literature reports poor larvicidal activity of 1,8-cineole. 1,8-cineole performed poorly against larvae of Aedes aegypti and Aedes albopictus with LC50 - 450 mg/cm3 (Cheng et al., 2009), but failed to show any larval mortality against Ae. aegypti larvae at 100 mg/cm3 (Chantraine et al., 1998). Superior control activity was reported for citral against housefly adults in fumigation (LC50, 13 mg/cm3) and topical toxicity (LC50, 61 mg/fly) assay (Rice and Coat, 1994). Similarly, appreciable activity of citral against housefly adult in fumigation assay (50 mg/cm3, 80 percent mortality) was observed by Lee et al. (2003), while topical toxicity activity (LC50, 260 mg/fly) was reported by Lee et al. (1997). Limonene showed good fumigation activity against housefly adults with 100 percent mortality at 50 mg/cm3 (Lee et al., 2003). Moderate activity of limonene was observed in a larvicidal assay against mosquito larvae, with a LC50 of 18.1 and 32.7 mg/cm3 against Ae. aegypti and Ae. albopictus, respectively (Cheng et al., 2009). The results were in variation with the present study where poor control activity by limonene was observed. From the consideration of various literature studies, it appears that oxygenated monoterpenes (1, 8-cineole and limonene), although perform superiorly in fumigation assay, could not execute similarly in other types of bioassays, where non-oxygenated monoterpenes outperformed them repeatedly. This could explain higher efficacy of menthol and menthone in fumigation assay in present study. Structure–toxicity investigations by Abdelgaleil et al. (2009) revealed ketone monoterpenes to have high contact toxicity while monoterpene ether was the most potent fumigant against stored grain insects. In a recent study by Pavela (2011), higher biological efficiency of phenols (e.g. thymol, carvacrol, 2-ethylphenol) over other terpenoids were stated. Further, phenols were also reported for their activity as acute toxicant, antifeedant, anti-ovipositional, and growth inhibitory effects. Significant activity of phenolic terpenes over other monoterpenes was also ascertained by the findings of other studies (Amer and Mehlhorn, 2006; Pavela, 2009). The discussion of the literature studies with variable insecticidal activity of monoterpenes also suggested dependence of insecticidal activity of monoterpenes on type of insects and their life stages (Abdelgaleil et al., 2009; Cheng et al., 2009). This could be observed from the study of Cheng et al. (2009), which reported high activity of limonene against larvae of Ae. aegypti and Ae. albopictus, whereas for the same sets of condition 1,8-cineole remained comparatively ineffective. Similar response was observed in the present study where performance of monoterpenes was found to be dependent upon life stages (larvae and pupae) of housefly. Activity of monoterpenes was also influenced by bioassay types. This fact was illustrated by the dendodgrams for various assays in the present study. In contact toxicity assay against housefly larvae, acivity of citral was grouped with menthone. However, in fumigation assay, activity of citral depicted similarity with menthol, while menthone showed comparable activity with that of limonene. Overall higher efficacy of menthol and menthone (major monoterpenes of M. x piperita essential oil) in various bioassays, obtained in the present study are in agreement with the results of essential oil activity obtained earlier (Kumar et al., 2011). In earlier studies, the essential oil of M. x piperita was found to be most effective followed by E. globulus. The present study showed
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superior performance of menthone and menthol, followed by 1,8-cineole. The results suggest that the higher efficacy of M. x piperita oil may be attributed to menthol and menthone. Dependency of essential oil insecticidal activity on their major component was further supported by higher fumigation activity of citral, which formed major component of C. citratus essential oil. The essential oil of C. citratus had earlier shown a very good efficacy as housefly fumigant (Kumar et al., 2013). From the results of present study, it is expected that monoterpenes could be used successfully as control agent to counter houseflies. Earlier, Palacios et al. (2009b) reported competence of C. sinensis (LC50, 3.9 mg/dm3) and 1,8-cineole (LC50, 3.3 mg/dm3) with commercially used chemical insecticide, dimethyl 2, 2-dichlorovinyl phosphate (LC50, 0.5 mg/dm3) as control agent against housefly. Although, monoterpenes may require higher doses to produce the effectiveness similar to chemical insecticides, the same could be countered with development of characteristics formulation. The results from the present study could be used for development of various formulations based on monoterpenes. Such formulations with overall control efficacy against different life stages of housefly are expected to effectively reduce housefly population.
Acknowledgments The authors acknowledge Mr. Satendar Singh (IIT Delhi, India) for their help in experimental work. Partial financial support from KVIC Interphase project is gratefully acknowledged.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2013.11.013. References Abdelgaleil, S.A.M., Mohamed, M.I.E., Badawy, M.E.I., El-arami, S.A.A., 2009. Fumigant and contact toxicities of monoterpenes to Sitophilus oryzae (L.) and Tribolium castaneum (Herbst) and their inhibitory effects on acetylcholinesterase activity. J. Chem. Ecol. 35, 518–525. Abbott, W.S., 1925. A method of computing the effectiveness of an insecticide. J. Econ. Entomol. 18, 265–267. Amer, A., Mehlhorn, H., 2006. Larvicidal effects of various essential oils against Aedes, Anopheles, and Culex larvae (Diptera, Culicidae). Parasitol. Res. 99, 466–472. Chantraine, J.M., Laurent, D., Ballivian, C., Saavedra, G., 1998. Insecticidal activity of essential oils on Aedes aegypti larvae. Phytother. Res. 12, 350–354. Cheng, S.S., Huang, C.G., Chen, Y.J., Yu, J.J., Chen, W.J., Chang, S.T., 2009. Chemical compositions and larvicidal activities of leaf essential oils from two eucalyptus species. Bioresour. Technol. 100, 452–456. Finney, D.J., 1971. Probit Analysis, third ed. Cambridge University Press, London. Fotedar, R., 2001. Vector potential of houseflies (Musca domestica) in the transmission of Vibriocholerae in India. Acta Trop. 78, 31–34. Hummelbrunner, L.A., Isman, M.B., 2001. Acute, sublethal, antifeedant, and synergistic effects of monoterpenoid essential oil compounds on the tobacco cutworm, Spodoptera litura (Lep., Noctuidae). J. Agric. Food Chem. 49, 715–720. Kabir, K.E., Choudharya, M.I., Ahmed, S., Tariq, R.M., 2013. Growth-disrupting, larvicidal and neurobehavioral toxicity effects of seed extract of Seseli diffusum against Aedes aegypti (L.) (Diptera: Culicidae). Ecotoxicol. Environ. Saf. 90, 52–60. Kumar, P., Mishra, S., Malik, A., Satya, S., 2011. Repellent, larvicidal and pupicidal properties of essential oils and their formulations against the housefly Musca domestica. Med. Vet. Entomol. 25, 302–310. Kumar, P., Mishra, S., Malik, A., Satya, S., 2012a. Insecticidal evaluation of essential oils of Citrus sinensis L. (Myrtales: Myrtaceae) against housefly, Musca domestica L. (Diptera: Muscidae). Parasitol. Res. 110, 1929–1936. Kumar, P., Mishra, S., Malik, A., Satya, S., 2012b. Efficacy of Mentha x piperita and Mentha citrata essential oils against housefly, Musca domestica L. Ind. Crops Prod. 39, 106–112. Kumar, P., Mishra, S., Malik, A., Satya, S., 2012c. Compositional analysis and insecticidal activity of Eucalyptus globules (family: Myrtaceae) essential oil against housefly (Musca domestica). Acta Trop. 122, 212–218.
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Kumar, P., Mishra, S., Malik, A., Satya, S., 2013. Housefly (Musca domestica L.) control potential of Cymbopogon citratus Stapf. (Poales: Poaceae) essential oil and monoterpenes (citral and 1,8-cineole). Parasitol. Res. 112, 69–76. Lee, S., Peterson, C.J., Coats, J.R., 2003. Fumigation toxicity of monoterpenoids to several stored product insects. J. Stored Prod. Res. 39, 77–85. Lee, S., Tsao, R., Peterson, C., Coats, J.R., 1997. Insecticidal activity of monoterpenes to western corn root worm (Coleoptera: Chrysomelidae), two spotted spider mite (Acari: Tetanychidae), and housefly (Diptera: Muscidae). J. Econ. Entomol. 90, 883–892. Nathan, S.S., Choia, M.Y., Seoa, H.Y., Paika, C.H., Kalaivania, K., Kim, J.D., 2008. Effect of azadirachtin on acetylcholinesterase (AChE) activity and histology of the brown planthopper Nilaparvata lugens (Sta˚l). Ecotoxicol. Environ. Saf. 70, 244–250. Novartis, 2013. Fly Control in Livestock and Poultry Production. 〈http://www. flycontrol.novartis.co.uk/antifly/en/index.shtml〉. Palacios, S.M., Bertoni, A., Rossi, Y., Santander, R., 2009a. Efficacy of essential oils from edible plants as insecticides against the house fly, Musca domestica L. Molecules 14, 1938–1947.
Palacios, S.M., Bertoni, A., Rossi, Y., Santander, R., 2009b. Insecticidal activity of essential oils from native medicinal plants of Central Argentina against the housefly, Musca domestica (L.). Parasitol. Res. 106, 207–212. Pavela, R., 2007. Lethal and sublethal effects of Thyme oil (Thymus vulgaris L.) on the house fly (Musca domestica Lin.). J. Essent. Oil Bear. Plants 10, 346–356. Pavela, R., 2009. Larvicidal property of essential oils against Culex quinquefasciatus say (Diptera: Culicidae). Ind. Crop Prod. 30, 311–315. Pavela, R., 2011. Insecticidal properties of phenols on Culex quinquefasciatus say and Musca domestica L. Parasitol. Res. 109, 1547–1553. Rice, P.J., Coats, J.R., 1994. Insecticidal properties of several monoterpenoids to the housefly (Diptera: Muscidae), red flour beetle (Coleoptera: Tenebrionidae), and southern corn rootworm (Coleoptera: Chrysomelidae). J. Econ. Entomol. 87, 1172–1179. Roitberg, B.D., Isman, M.B. (Eds.), 1992. Insect Chemical Ecology: An Evolutionary Approach. Chapman & Hall, New York. Samarasekera, R., Weerasinghe, I.S., Hemalal, K.D.P., 2008. Insecticidal activity of menthol derivatives against mosquitoes. Pest Manage. Sci. 64, 290–295. SPSS, 2008. Statistical product and service solution. System User’s Guide, version 17.5.
Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Biocontrol potential of essential oil monoterpenes against housefly, Musca domestica (Diptera: Muscidae) Peeyush Kumar, Sapna Mishra, Anushree Malik n, Santosh Satya Applied Microbiology Laboratory, Centre for Rural Development & Technology, Indian Institute of Technology Delhi, New Delhi 110016, India
art ic l e i nf o
a b s t r a c t
Article history: Received 17 September 2013 Received in revised form 11 November 2013 Accepted 18 November 2013
Housefly (Musca domestica L.), one of the most common insects in human settlements, has been associated as vectors for various food-borne pathogens, causing food spoilage and disease transmission. The control of housefly was attempted using plant monoterpenes; menthone, menthol, menthyl acetate, limonene, citral and 1,8-cineole, against different life stages of housefly. Bioefficacy against housefly adults revealed highest repellent activity by menthol (95.6 percent) and menthone (83.3 percent). Against housefly larvae, menthol with an LC90 of 0.02 ml/cm2 in contact toxicity assay and menthone with a LC90 value of 5.4 ml/L in fumigation assay were found to be most effective control agent. With respect to pupicidal activity, superior performance was shown by menthol, citral and 1,8-cineole in contact toxicity assay and citral and 1,8-cineole in fumigation assay. Limonene was found to be the poorest performer in all the assays. Overall, highest efficacy observed for menthol and menthone in various bioassays was in agreement with the results of essential oil activity obtained previously. Significant activity of monoterpenes against various life stages of housefly demonstrates their potential as excellent insecticides with prospects of monoterpenes being developed into eco-friendly and acceptable products for housefly control. & 2013 Elsevier Inc. All rights reserved.
Keywords: Musca domestica Essential oil Monoterpenes Bioefficacy Repellency
1. Introduction The housefly (Musca domestica L.), one of the most common insects in human settlements, has been associated as vectors for various food borne pathogens. Houseflies breed in rotting vegetable matter or animal feces, from where they acquire and transmit pathogenic organisms to foodstuffs, causing food spoilage and disease transmission. On a conservative estimate, houseflies are associated with vectoring of more than 100 etiological agents of bacterial, protozoan, and viral diseases (Fotedar, 2001; Kumar et al., 2012a). Conventional control of housefly is based on the use of chemical insecticides. However, these chemicals are not only detrimental to animal and human health but also cause various types of soil and water pollution. Besides, gradually insects tend to develop resistance against these insecticides leading to pest resurgence (Kumar et al., 2011, 2012a. Additionally, these insecticides are toxic to a large spectrum of animal species, thus, also killing non-target organisms. Efforts are thus being made world over to replace these synthetic chemicals with alternatives which are safer and do not cause any toxicological effects on the environment.
n
Corresponding author. Fax: þ 91 11 26591121. E-mail address: [email protected] (A. Malik).
0147-6513/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2013.11.013
In our search for housefly control agents compatible with human surroundings and food materials, essential oils and terpenoids could be a good alternative. Terpenoids are major components of plant essential oils. Terpenoids mostly constitute of monoterpenes and sesquiterpenes and are supposedly responsible for pesticidal activity of essential oils. The insecticidal activity of monoterpenes has been reported against various agricultural and household pests with a potential effect as contact toxicant, fumigant, repellent and antifeedant (Lee et al., 2003; Abdelgaleil et al., 2009; Nathan et al., 2008). Rice and Coats (1994) investigated a number of monoterpenes and their derivatives in different bioassays against adult housefly. Similarly, Palacios et al. (2009a) reported potent fumigant activity of various monoterpenes against adult housefly while their repellency activity was demonstrated by Roitberg and Isman (1992). The above literature studies establish pesticidal potential of monoterpenes against housefly. However, most of these studies have been conducted against adult housefly, which at a given time form only 15 percent of the total housefly population (Novartis, 2013). In comparison, the investigations on use of monoterpenes for control of other stages of housefly remain neglected. Moreover, most of the studies in literature reports have utilized fumigation assay for housefly control. Although breeding sites of housefly (larvae or pupae) may create a microhabitat, ensuring fumigation activity of monoterpenes; fumigation assay against housefly adults may be an impractical approach, as in real life situation housefly adults rarely remain confined within a space to allow fumigation
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action of essential oils or monoterpenes to take place. In view of the above facts, the present study was conducted to evaluate the activity of six monoterpenes; menthone, menthol, menthyl acetate, limonene, citral and 1,8-cineole, against different stages of housefly. The activity was assessed through repellency (against adults) and contact toxicity and fumigation (larvae and pupae). Monoterpenes, menthone, menthol and menthyl acetate were chosen as they formed the major components of Mentha x piperita (peppermint) essential oil, which was found to be highly effective against housefly in earlier studies (Kumar et al., 2011, 2012b. Limonene is principal constituent of Citrus sinensis (orange) essential oil, which was reported to be most potent against housefly adults (Palacios et al., 2009a). Citral and 1,8-cineole are major components of Cymbopogon citratus (lemongrass) and Eucalyptus globulus (blue gum), respectively. The insecticidal activities of both the essential oils against housefly have been reported in earlier studies (Kumar et al., 2011, 2012c, 2013.
2. Materials and Methods
2.5. Fumigation assay Fumigation assay was performed in a 1 L conical flask (Kumar et al., 2013). Cotton swabs impregnated with different concentrations of monoterpenes (1, 3, 5, 7, 10 ml/L of air) were attached to the cork of the flask, while larvae/pupae were placed at the bottom surface of conical flask. Control was treated with acetone alone. Each treatment incorporated three replicates. During the assay, flasks were kept at the temperature of 28 7 2 1C and RH of 65 percent. Larvicidal assay was done with twenty lab reared larvae (2nd instars) placed in conical flask along with larval diet. Any change in treated larvae or mortality was observed till 48 h. Pupicidal assay was carried out with twenty pupae (2–3 days old). Observations for adult emergence in treated pupae were made till six days.
2.6. Statistical analysis The repellency data of essential oils was abbot corrected by accounting the number of flies repelled in the control (Abbott, 1925). The values for LC50, LC90, chi-square and 95 percent-confidence intervals for each regression coefficient were calculated by using probit analysis (Finney, 1971). A two-way analysis of variance (ANOVA) was used to analyze the effect of varying doses and exposure periods on larval mortality. All statistical analysis including preparation of dendogram was performed using the statistical software SPSS version 17.0 (SPSS, 2008).
2.1. Materials The monoterpenes (menthone, menthol, menthyl acetate, limonene, citral and 1,8-cineole) were purchased from Sigma Aldrich, India.
2.2. Housefly Adult houseflies collected from field by sweep net method, were reared in cylindrical boxes containing a diet of groundnut oil cake and wheat bran (1:3), according to the method described earlier (Kumar et al., 2011). Hatched larvae were transferred individually to cylindrical vials (28 12 mm2) containing a larval diet (constituents: 2 g groundnut oil cake, 5 g wheat bran, 2 g milk powder, 1 g honey mixed with 10 ml of water) which was changed daily until larvae reached the pupal stage. For repellency bioassay, field flies were used while larvicidal and pupicidal bioassay were done with larvae and pupae obtained from rearing of field flies. 2.3. Repellency assay The repellent effect of different monoterpenes was evaluated against field collected adult houseflies, irrespective of their sex. Repellency assay was performed in a repellency chamber described earlier (Kumar et al., 2011). Outer chamber of the repellency chamber was connected to the inner chamber through an opening (dia., 60 mm). Adult flies (50) were placed in the outer chamber along with petri plate (dia., 90 mm) containing filter paper impregnated with monoterpene solutions ( E 0.16 μl/cm2) diluted in acetone. Control was treated with 0.5 ml of acetone. Treated and control discs were air-dried for 5 min to evaporate the solvent completely, before experiment initiation. Houseflies moving from outer to inner chamber were counted as being repelled. Three replicates of each monoterpene treatment were performed.
2.4. Contact toxicity assay
3. Results 3.1. Repellency assay The repellency activity against housefly adults varied significantly among different monoterpenes (F¼93.9; df ¼ 5; Po 0.001). Also, significant variation between monoterpenes was observed for different time of observation (F¼56.7; df ¼7; Po 0.001). Repellency assay with different monoterpenes is represented in Fig. 1. Menthol was found to be most superior repellent (95.6 percent), while limonene was a poor performer with only 38 percent of overall repellency after 2 h of observation. The figure clearly showed almost similar trend in percentage repellency with monoterpenes of menthone (83.3 percent) and citral (76.0 percent) and menthyl acetate (67.3 percent) and 1,8-cineole (64.0 percent). Dendogram based on the repellency results for different monoterpenes showed a cluster formed by monoterpenes (menthone, citral, menthyl acetate and 1,8-cineole), indicating close repellency relationship between them (Suppl. Fig. 1). The higher repellency activity of menthol was also reflected by its RT50, the time for repellency of 50 percent of adult flies, which was 16.28 min (Suppl. Table 1). Menthone and citral with RT50 of 21.39 and 33.54 min, respectively, showed adequate housefly repellency activity, as well as followed the repellency activity trend reflected by percentage repellency of adult housefly using monoterpenes.
The contact toxicity assay was performed in a Petri plate (dia., 90 mm) according to the method described by Kumar et al. (2013). Different concentrations of monoterpenes (0.016, 0.047, 0.079, 0.11 and 0.16 μl/cm2) were prepared by mixing their variable volumes with 0.5 ml of acetone and applied on the filter paper using a micropipette. Control Petri plate was treated with acetone alone. Treated filter paper was air dried for 5 min, before introduction of larvae/pupae to Petri plates. Three replicates of each oil treatment were performed. Petri plates were kept at the temperature of 28 7 2 1C and relative humidity (RH) of 65 percent. For each larvicidal bioassay, twenty larvae (2nd instars) were kept on filter paper along with larval diet and the treated larvae were observed for any change in appearance and mobility for next four days. Assay on housefly pupae was performed with twenty pupae (2–3 days old). Any adult emergence in the control and treated pupae were observed till six days. Activity of oil on housefly pupae was calculated (Kumar et al., 2011) as the percentage reduction in adult emergence or inhibition rate. Percentage inhibition rate (PIR) was calculated as: %IR or PIR ¼
Cn T n 100 Cn
where Cn is the number of newly emerged insects in the untreated (control) Petri plates and Tn is the number of insects in the treated Petri plates.
Fig. 1. Repellency efficacy (%) of different monoterpenes (0.16 ml/cm2) against adult housefly, Musca domestica, in a repellency chamber experiment.
P. Kumar et al. / Ecotoxicology and Environmental Safety 100 (2014) 1–6
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Table 1 Lethal concentrations of essential oil monoterpenes against housefly larvae in contact toxicity assay. Monoterpenes
Day 1
Day 2
Day 3
LC50 (LCI–UCI)a
LC90 (LCI–UCI)a
Chi-square (χ2)
LC50 (LCI–UCI)a
LC90 (LCI–UCI)a
Chi-square (χ2)
LC50 (LCI–UCI)a
LC90 (LCI–UCI)a
Chi-square (χ2)
Menthol
–b
0.785
–
0.020 (–)
2.49
–
–
–
Menthyl acetate
0.038 (0.0–0.067)c 0.023 (0.0–0.066) 0.068 (0–0.133) 0.033 (–) 0.111 (0.058–1.385)
0.051 (0.018–0.106) 0.179 (0.133–0.350) 0.144 (0.095–1.154) 0.295 (0.188–2.677) 0.194 (–) 0.356 (0.217–12.181)
0.684
0.011 (0.0–0.035)
0.096 (0.073–0.151)
1.433
–
0.663
0.115 (–)
3.037
0.200 (0.135–0.946)
0.915
0.004 (0.0–0.025) –
0.079 (0.55–0.141) 0.064
0.120 (–) 0.151 (–)
1.211 0.631
– 0.0 (0.0–0.018)
Menthone Limonene Citral Cineole
a b c
3.304 0.493 1.276 0.178
0.001 (0.0–0.048) – 0.012 (–)
0.109 (0.081–0.197) 0.033 (–) 0.043 (0.028–0.088)
1.173 1.514 2.101 0.850
LC50 and LC90, values in μl/cm2; LCI, lower limit of 95% confidence interval; UCI, upper limit of 95% confidence interval. –, the values could not be obtained during statistical analysis. 0.0, negative value obtained through statistical analysis.
3.2. Larvicidal assay through contact toxicity The percent mortality of housefly larvae with different monoterpenes was highly significant at different concentration (F¼76.66; df¼ 4, 89; Po0.0001) and time (F¼30.26; df¼17, 89; Po0.0001). Significant results were also obtained for interaction between concentration and time (F¼38.28; df¼ 68, 89; Po0.0001). Activity of different monoterpenes in contact toxicity assay is represented in terms of lethal concentrations (Table 1). The monoterpene concentrations required to kill housefly larvae varied with time of observation. For day 2, the values of lethal concentration, LC90 was lowest for menthol (LC90, 0.02 ml/cm2), followed by citral (LC90, 0.092 ml/cm2), menthyl acetate (LC90, 0.096 ml/cm2), menthone (LC90, 0.115 ml/cm2) and 1,8-cineole (LC90, 0.151 ml/cm2). Limonene with a LC90 value of 0.2 ml/cm2 was observed to be least effective. The statistical evaluation of insecticidal activity revealed similarity in activity for menthone and citral and menthyl acetate and limonene (Suppl. Fig. 2). 3.3. Larvicidal assay through fumigation Housefly larvicidal activity through fumigation toxicity varied significantly between different monoterpenes (Po 0.01). Significant results were also obtained for the percent mortality of housefly larvae for different concentrations of monoterpenes (F¼ 102.76; df ¼ 4, 179; Po 0.0001), exposure period (F¼126.67; df ¼35, 179; P o0.0001) and interaction between concentration and time (F¼ 42.17; df ¼140, 179; P o0.0001). Menthol was found to be the most effective fumigant with an LC90 value of 5.4 ml/L, at 32 h (Table 2). At 32 h, values of LC90 for different monoterpenes varied between 5.4 and 18.53 ml/L. Observation of relationship between monoterpenes based on their fumigation toxicity resulted in the formation of two distinct groups; those with higher activity (menthol, citral, 1,8-cineole, menthyl acetate) and another with comparatively lower larvicidal activity (menthone, limonene) (Suppl. Fig. 3).
indicate PIR values at lower concentrations. The box plot indicates absolute inhibition in adult emergence (PIR-100 percent) with menthol, citral and 1,8-cineole, at their highest concentration. Value of PIR at different tested concentrations varied between 47.3 and 100 percent for menthol, while for menthyl acetate and menthone, it was 36.8–73.7 percent and 42.1–94.7 percent, respectively. For limonene, PIR varied between 36.8 and 89.5 percent. Citral and 1,8-cineole were observed to have higher PIR values even at lower concentration, implying suppression of adult emergence at very low concentrations. Although, menthol and menthone showed comparatively low values for PIR at lower concentrations, pupae treated with these monoterpenes showed high incidence of incomplete emergence and malformation in emergence of adult flies. Moreover, the emerged flies showed overall stunted growth. 3.5. Pupicidal assay through fumigation In the fumigation assay against housefly pupae, significant results were observed for growth inhibition of housefly pupae for different monoterpenes (F¼20.98; df ¼5, 29; P o0.0001), and at different concentrations of monoterpenes (F¼14.97; df ¼ 4, 29; Po 0.0001). From the box plot, the highest effectivity was observed for 1,8-cineole with PIR values varying between 90 and 100 percent (Fig. 3). Asterisk in the box plot represents 90 percent PIR observed at lowest concentration of 1,8-cineole. The activity of 1,8-cineole was followed closely by citral (88.9–100 percent). Menthol (73.7–100 percent), menthone (68.4–100 percent), menthyl acetate (57.9–89.5 percent) and limonene (36.8–89.5 percent) also showed appreciable pupicidal activity. Malformation, reduced size and stunted growth was observed in emerged adult flies obtained from pupae treated with menthol, menthone and menthyl acetate.
4. Discussion 3.4. Pupicidal assay through contact toxicity Pupicidal assay through contact toxicity showed significant variation in growth inhibition for housefly pupae with different monoterpenes (F¼27.31; df ¼ 5, 29; Po0.0001), and with variation in concentration of monoterpenes (F¼29.19; df ¼4, 29; P o0.0001). Values of PIR for monoterpenes treated housefly pupae are represented through a box plot (Fig. 2). Horizontal lines on the box and stem of the box indicate PIR values for a particular concentration. Further, the lower horizontal lines in a vertical bar
The present study has shown higher activity of menthol in almost all the assays against housefly. The result is supported by the study of Rice and Coats (1994), who investigated a number of monoterpenes and their derivatives in fumigation assay against housefly adults and reported higher activity of menthol (LC50, 3.6 mg/cm3) than menthone (LC50,13.7 mg/cm3). The higher fumigation efficacy of menthol was also reflected in the study of Lee et al. (2003), which reported 100 percent mortality of adult housefly at 50 mg/cm3. Samarasekera et al. (2008) found superior repellent
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Table 2 Lethal concentrations of essential oil monoterpenes against housefly larvae in fumigation assay. Statistical values
Time (h)
Monoterpenes Menthol
Menthyl acetate
Menthone
Limonene
Citral
Cineole
LC50 (LCI–UCI)a LC90 (LCI–UCI)a Chi-square (χ2)
8
12.69 (–)b 17.26 (–) 0.614
12.65 (–) 18.02 (–) 2.226
12.69 (–) 17.27 (–) 0.62
– – –
12.69 (–) 17.27 (–) 0.620
12.69 (–) 17.27 (–) 0.614
LC50 (LCI–UCI) LC90 (LCI–UCI) Chi-square (χ2)
16
10.03 (–) 24.51 (–) 0.156
14.19 (–) 21.28 (–) 0.102
12.28 (–) 24.47 (–) 0.921
11.72 (9.00–40.05) 17.46 (12.45–81.02) 0.870
9.93 (–) 22.29 (–) 0.126
10.60 (7.94–29.49) 17.69 (12.33–66.59) 0.834
LC50 (LCI–UCI) LC90 (LCI–UCI) Chi-square (χ2)
24
0.39 (–) 14.76 (–) 0.282
8.67 (–) 20.38 (–) 0.722
2.93 (–) 16.77 (–) 0.281
9.30 (6.18–322.14) 19.85 (12.42–1211.15) 0.686
0.99 (–) 15.43 (–) 0.110
2.93 (–) 16.77 (–) 0.282
LC50 (LCI–UCI) LC90 (LCI–UCI) Chi-square (χ2)
32
0.43 (0.0–2.81)c 7.11 (4.84–23.19) 1.258
0.65 (0.0–2.47) 5.40 (3.68–13.24) 1.339
5.19 (0.0–25.91) 16.09 (10.24–965.04) 0.029
7.08 (–) 18.53 (–) 0.767
1.08 (0.0–2.96) 6.44 (4.55–14.41) 0.305
1.45 (–) 14.19 (–) 0.336
LC50 (LCI–UCI) LC90 (LCI–UCI) Chi-square (χ2)
40
– 4.21 (2.17–20.28) 0.533
– – –
2.13 (0.0–4.60) 10.61 (7.28–43.65) 0.587
5.19 (0.0–25.91) 16.09 (10.24–965.24) 0.029
0.24 (0.0–2.21) 5.09 (3.30–13.99) 1.497
0.26 (0.0–258) 6.50 (4.37–19.58) 0.430
LC50 (LCI–UCI) LC90 (LCI–UCI) Chi-square (χ2)
48
– – –
– 10.79 (–) 0.236
– – –
1.99 (–) 14.78 (–) 0.153
– 3.31 (–) 0.772
– 2.38 (–) 0.367
a b c
LC50 and LC90, values in μl/L; LCI, lower limit of 95% confidence interval; UCI, upper limit of 95% confidence interval. –, the values could not be obtained during statistical analysis. 0.0, negative value obtained through statistical analysis.
Fig. 2. Box plot representing values of percentage inhibition rate (PIR) against housefly pupae for different monoterpenes in a contact toxicity assay [♯Horizontal lines on the box and stem of the box indicates PIR values for particular concentrations; €the lower horizontal lines in a vertical bar indicates PIR values at lower concentrations].
activity of menthol against Anopheles tessellatus (KD50, 0.54 mg/ml) and Culex quinquefasciatus (KD50, 0.5 mg/ml) compared to menthone against An. tessellatus (KD50, 4.31 mg/ml) and menthyl acetate against Cx. quinquefasciatus (KD50, 1.27 mg/ml). Similarly, Pavela (2011) reported significant effect of thymol (LD50, 30 μg/ml), carvacrol (LD50, 36 μg/ml), and other phenols against larvae of Cx. quinquefasciatus. In their study, higher bio-efficiency of phenol over other terpenoids was affirmed, ascertaining higher efficacy of menthol obtained in the present study. However, the higher activity of menthol shown in this study was contradicted by the study of Palacios et al. (2009b), which reported menthol as poor fumigant against housefly adults (LC50 4100 mg/dm3) than menthone (LC50, 8.6 mg/dm3) and 1,8-cineole (LC50, 3.3 mg/dm3). The variation in results for above studies highlights the effect of age and strain
Fig. 3. Box plot representing values of percentage inhibition rate (PIR) against housefly pupae for different monoterpenes in a fumigation toxicity assay [♯Horizontal lines on the box and stem of the box indicate PIR values for a particular concentrations; €the lower horizontal lines in a vertical bar indicate PIR values at lower concentrations; *100% PIR observed at four upper concentrations, represented by bar, asterisk represents 90% PIR observed at lowest concentration of 1,8cineole].
variation of target insects and disparity in bioassay methodology on efficacy of control agents. The present study showed various incidences of incomplete emergence of adult flies from pupal case as well as stunted growth for emerged flies with application of menthol and menthone in pupicidal assay. Similar observations were reported with sub-lethal doses of essential oils for control of various other insets (Hummelbrunner and Isman, 2001; Pavela, 2007). Hummelbrunner and Isman (2001) reported growth inhibition, weight loss and high agitation in insects' larvae at sub-lethal doses of essential oils. The effect of sub-lethal doses on housefly adults showed decreased longevity, increased natality and higher mortality in F1 larvae and adults (Pavela, 2007). The above discussion suggests that sub-lethal dose of
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essential oils or monoterpenes might manifest overall biocontrol by affecting insects' behavior. Similarly, Kabir et al. (2013) concluded that growth-disrupting and neurobehavioral toxicity effects of plant control agents may be a valuable approach in integrated vector management. 1,8-cineole has been reported to have an appreciable activity against housefly adults in fumigation [100 percent mortality at 50 mg/cm3 (Lee et al., 2003) and LC50 of 3.3 mg/dm3 (Palacios et al., 2009a)] and topical toxicity assay [LC50, 240 mg/fly (Lee et al., 1997)]. However, in contrast to the present study, the literature reports poor larvicidal activity of 1,8-cineole. 1,8-cineole performed poorly against larvae of Aedes aegypti and Aedes albopictus with LC50 - 450 mg/cm3 (Cheng et al., 2009), but failed to show any larval mortality against Ae. aegypti larvae at 100 mg/cm3 (Chantraine et al., 1998). Superior control activity was reported for citral against housefly adults in fumigation (LC50, 13 mg/cm3) and topical toxicity (LC50, 61 mg/fly) assay (Rice and Coat, 1994). Similarly, appreciable activity of citral against housefly adult in fumigation assay (50 mg/cm3, 80 percent mortality) was observed by Lee et al. (2003), while topical toxicity activity (LC50, 260 mg/fly) was reported by Lee et al. (1997). Limonene showed good fumigation activity against housefly adults with 100 percent mortality at 50 mg/cm3 (Lee et al., 2003). Moderate activity of limonene was observed in a larvicidal assay against mosquito larvae, with a LC50 of 18.1 and 32.7 mg/cm3 against Ae. aegypti and Ae. albopictus, respectively (Cheng et al., 2009). The results were in variation with the present study where poor control activity by limonene was observed. From the consideration of various literature studies, it appears that oxygenated monoterpenes (1, 8-cineole and limonene), although perform superiorly in fumigation assay, could not execute similarly in other types of bioassays, where non-oxygenated monoterpenes outperformed them repeatedly. This could explain higher efficacy of menthol and menthone in fumigation assay in present study. Structure–toxicity investigations by Abdelgaleil et al. (2009) revealed ketone monoterpenes to have high contact toxicity while monoterpene ether was the most potent fumigant against stored grain insects. In a recent study by Pavela (2011), higher biological efficiency of phenols (e.g. thymol, carvacrol, 2-ethylphenol) over other terpenoids were stated. Further, phenols were also reported for their activity as acute toxicant, antifeedant, anti-ovipositional, and growth inhibitory effects. Significant activity of phenolic terpenes over other monoterpenes was also ascertained by the findings of other studies (Amer and Mehlhorn, 2006; Pavela, 2009). The discussion of the literature studies with variable insecticidal activity of monoterpenes also suggested dependence of insecticidal activity of monoterpenes on type of insects and their life stages (Abdelgaleil et al., 2009; Cheng et al., 2009). This could be observed from the study of Cheng et al. (2009), which reported high activity of limonene against larvae of Ae. aegypti and Ae. albopictus, whereas for the same sets of condition 1,8-cineole remained comparatively ineffective. Similar response was observed in the present study where performance of monoterpenes was found to be dependent upon life stages (larvae and pupae) of housefly. Activity of monoterpenes was also influenced by bioassay types. This fact was illustrated by the dendodgrams for various assays in the present study. In contact toxicity assay against housefly larvae, acivity of citral was grouped with menthone. However, in fumigation assay, activity of citral depicted similarity with menthol, while menthone showed comparable activity with that of limonene. Overall higher efficacy of menthol and menthone (major monoterpenes of M. x piperita essential oil) in various bioassays, obtained in the present study are in agreement with the results of essential oil activity obtained earlier (Kumar et al., 2011). In earlier studies, the essential oil of M. x piperita was found to be most effective followed by E. globulus. The present study showed
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superior performance of menthone and menthol, followed by 1,8-cineole. The results suggest that the higher efficacy of M. x piperita oil may be attributed to menthol and menthone. Dependency of essential oil insecticidal activity on their major component was further supported by higher fumigation activity of citral, which formed major component of C. citratus essential oil. The essential oil of C. citratus had earlier shown a very good efficacy as housefly fumigant (Kumar et al., 2013). From the results of present study, it is expected that monoterpenes could be used successfully as control agent to counter houseflies. Earlier, Palacios et al. (2009b) reported competence of C. sinensis (LC50, 3.9 mg/dm3) and 1,8-cineole (LC50, 3.3 mg/dm3) with commercially used chemical insecticide, dimethyl 2, 2-dichlorovinyl phosphate (LC50, 0.5 mg/dm3) as control agent against housefly. Although, monoterpenes may require higher doses to produce the effectiveness similar to chemical insecticides, the same could be countered with development of characteristics formulation. The results from the present study could be used for development of various formulations based on monoterpenes. Such formulations with overall control efficacy against different life stages of housefly are expected to effectively reduce housefly population.
Acknowledgments The authors acknowledge Mr. Satendar Singh (IIT Delhi, India) for their help in experimental work. Partial financial support from KVIC Interphase project is gratefully acknowledged.
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