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Industrial Crops and Products
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fficacy of Mentha × piperita and Mentha citrata essential oils against housefly,usca domestica L.
eeyush Kumar, Sapna Mishra, Anushree Malik ∗, Santosh Satyapplied Microbiology Laboratory, Centre for Rural Development & Technology, Indian Institute of Technology Delhi, New Delhi 110016, India
r t i c l e i n f o
rticle history:eceived 30 September 2011eceived in revised form 6 January 2012ccepted 16 February 2012
The essential oils of two Mentha species: peppermint, Mentha piperita, and bergamot mint, Mentha cit-rata (both, Lamiales: Lamiaceae) were evaluated for its chemical composition and insecticidal activityagainst the housefly, Musca domestica L. (Diptera: Muscidae). The gas chromatographic–mass spectrom-etry (GC–MS) analysis of M. × piperita revealed menthol (26.53%), menthone (25.83%), menthyl acetate(7.35%) while M. citrata oil showed linalool acetate (26.69%) and d-linalool (24%) as major constituents.Analysis of M. × piperita oil vapour depicted increased menthone content while M. citrata oil vapourshowed increase in linalool acetate and d-linalool content as compared to respective oils. Insecticidalactivity of oils was assessed against larvae and pupae of housefly, through two different bioassays: con-tact toxicity and fumigation. For the larvicidal assay, lethal concentration, LC50 of M. × piperita oil, was0.54 �l/cm2 (contact toxicity) and 48.4 �l/L (fumigation) while for M. citrata oil, it was 1.39 �l/cm2 (con-tact toxicity) and 61.9 �l/L (fumigation). Oil treated larvae showed swollen spinose areas and proliferationof spinous cells in the intersegmental region, and bleb formation at the anterior region. M. × piperita oiltreated larvae showed significantly higher and acute proliferation of spinose cells and bleb formation
than M. citrata oil. Pupicidal effectivity was measured in terms of percentage inhibition rate (PIR). For,M. × piperita oil, PIR was 100% for both contact toxicity and fumigation toxicity assay while M. citrata oilshowed PIR of 68% and 57% for contact toxicity and fumigation assay, respectively. The present studyestablished higher pupicidal and larvicidal efficacy of M. × piperita than M. citrata essential oil in contacttoxicity as well as fumigation assay. The study demonstrates potentiality of both the Mentha oils as acontrol agent against housefly with M. piperita being better candidate among the two.
. Introduction
Mentha × piperita L. (peppermint oil) is commercially the mostmportant mint species. Peppermint oil is one of the most popularnd widely used essential oil, with use in various medical condi-ion such as, to relieve skin irritation, sun burn, sore throat, fever,
uscle aches and in nasal congestion and also in perfumery and asavouring agent (Kumar et al., 2011a). M. × piperita L. nothosubsp.itrata (Ehrh.) Briq. is also known as Mentha citrata Ehrh. The oilf M. citrata Ehrh. is used for stomach aches, nausea, parasites andther digestive disorders (Seidemann, 2005). Besides their medic-nal properties, oils of M. × piperita L. and M. citrata Ehrh. and itsomponents are also reported for its antibacterial, antifungal, and
nsecticidal properties (Bakkali et al., 2008; Kumar et al., 2011a).nsecticidal activity of both the Mentha species has widely beennvestigated against mosquitoes and grain storage pests (Ansari
et al., 2000; Yang and Ma, 2005; Amer and Mehlhorn, 2006; Kumaret al., 2011a) but has been less explored against other vectors, suchas housefly.
The housefly, Musca domestica (L.), is mechanical carrier of morethan 100 human and animal intestinal diseases and is responsiblefor protozoan, bacterial, helminthic, and viral infections by trans-mission of various pathogens (Malik et al., 2007; Palacios et al.,2009a). The housefly also aids to food contamination and is cate-gorized by the U.S. Food and Drug Administration as an importantcontributing factor in the dissemination of various infectious dis-eases such as cholera, shigellosis, and salmonellosis (Palacios et al.,2009b). Insecticidal activity of several essential oils including Men-tha oil (Pavela, 2008; Palacios et al., 2009a; Kumar et al., 2011b) andmonoterpenes (Coats et al., 1991; Palacios et al., 2009a,b) has beenevaluated against housefly using topical toxicity, fumigation andcontact toxicity assays. Most of the above studies have been doneon the housefly adults while other stage of housefly life cycle, viz.
larvae and pupa remains neglected, even though, only 15% of totalhousefly population exists as adult. In the present study, the oils ofM. × piperita L., and M. citrata Ehrh. have been tested against house-fly larvae and pupae, in contact toxicity and fumigation assay along
ith elucidation of the chemical compositions of the essential oils.ajority of the studies describe the chemical composition of only
he liquid phase (oil) although several activities like repellency,umigation efficacy, etc. are directly mediated by vapour phase. Inhe present study, apart from the identification of chemical compo-ents of liquid oils through GC–MS, the volatile components of oilsere also evaluated through SPME/GC–MS to deduce a correlation
n activity for different assays.
. Materials and methods
.1. Essential oils
Essential oils of M. piperita and M. citrata were purchased fromanta Chemicals Pvt. Ltd., Khari Bowli, New Delhi, India and weretored in plastic bottles at 4 ◦C.
.2. Housefly
Adult houseflies were collected from the garbage sitef the Indian Institute of Technology, Delhi, India, using aweep net method. Houseflies were reared in cylindrical boxes90 mm × 140 mm) containing a diet of groundnut oil cake andheat bran (1:3), pasted on its inner surface at a controlled envi-
onment at 28 ± 2 ◦C temperature and 65% relative humidity (RH) in growth chamber. Hatched larvae were transferred individually toylindrical vials (28 mm × 12 mm) containing a semi-synthetic dietconstituents: 2 g groundnut oil cake, 5 g wheat bran, 2 g milk pow-er, 1 g honey mixed with 10 ml of water); this diet was changedaily until larvae reached the pupal stage to avoid any contamina-ion. Larvae and pupae obtained were used for different bioassays.
.3. Chemical composition analysis
.3.1. Gas chromatographic–mass spectrometry (GC–MS)Chemical composition of these essential oils was determined
y GC–MS using with a flame ionization detection (FID) detec-or fitted with a 60 m × 0.25 mm × 0.25 �m WCOT column coatedith diethylene glycol (AB-Innowax 7031428, Japan). Helium was
carrier gas at a flow rate of 3 ml/min. at a column pressure of55 kPa. Both injector and detector temperatures were maintainedt 260 ◦C. Samples (0.2 �l) were injected into the column with aplit ratio of 80:1. Component separation was achieved follow-ng a linear temperature programme of 60–260 ◦C at 3 ◦C/min andhen held at 260 ◦C for 10 min, with a total run time of 50 min. Theercentage composition was calculated using peak normalizationethod assuming equal detector response. The samples were then
nalysed on same Shimadzu instrument fitted with the same col-mn and following the same temperature programme as above andhe MS parameters used were: ionization voltage (EI) 70 eV, peakidth 2 s, mass range 40–850 m/z and detector voltage 1.5 V. Ana-
ytes profile was characterized from their mass spectral data usingational Institute of Standards and Technology (NIST12 or NIST62)nd Wiley 229 mass spectrometry libraries.
.3.2. Headspace-solid phase microextraction (HS-SPME)Solid phase microextraction (SPME) with GC–MS has been
sed for the determination of the volatile chemical compo-ents of essential oils. Sample extractions for SPME were doney using a divinyl benzene/carboxen/poly dimethyl siloxaneDVB/CAR/PDMS) coated fibre (Supelco, France) and a manual SPMEolder (Sigma–Aldrich, India). In a blank run, the fibre was exposed
o the GC inlet for 5 min for thermal desorption at the temperaturef 260 ◦C, before sampling of oils. Essential oils (2 ml) were placedn 10 ml sample vial and the SPME fibre was exposed to each sam-le for 5 min by manually penetrating the septum, present near
Products 39 (2012) 106– 112 107
cap of the vial, in a headspace sampling. When the sampling wasfinished, the fibre was withdrawn into the needle and transferredto the injection port of GC–MS (Shimadzu GC-MS QP2010 Plus,Japan) system, operating in the same conditions used as above bothfor the identification and the quantification of the oil constituents.An AB-Innowax 7031428 capillary column (60.0 m × 0.25 mm i.d.,0.25 �m film thicknesses) was used for the separation of the samplecomponents.
2.4. Contact toxicity assay
2.4.1. Larvicidal bioassaysHousefly larvae were obtained by rearing the field-collected flies
as described above. For each larvicidal bioassay, 20 larvae (thirdinstars) were placed on a filter paper (in Petri plate) containing adiet of 2 g groundnut oil cake, 5 g wheat bran, 2 g milk powder and1 g honey mixed with 10 ml water (Kumar et al., 2011b). Differentvolumes of each essential oil were mixed with 0.5 ml of acetone tocorrespond to the oil concentrations of 0.16, 0.25, 0.50, 1.01 and2.01 �l/cm2, and were applied to the diet (using a micropipette) ina pour-on treatment. Three replicates of each oil treatment wereperformed. Control filter paper was sprayed with acetone. Beforeputting on larvae on the treated filter paper/diet was air dried for5 min. Larvae were observed for any change in appearance andmobility for 4 days. Larval mortality was assessed by withering andthe development of a brownish appearance (Kumar et al., 2011b).
2.4.2. Pupicidal bioassaysHousefly pupae were obtained by rearing the field-collected
flies. For each pupicidal bioassay, 20 pupae (aged 3 days) wereplaced on a filter paper in a Petri plate. Different concentrationsof oils, 0.16, 0.25, 0.50, 1.01 and 2.01 �l/cm2 were sprayed on tofilter paper, in different treatments. Treated filter paper/diet wasair dried for 5 min, before the introduction of pupae. Three repli-cates of each oil treatment were performed. Control treatmentswere sprayed with acetone only. All bioassays were performedat 28 ± 2 ◦C and RH 65 ± 5%. The rate of pupicidal activity of theoil was calculated as the percentage reduction in adult emergenceor inhibition rate (Kumar et al., 2011b). Inhibition rate (% IR) wascalculated as:
% IR = Cn − Tn
Cn× 100
where Cn is the number of newly emerged insects in the untreated(control) Petri plates and Tn is the number of insects in the treatedPetri plates.
2.5. Fumigation assay
Fumigation assays were performed in a 1 L conical flask. Cot-ton swabs impregnated with different concentration of oils wereplaced, attached to the cork of flask while larvae or pupa were placeat the bottom surface of conical flask.
placed at the bottom of conical flak along with a diet of 2 g ground-nut oil cake, 5 g wheat bran, 2 g milk powder and 1 g honey mixedwith 10 ml water. Different concentrations of oils taken were 40, 50and 70 �l/L of air. Three replicates of each oil treatment were per-
formed while control was treated with acetone. All bioassays wereperformed at 28 ± 2 ◦C and RH 65 ± 5%. The observations were madeup to 48 h, during which any change in mobility and appearance oflarvae were noted down.
1 ps and Products 39 (2012) 106– 112
2
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Table 1Chemical composition of M. × piperita and M. citrata oil by GC–MS.
.5.2. Pupicidal bioassaysPupicidal bioassays were performed with 20 housefly pupae
aged 3 days) obtained through rearing of housefly under opti-um condition. Three concentrations of oils, 40, 50 and 70 �l/L
f air were assessed in fumigation assay. Each treatment was repli-ated thrice while control was acetone treated. All bioassays wereerformed at 28 ± 2 ◦C and RH 65 ± 5%. Observations were madeill 6 days and activity of the oil was adjudged by calculation ofercentage inhibition rate of adult emergence (mentioned above).
.6. Scanning electron microscopy (SEM) for housefly larvae
Larvae treated through contact toxicity of M. × piperita and M.itrata were used for SEM image analysis. Oil treated dead larvaend live larva from control were prepared for SEM visualizationy primary fixation in 2.5% glutaraldehyde in distilled water for–2 h. Thereafter, it was washed subsequently with distilled wateror 10–20 min, followed by secondary fixation with 1–4% osmiumetroxide in distilled water for 1–2 h. Secondary fixation is followedy washing with distilled water (10–20 min) and then serial dehy-ration with 25, 50, 75, 90, and 100% ethanol for 10 min each. Afterehydration, larvae were subjected to critical point drying (CPD) inexamethylene disilazane (HMDS). All the process was performedt room temperature. Larva was mounted on silver stub for SEMbservation and gold-covered by cathodic spraying (Polaron gold).orphology of housefly larva was observed on a scanning elec-
ronic microscope (ZEISS EVO 50). The SEM observation was donender the following analytical condition: EHT ¼ 20.00 kV, WD ¼.5 mm, Signal A ¼ SE1.
.7. Statistical analysis
Data obtained from each dose and time response bioassay wereubjected to regression analysis by probit to generate values forC50, LC90 and LT50 (Finney, 1971; SPSS, 2008). The effect of varyingoses vs. different exposure periods on larval mortality and pupal
nhibition rate was analysed with two-way analysis of variance. Ane-way analysis of variance was performed to compare the effectsf exposure period for each dose tested. All statistical analyses wereerformed using the statistical software SPSS version 17.0 (SPSS,008). The LC50, LC90, chi-square and 95%-confidence intervals forach regression coefficient was calculated by using probit analysisFinney, 1971).
. Results and discussion
.1. Chemical composition analysis
.1.1. GC–MS analysis of oil componentsInsecticidal activities of essential oils are influenced by types as
ell as content of each monoterpenes. Monoterpenoid constituentf oils vary according to geographical location, agrochemical prac-ices, and extraction procedure (Kumar et al., 2011a), whicharrants investigation of chemical composition of each oil to dis-
ern its bio-efficacy. Qualitative and quantitative analysis of the. × piperita and M. citrata oil used in the present study is listed in
able 1. M. × piperita essential oil was characterized by the presencef 40 compounds which formed 98.88% of the total oil compo-ents. The major components identified in the oil were, menthol26.53%), menthone (25.83%) and menthyl acetate (7.35%). Mentholnd menthone are reported to be two of the major components of. × piperita oil responsible for its various properties (Gul, 1994).
evertheless, the content of menthol (30–55%) and menthone
14–32%) has been reported to vary widely (ESCOP, 1997). Anal-sis of M. citrata essential oil revealed 48 compounds, accountingor 94.42% of the total oil components (Table 1) with linalool acetate
Total 98.88 94.42
a Kovat Indices.
(26.69%) and d-linalool (24%) as principal components, which is inagreement with the study of Hilton et al. (1995).
3.1.2. SPME analysis of oil vapourThe vapour profile of M. × piperita was represented by 20 com-
pounds which formed 99.5% of the total vapour composition(Table 2) with menthone (35.54%), menthol (24.01%) and cineole(9.24%) as major components. Vapour profile of M. × piperita oilshowed increase in menthone while slight decrease in mentholcontent compared to its essential oil. M. citrata oil vapour wascontributed by 20 compounds which was 94.5% of the total com-ponents. Linalool acetate (39.24%) and linalool (36.14%) were themain constituents of M. citrata oil vapour (Table 2). For M. citrata
oil, major components in oil and vapour remained same: linalooland linalool acetate. However, both the components showed sig-nificant increase in their percentage composition in vapour thanoil.
P. Kumar et al. / Industrial Crops and
Table 2Chemical composition of M. × piperita and M. citrata oil vapour by SPME/GC–MS.
better performance compared to the study by Pavela (2008) against
TL
TL
a Kovat Indices.
Few studies have reported a comparison between vapour andil profile of essential oil (Rohloff, 1999; Tyagi and Malik, 2011).yagi and Malik (2011) reported significant reduction in mentholnd iso-menthone content from oil to vapour for M. × piperita oil.imilarly, the study by Rohloff (1999) described the decrease inenthol content from 46.6% (oil) to 16.7% (vapour). However, in
he same study, menthone and 1,8-cineole showed increase in theirercentage from oil to vapour. The result obtained in the presenttudy, showing enrichment of menthone and 1,8-cineole in vapourhase is in agreement with Rohloff (1999), however, such signif-
cant reduction in menthol was not observed in this study. In the
nalysis of M. citrata oil vapour, linalool (51%), carvone (23.42) and-octanol (10.1%) were found to be major components (Sartorattond Augusto, 2003).
able 3ethal concentrations of M. × piperita and M. citrata essential oils against housefly larvae
Oils Days LC50 (�l/cm2) 95% C.I.
M. × piperita Day 1 3.39 2.26–11.66
Day 2 1.32 1.08–1.68
Day 3 0.88 0.69–1.09
Day 4 0.54 0.42–0.68
M. citrata Day 1 4.25 2.61–6.81
Day 2 2.98 2.04–8.37
Day 3 2.02 1.51–3.49
Day 4 1.39 1.09–1.88
able 4ethal concentrations of M. × piperita and M. citrata essential oils against housefly larvae
Oils Days LC50 (�l/L) 95% C.I.
M. × piperita Day 1 62.6 59.58–66.31
Day 2 48.4 45.47–51.13
M. citrata Day 1 79.5 72.91–94.16
Day 2 61.9 57.78–67.64
Products 39 (2012) 106– 112 109
3.2. Bioassay
3.2.1. Larvicidal assayMentha essential oils have been known for its insecticidal value
against several insects and vectors (Kumar et al., 2011a), how-ever, reports on larvicidal and pupicidal activities are relativelyscanty. In the present study, larvicidal activity of M. × piperita andM. citrata through contact toxicity assay were found to be doseand time dependent. Mortality of larvae was significant with dif-ferent concentrations (F = 5.01; df = 4; P < 0.01) and time (F = 2.43;df = 3; P < 0.05). Lethal concentration, LC50, of M. × piperita oil variedbetween 3.39 and 0.54 �l/cm2 for different observation days whileLC90 for the same was between 6.09 and 1.18 �l/cm2 (Table 3).
Larvicidal assay of M. citrata oil treatment was significant withdifferent concentration (F = 4.11; df = 4; P < 0.02) and time (F = 2.91;df = 3; P < 0.05). Lethal concentration, LC50 varied between 4.25 and1.39 �l/cm2 for different days of the observation (Table 3). LC90for different observation days was between 7.06 and 2.99 �l/cm2.Lethal time, LT50, to kill 50% of housefly larvae was 1.5 and 3.3 daysfor M. × piperita and M. citrata, respectively, at the concentration of2.01 �l/cm2.
Fumigant assay for housefly larvae with M. × piperita oil was sig-nificant between different concentrations (F = 6.25; df = 2; P < 0.05).The three tested concentrations of the oil (40–70 �l/L of air)resulted in 23.3–95% larval mortality at 48 h of oil exposure. Lethalconcentration, LC50 for 24 h and 48 h was 62.6 and 48.4 �l/L, respec-tively (Table 4). With M. citrata oil different concentration (F = 2.7;df = 2; P < 0.05) showed highest larval mortality of 66.7% with70 �l/L at 48 h of exposure period. Lethal concentration, LC50 ofthe M. citrata oil was 79.5 �l/L at 24 h while at 48 h it was found tobe 61.9 �l/L of air.
Efforts were made to compare the lethal dose obtained in thepresent study with the similar studies reported earlier in the lit-erature. Relatively lower lethal dose have been reported for M.piperita against housefly larvae in contact toxicity assay (Kumaret al., 2011b) and for M. citrata against Spodoptera littoralis larvae intopical toxicity assay (Pavela, 2005). On the other hand, with regardto fumigation assay, LC50 of M. piperita against housefly larvae inthe present study (LC50 = 48.4 �l/L or �g/cm3) was comparable tothat obtained by Amer and Mehlhorn (2006) against Aedes aegyptilarvae (50 �g/cm3) while for M. citrata, the present study showed
housefly adults (LC50 = >80 �g/cm3). In fact, it is difficult to makeexact comparisons with other studies due to large variation inoil composition, target insect, mode/scale of experimentation,
Table 5Percentage inhibition rate (PIR) against housefly pupae with different concentra-tions of M. × piperita and M. citrata essential oils in contact toxicity assay.
Oil concentration (�l/cm2) PIR
M. × piperita M. citrata
0.16 54.5 22.70.25 63.6 31.80.50 77.3 40.9
Fe
10 P. Kumar et al. / Industrial Cro
ifferent exposure regimes/times and concentrations employedKumar et al., 2011a). Due to variation in any of these parameters,he resulting insecticidal activity of given oil would vary substan-ially.
Although larger lethal dose requirement of essential oils haseen implicated, other important effects at sub-lethal dosesay play significant role in overall insect bio-control (Pavela,
007). Sub-lethal doses of essential oils have been indicated forrowth inhibition, weight loss and high agitation in insects’ lar-ae (Hummelbrunner and Isman, 2001). Pavela (2007) studied theffects of the thyme oil sub-lethal doses on vitality and fecundityf adults and on the F1 generation vitality and reported decreasedn longevity, increased natality, as well as, higher mortality in F1arvae and adults. The above discussion suggests that even if largerose of essential oils may be required for insects’ lethality, con-equence of sub-lethal dose on their behaviour, although not veryrominent, might manifest overall biocontrol.
.2.2. Pupicidal assay
Pupicidal bioassays of houseflies with M. × piperita and M. citrata
t different concentration showed diverse efficacy. The percentagenhibition rate (PIR), calculated after 6 days, at different concen-rations, varied between 54 and 100% for M. × piperita while for
ig. 1. SEM micrograph showing housefly larvae with the application of M. × piperita on
ntire larva, (e) intersegmental region, and (f) anterior region.
1.01 100.0 54.52.01 100.0 68.2
M. citrata, it was between 23 and 68% (Table 5). High activityof M. × piperita oil against housefly pupae was also observed infumigation assay, with PIR value of 100% at all the concentrations(Table 6). M. citrata oil was relatively poor performer with PIR valuebetween 25 and 57%, for different concentrations of oil tested. Phy-tochemicals and essential oils have shown growth inhibiting effectson various developmental stages of insects (Regnault-Roger et al.,2004; Malik et al., 2007), including prolongation of instar and pupae
durations, inhibition of larval and pupal molting and morphologi-cal abnormalities and mortality especially during molting (Shaalanet al., 2005; Kumar et al., 2011a). Ansari et al. (2000) reported sub-stantial reduction in fecundity and fertility for the emerged female
(a) entire larva, (b) intersegmental region, (c) anterior region, and M. citrata on (d)
P. Kumar et al. / Industrial Crops and Products 39 (2012) 106– 112 111
Table 6Percentage inhibition rate (PIR) against housefly pupae with different concentra-tions of M. × piperita and M. citrata essential oils in fumigation toxicity assay.
Oil concentration (�l/L) PIR
M. × piperita M. citrata
iKfa
owdmeaRaatethtffl1fsoifo
3
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is
40 100 25.050 100 35.7
n M. × piperita oil treated larvae of different mosquito species.umar et al. (2011b) reported 100% inhibition of adult emergence
rom pupae of M. domestica through treatment with M. × piperitand M. citrata oil.
Overall, the bioassay results showed that M. × piperita essentialil was more effective larvicide in terms of both oil concentration asell as time required for activity, than M. citrata oil. Moreover, theifference in activity between M. × piperita and M. citrata oil wasuch pronounced for pupicidal than larvicidal assay. The higher
fficacy of M. × piperita oil may be attributed to menthol, menthonend pulegone which were present in appreciable amount in its oil.ice and Coats (1994a) investigated a number of monoterpenesnd their derivatives in fumigation bioassay on adult houseflynd reported good activity of menthol (LC50 ≈ 3.6 �g/cm3), men-hone (13.7 �g/cm3) and pulegone (9.2 �g/cm3). Similarly, Palaciost al. (2009a) also reported potent fumigant activity of men-hone (LC50 ≈ 8.6 �g/cm3) and pulegone (1.7 �g/cm3) against adultousefly. Likewise, the activity of M. citrata oil could be attributedo its major components; linalool and linalyl acetate. Appreciableumigant activity of linalool has been reported against adult house-y by Palacios et al. (2009b) and Rice and Coats (1994b), at LC50 of3.6 and 6.8 �g/cm3, respectively. The activity of linalyl acetate wasound to be relatively better at the LC50 value of 4.8 �g/cm3 for theame assay (Rice and Coats, 1994b). The higher efficacy of M. citratail obtained in the fumigation assay also corresponds with signif-cant increase of linalool and linalyl acetate in its vapour phase,urther suggesting them to be the active insecticidal componentsf M. citrata oil.
.3. Scanning electron microscopy (SEM) for housefly larvae
Higher larvicidal efficacy of M. × piperita over M. citrata was alsostablished by the scanning electron microscopy. Fig. 1 shows theffect of M. × piperita and M. citrata essential oils on the houseflyarvae. Fig. 1a and d shows an entire larva after application with
. × piperita and M. citrata essential oils, respectively. The differ-nce in effect of both the oils on larvae could be discerned by thebservation of anterior region and spinose ring between segments.ig. 1a reveals highly affected anterior region and spinose ring in. × piperita treated larva as compared to M. citrata treated larva
n Fig. 1d. Fig. 1b and e reveals swollen spinose areas at the inter-egmental region of larva. Both the larvae show proliferation ofpinose cells but proliferation is more acute with treatment with. × piperita (Fig. 1b). Fig. 1c and f shows anterior region of the larva
reated with M. × piperita and M. citrata, respectively. Fig. 1c showsore and prominent blebs in anterior region of the housefly larva
ompared to Fig. 1f. Thus, the SEM image for housefly larvae con-orms the above finding of essential oils of M. × piperita being moreffective larvicide than M. citrata. Comparing the image obtainedor treated larvae with SEM image for control larvae (Fig. 2), theater did not reveal any visible surface distortion (Fig. 2a). Controlarvae do not show any proliferation in intersegmental region orn spinose ring (Fig. 2b) and also devoid of any blebs in its anterior
egion, so prominently characteristic of treated larvae.
The morphological damage observed in the treated larvae wasn agreement with the previous reports. Sukontason et al. (2004)tudied surface structural changes in housefly larvae by application
Fig. 2. SEM micrograph showing housefly larvae without any oil application (con-trol).
of eucalyptol through SEM image analysis and observed bleb forma-tion, deformation in the integument and intersegmental spines oftreated larvae. Aberration in integument, larval midgut, and mus-cles was reported by Assar and El-Sobky (2003) who investigatedeffect of plant extract of Eichhornia crassipes against Culex pipienslarvae. Similarly, plant extract of Kaempferia galanga demonstratedsever damage to the surface morphology on Cx. quinquefasciatus
larvae (Insun et al., 1999). Abdel-Shafy et al. (2009) concluded thatthe plant extracts by its penetration to the larvae gut or epithe-lial gut cells or by ingestion through the cuticle is responsible forsurface aberration. Although, the mode of action of the essential
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12 P. Kumar et al. / Industrial Cro
ils or their constituents is not clearly known, report suggestsomparative absorption in larvae midgut for larvicidal activity ofarious Mentha species (Rey et al., 1999). However, as differenthemical constituents have different absorptive capacity, this ledo their varied efficacy. In lepidopteran larvae, terpenes (drimaneesquiterpines) block the stimulatory effects of glucose and inos-tol on chemosensory receptor cells located on the mouthpartsGershenzon and Dudareva, 2007; Rattan, 2010) while monoter-enoids have been reported for their neurotoxicity against houseflyCoats et al., 1991).
. Conclusions
The present study established higher pupicidal and larvicidalfficacy of M. × piperita essential oil than M. citrata in contact toxic-ty as well as fumigation assay. These activities could be attributedo the major components present in the respective oils. Moreover,ncrease in efficacy of both the oils in fumigation assay, was ascribedo the enrichment of the active components in the vapour phases demonstrated by the compositional analysis of the essential oilapours by SPME–GC–MS. These results were further confirmedy the severe morphological damages in treated larvae recordedhrough scanning electron microscopy. Hence the analytical, micro-copic and mortality data based evidence together established theotential of tested Mentha oils as larvicidal and pupicidal agentsgainst housefly.
cknowledgements
The authors acknowledge the technical support provided by Mr.jai Kumar (AIRF JNU, India) for GC–MS and SPME analysis and Mr.abal Singh and Mr. Satendar Singh (IIT Delhi, India) for their helpn experimental work.
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