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Fumigant toxicity of Lavandula spica essential oil and linalool on different life stages of Tribolium confusum (Coleoptera: Tenebrionidae)
Journal Pre-proofs Full length article Fumigant toxicity of Lavandula spica essential oil and linalool on different life stages of Tribolium confusum (Coleoptera: Tenebrionidae) Lynda Kheloul, Sylvia Anton, Christophe Gadenne, Abdellah Kellouche PII: DOI: Reference:
S1226-8615(19)30737-X https://doi.org/10.1016/j.aspen.2020.02.008 ASPEN 1516
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Journal of Asia-Pacific Entomology
Received Date: Revised Date: Accepted Date:
13 November 2019 23 January 2020 14 February 2020
Please cite this article as: L. Kheloul, S. Anton, C. Gadenne, A. Kellouche, Fumigant toxicity of Lavandula spica essential oil and linalool on different life stages of Tribolium confusum (Coleoptera: Tenebrionidae), Journal of Asia-Pacific Entomology (2020), doi: https://doi.org/10.1016/j.aspen.2020.02.008
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Fumigant toxicity of Lavandula spica essential oil and
2
linalool on different life stages of Tribolium confusum
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(Coleoptera: Tenebrionidae)
4
Lynda Kheloula, Sylvia Antonb, Christophe Gadenneb, Abdellah Kellouchea
5
a
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des variations climatiques. Faculté des sciences biologiques et des sciences
7
agronomiques, Université Mouloud Mammeri, Tizi-Ouzou 15000, Algeria
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[email protected]; [email protected]
9
b
Laboratoire de production, sauvegarde des espèces menacées et des récoltes. Influence
UMR IGEPP INRAE/Agrocampus Ouest/Université Rennes 1, Agrocampus Ouest,
10
49054 Angers cedex 01, France
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[email protected]; [email protected]
12
Correspondence
to
be
sent
to :
13
INRA/AgrocampusOuest/Université Rennes 1, AgrocampusOuest, 2, rue Le Nôtre, 49054
14
Angers cedex 01, France. email : [email protected]
15 16 17 18 19 20 21 22 23 1
Sylvia
Anton,
UMR
IGEPP
24
Abstract
25
The confused flour beetle, Tribolium confusum, is a common and severe pest of stored
26
products. Here, using fumigation tests during four different exposure times, we evaluated the
27
toxicity of different doses of essential oil of spike lavender, Lavandula spica and one of its
28
major constituents, linalool, on different life stages of T. confusum under laboratory
29
conditions. The toxicity of the L. spica oil and linalool varied as a function of the
30
developmental stage and treatment duration. Young larvae (L1) were the most susceptible to
31
toxic effects, with LC50 = 19.535μl/L of air for L. spica oil and LC50 =14.198 μl/L of air for
32
linalool after 24h of exposure, whereas older larvae (L8) were affected only very little by
33
fumigation. Linalool caused higher egg mortality than L. spica oil at equivalent doses, but
34
lower mortality in pupae and adults. Emergence of intact adult insects from surviving eggs,
35
larvae and pupae was further reduced as a function of dose and exposure time to both L.
36
spica oil and linalool compared to control-treated insects. Our results show that L. spica oil
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and linalool might be suitable for biological control of T. confusum, but tests at a larger scale
38
are necessary to confirm our results.
39 40
Keywords: Tribolium confusum; Lavandula spica; essential oil; linalool; fumigation; toxicity.
41 42 43
2
44
Introduction
45
Insects pests are the main cause of post-harvest losses because they depreciate food in
46
storage and are able to destroy a whole stock in a very short period of time (Ngamo and
47
Hance, 2007).. In in traditional maize storage systems in Togo, for example, insects accounted
48
for 80-90% of losses (Pantenius, 1988). Among pests of stored products, Tribolium confusum
49
(du Val) is one of the major insect pests of cereals and derivatives, infesting particularly mills,
50
silos and other storage facilities. This beetle causes considerable damage both at the larval and
51
adult stages by feeding on and contaminating food with their faeces, exuviae, dead insects and
52
the strong noxious odour they produce (Timothy, 2009), leading to a serious loss of market
53
value (Trematerra et al., 2011).
54
The techniques used currently to control stored-product pests rely largely on the use of
55
synthetic insecticides, such as phosphine and sulfuryl fluoride applied by fumigation (Lienard
56
and Seck, 1994 ; Arthur, 1996; Campbell et al ; 2010). However, chemical control causes
57
numerous problems such as toxicity for vertebrates including humans, environmental
58
pollution, high costs and the development of insect resistance (Arthur, 1996; Gueye et al.,
59
2011). The resistance of insects to synthetic pesticides is one of the main problems of frequent
60
application of these products. In Brazil, for example, populations of Tribolium castaneum
61
(Herbst) have shown a high degree of tolerance to phosphine, explained by the ability of these
62
insects to reduce their breathing rate (Pimentel et al., 2008). Because of, the development of
63
resistances and efforts to reduce environmental pollution, there is an urgent need to find
64
natural and sound alternative protection strategies to reduce the application of synthetic
65
insecticides. Plant essential oils are among the most promising alternatives to chemical
66
insecticides. Essential oils are complex natural mixtures of volatile organic compounds
67
produced as secondary metabolites in plants, which are constituted by terpenes, terpenoids
68
and phenol-derived aromatic components and aliphatic components (Bakkali et al., 2008).
3
69
They have been reported to have toxic, repellent, antifeedant, antibacterial, antifungal,
70
reproduction and growth inhibitory effects against insect pests (Regnault-Roger et al., 2012;
71
Ebadollahi and Jalali Sendi, 2015; Tu et al., 2018; Hu et al., 2019). In most cases, active
72
compounds of essential oils have low toxicity for vertebrates and short environmental
73
persistence, which makes them good candidates for eco-friendly insect management
74
(Regnault-Roger et al., 2012). A variety of essential oils has been examined for their
75
efficiency to control stored product pests with largely varying results, depending on the type
76
of essential oil and the insect species (e.g. Kellouche and Soltani, 2004; Stamopoulos et al.,
77
2007; Kellouche et al., 2010; Haouas et al., 2012; Regnault-Roger et al., 2012).
78
Spike lavender, Lavandula spica, is an aromatic plant of the Lamiaceae family largely
79
cultivated in the Mediterranean area (Muñoz-Bertomeu et al., 2007) and its main constituent,
80
linalool has been shown to be a potential alternative to synthetic insecticides against stored-
81
product pests such as Callosobruchus maculatus (Fabricius) and Rhyzopertha dominica
82
(Fabricius) (Davoudi et al., 2011). However, the effects of L. spica essential oil on the
83
different developmental stages of T. confusum, have only partially been investigated so far
84
and information on the efficiency of linalool against T. confusum is limited (Stamopoulos et
85
al., 2007; Saglam and Özder, 2013).
86
The aim of the current study was to evaluate fumigant toxicity of L. spica essential oil
87
and its main component, linalool, against eggs, young larvae, (L1) old larvae (L8), pupae and
88
adults of T. confusum under laboratory conditions.
89 90
Materials and methods
91
Insect rearing
4
92
The original strain was collected from a mill in the region of Azazga (Tizi-Ouzou,
93
Algeria) and has been maintained in our laboratory (Laboratoire de production, sauvegarde
94
des espèces menacées et des récoltes. Influence des variations climatiques. Faculté des
95
Sciences Biologiques et des Sciences Agronomiques, Université Mouloud Mammeri, Tizi-
96
Ouzou 15000, Algeria) since 2014. Eggs and young larvae of T. confusum were obtained from
97
cultures reared on a diet of wheat flour inside a dark incubator set at 30 ± 2° C and 70 ± 5 %
98
RH (Haouas et al., 2012). The old larvae, pupae and adults were taken from cultures reared on
99
a diet of medium-grain semolina under the same conditions.
100
For bioassays, we used 24 h-old eggs, one to two days-old young larvae (1st larval
101
instar: L1), 27 day-old larvae (8th larval instar: L8), one to three days-old pupae and adults
102
aged between one and seven days. All experiments were carried out under the same
103
conditions as the rearing.
104 105
Life cycle of T. confusum on semolina substrate
106
In order to follow the effects of fumigation on defined developmental stages, we recorded
107
in detail the life cycle of T. confusum on medium-grain semolina substrate under the same
108
laboratory conditions as the insect rearing. Adults were introduced in wheat flour for
109
oviposition and were removed after 24 h. The flour was then sifted with a 250 μm mesh sieve
110
to collect the eggs. Larvae started to hatch between 4 and 7 days later and we followed the
111
development of an homogeneous group of 60 L1 larvae, hatched after 4 days of egg
112
incubation. Each L1 larva was transferred into a small petri dish containing 0.5 g of medium-
113
grain semolina and the development was followed daily. At the L4 instar, larvae were
114
transferred into a new dish with 0.5 g of medium-grain semolina and the development was
115
further observed daily. Moulting was noted for each change of larval instar and the pupae and
116
exuviae were removed. 5
117 118
Essential oil and compound
119
The essential oil of L. spica flowers from Spain was purchased from the French
120
laboratory Phytosun Arôms (Laboratoires Omega Pharma, Chatillon, France). Previous
121
chemical analysis of the same oil used in the present study revealed linalool (43.79%),
122
eucalyptol (31.80%) and camphor (11.98%) as major constituents (Kheloul et al., 2019).
123
Linalool of 97% purity was provided by Sigma-Aldrich (Saint-Quentin Fallavier, France).
124 125
Fumigation tests
126
Fumigation tests were done in 64 ml Plexiglas containers with screw caps. A filter paper
127
disk (2 cm diameter, Whatman No 1) was fixed to the inner side of the screw cap with a
128
cotton thread and impregnated with a defined amount of pure essential oil, and the
129
corresponding doses per volume of air were calculated. Twenty individuals of each
130
developmental stage were introduced into a container without substrate for eggs and pupae,
131
with 0.1 g of semolina for L1 larvae and with 3 g of semolina for L8 larvae and adults. Eggs
132
and L1 larvae were deposited on a small lid (3 cm diameter, 1 cm high) inside the container
133
and the other stages were directly deposited into the container.
134
For young larvae the following doses were applied: 7.81, 15.63, 31.25 and 62.5 μl/L of
135
air. For all other stages, 62.5, 125, 250, 500 or 1000 μl/L of air doses were applied, and
136
containers without essential oil application served as controls. Five replicates were performed
137
for each dose including controls. Mortality was recorded after 24, 48, 72 or 96 h exposure
138
periods.
139
Twenty-four hours after the end of the exposure period, the number of dead insects was
140
determined for larvae and adults. Larvae and adults were considered dead if no movement of 6
141
antennae and legs were observed under a stereomicroscope. The number of larvae hatched
142
from eggs, and of adults hatched from pupae were determined 7 days after treatment. All non-
143
hatched eggs and pupae were considered dead. The observed mortality was corrected only for
144
the egg stage, taking the mortality in control experiments into account, using Abbott’s
145
correction formula (Abott, 1925). For the other life stages there was no mortality in the
146
control experiments.
147
The larval development of surviving eggs and the development of surviving treated
148
larvae were followed up until adult emergence, and the percentage of adults emerging from
149
surviving individuals of each treated developmental stage was calculated. For treated pupae,
150
the percentage of intact (undamaged) adults emerging was determined, because pupal
151
treatment elicited malformations and also mosaics (adultoids) in some surviving pupae,
152
depending on the dose of the treatment and exposure time. However these malformed insects
153
did not survive more than 48 h.
154
Another experiment was designed to assess the median lethal concentration (LC50) after
155
24 h exposure. For each test, six concentrations of the oil, each with five replicates and twenty
156
individuals per replicate, were used. Control insects were kept in the same condition without
157
any essential oil. Mortality was determined as described in the previous experiment. The LC50
158
after 24 h was calculated for the 1st instar larvae, pupae and adults treated with L. spica
159
essential oil using respectively the following doses (7.81; 15.63; 23.44; 31.25; 46.88; 62.5
160
μl/L of air), (250; 375; 437.5; 500; 562.5; 625 μl/L of air) and (156.25; 250; 312.5; 375;
161
437.5; 500 μl/L). In the case of linalool, the LC50 was calculated for eggs and 1st instar larvae
162
using respectively the following doses (62.5; 125; 187.5; 250; 500; 562.5 μl/L of air) and
163
(7.81; 15.63; 23.44; 31.25; 62.5; 125 μl/L of air). In the other developmental stages, even very
164
high doses did not lead to sufficient mortality to calculate LC50 values.
165
7
166
Data analyses
167
The mortality data were submitted to an analysis of variance, using Stat Box Pro
168
(version 6.40) (Grimmer software, France). When the treatment effect was significant, the
169
analysis was completed with the Newman-Keuls test at 5% probability. A probit analysis
170
allowed estimating the LC50 and 95% confidence limits after 24 h exposure, using the SPSS
171
version 20 software package.
172 173
Results
174
Developmental cycle of T. confusum on medium-grain semolina
175
The total development duration from the egg to the adult beetle took 43.07 ± 0.29 days on
176
medium grain semolina. The larval development, comprising 8 instars, took 32.52 ± 0.28 days
177
and the pupal instar lasted for 6.55 ± 0.07 days (Supplementary Table 1).
178 179
Mortality
180
Fumigation with L. spica essential oil and linalool revealed toxicity as a function of the
181
developmental stage of T. confusum, the tested dose and the time of exposure. A variance
182
analysis using 2 classification criteria revealed that dose and duration of exposure, as well as
183
their interaction, act with a high degree of significance on the mortality of the different
184
developmental stages (Table 1).
185 186 187 188 8
189 190
Table 1. Analyses of variance for Tribolium confusum mortality as a function of L. spica oil and linalool dose and treatment duration (time).
191 Mortality (L.spica) Developmental stage Egg
L1
L8
Pupa
Adult
Mortality (linalool)
Factor
df
F
p
df
F
p
dose
4
222.756
<0,01
4
1056.77
<0,01
time
3
49.943
<0,01
3
2.139
0.10035
dose × time
12
17.455
<0,01
12
3.218
<0,01
dose
4
1,385.937
<0,01
4
569.401
<0,01
time
3
369.365
<0,01
3
94.664
<0,01
dose × time
12
81.117
<0,01
12
14.759
<0,01
dose
4
145
<0,01
5
43.094
<0,01
time
3
66.461
<0,01
3
17.731
<0,01
dose × time
12
33.471
<0,01
15
10.265
<0,01
dose
4
2347.385
<0,01
5
426.547
<0,01
time
3
95.455
<0,01
3
336.029
<0,01
dose × time
12
53.115
<0,01
15
70.826
<0,01
dose
4
989.589
<0,01
5
79.933
<0,01
time
3
7.165
<0,01
3
31.159
<0,01
dose × time
12
3.286
<0,01
15
20.911
<0,01
192 193 194
The L1 larvae showed the highest mortality upon exposure with L. spica essential oil
195
(Figure 1B). Whereas the highest applied dose for L1 larvae (62.5 μl/L of air) elicited almost
196
100% mortality already after 24h of exposure, this dose elicited only very low mortality in the
197
other developmental stages (Figure 1). The highest tested dose (500 μl/L of air) caused 100%
198
mortality after 48 h in pupae (Figure 1D), whereas it took 96 h for a similar effect in eggs
199
(Figure 1A). L8 larvae were a lot more resistant against exposure with L. spica essential oil:
200
the highest dose caused less than 40% mortality even after 96 h of exposure (Figure 1C). For
201
the adults, the two lowest applied doses (62.5 and 125 μl/L of air) did hardly elicit any
202
mortality, but at the higher doses, close to 95% mortality was observed after 48 h of
203
fumigation (Figure 1E).
9
204
Linalool also proved very toxic against eggs of T. confusum with almost 100% of
205
mortality recorded with the dose 250 μl/L of air already after 24 h of exposure (Figure 2A).
206
Linalool is also toxic for 1st instar larvae: even at the lowest tested dose almost 100%
207
mortality was observed after 72 h of exposure (Figure 2B). For 8th instar larvae, the results
208
show that with high doses, recorded mortalities remain largely below 50% (Figure 2C). For
209
the pupal stage, linalool caused high rates of mortality from 250 μl/L of air on at 96 h
210
exposure (Figure 2D). Fumigation with linalool alone elicited much lower mortality rates than
211
the essential oil in adults. Mortality at doses up to 500 μl/L of air was negligible and even at a
212
dose of 1000 μl/L of air, only 25% of the adult insects died after an exposure of 96 h (Figure
213
2E).
214
Probit analysis after 24 h for L. spica essential oil and linalool revealed that L1 larvae
215
(LC50 = 19.535μl/L of air for L. spica oil; LC50 =14.198 μl/L of air for linalool) were more
216
susceptible than the other instars (Table 2).
217 218 219 220 221 222
Table 2. Probit analysis of fumigant toxicity data of Lavandula spica essential oil and linalool against Tribolium confusum life stages after 24 h of exposure. LC50 values are considered significantly different when the 95% confidence limits fail to overlap. The highest tested dose (still leading to less than 50% mortality) is provided for developmental stages with very low mortality. Life stage
Dose LC50 (μl/L of air)
95% confidence limit
Intercept
Slope
> 500 19.535
(17.892 – 21.188)
- 4.516
3.498
4
6.617 (1)
L8 Pupa Adult Linalool Egg L1
> 500 444.824 176.186
(370.903-522.358) (134.985 - 207.149)
-16.448 -4.808
6.211 2.141
4 4
23.419(2) 2.895 (1)
128.404 14.198
(90.878-165.494) (1.841-26.500)
-7.094 -1.355
3.364 1.176
4 4
16.318(2) 17.832(2)
L8 Pupa Adult
> 1000 > 1000 > 1000
-
-
-
-
-
L .spica Egg L1
223 224 225
df
Chi-square
Since the significance level is greater than 0.15, no heterogeneity factor is used in the calculation of confidence limits. (1)
10
226 227
Since the significance level is lower than 0.15, a heterogeneity factor is used in the calculation of confidence limits. (2)
228 229
Adult emergence of surviving insects after treatment at different developmental stages
230
Fumigation had different effects on the emergence of adults in surviving insects treated at
231
different developmental stages with different doses and exposure times. Variance analysis
232
revealed a highly significant effect of the factor dose and the factor exposure time, as well as
233
their interaction on the emergence of adults after treatments of earlier developmental stages
234
(Table 3).
235 236 237
Table 3. Analyses of variance for Tribolium confusum intact adult emergence as a function of L. spica and linalool dose and treatment duration (time).
238 Adult emergence (L.spica) Developmental stage Egg
L1
L8
Pupa
Adult emergence (linalool)
Factor
df
F
p
df
F
p
dose
4
367.657
<0,01
4
819.204
<0,01
time
3
63.209
<0,01
3
1.29
0,28296
dose × time
12
15.148
<0,01
12
1.949
0,04033
dose
4
1,884.328
<0,01
4
435.891
<0,01
time
3
158.313
<0,01
3
45.946
<0,01
dose × time
12
48.16
<0,01
12
10.512
<0,01
dose
4
64.382
<0,01
5
90.251
<0,01
time
3
28.713
<0,01
3
46.697
<0,01
dose × time
12
14.069
<0,01
15
17.659
<0,01
dose
4
1945.892
<0,01
5
554.195
<0,01
time
3
43.708
<0,01
3
310.174
<0,01
dose × time
12
23.491
<0,01
15
52.583
<0,01
239 240
The treatment with L. spica and linalool affected the emergence of adults from surviving
241
eggs and larvae in a similar way as mortality (Figures 3, 4). A large proportion of adult
242
insects hatched for the treated stages and doses with low mortality, whereas higher treatment
243
doses at susceptible stages lead to low emergence rates (Figures 3, 4). Especially for treated 11
244
1st instar larvae, the number of emerging adults was further decreased as compared to the
245
number of surviving insects with slightly stronger reductions for L. spica oil than for linalool
246
(Figures 3B, 4B). Most surviving L8 larvae from fumigation with L. spica reach the adult
247
stage even at the highest treatment dose (Figure 3C). The percentage of adults hatching from
248
larvae treated at L8 with linalool are mainly affected by the 1000 µl dose at 72 and 96 h
249
exposure (Figure 4C). Insects treated at the pupal instar with high doses and long duration,
250
resulted in a larger percentage of emerging intact adults for linalool than for L. spica oil, and
251
consequently less malformed adults and mosaics (Figure 3D, 4D).
252 253
Discussion
254
To know the precise age of the tested insects at each developmental stage, it was
255
necessary to study the life cycle of T. confusum and the mean duration of each stage in our
256
laboratory conditions. The development of T. confusum on semolina under our conditions was
257
slightly shorter compared to the mean development time reported on flour at a slightly lower
258
temperature (Stamopoulos et al., 2007).
259
We then estimated in this study the mortality effects of essential oil of L. spica and
260
linalool by fumigation against eggs, larvae, pupae and adults. Our data showed that the toxic
261
effects of L. spica essential oil and linalool depend on the developmental stage, the applied
262
dose and the duration of exposure. A relatively high dose (500 μl/L of air) and a long
263
exposure time (96 h) with L. spica oil is necessary to kill 100% of the eggs of T. confusum,
264
whereas half the concentration (250 μl/L of air) of linalool alone has the same effect. This
265
indicates that linalool might be to a large part responsible for the toxic effect on eggs, because
266
the used L. spica oil contains 43.79 % of linalool (Kheloul et al., 2019). The same exposure
267
time and similarly high doses (196.9 μl/L of air) of Pimpinella anisum are necessary to cause
268
100% egg mortality in T. confusum (Tunç et al., 2000). Studies on other plant essential oils,
12
269
such as Allium sativum, Betula lenta and Cinnamonum zeylanicum showed a better efficiency,
270
killing 100% of the eggs at a dose of 20 μl/L of air and within 24 h (I şıkber et al., 2009).
271
Essential oils from Allium cepa and Foeniculum vulgare killed 100% of T. confusum eggs at
272
an intermediate dose and exposure time (100 μl/L of air, 72 h) (Karci and Isikber, 2007) and
273
around 10 g of Laurus nobilis essential oil/h caused 90% mortality of T. confusum eggs,
274
whereas rosemary oil was not toxic for eggs (Isikber et al., 2006). Due to very different
275
compounds dominating the different oils tested in the cited studies (though not provided in all
276
studies), it is difficult to correlate egg toxicity of the different plant oils with the action of
277
specific compounds. The generally relatively high resistance of T. confusum eggs to
278
fumigation with essential oils might be due to the lack of aeropyles and micropyles in their
279
eggs, as well as a thick endochorion layer and a greater intrachorionic meshwork as compared
280
to other insect eggs, which obstruct the diffusion of vapors into the egg (Gautam et al., 2015).
281
In addition respiration rates are low at the egg stage, reducing gas exchange and consequently
282
diffusion of volatiles into the egg (Emekci et al., 2002)
283
Comparison of LC50 doses at 24 h exposure, obtained for different developmental stages
284
with L. spica essential oil, shows that the oil is more efficient on young larvae (L1) than on
285
older larvae (L8) or other stages. This age-dependent effect has also been found with
286
Artemisia haussknechtii and Lavandula hybrida essential oils, with increasing LC50 doses
287
with the age of larvae of T. confusum, even though main compounds of these oils are different
288
from the main compounds found in our L. spica oil (Hashemi and Safavi, 2013; Theou et al.,
289
2013). 1st instar larvae were indeed also the most sensitive developmental stage to the linalool
290
treatment, with an LC50 of 14.198 μl/L of air. This confirms earlier studies, which had
291
identified young larval instars as the most susceptible to linalool (Stamopoulos et al., 2007).
292
Toxicity of essential oils and linalool thus seems to be stronger for younger than for older
293
larvae in this insect.
13
294
Our results also showed that L. spica essential oil is more toxic for T. confusum pupae
295
than linalool, causing not only mortality but also malformations of the adults hatching from
296
surviving pupae and mosaics and consequently reducing the percentage of emergence of
297
intact adults. Thus for pupae, contrary to eggs, linalool might not be the most toxic compound
298
within the oil. Similar toxicity for pupae as for L. spica oil was also observed in treatments
299
with another lavender essential oil, L. hybrida (Theou et al., 2013). When adults were treated
300
with L. spica essential oil, marked toxicity started at 250 μl/L of air with an LC50 of 176.19
301
μl/L of air following a 24 h exposure. Linalool, however, caused only a low percentage of
302
mortality in adults, comparable to an earlier study in the same species, where a dose of 100
303
μl/L of air applied for 24 h elicited low rates of mortality in eggs, old larvae, pupae, and
304
adults, whereas young larvae had not been tested (Saglam and Özder, 2013). Similarly, low
305
toxicity of fumigation with linalool was found for adult T. castaneum (Rozman et al., 2007).
306
Thus other compounds present in the essential oil might be responsible for the observed toxic
307
effect or different compounds may act synergistically.
308
Essential oils of different lavender species have been previously shown to vary
309
substantially in their toxicity on adults of the related species T. castaneum. Whereas
310
Lavandula stoechas oil showed an LC50 at less than 40 μl/L of air after 24 h (Ebadollahi et al.,
311
2010), Lavandula angustifolia had only very minor toxic effects, but details on the used doses
312
are not provided (Pugazhvendan et al., 2012). However, L. angustifolia was highly toxic to a
313
different coleopteran stored product pest, Sitophilus granarius (L.) (Germinara et al., 2017).
314
Overall, effects of different oils on the same species and the same oil on different insect
315
species are highly variable, indicating that tests of different oils have to be performed for each
316
pest insect species..The highly variable effects of essential oils on different insects and even
317
different developmental stages of the same insect can be explained by strong differences in
318
metabolic activity and the importance of targets of active compounds. Certain constituents of
14
319
aromatic essential oils are known to have neurotoxic effects, whereas others are thought to act
320
on insect growth regulators, i.e. hormones (Isman, 2000; Tandon et al., 2008; Regnault-Roger
321
et al., 2012)..
322
Our study revealed that L. spica and linalool showed toxicity on certain stages and
323
ineffectiveness against other stages of T. confusum. They also caused malformations in adults
324
hatching from treated pupae, further reducing reproductive success through non-viable adults.
325
Thus the tested volatiles might represent a potential alternative to synthetic insecticides for
326
the control of the stored product pest T. confusum. Because of the long life duration of adult
327
beetles, relatively high doses also causing adult mortality would be needed at least in initial
328
treatments. Due to the strong odour of the used volatiles, direct treatments of stored products
329
are difficult to imagine, but treatments of storage surfaces might be more suitable instead, at
330
the same time reducing absorption of essential oils by the stored products, which could in turn
331
reduce efficacy (Arthur et al., 2011). Further studies are necessary to explore potential
332
practical application of L. spica essential oil and its main component including safety tests for
333
vertebrates and the environment and development of formulations.
334 335 336 337
Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
338 339
References
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Kellouche, A., Soltani, N., 2004. Activité biologique des poudres de cinq plantes et de l’huile essentielle d’une d’entre elles sur Callosobruchus maculatus (F.). Int. J. Trop. Insect Sci. 24, 211. https://doi.org/10.1079/IJT200420 Kheloul, L., Kellouche, A., Bréard, D., Gay, M., Gadenne, C., Anton, S., 2019. Trade-off between attraction to aggregation pheromones and repellent effects of spike lavender essential oil and its main constituent linalool in the flour beetle Tribolium confusum. Entomol. Exp. Appl. 167, 826–834. https://doi.org/10.1111/eea.12831 Lienard, V., Seck, D., 2011. Revue des methodes de lutte contre Callosobruchus maculatus (F.) (Coleoptera: Bruchidae), ravageur des graines de niébé ( Vigna unguiculata (L.) Walp) en Afrique tropicale. Int. J. Trop. Insect Sci. 15, 301–311. Muñoz-Bertomeu, J., Arrillaga, I., Segura, J., 2007. Essential oil variation within and among natural populations of Lavandula latifolia and its relation to their ecological areas. Biochem. Syst. Ecol. 35, 479–488. https://doi.org/10.1016/j.bse.2007.03.006 Pantenius, C.U., 1988. Storage losses in traditional maize granaries in Togo. Int. J. Trop. Insect Sci. 9, 725–735. https://doi.org/10.1017/S1742758400005610 Pimentel, M.A.G., Faroni, L.R.D., Batista, M.D., Silva, F.H. da, 2008. Resistance of storedproduct insects to phosphine. Pesqui. Agropecuária Bras. 43, 1671–1676. https://doi.org/10.1590/S0100-204X2008001200005 Pugazhvendan, S.R., Ross, P.R., Elumalai, K., 2012. Insecticidal and repellant activities of plants oil against stored grain pest, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae). Asian Pac. J. Trop. Dis. 2, S412–S415. https://doi.org/10.1016/S2222-1808(12)60193-5 Regnault-Roger, C., Vincent, C., Arnason, J.T., 2012. Essential oils in insect control: low-risk products in a high-stakes world. Annu. Rev. Entomol. 57, 405–424. https://doi.org/10.1146/annurev-ento-120710-100554 Rozman, V., Kalinovic, I., Korunic, Z., 2007. Toxicity of naturally occurring compounds of Lamiaceae and Lauraceae to three stored-product insects. J. Stored Prod. Res. 43, 349–355. https://doi.org/10.1016/j.jspr.2006.09.001 Saglam, O., Özder, N., 2013. Fumigant toxicity of monoterpenoid compounds against the confused flour beetle, Tribolium confusum Jacquelin du Val. (Coleoptera: Tenebrionidae). Turk. J. Entomol. 37. Stamopoulos, D.C., Damos, P., Karagianidou, G., 2007. Bioactivity of five monoterpenoid vapours to Tribolium confusum (du Val) (Coleoptera: Tenebrionidae). J. Stored Prod. Res. 43, 571–577. https://doi.org/10.1016/j.jspr.2007.03.007 Tandon, S., Mittal, A.K., Pant, A.K., 2008. Insect growth regulatory activity of Vitex trifolia and Vitex agnus-castus essential oils against Spilosoma obliqua. Fitoterapia 79, 283– 286. https://doi.org/10.1016/j.fitote.2007.11.032 Theou, G., Papachristos, D.P., Stamopoulos, D.C., 2013. Fumigant toxicity of six essential oils to the immature stages and adults of Tribolium confusum. Hell. Plant Prot. J. 6, 29–39. Timothy, D., 2009. Effects of crowding on the loss in weight of sorghum flour and on the survival and development of adult confused flour beetle, Tribolium confusum in sorghum flour. N. Y. Sci. J. 2, 56–61. Trematerra, P., Stejskal, V., Hubert, J., 2011. The monitoring of semolina contamination by insect fragments using the light filth method in an Italian mill. Food Control 22, 1021– 1026. https://doi.org/10.1016/j.foodcont.2010.11.026 Tu, X.-F., Hu, F., Thakur, K., Li, X.-L., Zhang, Y.-S., Wei, Z.-J., 2018. Comparison of antibacterial effects and fumigant toxicity of essential oils extracted from different plants. Ind. Crops Prod. 124, 192–200. https://doi.org/10.1016/j.indcrop.2018.07.065
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Tunç, İ., Berger, B.M., Erler, F., Dağlı, F., 2000. Ovicidal activity of essential oils from five plants against two stored-product insects. J. Stored Prod. Res. 36, 161–168. https://doi.org/10.1016/S0022-474X(99)00036-3
18
450
Figure Legends
451
Figure 1. Mortality rates (mean ± SEM) of the different developmental stages of Tribolium
452
confusum treated by fumigation with Lavandula spica essential oil. (A) egg, (B) 1st instar L1,
453
(C) 8th instar L8, (D) pupa and (E) adults. N = 100 (5 x 20 insects) for each treatment. Bars
454
with the same letter are not significantly different.
455 456
Figure 2. Mortality rates (mean ± SEM) of the different developmental stages of Tribolium
457
confusum treated with linalool. (A) egg, (B) 1st instar L1, (C) 8th instar L8, (D) pupa and (E)
458
adults. N = 100 (5 x 20 insects) for each treatment. Bars with the same letter are not
459
significantly different.
460 461
Figure 3. Proportion of emerging intact adults (mean ± SEM) from insects surviving
462
treatments with L. spica essential oil of immature stages. Treatments at (A) egg, (B) 1st instar
463
L1, (C) 8th instar L8, (D) pupa. Bars with the same letter are not significantly different.
464 465
Figure 4. Proportion of emerging intact adults (mean ± SEM) from insects surviving
466
treatments with linalool of immature stages. Treatments at (A) egg, (B) 1st instar L1, (C) 8th
467
instar L8, (D) pupa. Bars with the same letter are not significantly different.
468 469 470 471 472 473 474 475 476
Declaration of interests ☐ x The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
19
477 478 479 480 481 482
Highlights
483
- Spike lavender toxicity was stage-, and exposure time-dependent in Tribolium confusum
484
- First instar larvae were most sensitive to lavender oil and its component linalool
485
- Adult emergence of most treated stages was reduced depending on dose or exposure time
486 487 488
20
S1226-8615(19)30737-X https://doi.org/10.1016/j.aspen.2020.02.008 ASPEN 1516
To appear in:
Journal of Asia-Pacific Entomology
Received Date: Revised Date: Accepted Date:
13 November 2019 23 January 2020 14 February 2020
Please cite this article as: L. Kheloul, S. Anton, C. Gadenne, A. Kellouche, Fumigant toxicity of Lavandula spica essential oil and linalool on different life stages of Tribolium confusum (Coleoptera: Tenebrionidae), Journal of Asia-Pacific Entomology (2020), doi: https://doi.org/10.1016/j.aspen.2020.02.008
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© 2020 Published by Elsevier B.V. on behalf of Korean Society of Applied Entomology.
1
Fumigant toxicity of Lavandula spica essential oil and
2
linalool on different life stages of Tribolium confusum
3
(Coleoptera: Tenebrionidae)
4
Lynda Kheloula, Sylvia Antonb, Christophe Gadenneb, Abdellah Kellouchea
5
a
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des variations climatiques. Faculté des sciences biologiques et des sciences
7
agronomiques, Université Mouloud Mammeri, Tizi-Ouzou 15000, Algeria
8
[email protected]; [email protected]
9
b
Laboratoire de production, sauvegarde des espèces menacées et des récoltes. Influence
UMR IGEPP INRAE/Agrocampus Ouest/Université Rennes 1, Agrocampus Ouest,
10
49054 Angers cedex 01, France
11
[email protected]; [email protected]
12
Correspondence
to
be
sent
to :
13
INRA/AgrocampusOuest/Université Rennes 1, AgrocampusOuest, 2, rue Le Nôtre, 49054
14
Angers cedex 01, France. email : [email protected]
15 16 17 18 19 20 21 22 23 1
Sylvia
Anton,
UMR
IGEPP
24
Abstract
25
The confused flour beetle, Tribolium confusum, is a common and severe pest of stored
26
products. Here, using fumigation tests during four different exposure times, we evaluated the
27
toxicity of different doses of essential oil of spike lavender, Lavandula spica and one of its
28
major constituents, linalool, on different life stages of T. confusum under laboratory
29
conditions. The toxicity of the L. spica oil and linalool varied as a function of the
30
developmental stage and treatment duration. Young larvae (L1) were the most susceptible to
31
toxic effects, with LC50 = 19.535μl/L of air for L. spica oil and LC50 =14.198 μl/L of air for
32
linalool after 24h of exposure, whereas older larvae (L8) were affected only very little by
33
fumigation. Linalool caused higher egg mortality than L. spica oil at equivalent doses, but
34
lower mortality in pupae and adults. Emergence of intact adult insects from surviving eggs,
35
larvae and pupae was further reduced as a function of dose and exposure time to both L.
36
spica oil and linalool compared to control-treated insects. Our results show that L. spica oil
37
and linalool might be suitable for biological control of T. confusum, but tests at a larger scale
38
are necessary to confirm our results.
39 40
Keywords: Tribolium confusum; Lavandula spica; essential oil; linalool; fumigation; toxicity.
41 42 43
2
44
Introduction
45
Insects pests are the main cause of post-harvest losses because they depreciate food in
46
storage and are able to destroy a whole stock in a very short period of time (Ngamo and
47
Hance, 2007).. In in traditional maize storage systems in Togo, for example, insects accounted
48
for 80-90% of losses (Pantenius, 1988). Among pests of stored products, Tribolium confusum
49
(du Val) is one of the major insect pests of cereals and derivatives, infesting particularly mills,
50
silos and other storage facilities. This beetle causes considerable damage both at the larval and
51
adult stages by feeding on and contaminating food with their faeces, exuviae, dead insects and
52
the strong noxious odour they produce (Timothy, 2009), leading to a serious loss of market
53
value (Trematerra et al., 2011).
54
The techniques used currently to control stored-product pests rely largely on the use of
55
synthetic insecticides, such as phosphine and sulfuryl fluoride applied by fumigation (Lienard
56
and Seck, 1994 ; Arthur, 1996; Campbell et al ; 2010). However, chemical control causes
57
numerous problems such as toxicity for vertebrates including humans, environmental
58
pollution, high costs and the development of insect resistance (Arthur, 1996; Gueye et al.,
59
2011). The resistance of insects to synthetic pesticides is one of the main problems of frequent
60
application of these products. In Brazil, for example, populations of Tribolium castaneum
61
(Herbst) have shown a high degree of tolerance to phosphine, explained by the ability of these
62
insects to reduce their breathing rate (Pimentel et al., 2008). Because of, the development of
63
resistances and efforts to reduce environmental pollution, there is an urgent need to find
64
natural and sound alternative protection strategies to reduce the application of synthetic
65
insecticides. Plant essential oils are among the most promising alternatives to chemical
66
insecticides. Essential oils are complex natural mixtures of volatile organic compounds
67
produced as secondary metabolites in plants, which are constituted by terpenes, terpenoids
68
and phenol-derived aromatic components and aliphatic components (Bakkali et al., 2008).
3
69
They have been reported to have toxic, repellent, antifeedant, antibacterial, antifungal,
70
reproduction and growth inhibitory effects against insect pests (Regnault-Roger et al., 2012;
71
Ebadollahi and Jalali Sendi, 2015; Tu et al., 2018; Hu et al., 2019). In most cases, active
72
compounds of essential oils have low toxicity for vertebrates and short environmental
73
persistence, which makes them good candidates for eco-friendly insect management
74
(Regnault-Roger et al., 2012). A variety of essential oils has been examined for their
75
efficiency to control stored product pests with largely varying results, depending on the type
76
of essential oil and the insect species (e.g. Kellouche and Soltani, 2004; Stamopoulos et al.,
77
2007; Kellouche et al., 2010; Haouas et al., 2012; Regnault-Roger et al., 2012).
78
Spike lavender, Lavandula spica, is an aromatic plant of the Lamiaceae family largely
79
cultivated in the Mediterranean area (Muñoz-Bertomeu et al., 2007) and its main constituent,
80
linalool has been shown to be a potential alternative to synthetic insecticides against stored-
81
product pests such as Callosobruchus maculatus (Fabricius) and Rhyzopertha dominica
82
(Fabricius) (Davoudi et al., 2011). However, the effects of L. spica essential oil on the
83
different developmental stages of T. confusum, have only partially been investigated so far
84
and information on the efficiency of linalool against T. confusum is limited (Stamopoulos et
85
al., 2007; Saglam and Özder, 2013).
86
The aim of the current study was to evaluate fumigant toxicity of L. spica essential oil
87
and its main component, linalool, against eggs, young larvae, (L1) old larvae (L8), pupae and
88
adults of T. confusum under laboratory conditions.
89 90
Materials and methods
91
Insect rearing
4
92
The original strain was collected from a mill in the region of Azazga (Tizi-Ouzou,
93
Algeria) and has been maintained in our laboratory (Laboratoire de production, sauvegarde
94
des espèces menacées et des récoltes. Influence des variations climatiques. Faculté des
95
Sciences Biologiques et des Sciences Agronomiques, Université Mouloud Mammeri, Tizi-
96
Ouzou 15000, Algeria) since 2014. Eggs and young larvae of T. confusum were obtained from
97
cultures reared on a diet of wheat flour inside a dark incubator set at 30 ± 2° C and 70 ± 5 %
98
RH (Haouas et al., 2012). The old larvae, pupae and adults were taken from cultures reared on
99
a diet of medium-grain semolina under the same conditions.
100
For bioassays, we used 24 h-old eggs, one to two days-old young larvae (1st larval
101
instar: L1), 27 day-old larvae (8th larval instar: L8), one to three days-old pupae and adults
102
aged between one and seven days. All experiments were carried out under the same
103
conditions as the rearing.
104 105
Life cycle of T. confusum on semolina substrate
106
In order to follow the effects of fumigation on defined developmental stages, we recorded
107
in detail the life cycle of T. confusum on medium-grain semolina substrate under the same
108
laboratory conditions as the insect rearing. Adults were introduced in wheat flour for
109
oviposition and were removed after 24 h. The flour was then sifted with a 250 μm mesh sieve
110
to collect the eggs. Larvae started to hatch between 4 and 7 days later and we followed the
111
development of an homogeneous group of 60 L1 larvae, hatched after 4 days of egg
112
incubation. Each L1 larva was transferred into a small petri dish containing 0.5 g of medium-
113
grain semolina and the development was followed daily. At the L4 instar, larvae were
114
transferred into a new dish with 0.5 g of medium-grain semolina and the development was
115
further observed daily. Moulting was noted for each change of larval instar and the pupae and
116
exuviae were removed. 5
117 118
Essential oil and compound
119
The essential oil of L. spica flowers from Spain was purchased from the French
120
laboratory Phytosun Arôms (Laboratoires Omega Pharma, Chatillon, France). Previous
121
chemical analysis of the same oil used in the present study revealed linalool (43.79%),
122
eucalyptol (31.80%) and camphor (11.98%) as major constituents (Kheloul et al., 2019).
123
Linalool of 97% purity was provided by Sigma-Aldrich (Saint-Quentin Fallavier, France).
124 125
Fumigation tests
126
Fumigation tests were done in 64 ml Plexiglas containers with screw caps. A filter paper
127
disk (2 cm diameter, Whatman No 1) was fixed to the inner side of the screw cap with a
128
cotton thread and impregnated with a defined amount of pure essential oil, and the
129
corresponding doses per volume of air were calculated. Twenty individuals of each
130
developmental stage were introduced into a container without substrate for eggs and pupae,
131
with 0.1 g of semolina for L1 larvae and with 3 g of semolina for L8 larvae and adults. Eggs
132
and L1 larvae were deposited on a small lid (3 cm diameter, 1 cm high) inside the container
133
and the other stages were directly deposited into the container.
134
For young larvae the following doses were applied: 7.81, 15.63, 31.25 and 62.5 μl/L of
135
air. For all other stages, 62.5, 125, 250, 500 or 1000 μl/L of air doses were applied, and
136
containers without essential oil application served as controls. Five replicates were performed
137
for each dose including controls. Mortality was recorded after 24, 48, 72 or 96 h exposure
138
periods.
139
Twenty-four hours after the end of the exposure period, the number of dead insects was
140
determined for larvae and adults. Larvae and adults were considered dead if no movement of 6
141
antennae and legs were observed under a stereomicroscope. The number of larvae hatched
142
from eggs, and of adults hatched from pupae were determined 7 days after treatment. All non-
143
hatched eggs and pupae were considered dead. The observed mortality was corrected only for
144
the egg stage, taking the mortality in control experiments into account, using Abbott’s
145
correction formula (Abott, 1925). For the other life stages there was no mortality in the
146
control experiments.
147
The larval development of surviving eggs and the development of surviving treated
148
larvae were followed up until adult emergence, and the percentage of adults emerging from
149
surviving individuals of each treated developmental stage was calculated. For treated pupae,
150
the percentage of intact (undamaged) adults emerging was determined, because pupal
151
treatment elicited malformations and also mosaics (adultoids) in some surviving pupae,
152
depending on the dose of the treatment and exposure time. However these malformed insects
153
did not survive more than 48 h.
154
Another experiment was designed to assess the median lethal concentration (LC50) after
155
24 h exposure. For each test, six concentrations of the oil, each with five replicates and twenty
156
individuals per replicate, were used. Control insects were kept in the same condition without
157
any essential oil. Mortality was determined as described in the previous experiment. The LC50
158
after 24 h was calculated for the 1st instar larvae, pupae and adults treated with L. spica
159
essential oil using respectively the following doses (7.81; 15.63; 23.44; 31.25; 46.88; 62.5
160
μl/L of air), (250; 375; 437.5; 500; 562.5; 625 μl/L of air) and (156.25; 250; 312.5; 375;
161
437.5; 500 μl/L). In the case of linalool, the LC50 was calculated for eggs and 1st instar larvae
162
using respectively the following doses (62.5; 125; 187.5; 250; 500; 562.5 μl/L of air) and
163
(7.81; 15.63; 23.44; 31.25; 62.5; 125 μl/L of air). In the other developmental stages, even very
164
high doses did not lead to sufficient mortality to calculate LC50 values.
165
7
166
Data analyses
167
The mortality data were submitted to an analysis of variance, using Stat Box Pro
168
(version 6.40) (Grimmer software, France). When the treatment effect was significant, the
169
analysis was completed with the Newman-Keuls test at 5% probability. A probit analysis
170
allowed estimating the LC50 and 95% confidence limits after 24 h exposure, using the SPSS
171
version 20 software package.
172 173
Results
174
Developmental cycle of T. confusum on medium-grain semolina
175
The total development duration from the egg to the adult beetle took 43.07 ± 0.29 days on
176
medium grain semolina. The larval development, comprising 8 instars, took 32.52 ± 0.28 days
177
and the pupal instar lasted for 6.55 ± 0.07 days (Supplementary Table 1).
178 179
Mortality
180
Fumigation with L. spica essential oil and linalool revealed toxicity as a function of the
181
developmental stage of T. confusum, the tested dose and the time of exposure. A variance
182
analysis using 2 classification criteria revealed that dose and duration of exposure, as well as
183
their interaction, act with a high degree of significance on the mortality of the different
184
developmental stages (Table 1).
185 186 187 188 8
189 190
Table 1. Analyses of variance for Tribolium confusum mortality as a function of L. spica oil and linalool dose and treatment duration (time).
191 Mortality (L.spica) Developmental stage Egg
L1
L8
Pupa
Adult
Mortality (linalool)
Factor
df
F
p
df
F
p
dose
4
222.756
<0,01
4
1056.77
<0,01
time
3
49.943
<0,01
3
2.139
0.10035
dose × time
12
17.455
<0,01
12
3.218
<0,01
dose
4
1,385.937
<0,01
4
569.401
<0,01
time
3
369.365
<0,01
3
94.664
<0,01
dose × time
12
81.117
<0,01
12
14.759
<0,01
dose
4
145
<0,01
5
43.094
<0,01
time
3
66.461
<0,01
3
17.731
<0,01
dose × time
12
33.471
<0,01
15
10.265
<0,01
dose
4
2347.385
<0,01
5
426.547
<0,01
time
3
95.455
<0,01
3
336.029
<0,01
dose × time
12
53.115
<0,01
15
70.826
<0,01
dose
4
989.589
<0,01
5
79.933
<0,01
time
3
7.165
<0,01
3
31.159
<0,01
dose × time
12
3.286
<0,01
15
20.911
<0,01
192 193 194
The L1 larvae showed the highest mortality upon exposure with L. spica essential oil
195
(Figure 1B). Whereas the highest applied dose for L1 larvae (62.5 μl/L of air) elicited almost
196
100% mortality already after 24h of exposure, this dose elicited only very low mortality in the
197
other developmental stages (Figure 1). The highest tested dose (500 μl/L of air) caused 100%
198
mortality after 48 h in pupae (Figure 1D), whereas it took 96 h for a similar effect in eggs
199
(Figure 1A). L8 larvae were a lot more resistant against exposure with L. spica essential oil:
200
the highest dose caused less than 40% mortality even after 96 h of exposure (Figure 1C). For
201
the adults, the two lowest applied doses (62.5 and 125 μl/L of air) did hardly elicit any
202
mortality, but at the higher doses, close to 95% mortality was observed after 48 h of
203
fumigation (Figure 1E).
9
204
Linalool also proved very toxic against eggs of T. confusum with almost 100% of
205
mortality recorded with the dose 250 μl/L of air already after 24 h of exposure (Figure 2A).
206
Linalool is also toxic for 1st instar larvae: even at the lowest tested dose almost 100%
207
mortality was observed after 72 h of exposure (Figure 2B). For 8th instar larvae, the results
208
show that with high doses, recorded mortalities remain largely below 50% (Figure 2C). For
209
the pupal stage, linalool caused high rates of mortality from 250 μl/L of air on at 96 h
210
exposure (Figure 2D). Fumigation with linalool alone elicited much lower mortality rates than
211
the essential oil in adults. Mortality at doses up to 500 μl/L of air was negligible and even at a
212
dose of 1000 μl/L of air, only 25% of the adult insects died after an exposure of 96 h (Figure
213
2E).
214
Probit analysis after 24 h for L. spica essential oil and linalool revealed that L1 larvae
215
(LC50 = 19.535μl/L of air for L. spica oil; LC50 =14.198 μl/L of air for linalool) were more
216
susceptible than the other instars (Table 2).
217 218 219 220 221 222
Table 2. Probit analysis of fumigant toxicity data of Lavandula spica essential oil and linalool against Tribolium confusum life stages after 24 h of exposure. LC50 values are considered significantly different when the 95% confidence limits fail to overlap. The highest tested dose (still leading to less than 50% mortality) is provided for developmental stages with very low mortality. Life stage
Dose LC50 (μl/L of air)
95% confidence limit
Intercept
Slope
> 500 19.535
(17.892 – 21.188)
- 4.516
3.498
4
6.617 (1)
L8 Pupa Adult Linalool Egg L1
> 500 444.824 176.186
(370.903-522.358) (134.985 - 207.149)
-16.448 -4.808
6.211 2.141
4 4
23.419(2) 2.895 (1)
128.404 14.198
(90.878-165.494) (1.841-26.500)
-7.094 -1.355
3.364 1.176
4 4
16.318(2) 17.832(2)
L8 Pupa Adult
> 1000 > 1000 > 1000
-
-
-
-
-
L .spica Egg L1
223 224 225
df
Chi-square
Since the significance level is greater than 0.15, no heterogeneity factor is used in the calculation of confidence limits. (1)
10
226 227
Since the significance level is lower than 0.15, a heterogeneity factor is used in the calculation of confidence limits. (2)
228 229
Adult emergence of surviving insects after treatment at different developmental stages
230
Fumigation had different effects on the emergence of adults in surviving insects treated at
231
different developmental stages with different doses and exposure times. Variance analysis
232
revealed a highly significant effect of the factor dose and the factor exposure time, as well as
233
their interaction on the emergence of adults after treatments of earlier developmental stages
234
(Table 3).
235 236 237
Table 3. Analyses of variance for Tribolium confusum intact adult emergence as a function of L. spica and linalool dose and treatment duration (time).
238 Adult emergence (L.spica) Developmental stage Egg
L1
L8
Pupa
Adult emergence (linalool)
Factor
df
F
p
df
F
p
dose
4
367.657
<0,01
4
819.204
<0,01
time
3
63.209
<0,01
3
1.29
0,28296
dose × time
12
15.148
<0,01
12
1.949
0,04033
dose
4
1,884.328
<0,01
4
435.891
<0,01
time
3
158.313
<0,01
3
45.946
<0,01
dose × time
12
48.16
<0,01
12
10.512
<0,01
dose
4
64.382
<0,01
5
90.251
<0,01
time
3
28.713
<0,01
3
46.697
<0,01
dose × time
12
14.069
<0,01
15
17.659
<0,01
dose
4
1945.892
<0,01
5
554.195
<0,01
time
3
43.708
<0,01
3
310.174
<0,01
dose × time
12
23.491
<0,01
15
52.583
<0,01
239 240
The treatment with L. spica and linalool affected the emergence of adults from surviving
241
eggs and larvae in a similar way as mortality (Figures 3, 4). A large proportion of adult
242
insects hatched for the treated stages and doses with low mortality, whereas higher treatment
243
doses at susceptible stages lead to low emergence rates (Figures 3, 4). Especially for treated 11
244
1st instar larvae, the number of emerging adults was further decreased as compared to the
245
number of surviving insects with slightly stronger reductions for L. spica oil than for linalool
246
(Figures 3B, 4B). Most surviving L8 larvae from fumigation with L. spica reach the adult
247
stage even at the highest treatment dose (Figure 3C). The percentage of adults hatching from
248
larvae treated at L8 with linalool are mainly affected by the 1000 µl dose at 72 and 96 h
249
exposure (Figure 4C). Insects treated at the pupal instar with high doses and long duration,
250
resulted in a larger percentage of emerging intact adults for linalool than for L. spica oil, and
251
consequently less malformed adults and mosaics (Figure 3D, 4D).
252 253
Discussion
254
To know the precise age of the tested insects at each developmental stage, it was
255
necessary to study the life cycle of T. confusum and the mean duration of each stage in our
256
laboratory conditions. The development of T. confusum on semolina under our conditions was
257
slightly shorter compared to the mean development time reported on flour at a slightly lower
258
temperature (Stamopoulos et al., 2007).
259
We then estimated in this study the mortality effects of essential oil of L. spica and
260
linalool by fumigation against eggs, larvae, pupae and adults. Our data showed that the toxic
261
effects of L. spica essential oil and linalool depend on the developmental stage, the applied
262
dose and the duration of exposure. A relatively high dose (500 μl/L of air) and a long
263
exposure time (96 h) with L. spica oil is necessary to kill 100% of the eggs of T. confusum,
264
whereas half the concentration (250 μl/L of air) of linalool alone has the same effect. This
265
indicates that linalool might be to a large part responsible for the toxic effect on eggs, because
266
the used L. spica oil contains 43.79 % of linalool (Kheloul et al., 2019). The same exposure
267
time and similarly high doses (196.9 μl/L of air) of Pimpinella anisum are necessary to cause
268
100% egg mortality in T. confusum (Tunç et al., 2000). Studies on other plant essential oils,
12
269
such as Allium sativum, Betula lenta and Cinnamonum zeylanicum showed a better efficiency,
270
killing 100% of the eggs at a dose of 20 μl/L of air and within 24 h (I şıkber et al., 2009).
271
Essential oils from Allium cepa and Foeniculum vulgare killed 100% of T. confusum eggs at
272
an intermediate dose and exposure time (100 μl/L of air, 72 h) (Karci and Isikber, 2007) and
273
around 10 g of Laurus nobilis essential oil/h caused 90% mortality of T. confusum eggs,
274
whereas rosemary oil was not toxic for eggs (Isikber et al., 2006). Due to very different
275
compounds dominating the different oils tested in the cited studies (though not provided in all
276
studies), it is difficult to correlate egg toxicity of the different plant oils with the action of
277
specific compounds. The generally relatively high resistance of T. confusum eggs to
278
fumigation with essential oils might be due to the lack of aeropyles and micropyles in their
279
eggs, as well as a thick endochorion layer and a greater intrachorionic meshwork as compared
280
to other insect eggs, which obstruct the diffusion of vapors into the egg (Gautam et al., 2015).
281
In addition respiration rates are low at the egg stage, reducing gas exchange and consequently
282
diffusion of volatiles into the egg (Emekci et al., 2002)
283
Comparison of LC50 doses at 24 h exposure, obtained for different developmental stages
284
with L. spica essential oil, shows that the oil is more efficient on young larvae (L1) than on
285
older larvae (L8) or other stages. This age-dependent effect has also been found with
286
Artemisia haussknechtii and Lavandula hybrida essential oils, with increasing LC50 doses
287
with the age of larvae of T. confusum, even though main compounds of these oils are different
288
from the main compounds found in our L. spica oil (Hashemi and Safavi, 2013; Theou et al.,
289
2013). 1st instar larvae were indeed also the most sensitive developmental stage to the linalool
290
treatment, with an LC50 of 14.198 μl/L of air. This confirms earlier studies, which had
291
identified young larval instars as the most susceptible to linalool (Stamopoulos et al., 2007).
292
Toxicity of essential oils and linalool thus seems to be stronger for younger than for older
293
larvae in this insect.
13
294
Our results also showed that L. spica essential oil is more toxic for T. confusum pupae
295
than linalool, causing not only mortality but also malformations of the adults hatching from
296
surviving pupae and mosaics and consequently reducing the percentage of emergence of
297
intact adults. Thus for pupae, contrary to eggs, linalool might not be the most toxic compound
298
within the oil. Similar toxicity for pupae as for L. spica oil was also observed in treatments
299
with another lavender essential oil, L. hybrida (Theou et al., 2013). When adults were treated
300
with L. spica essential oil, marked toxicity started at 250 μl/L of air with an LC50 of 176.19
301
μl/L of air following a 24 h exposure. Linalool, however, caused only a low percentage of
302
mortality in adults, comparable to an earlier study in the same species, where a dose of 100
303
μl/L of air applied for 24 h elicited low rates of mortality in eggs, old larvae, pupae, and
304
adults, whereas young larvae had not been tested (Saglam and Özder, 2013). Similarly, low
305
toxicity of fumigation with linalool was found for adult T. castaneum (Rozman et al., 2007).
306
Thus other compounds present in the essential oil might be responsible for the observed toxic
307
effect or different compounds may act synergistically.
308
Essential oils of different lavender species have been previously shown to vary
309
substantially in their toxicity on adults of the related species T. castaneum. Whereas
310
Lavandula stoechas oil showed an LC50 at less than 40 μl/L of air after 24 h (Ebadollahi et al.,
311
2010), Lavandula angustifolia had only very minor toxic effects, but details on the used doses
312
are not provided (Pugazhvendan et al., 2012). However, L. angustifolia was highly toxic to a
313
different coleopteran stored product pest, Sitophilus granarius (L.) (Germinara et al., 2017).
314
Overall, effects of different oils on the same species and the same oil on different insect
315
species are highly variable, indicating that tests of different oils have to be performed for each
316
pest insect species..The highly variable effects of essential oils on different insects and even
317
different developmental stages of the same insect can be explained by strong differences in
318
metabolic activity and the importance of targets of active compounds. Certain constituents of
14
319
aromatic essential oils are known to have neurotoxic effects, whereas others are thought to act
320
on insect growth regulators, i.e. hormones (Isman, 2000; Tandon et al., 2008; Regnault-Roger
321
et al., 2012)..
322
Our study revealed that L. spica and linalool showed toxicity on certain stages and
323
ineffectiveness against other stages of T. confusum. They also caused malformations in adults
324
hatching from treated pupae, further reducing reproductive success through non-viable adults.
325
Thus the tested volatiles might represent a potential alternative to synthetic insecticides for
326
the control of the stored product pest T. confusum. Because of the long life duration of adult
327
beetles, relatively high doses also causing adult mortality would be needed at least in initial
328
treatments. Due to the strong odour of the used volatiles, direct treatments of stored products
329
are difficult to imagine, but treatments of storage surfaces might be more suitable instead, at
330
the same time reducing absorption of essential oils by the stored products, which could in turn
331
reduce efficacy (Arthur et al., 2011). Further studies are necessary to explore potential
332
practical application of L. spica essential oil and its main component including safety tests for
333
vertebrates and the environment and development of formulations.
334 335 336 337
Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
338 339
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18
450
Figure Legends
451
Figure 1. Mortality rates (mean ± SEM) of the different developmental stages of Tribolium
452
confusum treated by fumigation with Lavandula spica essential oil. (A) egg, (B) 1st instar L1,
453
(C) 8th instar L8, (D) pupa and (E) adults. N = 100 (5 x 20 insects) for each treatment. Bars
454
with the same letter are not significantly different.
455 456
Figure 2. Mortality rates (mean ± SEM) of the different developmental stages of Tribolium
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confusum treated with linalool. (A) egg, (B) 1st instar L1, (C) 8th instar L8, (D) pupa and (E)
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adults. N = 100 (5 x 20 insects) for each treatment. Bars with the same letter are not
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significantly different.
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Figure 3. Proportion of emerging intact adults (mean ± SEM) from insects surviving
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treatments with L. spica essential oil of immature stages. Treatments at (A) egg, (B) 1st instar
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L1, (C) 8th instar L8, (D) pupa. Bars with the same letter are not significantly different.
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Figure 4. Proportion of emerging intact adults (mean ± SEM) from insects surviving
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treatments with linalool of immature stages. Treatments at (A) egg, (B) 1st instar L1, (C) 8th
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instar L8, (D) pupa. Bars with the same letter are not significantly different.
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Declaration of interests ☐ x The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
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Highlights
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- Spike lavender toxicity was stage-, and exposure time-dependent in Tribolium confusum
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- First instar larvae were most sensitive to lavender oil and its component linalool
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- Adult emergence of most treated stages was reduced depending on dose or exposure time
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