Evaluation of the Toxicity of Satureja intermedia C. A. Mey Essential Oil to Storage and Greenhouse Insect Pests and a Predator Ladybird

Abstract

The use of chemical insecticides has had several side-effects, such as environmental contamination, foodborne residues, and human health threats. The utilization of plant-derived essential oils as efficient bio-rational agents has been acknowledged in pest management strategies. In the present study, the fumigant toxicity of essential oil isolated from Satureja intermedia was assessed against cosmopolitan stored-product insect pests: Trogoderma granarium Everts (khapra beetle), Rhyzopertha dominica (Fabricius) (lesser grain borer), Tribolium castaneum (Herbst) (red flour beetle), and Oryzaephilus surinamensis (L.) (saw-toothed grain beetle). The essential oil had significant fumigant toxicity against tested insects, which positively depended on essential oil concentrations and the exposure times. Comparative contact toxicity of S. intermedia essential oil was measured against Aphis nerii Boyer de Fonscolombe (oleander aphid) and its predator Coccinella septempunctata L. (seven-spot ladybird). Adult females of A. nerii were more susceptible to the contact toxicity than the C. septempunctata adults. The dominant compounds in the essential oil of S. intermedia were thymol (48.1%), carvacrol (11.8%), p-cymene (8.1%), and γ-terpinene (8.1%). The high fumigant toxicity against four major stored-product insect pests, the significant aphidicidal effect on A. nerii, and relative safety to the general predator C. septempunctata make terpene-rich S. intermedia essential oil a potential candidate for use as a plant-based alternative to the detrimental synthetic insecticides.

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foods
Article
Evaluation of the Toxicity of Satureja intermedia C. A.
Mey Essential Oil to Storage and Greenhouse Insect
Pests and a Predator Ladybird
Asgar Ebadollahi 1, * and William N. Setzer 2,3 ,*
1Moghan College of Agriculture and Natural Resources, University of Mohaghegh Ardabili,
Ardabil 56199-36514, Iran
2Department of Chemistry, University of Alabama in Huntsville, Huntsville, AL 35899, USA
3Aromatic Plant Research Center, 230 N 1200 E, Suite 100, Lehi, UT 84043, USA
*Correspondence: ebadollahi@uma.ac.ir (A.E.); wsetzer@chemistry.uah.edu (W.N.S.)
Received: 13 May 2020; Accepted: 21 May 2020; Published: 2 June 2020


Abstract:
The use of chemical insecticides has had several side-effects, such as environmental
contamination, foodborne residues, and human health threats. The utilization of plant-derived
essential oils as efficient bio-rational agents has been acknowledged in pest management strategies.
In the present study, the fumigant toxicity of essential oil isolated from Satureja intermedia was
assessed against cosmopolitan stored-product insect pests: Trogoderma granarium Everts (khapra
beetle), Rhyzopertha dominica (Fabricius) (lesser grain borer), Tribolium castaneum (Herbst) (red flour
beetle), and Oryzaephilus surinamensis (L.) (saw-toothed grain beetle). The essential oil had significant
fumigant toxicity against tested insects, which positively depended on essential oil concentrations
and the exposure times. Comparative contact toxicity of S. intermedia essential oil was measured
against Aphis nerii Boyer de Fonscolombe (oleander aphid) and its predator Coccinella septempunctata
L. (seven-spot ladybird). Adult females of A. nerii were more susceptible to the contact toxicity than
the C. septempunctata adults. The dominant compounds in the essential oil of S. intermedia were
thymol (48.1%), carvacrol (11.8%), p-cymene (8.1%), and
γ
-terpinene (8.1%). The high fumigant
toxicity against four major stored-product insect pests, the significant aphidicidal effect on A. nerii,
and relative safety to the general predator C. septempunctata make terpene-rich S. intermedia essential
oil a potential candidate for use as a plant-based alternative to the detrimental synthetic insecticides.
Keywords:
Aphis nerii;Coccinella septempunctata; plant-based insecticide; Oryzaephius surinamensis;
Rhyzopertha dominica;Tribolium castaneum;Trogoderma granarium
1. Introduction
The Khapra Beetle {Trogoderma granarium Everts (Coleoptera: Dermestidae)}, lesser grain borer
{Rhyzopertha dominica (Fabricius) (Coleoptera: Bostrichidae)}, red flour beetle {Tribolium castaneum
(Herbst) (Coleoptera: Tenebrionidae)}, and saw-toothed grain beetle {Oryzaephilus surinamensis (L.)
(Coleoptera: Silvanidae)} are among the most well-known and economically-important stored-product
pests with world-wide distribution. Along with direct damage due to feeding on various stored
products, the quality of products is strictly diminished because of their residues and mechanically
associated microbes [1–5].
Oleander aphid {Aphis nerii Boyer de Fonscolombe (Hemiptera: Aphididae)}, as a cosmopolitan
obligate parthenogenetic aphid, is a common insect pest of many ornamental plants comprising
several species of Asclepiadaceae, Apocynaceae, Asteraceae, Convolvulaceae, and Euphorbiaceae,
especially in greenhouse conditions. Along with direct damage, A. nerii is able to transmit pathogenic
Foods 2020,9, 712 ; doi:10.3390/foods9060712 www.mdpi.com/journal/foods

Foods 2020,9, 712 2 of 12
viruses to many plants [
6
–
8
]. The seven-spot ladybird beetle {Coccinella septempunctata L. (Coleoptera:
Coccinellidae)} is a natural enemy of various soft-bodied pests like aphids, thrips, and spider mites,
and is considered an important biocontrol agent for greenhouse crops [9–11].
The utilization of chemical insecticides is the main strategy in the management of insect pests.
However, there is a global concern about their numerous side effects including environmental pollution,
insecticide resistance, resurgence of secondary pests, and toxicity to non-target organisms ranging
from soil microorganisms to pollinator, predator and parasitoid insects, fish, and even humans [
12
–
14
].
Therefore, the search for eco-friendly and efficient alternative agents for insect pest management
is urgent.
Based on the low toxicity to mammals, rapid biodegradation in the environment, and very low
chance of insect pest resistance, the use of essential oils extracted from different aromatic plants has been
the motivating subject of many researchers in pest management strategies over the past decade [
15
–
18
].
Sixteen species of the Satureja genus from the Lamiaceae have been reported in the Iranian
flora, of which S. atropatana Bunge, S. bachtiarica Bunge, S. edmondi Briquet, S. intermedia C. A. Mey,
S. isophylla Rech., S. kallarica Jamzad, S. khuzistanica Jamzad, S. macrosiphonia Bornm., S. sahendica
Bornm., and S. rechingeri Jamzad are endemic to Iran [
19
]. S. intermedia, as a small delicate perennial
plant growing on rock outcrops, is among aromatic plants with considerable amount (1.45% (w/w))
of essential oil [
20
]. The essential oil of S. intermedia is rich in terpenes such as 1,8-cineole, p-cymene,
limonene,
γ
-terpinene,
α
-terpinene, thymol, and
β
-caryophyllene, which are classified in four main
groups; monoterpene hydrocarbons, oxygenated monoterpenoids, sesquiterpene hydrocarbons,
and oxygenated sesquiterpenoids [20–22]. Some important biological effects of S. intermedia essential
oil include antifungal, antibacterial, and antioxidant effects, and cytotoxic effects have been reported
in previous studies [
21
–
23
]. Although the susceptibility of insect pests to the essential oils isolated
from some Satureja species such as S. hortensis,S. montana L., S. parnassica Heldr. & Sart ex Boiss.,
S. spinosa L.
, and S. thymbra L. was documented in recent years [
24
–
26
], the insecticidal effects of
S. intermedia essential oil have not reported yet.
As part of a screening program for eco-friendly and efficient plant-derived insecticides,
the evaluation of the fumigant toxicity against four major Coleopteran stored-product insect pests
O. surinamensis,R. dominica,T. castaneum and T. granarium and the contact toxicity against a greenhouse
insect pest Aphis nerii of the essential oil of S. intermedia was the main objective of the present study.
Because of the importance of studying the effects of insecticides on the natural enemies of insect pests,
the toxicity of S. intermedia essential oil against C. septempunctata was also investigated.
2. Materials and Methods
2.1. Plant Materials and Essential Oil Extraction
Aerial parts (3.0 kg) of S. intermedia were gathered from the Heiran regions, Ardebil province,
Iran (38
◦
23
0
N, 48
◦
35
0
E, elevation 907 m). It was identified according to the keys provided by
Jamzad [
27
]. The voucher specimen was deposited in the Department of Plant Production, Moghan
College of Agriculture and Natural Resources, Ardabil, Iran. The fresh leaves and flowers were
separated and dried under shade within a week. One hundred grams of the specimen were poured
into a 2-L round-bottom flask and subjected to hydrodistillation using a Clevenger apparatus for 3 h.
The extraction was repeated in triplicate and the obtained essential oil was dried over anhydrous
Na2SO4and stored in a refrigerator at 4 ◦C.
2.2. Essential Oil Characterization
The chemical profile of the S. intermedia essential oil was evaluated using gas chromatography
(Agilent 7890B) coupled with mass-spectrometer (Agilent 5977A). The analysis was carried out by a
HP-5 ms capillary column (30 m
×
0.25 mm
×
0.25
µ
m). The temperature of the injector was 280
◦
C
and the column temperature adjusted from 50 to 280
◦
C using the temperature program: 50
◦
C

Foods 2020,9, 712 3 of 12
(hold for 1 min), increase to 100
◦
C at 8
◦
/min, increase to 185
◦
C at 5
◦
/min, increase to 280
◦
C at 15
◦
/min,
and hold at 280
◦
C for 2 min. The carrier gas was helium (99.999%) with flow rate of 1 mL/min. Essential
oil was diluted in methanol, and 1
µ
L solution was injected (split 1:10 at 0.75 min). The identification of
components was performed by comparing mass spectral fragmentation patterns and retention indices
with those reported in the databases [28–30].
2.3. Insects
The required colonies of Oryzaephilus surinamensis and Rhyzopertha dominica were reared on wheat
grains for several generations at the Department of Plant Production, Moghan College of Agriculture
and Natural Resources, University of Mohaghegh Ardabili (Ardabil province, Iran). Tribolium castaneum
and Trogoderma granarium adults were collected from infested stored wheat grains in Moghan region
(Ardabil province, Iran). Insects were identified by Asgar Ebadollahi. Fifty unsexed pairs of adult
insects were separately released onto wheat grains and removed from breeding container after 48 h.
Wheat grains contaminated with insect eggs were separately kept in an incubator at 25
±
2
◦
C, 65
±
5%
relative humidity and a photoperiod of 14:10 (L:D) h. Finally, one to fourteen-day-old adults of O.
surinamensis,R. dominica,T. castaneum and T. granarium were designated for fumigant bio-assays.
Aphis nerii and its natural predator Coccinella septempunctata were used to evaluate the contact
toxicity of the S. intermedia essential oil. Cohorts of apterous adult females of A. nerii and unsexed adults
of C. septempunctata were taken directly from homegrown oleander (Nerium oleander L.) and a chemically
untreated alfalfa (Medicago sativa L.) field (Moghan region, Ardabil province, Iran), respectively.
2.4. Fumigant Toxicity
The fumigant toxicity of S. intermedia essential oil was tested on adults of O. surinamensis,
R. dominica,T. castaneum, and T. granarium. To determine the fumigant toxicity of the essential oil,
filter papers (Whatman No. 1, 2
×
2 cm) were impregnated with essential oil concentrations and were
attached to the under surface of the screw cap of glass containers (340-mL) as fumigant chambers.
A series of concentrations (4.71–14.71, 7.06–20.88, 20.59–58.82, and 8.82–35.29
µ
L/L for O. surinamensis,
R. dominica,T. castaneum, and T. granarium, respectively) was organized to assess the toxicity of
S. intermedia essential oil after an initial concentration setting experiment for each insect species. Twenty
unsexed adults (1–14 days old) of each insect species were separately put into glass containers and
their caps were tightly affixed. The same conditions without any essential oil concentration were used
for control groups and each treatment was replicated five times. Insects mortality was documented 24,
48 and 72 h after initial exposure to the essential oil. Insects were considered dead when no leg or
antennal movements were observed [31].
2.5. Contact Toxicity
The contact toxicity of S. intermedia essential oil against the apterous adult females of A. nerii
and unsexed adults of C. septempunctata was tested through filter paper discs (Whatman No. 1), 9 cm
diameter, positioned in glass petri dishes (90
×
10 mm). Range-finding experiments were established
to find the proper concentrations for each insect. Concentrations ranging from 200 to 750
µ
g/mL for
A. nerii and from 500 to 1400
µ
g/mL for C. septempunctata were prepared via 1.00% aqueous Tween-80 as
an emulsifying agent. Each solution (200
µ
L) was applied to the surface of the filter paper. Ten insects
were separately released onto each treated disc, the dishes sealed with Parafilm
®
and kept at 25
±
2
◦
C,
65
±
5% relative humidity and a photoperiod of 16:8 h (light:dark). Except for the addition of essential
oil concentrations, all other procedures were unchanged for the control groups. Four replications
were made for each treatment and mortality was documented after 24 h. Aphids and ladybirds were
considered dead if no leg or antennal movements were detected when softly prodded [32,33].

Foods 2020,9, 712 4 of 12
2.6. Data Analysis
The mortality percentage was corrected using Abbott’s formula: Pt=[(Po
−
Pc)/(100
−
Pc)]
×
100, in which
Pt is the corrected mortality percentage, Po is the mortality (%) caused by essential oil concentrations
and Pc is the mortality (%) in the control groups [34].
Analysis of variance (ANOVA) and Tukey’s test at p=0.05 were used to statistically identify the
effects of independent factors (essential oil concentration and exposure time) on insect mortality and
the differences among mean mortality percentage of insects, respectively. Probit analysis was used to
estimate LC
50
and LC
95
values with 95% fiducial limits, the data heterogeneity and linear regression
information using SPSS 24.0 software package (Chicago, IL, USA).
3. Results
3.1. Chemical Composition of Essential Oil
The chemical composition of S. intermedia essential oil is presented in Table 1. A total of
47 compounds were identified in the essential oil, in which the phenolic monoterpenoids thymol (48.1%)
and carvacrol (11.8%), along with p-cymene (8.1%),
γ
-terpinene (8.1%), carvacryl methyl ether (4.0%),
α
-pinene (2.7%), and
β
-caryophyllene (2.4%) were dominants. Terpenoids were the most abundant
components (98.6%), especially monoterpene hydrocarbons (20.5%) and oxygenated monoterpenoids
(68.4%) with only minor amounts of phenylpropanoids or fatty acid-derived compounds.
Table 1. Chemical composition of the essential oil isolated from aerial parts of Satureja intermedia.
RIcalc RIdb Compound % RIcalc RIdb Compound %
929 932 α-Pinene 2.7 1384 1387 β-Bourbonene 0.1
984 974 1-Octen-3-ol 0.3 1389 1379 Geranyl acetate tr
990 988 Myrcene 0.4 1423 1417 β-Caryophyllene 2.4
1016 1020 p-Cymene 8.1 1428 1431 β-Gurjunene 0.1
1034 1024 Limonene 0.5 1432 1442 α-Maaliene 0.1
1037 1026 1,8-Cineole 1.7 1438 1439 Aromadendrene 0.7
1060 1054 γ-Terpinene 8.1 1454 1452 α-Humulene 0.3
1066 1065 cis-Sabinene hydrate 0.4 1476 1478 γ-Muurolene 0.5
1083 1086 Terpinolene 0.2 1487 1489 β-Selinene 0.2
1083 1089 p-Cymenene 0.2 1496 1496 Viridiflorene 0.7
1092 1095 Linalool 0.2 1500 1500 α-Muurolene 0.2
1094 1098 trans-Sabinene hydrate 0.1 1510 1505 β-Bisabolene 1.3
1121 1128 allo-Ocimene 0.2 1515 1513 γ-Cadinene 0.3
1164 1165 Borneol 0.4 1523 1522 δ-Cadinene 0.7
1176 1174 Terpinen-4-ol 0.8 1530 1533 trans-Cadina-1,4-diene 0.1
1187 1191 Hexyl butyrate 0.1 1535 1537 α-Cadinene tr
1239 1241 Carvacryl methyl ether 4.0 1540 1544 α-Calacorene 0.3
1284 1282 (E)-Anethole 0.7 1557 1553 Thymohydroquinone 0.5
1290 1289 Thymol 48.1 1578 1577 Spathulenol 0.9
1298 1298 Carvacrol 11.8 1581 1582 Caryophyllene oxide 0.8
1340 1340 Piperitenone tr Monoterpene hydrocarbons 20.5
1346 1346 α-Terpinyl acetate 0.1 Oxygenated monoterpenoids 68.4
1349 1349 Thymyl acetate 0.2 Sesquiterpene hydrocarbons 8.0
1357 1356 Eugenol 0.1 Oxygenated sesquiterpenoids 1.7
1365 1373 α-Ylangene 0.1 Phenylpropanoids 0.8
1371 1374 α-Copaene 0.2 Others 0.4
1376 1372 Carvacryl acetate 0.1 Total identified 99.8
RI
calc
=Retention index determined with respect to a homologous series of n-alkanes on a HP-5 ms column;
RIdb =Retention index from the databases [28–30]; tr =trace (<0.05%).
3.2. Fumigant Toxicity
Analysis of variance (ANOVA) revealed that the tested concentrations of S. intermedia essential oil
(F=239.462 and p<0.0001 for O. surinamensis,F=223.629 and p<0.0001 for R. dominica,F=169.615
and p<0.0001 for T. castaneum, and F=89.032 and p<0.0001 for T. granarium with df =4, 45) and the

Foods 2020,9, 712 5 of 12
considered exposure times (F=212.855 and p<0.0001 for O. surinamensis,F=281.180
and p<0.0001
for R. dominica,F=84.705 and p<0.0001 for T. castaneum, and F=84.501 and p<0.0001 for T. granarium
with df =2, 45) had significant effects on the mortality of all insect pests. According to Figure 1
and relatively high R
2
values, there is a positive correlation between the fumigation of essential oil
concentrations and the mortality of four storage insect pests at all exposure times. Furthermore, the
steep slopes indicate a homogenous toxic response among beetles to the essential oil.
Foods 2020, 9, x FOR PEER REVIEW 5 of 12
3.2. Fumigant Toxicity
Analysis of variance (ANOVA) revealed that the tested concentrations of S. intermedia essential
oil (F = 239.462 and p < 0.0001 for O. surinamensis, F = 223.629 and p < 0.0001 for R. dominica, F = 169.615
and p < 0.0001 for T. castaneum, and F = 89.032 and p < 0.0001 for T. granarium with df = 4, 45) and the
considered exposure times (F = 212.855 and p < 0.0001 for O. surinamensis, F = 281.180 and p < 0.0001
for R. dominica, F = 84.705 and p < 0.0001 for T. castaneum, and F = 84.501 and p < 0.0001 for T. granarium
with df = 2, 45) had significant effects on the mortality of all insect pests. According to Figure 1 and
relatively high r
2
values, there is a positive correlation between the fumigation of essential oil
concentrations and the mortality of four storage insect pests at all exposure times. Furthermore, the
steep slopes indicate a homogenous toxic response among beetles to the essential oil.
Figure 1. Concentration–response lines of contact and fumigant toxicity of Satureja intermedia
essential oil against Aphis nerii and Coccinella septempunctata, and Oryzaephilus surinamensis,
Rhyzopertha dominica, Tribolium castaneum, and Trogoderma granarium, respectively.
According to Table 2, an obvious difference in the mean mortality percentage of all tested storage
insect pests was detected, as essential oil concentration and exposure time were increased. For
Figure 1.
Concentration–response lines of contact and fumigant toxicity of Satureja intermedia essential
oil against Aphis nerii and Coccinella septempunctata, and Oryzaephilus surinamensis, Rhyzopertha
dominica, Tribolium castaneum, and Trogoderma granarium, respectively.
According to Table 2, an obvious difference in the mean mortality percentage of all tested storage
insect pests was detected, as essential oil concentration and exposure time were increased. For example,
25.00% mortality of O. surinamensis adults was observed at 4.71
µ
L/L and 24-h exposure time, which
had increased to 80.00% and 100% at 14.71 µL/L after 24 and 72 h, respectively. It is apparent that the

Foods 2020,9, 712 6 of 12
essential oil of S. intermedia gave at least 90% mortality against all tested stored-product insect pests at
58.82 µL/L after 72 h (Table 2).
Table 2.
Mean mortality
±
SE of the adults of Oryzaephilus surinamensis,Rhyzopertha dominica,Tribolium
castaneum, and Trogoderma granarium exposed to the fumigation of Satureja intermedia essential oil after
24, 48, and 72 h.
Insect Time (h) Concentration (µL/L)
4.71 6.18 8.24 11.18 14.71
O.
surinamensis
24 25.00 ±0.41 j38.75 ±0.63 i50.00 ±0.41 g60.00 ±0.41 f80.00 ±0.41 d
48 41.25 ±0.48 h57.50 ±0.29 f,g 70.00 ±0.41 e81.25 ±0.48 d93.75 ±0.48 c
72 53.75 ±0.48 g68.75 ±0.48 e80.00 ±0.58 d96.25 ±0.48 b100.00 ±0.00 a
7.06 9.12 12.35 16.18 20.88
R. dominica
24 25.00 ±0.41 l33.75 ±0.48 k46.25 ±0.48 i58.75 ±0.29 h75.00 ±0.58 e
48 33.75 ±0.48 k43.75 ±0.48 j56.25 ±0.48 h67.50 ±0.29 g82.50 ±0.29 c
72 57.50 ±0.29 h70.00 ±0.41 f78.75 ±0.25 d88.75 ±0.48 b97.50 ±0.29 a
20.59 27.06 34.71 45.29 58.82
T. castaneum
24 23.75 ±0.48 k38.75 ±0.48 i46.25 ±0.48 g60.00 ±0.41 e76.25 ±0.25 c
48 35.00 ±0.58 j50.00 ±0.58 f58.75 ±0.63 e71.25 ±0.48 d82.50 ±0.50 b
72 43.75 ±0.48 h60.00 ±0.41 e71.25 ±0.25 d83.75 ±0.63 b90.00 ±0.50 a
8.82 12.53 17.68 25.00 35.29
T. granarium
24 22.50 ±0.48 j35.00 ±0.29 i42.50 ±0.25 h50.00 ±0.41 g75.00 ±0.29 c
48 37.50 ±0.25 i45.00 ±0.29 h55.00 ±0.29 f70.00 ±0.41 d87.50 ±0.48 b
72 47.50 ±0.25 g62.50 ±0.48 e77.50 ±0.48 c87.50 ±0.48 b100.00 ±0.00 a
Data that do not have the same letters are statistically significant different at p=0.05 based on Tukey’s test. Each
datum represents mean ±SE of four replicates with eighty adult insects.
Based on lower LC
50
values of those stored-product insect pests tested, O.surinamensis was
significantly the most susceptible insect to the essential oil of S. intermedia at all time intervals. In contrast,
the adults of T. castaneum with highest LC
50
and LC
95
values were the most tolerant to fumigation with
S. intermedia essential oil. Furthermore, the susceptibility of insect pests to the fumigation of S. intermedia
essential oil followed in the order: O. surinamensis >R. dominica >T. granarium >T. castaneum (Table 3).
Table 3.
Probit analysis of the data obtained from fumigation of Satureja intermedia essential oil on the
adults of Oryzaephilus surinamensis,Rhyzopertha dominica,Tribolium castaneum, and Trogoderma granarium.
Insect Time (h) LC50 with 95% Confidence
Limits (µL/L)
LC90 with 95% Confidence
Limits (µL/L)
χ2
(df =3) Slope ±SE Sig. *
O. surinamensis
24 8.151 (7.396–8.970) 23.177 (18.675–32.578) 1.99 2.824 ±0.344 0.574
48 5.542 (4.853–6.119) 13.710 (11.971–16.756) 1.288 3.258 ±0.378 0.732
72 4.716 (4.143–5.174) 9.200 (8.413–10.405) 5.134 4.415 ±0.504 0.162
R. dominica
24 12.825 (11.661–14.189) 36.901 (29.147–54.0970) 0.885 2.792 ±0.356 0.829
48 10.398 (9.265–11.454) 30.455 (24.687–42.838) 1.056 2.746 ±0.358 0.788
72 6.358 (5.126–7.296) 15.970 (14.160–19.138) 2.488 3.204 ±0.432 0.477
T. granarium
24 20.489 (18.114–23.612) 81.507 (58.604–140.911) 4.233 2.137 ±0.283 0.237
48 13.654 (11.811–15.364) 49.192 (38.852–71.499) 3.978 2.302 ±0.289 0.264
72 9.785 (6.082–12.258) 24.075 (18.870–42.027) 5.842 3.277 ±0.360 0.12
T. castaneum
24 35.612 (32.538–39.070) 95.948 (77.352–135.744) 0.967 2.977 ±0.376 0.809
48 28.048 (24.747–30.916) 80.251 (65.751–111.454) 0.297 2.807 ±0.378 0.961
72 22.861 (19.648–25.415) 57.584 (50.068–71.481) 0.139 3.194 ±0.405 0.987
* Since the significance level is greater than 0.05, no heterogeneity factor is used in the calculation of confidence
limits. The number of insects for calculation of LC
50
values is 200 for T. granarium and 400 for other insects in
each time.
3.3. Contact Toxicity
The tested concentrations of S. intermedia essential oil demonstrated significant contact toxicity
on both A. nerii (F=27.682, df =4, 15; p<0.0001) and C. septempunctata (F=35.607, df =4, 15;
p<0.0001
). A positive correlation between essential oil concentrations and the mortality of A. nerii
and C. septempunctata in the contact assay is also apparent, based on the high R
2
values (Figure 1).
Comparisons of the mean mortality percentage of A. nerii and its predator C. septempunctata caused by

Foods 2020,9, 712 7 of 12
S. intermedia essential oil are shown in Table 4. The mortality percentages of both insects increased
with increasing essential oil concentrations, but their susceptibility to the essential oil was noticeably
different. For example, 62.50% mortality was documented for A. nerii at 500
µ
g/mL essential oil
concentration while its predator C. septempunctata was more tolerant and exhibited only 17.50%
mortality at this concentration (Table 4).
Table 4.
Mean mortality
±
SE of the adults of Aphis nerii and Coccinella septempunctata exposed to the
different concentration of Satureja intermedia essential oil after 24 h.
Insect Concentration (µg/mL)
200 300 400 500 750
A. nerii 22.50 ±0.25 e32.50 ±0.25 d40.00 ±0.41 c62.50 ±0.25 b77.50 ±0.75 a
500 700 900 1100 1400
C. septempunctata 17.50 ±0.48 e30.00 ±0.41 d45.00 ±0.29 c62.50 ±0.48 b80.00 ±0.41 a
Data that do not have the same letters are statistically significant different at p=0.05 based on Tukey’s test. Each
datum represents mean ±SE of four replicates with eighty adult insects.
The results of the probit analysis for the contact toxicity of S. intermedia essential oil against A.
nerii and C. septempunctata adults are shown in Table 5. According to low LC
50
and LC
95
values, the
adult females of A. nerii were more susceptible to contact toxicity of S. intermedia essential oil than the
adults of C. septempunctata.
Table 5.
Probit analysis of the data obtained from contact toxicity of Satureja intermedia essential oil on
the adults of Aphis nerii and Coccinella septempunctata.
Insect LC50 with 95%
Confidence Limits (µg/mL)
LC90 with 95%
Confidence Limits (µg/mL)
χ2
(df =3) Slope ±SE Sig. *
A. nerii 418.379 (379.586–464.130) 1224.788 (975.704–1738.840) 4.363 2.747 ±0.318 0.225
C. septempunctata 913.722 (853.739–980.799) 1908.099 (1652.748–2352.473) 1.932 4.008 ±0.413 0.587
* Since the significance level is greater than 0.05, no heterogeneity factor is used in the calculation of confidence
limits. The number of insects for calculation of LC50 values is 240 for each insect.
4. Discussion
The susceptibility of O. surinamensis,R. dominica,T. castaneum and T. granarium adults to the
essential oil of S. intermedia with 24-h LC
50
values of 8.151, 12.825, 20.489, and 35.612
µ
L/L, respectively,
was distinguished in the present study. The fumigant toxicity of some plant-derived essential oils
against O. surinamensis,R. dominica,T. castaneum and T. granarium has been documented in previous
studies; it was found that the essential oils of Agastache foeniculum (Pursh) Kuntze, Achillea filipendulina
Lam., and Achillea millefolium L. with respective 24-h LC
50
values of 18.781, 12.121, and 17.977
µ
L/L, had
high toxicity on the adults of O. surinamensis [
31
,
34
–
36
]. The adults of R. dominica were also susceptible
to the fumigation of essential oils extracted from Eucalyptus globulus Labill (24-h LC
50
=3.529
µ
L/L),
Lavandula stoechas L. (24-h LC
50
=5.660
µ
L/L), and Apium graveolens L. (
24-h LC50 =53.506 µL/L) [37,38]
.
The fumigation of the essential oils of Lippia citriodora Kunth (24-h LC
50
=37.349
µ
L/L), Melissa officinalis
L. (24-h LC
50
=19.418
µ
L/L), and Teucrium polium L. (24-h LC
50
=20.749
µ
L/L) resulted in significant
mortality in T. castaneum [
39
–
41
]. The essential oils of Schinus molle L. (48-h LC
50
=806.50
µ
L/L) and
Artemisia sieberi Besser (24-h LC
50
=33.80
µ
L/L) also had notable fumigant toxicity against the adults
of T. granarium [
42
,
43
]. The toxicity of all the above-mentioned essential oils was augmented when
the exposure time was prolonged. These findings support the results regarding the time-dependent
susceptibility of O. surinamensis,R. dominica,T. castaneum and T. granarium to plant essential oils.
The differences in observed LC
50
values are likely due to the differences in the essential oil compositions
from the different plant species and possibly to differences in the experimental conditions. Furthermore,
the S. intermedia essential oil with low 24-h LC
50
value was more toxic on O. surinamensis than
A. foeniculum,A. filipendulina, and A. millefolium essential oils, on R. dominica than A. graveolens essential
oil, on T. castaneum than Lippia citriodora essential oil, and on T. granarium than S. molle essential oil.

Foods 2020,9, 712 8 of 12
The terpenes, especially thymol, carvacrol, p-cymene and
γ
-terpinene, were recognized as the
main components of S. intermedia essential oil in the present study. In the study of Sefidkon and
Jamzad, thymol (32.3%),
γ
-terpinene (29.3%), p-cymene (14.7%), elemicin (4.8%), limonene (3.3%),
and
α
-terpinene (3.3%) were the main components of S. intermedia essential oil [
20
]. In another study,
thymol (34.5%),
γ
-terpinene (18.2%), p-cymene (10.5%), limonene (7.3%),
α
-terpinene (7.1%), carvacrol
(6.9%), and elemicin (5.3%) were found to be major components in the essential oil of S. intermedia [
23
].
In the present study, however, limonene was a minor component (0.5%), and neither elemicin nor
α
-terpinene were detected. Ghorbanpour et al. reported the terpenes thymol (32.3%), p-cymene
(14.7%),
γ
-terpinene (3.3%), and carvacrol (1.0%), and the phenylpropanoid elemicin (4.8%) as the
main components in the essential oil of S. intermedia [
22
], while the concentrations of
γ
-terpinene
and carvacrol were much lower compared to the present findings. The differences in the chemical
profile of the plant essential oils are likely due to the internal and external factors such as seasonal
variation, geographical features, plant growth stage, and different extraction conditions [
19
,
44
,
45
]. The
insecticidal properties of several terpenes, especially monoterpene hydrocarbons and monoterpenoids,
which accounted for 88.9% of the S. intermedia essential oil in the present study, have been documented
in recent investigations. For example, insecticidal activities of p-cymene,
α
-pinene,
γ
-terpinene,
1,8-cineole, and limonene have been demonstrated against several detrimental insect pests [
46
–
50
].
Previous studies have also indicated that the monoterpenoids thymol and carvacrol had significant
toxicity against insect pests [
46
,
51
,
52
]. Accordingly, the insecticidal efficiency of S. intermedia essential
oil can be attributed to such components.
The contact toxicity of the essential oil of Eucalyptus globulus Labill. against A. nerii has been
reported by Russo et al. [
53
]. Although this is the only previous study to investigate the susceptibility of
A. nerii to a plant essential oil, its findings confirm the results of the present study about the possibility
of A. nerii management through plant essential oils. Indeed, the toxicity of S. intermedia essential oil was
evaluated for the first time in the present study against A. nerii and its natural enemy C. septempunctata.
The essential oil of S. intermedia was more toxic on A. nerii (LC
50
: 418
µ
g/mL) than the predator ladybird
C. septempunctata (LC
50
: 914
µ
g/mL), suggesting that the predator was more tolerant than the aphid
to S. intermedia essential oil, which is very valuable in terms of predator protection. Similar results
were obtained for controlling aphids [
54
,
55
] and some other insect pests [
56
–
58
] using plant-derived
essential oils along with protecting their predators. However, the destructive side-effects of some
essential oils on parasitoids have been reported [
59
–
61
]. Therefore, it is important to select efficient
pesticides with lower side effects on natural enemies at operative concentrations to the pests, which
has been achieved in the current study.
5. Conclusions
In conclusion, the terpene-rich essential oil of S. intermedia has significant fumigant toxicity against
the adults of O. surinamensis,R. dominica,T. castaneum, and T. granarium, and may be considered as
a natural effective fumigant on stored products. This bio-rational agent also has significant contact
toxicity on the adult females of A. nerii, one of the cosmopolitan insect pests of ornamental plants.
Furthermore, the predator ladybird C. septempunctata was more tolerant to the essential oil than the
aphid. Accordingly, S. intermedia essential oil can be nominated as an eco-friendly efficient insecticide
by decreasing the risks associated with the application of synthetic chemicals. However, the exploration
of any side-effects of the essential oil on other useful insects such as parasitoids and pollinators, its
phytotoxicity on the treated plants and crops, any adverse tastes or odors on stored products, and the
preparation of novel formulations to increase its stability in the environment for practical utilization
are needed.
Author Contributions:
Conceptualization, A.E.; methodology, A.E. and W.N.S.; validation, A.E. and W.N.S.;
formal analysis, A.E. and W.N.S.; investigation, A.E.; resources, A.E.; data curation, A.E.; writing—original draft
preparation, A.E.; writing—review and editing, A.E. and W.N.S.; project administration, A.E.; funding acquisition,
A.E. All authors have read and agreed to the published version of the manuscript.

Foods 2020,9, 712 9 of 12
Funding: This research was funded by the University of Mohaghegh Ardabili.
Acknowledgments:
W.N.S. participated in this work as part of the activities of the Aromatic Plant Research Center
(APRC, https://aromaticplant.org/). This study received financial support from the University of Mohaghegh
Ardabili, which is greatly appreciated.
Conflicts of Interest: The authors declare no conflict of interest.
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