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Insecticidal activities of Thymus vulgaris essential oil and its components (thymol and carvacrol) against larvae of lesser mealworm, Alphitobius diaperinus Panzer (Coleoptera: Tenebrionidae)

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Abstract

The insecticidal activity of Thymus vulgaris essential oil, thymol and carvacrol was evaluated in laboratory against different larval stages of lesser mealworm (Alphitobius diaperinus Panzer, Coleoptera, Tenebrionidae). The earlier and later larval stages were reared on diets containing 1 or 2% acetone solutions of tested compounds. Insecticidal activity of thyme essential oil and pure monoterpenes against A. diaperinus larvae depended on the dose and age of larvae. The growth of younger larvae was significantly affected, while those of older larval stage was less influenced and only by pure oil components. In young larvae the application 1% thyme oil, thymol and carvacrol, caused mortality of 50.0, 86.67 and 85 %, respectively. However, the mortality was less in old larvae (17.5, 27.5 and 27.5%, respectively). At the highest dose (2%) thyme oil, thymol and carvacrol killed 62.5, 91.67 and 97.5% of young larvae, respectively. These results showed that thymol and carvacrol were more active against A. diaperinus larvae than thyme oil, thus these two pure components (thymol and carvacrol) can effective control this pest.
0971-4693/94 US $ 5.00
Allelopathy Journal 30 (1): 129-142 (2012) International Allelopathy Foundation 2012
Tables: 5, Figs : 3
Insecticidal activities of Thymus vulgaris essential oil and its
components (thymol and carvacrol) against larvae of lesser
mealworm, Alphitobius diaperinus Panzer (Coleoptera:
Tenebrionidae)
M. SZCZEPANIK*, B. ZAWITOWSKA and A. SZUMNY1
Department of Invertebrate Zoology,
Nicolaus Copernicus University, Gagarina 9, 87-100 Toruń, Poland
E. Mail: mszczep@umk.pl
(Received in revised form: May 8, 2012)
ABSTRACT
The insecticidal activity of Thymus vulgaris essential oil, thymol and
carvacrol was evaluated in laboratory against different larval stages of lesser
mealworm (Alphitobius diaperinus Panzer, Coleoptera, Tenebrionidae). The earlier
and later larval stages were reared on diets containing 1 or 2% acetone solutions of
tested compounds. Insecticidal activity of thyme essential oil and pure monoterpenes
against A. diaperinus larvae depended on the dose and age of larvae. The growth of
younger larvae was significantly affected, while those of older larval stage was less
influenced and only by pure oil components. In young larvae the application 1%
thyme oil, thymol and carvacrol, caused mortality of 50.0, 86.67 and 85 %,
respectively. However, the mortality was less in old larvae (17.5, 27.5 and 27.5%,
respectively). At the highest dose (2%) thyme oil, thymol and carvacrol killed 62.5,
91.67 and 97.5% of young larvae, respectively. These results showed that thymol and
carvacrol were more active against A. diaperinus larvae than thyme oil, thus these two
pure components (thymol and carvacrol) can effective control this pest.
Key words: Allelochemicals, Alphitobius diaperinus, carvacrol, essential oils, lesser
mealworm, thymol, Thymus vulgaris
INTRODUCTION
Essential oils (EOs) and their major components, mainly the monoterpenoids are
potential source of ecologically safe botanical insecticides. These oils are formed by
aromatic plants as secondary metabolites and are widely used in medicine, food and
perfume industries and for crop protection. In nature, essential oils play major role in
protection of plants as antibacterials, antivirals, antifungals (10,15,26) and insecticides.
These secondary compounds are larvicidal, pupicidal and adulticidal, most being
repellents, oviposition deterrents, or antifeedants against both agricultural pests and
medically/veterinary important insects (2,23). They also may attract some insects to favour
the dispersion of pollens and seeds. Essential oils are very complex mixtures (contains
*
Correspondence author,
1
Department of Chemistry, Wroclaw University of Environmental and Life Sciences,
C.K. Norwida 25/27, 50-375 Wrocław, Poland
Szczepanik et al 130
about 20-60 components in different concentrations). They typically consists of two or
three major components present in high concentrations (20-70%) than other components
present in trace amounts (4). Generally, the major components determine the biological
properties of essential oils from which they were isolated. Carvacrol shows a strong
antimutagenic activity similar to Origanum onites L. essential oil, because carvacrol is the
main component in this oil (13). On the other hand, the insecticidal activity of mint
essential oils against Drosophila melanogaster larvae is not in accordance with their major
constituents (pulegone, menthone and carvone). Among the compounds studied, pulegone
was more toxic than Mentha pulegium essential oil containing 75.7% pulegone (8).
Thymol was 1.6-folds more toxic against malarial vector, Anopheles stephensi than the
essential oil of Trachyspermum ammi (23). Contrarily, the Majorana hortensis essential
oil was more toxic against Spodoptera littoralis and Aphis fabae than the isolated
compounds, γ-terpinene and terpinen-4-ol (1). Thus the activity of major components may
be modulated by other minor molecules and the biological activity of essential oil is due to
synergistic/antagonistic interactions of all molecules present.
The insecticidal activity of essential oils has been reviewed recently (25). The
sensitivity of different insect species to the same substance can vary. The effects of natural
plant-derived compounds on survivability, behaviour, growth and development of lesser
mealworm, Alphitobius diaperinus Panzer, a cosmopolitan pest inhabiting chicken and
broiler houses are little studied than other species of family Tenebrionidae (14,22,30). The
essential oils isolated from sassafras (Ocotea odorifera) and eucalyptus (Eucalyptus
viminalis) are insecticidal to lesser mealworm larvae, A. diaperinus (24). The extracts from
Ruta graveolens, Chenopodium ambrosioides and star anise, Illicium verum are very toxic
to A. diaperinus (21,33). Both natural terpenes and their synthetic derivatives are
deterrents to larvae and adults of A. diaperinus. While hydroxy-γ-spirolactones obtained
from the pulegone and bicyclic γ-hydroxy-δ-lactones obtained from isopulegol are good
feeding deterrents (31,32).
The A. diaperinus in poultry houses is controlled using chemical insecticides
(synthetic pyrethroids, organophosphates, chitin synthesis inhibitors), but complete control
is very difficult, due to resistance and inaccessibility of all developmental stages that occur
in soil (8,9). For its control, the use of plant extracts, including essential oils with known
insecticidal effects, may be an alternative method to the heavy use of insecticides. The
compounds in these extracts through a synergistic action may also enhance the activity of
insecticides used (28) and could also be used as flavour supplements to increase the feed
intake in poultry and hence becoming safe alternatives to antibiotic growth stimulators (6).
The European Union directives have prohibited the use of these stimulators in feed stuff
from January 2006. Introducing essential oils into poultry houses may help in improving
the poultry sanitary conditions and eliminate the poultry pathogens and parasites. The
chicken bred on litter sprayed with solutions of thymol and cinnamaldehyde had better
health and lower mortality (36).
Therefore, these studies were performed to compare the insecticidal activity of
thyme oil and its components, thymol and carvacrol against A. diaperinus larvae by
incorporating these compounds into the food of insects.
Insecticidal activities of thyme oil and its components 131
MATERIALS AND METHODS
I. Plant material
Fresh thyme (Thymus vulgaris L.) was supplied by Swedeponic Polska (Kraśnicza
Wola, Poland) Herbal Company. A voucher of appropriate specimen was deposited in
Herbarium, Department of Chemistry Wrocław University of Environmental and Life
Sciences. Only the leaves of dried plants (40oC) were used for distillation. The essential oil
from the dried leaves of Thymus vulgaris L. was obtained by hydrodistillation. The oil
contained 57.44% thymol and 2.80% carvacrol.
II. Preparation of thyme essential oil
A Deryng apparatus was used to extract the volatile compounds of thyme (34).
100 g dried thyme leaves were placed in 2 L round flask with 900 mL distilled water. It
was heated for 2 h after reaching the boiling point. The vapours were condensed by cold
refrigerant. After 2 h distillation, 1.1 mL of essential oil (containing the volatile
compounds) was collected in 2.5 mL vial and kept at -15oC until the GC- MS analyses
and biological tests were done.
III. Chromatographic analyses
The isolation, identification and quantification of volatile compounds was done
using a gas chromatograph (GC) coupled to a mass spectrometer (MS), a Saturn 2000 MS
Varian Chrompack with a DB-5 (5% phenyl methylpolysiloxane) 30 m x 0.25 mm ID x
0.25 µm film column (34). The MS was equipped with an ion-trap analyzer set at 1508 for
all analyses with an electron multiplier voltage of 1350 V. Scanning (1 scan s-1) was
performed in the range of 39–400 m/z using electron impact ionization at 70 eV. The
analyses were carried out using helium as carrier gas at flow rate of 1.0 mL min-1 in a split
ratio of 1:20 and the following program: (a) 80oC for 0 min; (b) rate of 5.0oC min-1 from
80 to 200oC; (c) rate of 25oC min-1 from 200 to 280oC and held for 5 min. The injector and
detector were held at 200 and 300oC, respectively. A one mL volume of sample was
analyzed. Most of the compounds were identified by using 3-analytical methods: (i).
Kovats indices (KI), (ii). GC-MS retention indices (authentic chemicals- standards (S) and
(iii). Mass spectra (authentic chemicals and NIST05 spectral library collection (MS). The
retention index standards used in this study consisted of a mixture of aliphatic
hydrocarbons ranging from C-5 through C-20 dissolved in hexane.
IV. Bioassays
Insects: In this study, laboratory reared strain of A. diaperinus collected from Poultry
Farm, Toruń (53°01N, 18°37E, Poland) were used. The colony was kept in glass
containers on a diet consisting of one part of oat flakes, one part of wheat bran, 0.01 part
of brewers΄ yeast and apple halves to maintain moisture levels at ca. 55%. The colony was
kept in a rearing chamber at +29°C in dark.
Ingestion toxicity and larval growth bioassay. The earlier and later larval stages were
used to assess the effect of T. vulgaris essential oil and the isolated components on larval
Szczepanik et al 132
mortality, growth and development. The culture method for the lesser mealworm described
by Rice and Lambkin was used (27) to obtain numerous larvae of same age. The body
weight of larvae was 2.25-3.09 mg [earlier larval stage (10-days old)], the second group of
larvae were 3-weeks old (body mass: 13.46-14.06 mg). The test oil and its major components
were added at 1 and 2% acetone solution into the diet. One g oat flakes was mixed with 1 ml
test solution or acetone as control. Oat flakes (dried at room temperature) were placed
together with 10-larvae in plastic containers with a capacity of 100ml. The containers were
transferred into the rearing chamber and kept under the same conditions for colony
maintenance. There were 4-replicates for each test, substance and concentration. Body
weight gain was recorded at 3-days interval and the mortality was assessed daily. Larvae
were considered dead, when they did not react to touching with a needle. When development
of larvae ended, the number of living pupae and their body weight were recorded. After
emergence of adults their number and body weight were also recorded.
Antifeedant no-choice bioassay. Feeding deterrent activity of test substances was assayed
using larvae of same age as done in larval growth bioassay. For the feeding assays, 1 and 2%
acetone solutions of test compounds were prepared. Oat flakes were used as test food. One g
oat flakes was dipped in either 1 ml test solution or in acetone alone as control. After
evaporation of solvent (30 min of air-drying), the oat flakes were weighed and placed in Petri
dishes (9 cm dia) together with 10 larvae. Four replicates of tests for each dose and larval
stage were done. The dishes were transferred into a rearing chamber and kept at 29 ± 1oC in
dark. After 3-days feeding, the remaining uneaten oat flakes were re-weighed and the mean
weight of food eaten was calculated. Absolute deterrence coefficients A based on the amount
of food consumed were calculated as under (12):
A= C-T/C+Tx100
Where, C: Weight of control, T: Weight of treated food consumed by insects.
Statistical analysis: The total mortality and deterrence coefficients were statistically
analysed by means of one-way analysis of variance (ANOVA) and Tukey test. t-Test was
used for comparison the mean body weight of treated and control larvae, the number of
pupae and adults and their mean body weight (11).
RESULTS AND DISCUSSION
Chemical composition of thyme essential oil
The chemical composition of essential oil is given in Table 1. Chromatographical
analysis proved that the thymol chemotype of thyme was used. Besides the predominant
thymol (57.44%), carvacrol (2.8%) as a monoterpenoic phenol was also present in the
essential oil. The important compounds analyzed in the distillate were also monoterpenoids:
γ
-terpinene, p-cymene, and sesquiterpene caryophyllene. The content of other terpenes and
terpenoids in oil was < 2%. Chemical composition and yield of essential oil was almost
identical to data of Baranauskienė et al., (5), where the thyme cultivated in Lithuania region
was used.
Insecticidal activities of thyme oil and its components 133
Table 1. Chemical composition of thyme essential oil analyzed by GC/MS and authentic chemical
comparison
No. Compound Retention time
(min)
(%) Identification
methods*
1 3-Hexen-1-ol 5.13 0.10 MS, KI, S
2 α -Thujene 6.75 1.48 MS, KI, S
3 α -Pinene 7.07 0.68 MS, KI, S
4 Camphene 7.6 0.25 MS, KI, S
5 1-Octen-3-ol 7.95 0.50 MS, KI, S
6 Sabinene 8.03 0.14 MS, KI, S
7 β - Myrcene 8.16 1.82 MS, KI, S
8 β -Pinene 8.33 1.82 MS, KI, S
9 3-Octanol 8.37 0.29 MS, KI, S
10 α - Phellandrene 8.94 0.33 MS, KI, S
11 α -Terpinene 9.19 2.37 MS, KI, S
12 p-Cymene 9.46 4.77 MS, KI, S
13 Limonene 9.57 0.29 MS, KI, S
14 Eucalyptol 9.72 0.35 MS, KI, S
15 γ -Terpinene 10.33 12.05 MS, KI, S
16 Sabinene hydrate trans isomer 10.78 1.33 MS, KI
17 Terpinolene 11.14 0.10 MS, KI, S
18 Linalool 11.30 1.24 MS, KI, S
19 Sabinene hydrate cis isomer 11.72 0.23 MS, KI
20 Camphor 13.4 0.11 MS, KI, S
21 Borneol 14.04 0.59 MS, KI, S
22 α -Terpineol 14.51 0.09 MS, KI, S
23 trans-Dihydrocarvone 14.65 0.05 MS, KI, S
24 Thymol methyl eter 15.15 0.35 MS, KI
25 Carvacrol methyl eter 15.44 1.10 MS, KI
26 Thymoquinon 16.09 0.08 MS, KI
27 Thymol 16.97 57.44 MS, KI, S
28 Carvacol 17.24 2.80 MS, KI, S
29 Thymol acetate 18.41 0.39 MS, KI
30 Caryophyllene 20.89 3.64 MS, KI, S
31 Germacrene D 22.46 1.00 MS, KI, S
32 Germacrene D-4-ol 24.96 0.28 MS, KI
33 τ- Cadinol 26.25 1.94 MS, KI
*MS: Mass spectrum, KI: Kovac index, S: Authentic chemicals (standard)
Growth of A. diaperinus larvae
Insecticidal activity of thyme essential oil and pure monoterpenes against
A. diaperinus larvae depended on the dose and age of larvae. Significant differences in the
growth of younger larvae were observed. The pure components decreased their body
weight than oil. Carvacrol solutions showed high activity. During experiments, a dose-
dependent manner of action was observed, but the differences between the doses were not
significant. Larvae reared on diet containing 1 or 2% carvacrol solutions weighed 7.26 and
6.4 mg, respectively, after 24 days. The body weight of larvae treated thymol was only
Szczepanik et al 134
slightly higher i.e. 8.06 and 9.14 mg for 1 or 2% concentrations, respectively (Fig. 1).
While the control larvae weighed 22.7 mg and entering the pupae stage. A much lower
reduction of the increase in body weight of larvae was observed with thyme essential oil
After 24 days of treatment, the larvae had over 2-folds higher body mass than larvae
treated with pure components. Depending on the dose of thyme oil (Fig. 1), larvae average
body mass was 16.81 and 14.54 mg i.e. 74.05 and 64.05% of controls, respectively (Table
2).
Table 2. Inhibitory effects of thyme oil and its components (thymol and carvacrol) on growth of
A. diaperinus larvae
Younger larvae- 24 DAT Older larvae- 15 DAT Compound Conc (%)
Body weight
(% of
control)
Reduction in
body weight
(%)
Body weight
(% of
control)
Reduction in
body weight
(%)
1% 74.05 a -25.95 a 96.07 a -3.93 a Thyme EO
2% 64.05 a -35.95 a 110.96 a +10.96 a
1% 35.50 b -64.50 b 85.77 ab -14.23 cb Thymol
2% 40.26 b -59.74 b 69.65 b -30.35 c
1% 31.98 b -68.02 b 96.26 a -3.74 b Carvacrol
2% 28.19 b -71.81 b 91.16 a -8.84 b
DAT: Days after treatment, EO: Essential oil, Means followed by the same letters within each
column are not significantly different (one-way ANOVA and Tukey test p<0.05)
The pure components slightly disturbed the growth of later larval stage. The
control larvae ended the development, 2-weeks after their average body weight was 21.16
mg. The thyme oil actually stimulated the growth of these larvae (Fig. 2A). Twelve days
after the start of experiment, the body weight of some larvae reared with 1% dose was
much higher than control and these larvae also developed into pupae with higher body
weight than control. With pure compounds small fluctuations occurred in body weight of
larvae. Larvae treated with thymol were relatively smaller. Two weeks after the start of
treatment with 1% thymol, carvacrol and thyme EO, the body mass of larvae was 85.77,
96.26 and 96.07 % of control, respectively. At higher dose of test substances, these values
were lower for pure compounds e.g. 69.65 % with thymol and 91.16% with carvacrol. In
2% thyme oil, the larvae body mass was increased (10.96%) than control (Table 2).
Ingestion toxicity of thyme oil and its components
Mortality of xenobiotic-treated larvae was depended mainly on the age of larvae
and to a lesser extent on the dose (Fig. 3). Pure compounds were more toxic than oil for
both larval stages. Mortality in younger larvae treated with 1 and 2% dose of thyme oil
was 50.0 and 62.5%, respectively. Older larvae susceptibility was very low and their
mortality was not different from control. Toxicity of thymol and carvacrol against younger
larvae was very high. Total mortality with 1 and 2% thymol was 86.67 and 91.67%,
respectively. Activity of carvacrol was similar to thymol and 2% doses caused high
mortality of 85.0 and 97.5 % (Fig. 3A). The mortality in older larvae treated with pure
monoterpenes was slightly higher than control, but these differences were not significant
except with 2% thymol (Fig.3B).
Insecticidal activities of thyme oil and its components 135
0
5
10
15
20
25
30
0 3 6 9 12 15 18 21 24 27 30
Days afte r treatment
Mean body weight (mg/larva)
Control
Thyme EO
Thymol
Carvacrol
0
5
10
15
20
25
30
0 3 6 9 12 15 18 21 24 27 30
Days after treatment
Mean body weight (mg/larva)
Control
Thy me EO
Thy mol
Carvacrol
Figure 1 A and B. Effects of thyme oil, thymol and carvacrol on the growth of younger larvae of
A. diaperinus : A: 1% Concentration, B: 2% Concentration. The standard error is indicated
on the bar.
A
B
Szczepanik et al 136
0
5
10
15
20
25
30
0 3 6 9 12 15 18
Mean body weight (mg/larva)
Days after treatment
Control
Thyme EO
Thymol
Carvacrol
0
5
10
15
20
25
30
0 3 6 9 12 15 18
Mean body weight (mg/larva)
Days after treatment
Control
Thyme EO
Thymol
Carvacrol
Figure 2 A and B. Effects of thyme oil, thymol and carvacrol on the growth of older larvae of
A. diaperinus: A: 1% Concentration, B: 2% Concentration. The standard error is indicated
on the bar.
A
B
Insecticidal activities of thyme oil and its components 137
a
b
cd cd
a
bc
d
d
0
20
40
60
80
100
120
Control Thyme EO Thymol Carvacrol
Total mortality (%)
Compound
1%
2%
aa
aa
aa
b
a
0
20
40
60
80
100
120
Control Thyme EO Thymol Carvacrol
Total mortality (%)
Compound
1%
2%
Figure 3 A and B. Mortality of A. diaperinus larvae treated with thyme oil, thymol and carvacrol:
A: Young larvae, B: Old larvae. Means followed by the same letters are not significantly
different (one-way ANOVA and Tukey test p<0.05). The standard error is indicated on the
bar.
A
B
Szczepanik et al 138
Development of A. diaperinus population
Application of thyme oil and its pure components in breeding of larvae A.
diaperinus affected the number of pupae, their body weight and survival (Table 3,4). The
monoterpenes were more active than essential oil and this activity was age- and dose-
dependent. The number of pupae developed from younger larvae treated with 1% thymol
and carvacrol was 13.33 and 15%, respectively. Higher doses of these oil components
caused drastic reduction in pupae number i.e. 91.67% (2% thymol) and 97.5% (2%
carvacrol). Their body weight was also lower than control. With 2% carvacrol solution the
body weight of pupae was very low (3.23 mg) than 18.89 mg in control. Survival of pupae
was relatively high. Differences between the number of pupae and adults were small
(<10%). Body weight of adults determined immediately after the emergence, corresponded
to body weight of pupae. The lowest body mass was observed in adults coming from trials
treated with both doses of carvacrol i.e. 2% solution of thymol and 2% solution of thyme
oil (Table 3).
Table 3. Effects of thyme oil and its components on number and body weight of pupae and adults
developed from treated younger larvae of A.diaperinus
Pupae Adults Compound
Conc
(%) Numbers
(%) ± SE
Body weight
(mg) ± SE
Numbers
(%) ± SE
Body weight
(mg) ± SE
Control 80.00 ± 10.80 18.89 ± 0.31 80.00 ± 10.80 15.48 ± 0.85
1% 50 ± 5.80∗ 16.38 ± 0.69 ΝS
43.30 ± 6.70∗ 14.21 ± 1.36 ΝS
Thyme EO
2% 37.50 ± 8.50∗∗ 15.67 ± 0.50∗ 22.5 ± 2.50∗∗ 12.33 ± 0.67∗
1% 13.33 ±2.23∗∗∗
18.24 ± 1.57 ΝS
13.33 ± 2.23∗∗∗
14.92 ± 0.94 ΝS
Thymol
2% 8.33 ±6.4∗∗∗ 14.63 ± 0.83 ∗ 5.0 ± 2.5∗∗∗ 12.85 ± 1.55∗
1% 15.00 ± 2.90** 12.53 ± 1.18** 10.00 ± 4.10*** 7.81 ± 2.76**
Carvacrol
2% 2.50 ± 2.50*** 3.23 ± 3.23*** 2.50 ± 2.50*** 2.60 ± 2.60***
EO: Essential oil, For comparison of means with the control the t-test was used. Differences
statistically significant at *p< 0.05; **p< 0.01; ***p<0.001; NS: Not significant
The effects of thyme oil and its components was not strong on the development of
older larvae. Pupae body weight was not significantly different from control, except with
2% thymol solution, when pupae weighed 14.86 mg compared to 16.46 mg in controls.
The pupae developed from larvae treated with both doses of thyme oil had higher body
mass compared to controls. Furthermore, adults developed from them also weighed more
(Table 4). The highest mortality (35.5%,) was observed in pupae developed from the
larvae treated with 2% carvacrol solution.
The thymol and carvacrol were more active against A. diaperinus larvae than
thyme oil. These monoterpenes from cymenole group, although they possess the same
chemical formula and molecular weight but they differed in position of hydroxyl group on
carbon ring. However, different position of hydroxyl group does not affect the activity of
these compounds against A. diaperinus larvae. Likewise, these compounds showed similar
activity against mosquito larvae, Ochlerotatus caspius and Culex pipiens (17,35), but their
similar effects on insects is not the rule. Thymol shows strong toxic properties when
applied to C. pipiens larvae, while carvacrol does not cause insects mortality (7). On the
Insecticidal activities of thyme oil and its components 139
Table 4. Effects of thyme oil and its components on number and body weight of pupae and adults
developed from treated older larvae of A. diaperinus
Pupae Adults Compound
Conc
(%) Numbers
(%) ± SE
Body weight
(mg) ± SE
Numbers
(%) ± SE
Body weight
(mg) ± SE
Control 87.5 ± 7.5 16.46 ± 0.27 80.00 ± 14.1 13.73 ± 0.35
1% 82.5 ± 8.5 ΝS 19.39 ± 0.30∗∗ 72.5 ± 9.5 ΝS 16.18 ± 0.57
Thyme EO
2% 90.0 ± 5.8 ΝS 18.34 ± 0.26∗ 83.3 ± 3.3 ΝS 16.64 ± 0.48
1% 72.5 ± 6.29 16.37 ± 0.31 ΝS 70.0 ± 4.78 14.25 ± 0.33 ΝS
Thymol
2% 57.5 ± 6.27∗∗ 14.86 ± 0.61∗∗ 27.5 ± 14.43∗∗∗ 11.17 ± 1.07
1% 70.0 ± 10.8* 17.94 ± 0.31* 65.0 ± 13.2* 14.88 ± 0.52 ΝS
Carvacrol
2% 77.5 ± 6.29∗ 16.31 ± 0.43 Ν S
40.0 ± 10.8∗∗ 12.98 ± 0.73 ΝS
EO: Essential oil, For comparison of means with the control the t-test was used. Differences
statistically significant at *p< 0.05; **p< 0.01; ***p<0.001; NS: Not significant
other hand, insecticidal activity of carvacrol against second instar larvae of Drosophila
melanogaster was stronger than thymol (16). Similary, carvacrol was more toxic than
thymol, when tested against Thecodiplosis japonensis larvae (20).
Thyme oil obtained by us contained 57.44% thymol and only 2.80% carvacrol. It
seems that thymol is major component responsible for the activity of this oil. Comparing
the activity of T. vulgaris oil with thymol, revealed that the oil was less active against A.
diaperinus larvae than the isolated components. This may suggest an antagonistic
interaction between them and other EO constituents. The content of biosynthetic
precursors of thymol and carvacrol, e.g. p-cymene and γ-terpinene can have an impact on
the relatively low activity of oil. Karpouhtsis et al., (16) tested the insecticidal and
genotoxic activities of essential oils of 3-Oregano taxa, found that Satureja thymbra oil
was richer in precursors (40.62% of total oil) than monoterpenoic phenols (thymol +
carvacrol constitutes 37.84% of oil) was most effective as insecticide aginst D.
melanogaster larvae. Thyme oil studied by us contained only 16.82% of precursors and
60.24% thymol and carvacrol. Components of essential oils may act synergistically in
some cases and cause stronger activity of oil when compared to isolated compounds. For
example, the Majorana hortensis oil exhibited stronger toxic effect against Spodoptera
littoralis larvae and adults of Aphis fabae than its major components, γ-terpinene and
terpinen-4-ol (1).
In our study we did not observe acute toxicity of thyme oil and its components.
All larvae were alive 24 h after treatment. The mortality after 3-days was 5-10% but the
total mortality in younger larvae exposed to pure components was high (above 85%).
Mortality of larvae treated with terpenes may have different reasons. These substances act
in many ways on various insects - as neurotoxins, growth regulators, antifeedants,
repellents etc. The total high mortality observed by us for younger larvae corresponded to
small gain in their body weight. It is not likely that the reduction in body weight gain is
caused by feeding deterrent activity of tested compounds. Low values of deterrence
coefficients, except with 2% carvacrol solution, do not indicate strong antifeedant activity
(Table 5).
Szczepanik et al 140
Table 5. Feeding deterrent activity of thyme oil and its components in no-choice test against A.
diaperinus larvae
Deterrence coefficients ± SΕα
Compound Conc (%)
Younger larvae Older larvae
1% -15.85± 6.30 a 26.04 ± 3.28 ab
Thyme EO
2% −1.87 ± 3.02 a 5.09 ± 5.67 ab
1% 4.69 ± 3.29 a 29.58 ± 7.68 ab
Thymol
2% 5.44 ± 2.31 a 51.28 ± 4.98 a
1% 2.81 ± 3.79 a 23.99 ± 13.41 ab
Carvacrol
2% 41.83 ± 3.23 b −2.18 ± 5.70 b
EO: Essential oil, aEach value represents the mean of four replicates, each set up with 10 insects (n
= 40). Means followed by the same letters within each column are not significantly different (one-
way ANOVA and Tukey test p<0.05).
In the no-choice tests, large fluctuations between the trials were observed (see SE
in Table 5). This may indicate that the reduction in body weight gain and high mortality
among the younger larvae were due to disturbances in digestion and absorption of food.
Age-related differences in larvae susceptibility may have resulted from the applied doses
of compounds. The presence of better developed mechanisms of detoxication in older
larvae can not be excluded. In our previous study, we observed the dose- and age-
dependent differences among the larvae A. diaperinus treated with star anise, Illicum
verum fruits essential oil, 7days old larvae followed by 14-days old were most sensitive
stages. Thirty days old larvae were most tolerant to test oil (33). The application of
thymol/thyme oil to chicken houses for poultry pest management does not contaminate
the poultry, laying hens and eggs (29). Moreover, application of phytogenic feed additive
containing essential oils may improve the broiler chickens nutrition (3).
CONCLUSIONS
Our study showed that the application of T. vulgaris EO against A. diaperinus
larvae was not effective. Significant reduction in pest population can be achieved with use
of pure components. Thymol and carvacrol inhibited the growth, particularly in younger
larvae and most of treated larvae did not survive. Thymol was strong deterrent against
adults of lesser mealworm (unpublished data). Therefore, thymol and carvacrol may have
potential as an alternative to chemical control and can be incorporated into IPM of this
pest.
ACKNOWLEDGEMENTS
This work was supported by Polish Ministry of Science and Higher Education
Grant N N310 146835. We also thank Dr. Ian N. Acworth, for the improvement of
English. For technical assistance in the laboratory we thank Ewelina Muszyńska and
Paulina Orszewska.
Insecticidal activities of thyme oil and its components 141
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