Comparative toxicity, growth inhibitory and biochemical effects of terpenes and phenylpropenes on Spodoptera littoralis (Boisd.)

Journal Pre-proofs Full length article Comparative toxicity, growth inhibitory and biochemical effects of terpenes and phenylpropenes on Spodoptera littoralis (Boisd.) Nagwa M.A. Al-Nagar, Hamdy K. Abou-Taleb, Mohamed S. Shawir, Samir A.M. Abdelgaleil PII: DOI: Reference:

S1226-8615(19)30285-7 https://doi.org/10.1016/j.aspen.2019.09.005 ASPEN 1450

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Journal of Asia-Pacific Entomology

Received Date: Revised Date: Accepted Date:

5 May 2019 5 September 2019 24 September 2019

Please cite this article as: N.M.A. Al-Nagar, H.K. Abou-Taleb, M.S. Shawir, S.A.M. Abdelgaleil, Comparative toxicity, growth inhibitory and biochemical effects of terpenes and phenylpropenes on Spodoptera littoralis (Boisd.), Journal of Asia-Pacific Entomology (2019), doi: https://doi.org/10.1016/j.aspen.2019.09.005

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Comparative toxicity, growth inhibitory and biochemical effects of terpenes and phenylpropenes on Spodoptera littoralis (Boisd.) Nagwa M. A. Al-Nagara, Hamdy K. Abou-Taleba, Mohamed S. Shawirb, Samir A. M. Abdelgaleilb,* aPlant

Protection Research Institute, Agricultural Research Center, Sabahia, Alexandria, Egypt

bDepartment

of Pesticide Chemistry, Faculty of Agriculture, 21545-El-Shatby, Alexandria University,

Alexandria, Egypt Corresponding author. E-mail address: [email protected] (S. A.M. Abdelgaleil)

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ABSTRACT Eleven monoterpenes, phenylpropenes and sesquiterpenes were evaluated for their insecticidal and growth inhibitory activities against the second and fourth larval instars of Spodoptera littoralis. Among the tested compounds, 1,8-cineole revealed the highest fumigant toxicity against the 2nd and 4th larval instars with LC50 values of 2.32 and 3.13 mg/L air, respectively. The monoterpenes, p-cymene, -terpinene, (-)-pinene and (−)carvone were highly toxic to both larval stages as their LC50 values ranged between 7.35 and 13.79 mg/L air against 2nd larval instar and between 14.66 and 32.02 mg/L air against 4th larval instar. In topical application assay against the 4th larval instar, (−)-carvone (LD50 = 0.15 mg/larva) and cuminaldehyde (LD50 = 0.27 mg/larva) were the most potent contact toxicants. In residual film assay, trans-cinnamaldehyde, (-)-citronellal and p-cymene showed the highest insecticidal activity against the 2nd larval instar, while -terpinene and (−)-carvone were most effective compounds against the 4th larval instar. Moreover, the tested compounds caused strong growth reduction of both larval stages with growth inhibition higher than 80% in the 2nd larval instar and higher than 70% in the 4th larval instar. On the other hand, (−)-carvone, cuminaldehyde and (Z,E)-nerolidol showed pronounced

inhibitory

effects

on

acetylcholinesterase

(AChE)

and

adenosine

triphosphatases (ATPases) activity of S. littoralis larvae. Cuminaldehyde (IC50 = 1.04 mM) and (Z,E)-nerolidol (IC50 = 0.02 mM) caused the highest inhibition of AChE and ATPases, respectively. Taken together, the results indicate that monoterpenes, phenylpropenes and phenylpropenes could be used to develop new botanical insecticides for S. littoralis management. Keywords:

Natural products: Fumigant toxicity; Contact toxicity; Residual toxicity;

Growth inhibition, AChE; ATPases; Spodoptera littoralis

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Introduction The increasing of public awareness to the risk on human health and environment associated with the continuous use of synthetic insecticides has promoted researchers and agrochemical companies to find natural alternatives with relatively low negative impact on non-target organisms and environment for insect control. In this regard, it is expected that the global market of biopesticides will increase up to 20% by 2025 (Isman, 2015). Botanicals constitute a major class of biopesticides and have been shown to possess a wide spectrum of biological activities. Essential oils and their major constituents, monoterpenes, phenylpropenes and sesquiterpenes, are among the most promising botanicals to be used as alternatives for insect control (Isman et al., 2011; Pavela and Benelli, 2016). The cotton leafworm, Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae), is a serious polyphagous moth attacking more than 85 host plants belonging to 40 plant families of economic importance including cotton, maize, potatoes, cereals, vegetables and ornamental plants. Although this insect is native in Africa (Tanani et al., 2015) it is distributed throughout the world (Pineda et al., 2007; Shairra and Nouh, 2014). S. littoralis causes a great loss in crop production in tropical and sub-tropical areas due to the reducing photosynthesis and market value of vegetables and ornamentals. As precautionary measure to prevent the possible spread out the insect within the temperate zone, EPPO has listed S. littoralis as an A2 quarantine pest (OEPP/EPPO, 2015). Monoterpenes, phenylpropenes and sesquiterpenes are usually present as major constituents in plant essential oils. It is will recognize that these compounds are involved in several ecological functions in plants, including protection against herbivores and pathogens, attraction of pollinators and growth inhibition of other plants or allelopathy (Dudarevaet al., 2006; Qualley and Dudareva, 2008). Enormous studies have been reported on the contact and fumigant toxicities of monoterpenes and phenylpropenes against stored 3

product insects (Abdelgaleil et al., 2009; Saad et al., 2018; Wang et al., 2018). However, few studies were described the insecticidal activity of monoterpenes, phenylpropenes and sesquiterpenes against S. littoralis. For example, eleven monoterpenes have been shown to possess different levels of fumigant and contact toxicities against the third larval instar of S. littoralis (Abdelgaleil, 2010). In addition, some monoterpenes and phenylpropenes, such as trans-ethyl cinnamate, thymol, carvacrol, trans-anethole, γ-terpinene, terpinen-4-ol and piperitone have been reported to exhibit contact toxicity against 3rd and 4th larval instars of S. littoralis (Abdelgaleil et al., 2008; Abbassy et al., 2009; Pavela, 2014). Moreover, sesquiterpenes were stated to possess topical and feeding toxicities against the larvae of S. littoralis (Srivastav et al., 1990; González et al., 1997; Handayani et al., 1997). However, no studies have been reported on the growth inhibitory effects of monoterpenes, phenylpropenes and sesquiterpenes against the larvae of S. littoralis. Moreover, to continue our studies on the chemistry and bioactivity of these three interesting groups of natural products (Abdelgaleil et al. 2009; Saad et al. 2012), we have selected eleven monoterpenes, phenylpropenes and sesquiterpenes belong to different chemical classes to evaluate their fumigant, contact and residual toxicities, and growth inhibitory effects against larvae of S. littoralis. The insecticidal and growth inhibitory activities of these compounds were not previously reported against 2nd and 4th larval instars of S. littoralis. The inhibitory effects of these compounds on the activities of acetylcholinesterase (AChE) and adenosine triphosphatases (ATPases) were also tested. Materials and methods Test insect A strain of cotton leafworm, Spodoptera littoralis Boisduval (Lepidoptera: Noctuidae) was reared at 26±2°C and 70±5% RH on Ricinus communis L. (Euphorbiaceae) leaves as 4

described by El-Defrawi et al. (1964). The second and fourth larval instars were used in this study because they are active feeders, easily handled, and the second instar represents early larval instars (1, 2 and 3 instars), while the fourth instar represents late larval instars (4, 5 and 6 instars) of S. littoralis. All the experiments were done under the same rearing condition. Chemicals Seven monoterpenes [cuminaldehyde (98%), (-)-carvone (98%), (-)-citronellal (95%), 1,8-cineole (99%),(-)-pinene (98%), -terpinene (85%) and p-cymene (99%)], two phenylpropenes [trans-cinnamaldehyde (99%) and eugenol (99%)] and two sesquiterpenes [farnesol (95%) and (Z,E)-nerolidol (98%)] were purchased from Sigma–Aldrich Chemical Co., Steinheim, Germany, and used in this study. Chemical structures of these compounds are shown in Figure 1. Methomyl (99.3%) and paraoxon (97.5%) were purchased from Sigma–Aldrich Chemical Co., St. Louis, MO, USA. A reference insecticide, chlorpyrifos (97%), was obtained from Kafr El-Zayat Pesticides and Chemicals Co., Egypt. All solvents used in bioassay and reagents used in biochemical studies were of HPLC grade. Fumigant toxicity assay The fumigation toxicity of monoterpenes, phenylpropenes and sesquiterpenes was carried out against S. littoralis larvae following the protocol described by Abdelgaleil (2010). Fumigation was done in 1 L capacity-glass jars. Preliminary tests were done to establish the appropriate concentrations of each tested compound. Different amounts of the tested compounds, without solvent dilution, were applied on filter paper pieces (23 cm, Whatman no. 1) affixed to the inner surface of screw covers of the glass jars. Jars containing 10 2nd or 4th larval instars were closed tightly with their covers. The

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monoterpenes, phenylpropenes and sesquiterpenes were evaluated at concentrations ranged between 0.2 and 100 l/L air. At least six concentrations for each compound were tested with four replicates for each concentration. Fresh castor bean leaves were introduced for larval feeding during exposure. The number of dead larvae was recorded after 24 h of treatment. The mortality percentages were subjected to probit analysis (Finney, 1971) to calculate LC50 values of each compound. Topical application assay The topical application method was used to evaluate the contact toxicity of monoterpenes, phenylpropenes and sesquiterpenes on the fourth larval instar of S. littoralis (Abbassy et al., 2009). Serial concentrations of each compound were first prepared in acetone. Then, a microapplicator was used to apply 1µl of test solution on the dorsum of larvae. The compounds were tested at concentrations ranged between 0.1 and 1 mg/larva. Chlorpyrifos, a reference insecticide, was tested at concentrations ranged between 0.15 and 6 µg/larva. The larvae were then moved to glass cups containing pieces of fresh castor bean leaves for feeding. Four replicates with 10 larvae in each replicate were used for each treatment. Mortality percentage was recorded at 24 h post treatment. The dose of each compound that causing 50% mortality (LD50) was calculated from regression lines (Finney, 1971). Residual film assay The insecticidal and growth inhibitory activities of monoterpenes, phenylpropenes and sesquiterpenes were evaluated against the second and fourth larval instars of S. littoralis using residual film method (Kubo and Nakanishi, 1977). Three concentrations (1000, 2000 and 4000 mg/L) of tested compounds were prepared in acetone. The castor bean leaf disks

6

(20 mm in diameter) were immersed in test solutions for 10 s and left to dry. Control disks were immersed in acetone only. Ten treated disks were transferred into a glass cup with 10 second and/or fourth larval instars. Four replicates of each concentration and control were used. Chlorpyrifos was used as reference insecticides and was tested under the same conditions. The larvae were allowed to feed on treated disks for two days and then untreated fresh leaf disks were introduced to larvae for three days until the end of experiment. Mortality percentages were calculated after 5 days of treatment. The larval gain weights were recorded after 5 days of treatment and growth inhibition were calculated from the following equation: Growth inhibition = [(CL - TL)/CL] × 100 where CL is the larval gain weight in control and TL is the larval gain weight the treatment. In vitro inhibition of acetylcholinesterase (AChE) activity assay Head capsules of the 4th and 5th larval instars of S. littoralis (6 g) were used as source of AChE. The inhibition of AChE was determined by the colorimetric method of Ellman et al. (1961) using acetylthiocholine iodide (ATChI) as substrate as described by Abdelgaleil et al. (2016). The compounds were tested at concentrations ranged between 0.01 and 20 mM. Protein was measured by the method of Lowry et al. (1951). Inhibition percentage of AChE activity was calculated from the following equation: AChE inhibition % = [1−SAT / SAC] × 100 where SAT is specific activity of the enzyme in the treatment and SAC is specific activity of the enzyme in the control. The concentrations of the tested compounds that inhibited the hydrolysis of substrate by 50% (IC50) were determined by a linear regression analysis. In vitro inhibition of adenosine triphosphatases (ATPases) activity assay 7

The 2nd larval instar of S. littoralis was used as source of ATPases. Total ATPases activity was determined using Koch et al. (1969) method as reported by Abdelgaleil et at. (2016). The compounds were tested at concentrations ranged between 0.005 and 1.0 mM. The inhibition percentages of ATPases activity and IC50 values of the tested compounds were calculated as previously described. Statistical analysis The software SPSS 21.0 was used in data analyses (SPSS, Chicago, IL, USA). Probit analysis (Finney, 1971) was applied to determine LC50, LD50 and IC50 values. The values of LC50, LD50 and IC50 were considered significantly different if the 95% confidence limits did not overlap. Mortality percentages and larval gain weights in residual film assay were subjected to one-way analysis of variance followed by Student–Newman–Keuls test (Cohort software Inc. 1985) to determine significant differences among mean values at the probability level of 0.05. Results Fumigant toxicity of compounds on S. littoralis larvae The results of fumigant toxicity of the tested monoterpenes, phenylpropenes and sesquiterpenes on the 2nd and 4th larval instars of S. littorallis are summarized Tables 1 and 2. The tested compounds showed different levels of potency against 2nd and 4th larval instars of S. littorallis. 1,8-Cineole was significantly the most toxic compound with LC50 value of 2.32 mg/L air, followed by p-cymene and -terpinene with LC50 values of 7.35 and 9.71 mg/L air, respectively. In addition, (-)-pinene (LC50 = 13.79 mg/L air) and (−)carvone (LC50 = 19.07 mg/L air) revealed strong fumigant toxicity. In contrary, the monoterpenes ((-)-citronellal and cuminaldehyde), sesquiterpenes (farnesol and (Z,E)8

nerolidol) and phenylpropenes (trans-cinnamaldehyde and eugenol) showed weak or no fumigant toxicity as their LC50 values were greater than 100 mg/L air. Similarly, 1,8cineole was significantly the most potent fumigant against the 4th larval instar, followed by (-)-pinene, p-cymene, -terpinene and (−)-carvone (Table 2). The LC50 values of these compounds were 3.13, 14.66, 21.25, 28.21 and 32.02 mg/L air, respectively. Comparing the LC50 values of compounds on the 2nd and 4th larval instars indicated that the 2nd larval instar was more susceptible than 4th larval instar to fumigant toxicity. Contact toxicity of compounds on S. littoralis larvae The toxicity of tested monoterpenes, phenylpropenes and sesquiterpenes on the 4th larval instar of S. littoralis by using topical application assay is presented in Table 3. The tested compounds displayed different levels of toxicity against the larvae. (−)-Carvone caused significantly the highest insecticidal activity against the larvae, followed by cuminaldehyde, eugenol, p-cymene and (Z,E)-nerolidol. The LD50 values for these compounds were 0.15, 0.27, 0.32, 0.36, and 0.41 mg/larva, respectively. In addition, farnesol, 1,8-cineole, (-)-pinene, (-)-citronellal and -terpinene exhibited a moderate toxic effect. The LD50 values for these compounds were 0.58, 0.62, 0.63 0.73, and 0.95 mg/larva, respectively. In contrast, trans-cinnamaldehyde gave a weak toxic effect since the LD50 value was higher than 1.0 mg/larva. The tested compounds were less toxic than a reference insecticide, chlorpyrifos. Residual toxicity of compounds on S. littoralis larvae The residual toxic effect of monoterpenes, phenylpropenes and sesquiterpenes on the 2nd and 4th larval instars of S. littoralis after 5 days of treatment is summarized in Table 4. The results showed that the tested compounds had different levels of residual toxicity. trans-Cinnamaldehyde (mortality = 70.0%), (-)-citronellal (mortality = 53.3%) and p9

cymene (mortality = 56.7%) exhibited the highest insecticidal activity against the 2nd larval instar at 1000, 2000 and 4000 mg/L, respectively. On the other hand, (−)-carvone (mortality = 90.0 %) exhibited the highest toxicity against the 4th larval instar, followed by terpinene (mortality = 80.0 %) and trans-cinnamaldehyde (mortality = 76.7 %) at 1000 mg/L. In addition, -terpinene, (−)-carvone and (-)-citronellal displayed the strongest toxicity at 2000 mg/L with 86.7, 80.0 and 83.3% mortalities, respectively. Moreover, terpinene, cuminaldehyde, eugenol, 1,8-cineole, (-)-citronellal, and (-)-pinene revealed the greatest insecticidal potency at 4000 mg/L with 93.3, 86.7, 83.3, 76.7, 76.7 and 70.0% mortalities, respectively. The tested compounds were less toxic than a reference insecticide, chlorpyrifos. Growth inhibitory effect of compounds on S. littoralis larvae Effect of monoterpenes, phenylpropenes and sesquiterpenes on the growth inhibition of S. littoralis 2nd and 4th larval instars after 5 days of treatment is presented in Table 5. In general, all of the tested compounds showed remarkable growth inhibition on both larval instars at the three tested concentrations (1000, 2000 and 4000 mg/L). In the case of the 2nd larval instar, all tested compounds caused growth inhibition greater than 80%. 1,8-Cineole was the most effective compound on the larval growth at the three tested concentrations with growth inhibition of 92.6, 93.6 and 96.9% at 1000, 2000 and 4000 mg/L, respectively. trans-Cinnamaldehyde, (-)-pinene and (Z,E)-nerolidol were the less effective compounds at 1000, 2000 and 4000 mg/L, respectively. On the other hand, (-)-pinene was the most potent growth inhibitor among the tested compounds against 4th larval instar, followed by ()-citronellal, 1,8-cineole, and -terpinene with growth inhibition of 93.2, 92.5, 91.3 and 89.5% at 1000 mg/L, respectively. Also, these compounds revealed the highest growth reduction at 2000 mg/L. Moreover, 1,8-cineole exhibited the greatest reduction of the

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growth inhibition, followed by (-)-pinene, eugenol, (-)-citronellal, and (−)-carvone with growth inhibition of 95.2, 94.3, 94.2, 93.0 and 91.7% at 4000 mg/L, respectively. Inhibitory effects of compounds on AChE and ATPases Based on the results of toxicity bioassay, three compounds, (−)-carvone, cuminaldehyde and (Z,E)-nerolidol, which showed the highest contact toxicity were selected to examine their in vitro inhibitory effects of AChE and ATPases. The tested compounds exhibited remarkable inhibitory effect on AChE enzyme activity of S. littoralis larvae (Table 6). Cuminaldehyde (IC50 = 1.04 mM) was significantly the most effective inhibitor of AChE activity, followed by (Z,E)-nerolidol (IC50 = 6.28 mM) and (−)-carvone (IC50 = 6.34 mM). On the other hand, these three compounds gave potent inhibitory effect on ATPases of S. littoralis larvae. (Z,E)-Nerolidol (IC50 = of 0.02 mM) caused the highest inhibitory effect, followed by cuminaldehyde (IC50 = 0.05 mM) and (−)-carvone (IC50 = 0.22 mM). Discussion The present study describes the fumigant toxicity of the tested monoterpens, phenylpropenes and sesquiterpenes against the 2nd and 4th larval instars of S. littoralis for the first time. It was obvious that the monoterpenes were more potent fumigants than phenylpropenes and sesquiterpenes on the two larval instars. Among the examined monoterpenes, the hydrocarbons ((-)-pinene, p-cymene, and -terpinene), ethers (1,8cineole) and ketones ((−)-carvone) showed higher fumigant toxicity than aldehydes ((-)citronellal and cuminaldehyde). The results also revealed that 1,8-cineole was more potent fumigant than ((−)-carvone and cuminaldehyde against 2nd and 4th larval instars of S. littoralis. These results are in agreement with those reported by Abdelgaleil (2010) who found that 1,8-cineole (LC50 = 4.17 mg/L) had higher fumigant toxicity than (−)-carvone

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(LC50 = 19.48 mg/L) and cuminaldehyde (LC50 > 100 mg/L) against the 3rd larval instar of this insect. In addition, some essential oils containing the tested monoterpenes as major constituents were shown to possess fumigant toxicity against the 3rd larval instar of S. littoralis. For example, the essential oils of Thuja occidentalis, Thymus mastichina and Origanum majorana which containing (-)-pinene, 1,8-cineole and p-cymene as major compounds, respectively, were highly toxic to the 3rd larval instar of S. littoralis with LC50 values of 6.5, 11.3 and 19.6 ml/m3, respectively. Moreover, other essential oils such as Nepeta cataria, Salvia sclarea, Pogostemon cablin, and Mentha pulegium were also shown to cause fumigant toxicity to this larval instar (Pavela, 2005). It has been also noticed that the 2nd larval instar of S. littoralis were more susceptible than the 4th larval instar to tested monoterpenes. These results are in agreement with other studies on the insecticidal activity of plant natural products and other biocides (Elbarky et al., 2008; El-Khayat et al., 2012; Khamis et al., 2016; Eesa et al., 2017). On the other hand, some of the tested monoterpenes and phenylpropenes have been described to show fumigant toxicity against other insect species. For instance, 1,8-cineole and (−)-carvone caused fumigant toxicity to the adults of Sitophilus oryzae (LC50 = 14.19 and 17.78 mg/L, respectively) and Tribolium castaneum (LC50 = 17.16 and 75.22 mg/L, respectively) (Abdelgaleil et al., 2009). Moreover, (-)α-pinene and p-cymene caused 90 to 100% mortality of Callosobruchus analis, S. oryzae, Stegobium paniceum and T. castaneum at a concentration of 10 µl/mL air (Brari and Thakur, 2015). The results of topical application assay indicated that all tested compounds caused acute toxicity against 4th larval instar of S. littoralis except trans-cinnamaldehyde. To the best of our knowledge, this is the first report on the topical toxicity of these compounds on 4th larval instar of S. littoralis. It was obvious from literature there are a few reported studies on the topical toxicity of monoterpenes, phenylpropenes and sesquiterpenes towards 12

different larval stages of S. littoralis. Abdelgaleil (2010) found that cuminaldehyde was more toxic than 1,8-cineole against the 3rd larvae stage with 70 and 40% mortality at 1 mg/larva. This finding is in agreement with the results of the current study as cuminaldehyde (LD50 = 0.27 mg/larva) had higher toxicity than 1,8-cineole (LD50 = 0.62 mg/larva). The contact toxicity of monoterpenes and phenylpropenes reported in this study is also supported by earlier studies in which trans-ethyl cinnamate, thymol, carvacrol, trans-anethole and piperitone revealed contact toxicity against the 3rd larval instar (Abdelgaleil et al., 2008; Pavela, 2014), and γ-terpinene and terpinen-4-ol caused contact toxicity against the 4th larval instar of S. littoralis (Abdelgaleil et al., 2008; Abbassy et al., 2009). It was obvious from the results of the current study that eugenol was more toxic than citronellal against 4th larval instar S. littoralis. However, Waliwitiya et al. (2005) stated that citronellal (LD50 = 404.9 µg/larva) was more toxic than eugenol (LD50 = 516.5 µg/larva) against the larvae of Agriotes obscurus. These findings indicated the toxicity of monoterpenes is highly depending on the insect species. The obtained results showed that the tested compounds caused different levels of residual toxicities on the 2nd and 4th larval instars of S. littoralis. It was also clear that the 4th larval instar was more susceptible to tested compounds than the 2nd larval instar. To the best of our knowledge, this is the first report on the residual toxicity of tested monoterpenes, phenylpropenes and sesquiterpenes against S. littoralis larvae except -terpinene which has been described to possess residual toxicity against the third larval instar of this insect (Abbassy et al., 2009). Among the previously examined sesquiterpenes, a sesquiterpenes lactone, encelin, (Srivastav et al., 1990) and sesquiterpenes with a dihydro-β-agarofuran (González et al., 1997) have been confirmed to show residual toxicity against S. littoralis. The residual toxicity of other monoterpenes has been observed against Tetranychus urticae (Badawy et al., 2010) and Drosophila suzukii (Park et al., 2016). 13

One of the most interesting finding in this study is the drastic reduction in the growth of 2nd and 4th larval instars of S. littoralis after residual exposure to the tested compounds. This finding indicates these compounds are efficient growth deterrents and can be used for control S. littoralis by delaying the insect development. None of the tested compounds were evaluated so far for their growth inhibition of S. littoralis larvae. However, some terpenoids, essential oils and plant extracts have been reported to inhibit the larval growth of S. littoralis (Zapata et al., 2009; Adel and Zaki, 2010; Pavela, 2011; Pavela and Vrchotova, 2013). Three of the tested compounds (cuminaldehyde, (−)-carvone and (Z,E)-nerolidol) represents the three classes of examined compounds were selected to study their inhibitory effects on two key target enzymes for insecticides, AChE and ATPases, to clarify a possible mode of action of these compounds. Although the inhibitory effect of these compounds on AChE from S. littoralis larvae was not reported the monoterpenes, cuminaldehyde and (−)carvone, have been shown to inhibit this enzyme from other insect and pest species. Abelgaleil et al. (2009) found that cuminaldehyde caused 65 and 70% inhibition of AChE isolated from S. oryzae at concentrations 0.01 and 0.05M, respectively, while (−)-carvone caused 30 and 45% inhibition at the same concentrations. These results are in agreement with the obtained data in the current study in which cuminaldehyde had higher AChE inhibition than (−)-carvone. On the contrary, cuminaldehyde caused less inhibition to AChE isolated T. castaneum than (−)-carvone with 70 and 25% inhibition at 0.05M, respectively. In accordance with our results, López and Pascual-Villalobos (2010) found that S-carvone induced high inhibition of AChE form stored product insects. The results of biochemical studies also revealed that cuminaldehyde, (−)-carvone and (Z,E)-nerolidol induced remarkable inhibition of ATPases. These compounds caused higher inhibition of ATPases than AChE. For example cuminaldehyde, (−)-carvone and (Z,E)-

14

nerolidol showed 29, 21 and 314-fold higher inhibition on ATPases than AChE, respectively.

These findings are in agreement with previous studies in which

monoterpenes, phenylpropenes and essential oils revealed higher inhibition to ATPases than AChE isolated from different insect species (Abdelgaleil et al., 2016; Abou-Taleb et al., 2016; Saad et al., 2018). Based on the available data in the literatures, it has been suggested that the mechanisms of action of monoterpenes and phenylpropenes might be correlated to the inhibition of AChE, GABA-gated chloride channels and octopamine receptors (Enan, 2001; Tak et al., 2016). Nevertheless, these compounds may also induce other effects, such as disruption in hormones and pheromones, and inhibition of cytochrome P450 monooxygenase (Garcia et al., 2005). However, the results of our current and previous studies indicated that the toxicity of monoterpenes and phenylpropenes to insects may be attributed to inhibition of ATPases. Overall, the current study explored the fumigant, contact and residual insecticidal potential of monoterpenes, phenylpropenes and sesquiterpenes against the larvae of S. littoralis. Also, these compounds are potent larval growth inhibitors. Some of the tested compounds, such as 1,8-cineole, -terpinene, (-)-pinene and showed potent fumigant toxicity and others such as, cuminaldehyde and eugenol showed promising contact toxicity, while (−)-carvone and p-cymene revealed strong fumigant and contact toxicities. These monoterpenes and phenylpropenes could be used as efficient bioinsecticides against the larvae of S. littoralis. Acknowledgments This work was partially supported by the Alexandria University Research Fund (ALEX-REP).

References 15

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Table 1. Fumigant toxicity of monoterpenes, phenylpropenes and sesquiterpenes on the 2nd larval instar of Spodoptera littoralis Compound Monoterpenes (−)-Carvone 1,8-Cineole (-)-Citronellal Cuminaldehyde p-Cymene (-)-Pinene

-Terpinene Phenylpropenes trans-Cinnamaldehyde Eugenol Sesquiterpenes Farnesol (Z,E)-Nerolidol

LC50a (µl/L) (95% confidence limits)

LC95b (µl/L) (95% confidence limits)

Slope ± SEc

Intercept ± SEd

(X2)e

29.07 (26.58 -33.50) 2.32 (1.85 -2.77) > 100 > 100 7.35 (6.70 -7.94) 13.79 (13.37 -14.20) 9.71 (9.13 - 11.01)

74.45 (55.74 -128.57) 9.24 (6.72 -16.32) 20.12 (17.29 - 25.02) 20.41 (19.21 – 22.13) 21.91 (19.19 -26.31)

4.03 ± 0.64

-5.89 ± 0.88

1.70

2.74 ± 0.23

-1.00 ± 0.12

7.16

3.76 ± 0.38

-3.26 ± 0.37

4.10

9.65 ± 0.80

-11.00 ± 0.91

1.95

4.65 ± 0.41

-4.49 ± 0.41

1.53

>100 > 100

-

-

-

-

> 100 > 100

-

-

-

-

a

The lethal concentration causing 50 % mortality after 24 h. The lethal concentration causing 95 % mortality after 24 h. c Slope of the concentration-mortality regression line ± standard error. d Intercept of the regression line ± standard error. e Chi square value. b

22

Table 2. Fumigant toxicity of monoterpenes, phenylpropenes and sesquiterpenes on the 4th larval instar of Spodoptera littoralis Compound

LC50a (µl/L) (95% confidence limits)

LC95b (µl/L) (95% confidence limits)

Slope ± SEc

Intercept ± SEd

(X2)e

32.02 (25.78 – 40.79)

86.20 (59.61 – 218.90)

3.83 ± 0.34

-5.76 ± 0.51

8.02

1,8-Cineole

3.13 (2.50- 3.94)

7.40 (5.71-13.60)

4.40 ± 0.40

-2.18±0.23

7.01

(-)-Citronellal Cuminaldehyde p-Cymene

> 100 > 100 21.25 (19.65 -23.49) 14.66 (14.11 -15.22) 28.21 (27.08 -29.47)

52.83 (42.32 -75.42) 24.18 (22.35 -26.93) 46.61 (42.21 – 54.05)

4.16 ± 0.51

-5.52 ± 0.64

2.36

7.57 ± 0.65

-8.83 ± 0.76

0.38

7.54 ± 0.81

-10.94 ± 1.16

2.37

> 100 > 100

-

-

-

-

> 100 > 100

-

-

-

-

Monoterpenes (−)-Carvone

(-)-Pinene

-Terpinene Phenylpropenes trans-Cinnamaldehyde Eugenol Sesquiterpenes Farnesol (Z,E)-Nerolidol a

The lethal concentration causing 50 % mortality after 24 h. The lethal concentration causing 95 % mortality after 24 h. c Slope of the concentration-mortality regression line ± standard error. d Intercept of the regression line ± standard error. e Chi square value. b

23

Table 3. Toxicity of monoterpenes, phenylpropenes and sesquiterpenes on the 4th larval instar of Spodoptera littoralis by topical application assay Compound Monoterpenes (−)-Carvone 1,8-Cineole (-)-Citronellal Cuminaldehyde p-Cymene (-)-Pinene -Terpinene Phenylpropenes trans-Cinnamaldehyde Eugenol Sesquiterpenes Farnesol (Z,E)-Nerolidol Chlorpyrifos

LD50a (mg/Larva) (95% confidence limits)

LD95b (mg/Larva) (95% confidence limits)

Slope ± SEc

Intercept ± SEd

(X2)e

0.15 (0.14 -0.16) 0.62 (0.58 - 0.68) 0.73 (0.68 -0.79) 0.27 (0.25 – 0.28) 0.36 (0.32 -0.39) 0.63 (0.58 -0.68) 0.95 (0.68 -11.44)

0.37 (0.33 – 0.43) 1.64 (1.38 -2.10) 1.66 (1.41 -2.10 ) 0.57 (0.50 -0.69) 0.94 (0.80 -1.19) 1.64 (1.38 -2.10) 2.76 (1.37 -77326.4)

4.16 ± 0.30 3.92 ± 0.38 4.60 ± 0.45 5.08 ± 0.58 3.96 ± 0.43 3.92 ± 0.38 3.68 ± 0.46

3.43±0.24 0.80 ±0.10 0.63± 0.11 2.90 ± 0.32 -175 ± 0.18 -0.80± 0.11 0.08 ± 0.10

5.30 0.50 0.32 2.36 1.25 0.50 5.31

> 1.0 0.32 (0.27 -0.46)

0.92 (0.61 -2.74)

3.64 ± 0.30

1.78 ± 0.16

4.28

0.58 (0.46 -0.75) 0.41 (0.36 -0.45) 0.001 (0.0008 -0.0014)

2.01 (1.33 -4.89) 1.51 (1.25 -1.93) 0.005 (0.0034 -0.0115)

3.07 ± 0.27 2.90 ± 0.23 2.36 ± 0.17

0.72 ± 0.10 1.13 ± 0.10 -0.05 ± 0.07

5.90 3.61 5.52

a

The lethal dose causing 50 % mortality after 24 h. The lethal dose causing 95 % mortality after 24 h. c Slope of the dose-mortality regression line ± standard error. d Intercept of the regression line ± standard error. e Chi square value. b

24

Table 4. Residual toxicity of monoterpenes, phenylpropenes and sesquiterpenes on the 2nd and 4th larval instar of Spodoptera littoralis after 5 days of treatmenta Mortality (%± SE)b

Compound 2nd

Control Monoterpenes (−)-Carvone 1,8-Cineole (-)-Citronellal Cuminaldehyde P-Cymene

(-)-Pinene -Terpinene

Phenylpropenes transCinnamaldehyde Eugenol Sesquiterpenes Farnesol (Z,E)-Nerolidol Chlorpyrifos

1000 mg/L 0.0 ± 0.00f

larval instar 2000 mg/L 0.0 ± 0.00f

4000 mg/L 0.0 ± 0.00e

1000 mg/L 0.0 ± 0.00d

4th larval instar 2000 mg/L 0.0 ± 0.00f

4000 mg/L 0.0 ± 0.00f

16.7 ± 3.34def 6.7 ± 3.34ef 63.3 ± 3.34bc 00.0 ± 0.00f 23.3 ± 3.34de 50.0 ± 5.78c 60.0 ± 5.78bc

40.0 ± 5.78bcd 20.0 ± 0.00de 53.3 ± 3.34b 00.0 ± 0.00f 36.7 ± 6.67bcd 33.3 ± 3.34bcd 46.7 ± 6.67bc

26.7 ± 3.34d 36.7 ± 6.67bcd 46.7 ± 3.34bcd 53.3 ± 3.34bc 56.7 ± 6.67b 53.3 ± 6.67bc 50.0 ± 5.78bc

90.0 ± 5.78ab 6.7 ± 3.34cd 13.3 ± 3.34cd 16.7 ± 3.34cd 20.0 ± 5.78cd 10.0 ± 5.78cd 80.0 ± 5.78b

80.0 ± 0.00b 36.7 ± 3.34cde 83.3 ± 8.83ab 26.7 ± 6.67de 50.0 ± 0.00c 43.3 ± 3.34cd 86.7 ± 6.67ab

50.0 ± 5.78d 76.7 ± 3.34bc 76.7 ± 3.34bc 86.7 ± 6.67abc 46.7 ± 3.34d 70.0 ± 5.78c 93.3 ± 3.34ab

70.0 ± 5.78b 16.7 ± 6.67def

43.3 ± 8.83bc 33.3 ± 3.34bcd

40.0 ± 5.78bcd 36.7 ± 3.34bcd

76.7 ± 8.83b 26.7 ± 6.67c

33.3 ± 3.34cde 43.3 ± 8.83cd

40.0 ± 5.78d 83.3 ± 3.34abc

26.7 ± 3.34d 33.3 ± 6.67d 100.0 ± 0.00a

13.3 ± 6.67ef 26.7 ± 3.34cde 100.0 ± 0.00a

26.7 ± 6.67d 33.7 ± 3.34cd 100.0 ± 0.00a

23.3 ± 3.34c 16.7 ± 3.34cd 100.0 ± 0.00a

20.0 ± 5.78e 20.0 ± 5.78e 100.0 ± 0.00a

36.7 ± 8.83d 20.0 ± 0.00e 100.0 ± 0.00a

a

Data are expressed as mean values ± SE from experiments with four replicates of 10 larvae each. b Mean values within a column sharing the same letter are not significantly different at the 0.05 probability level.

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Table 5. Growth inhibitory effect of monoterpenes, phenylpropenes and sesquiterpenes on the 2nd and 4th larval instars of Spodoptera littoralis after 5 days of feeding on treated leavesa Compound Control Monoterpenes (−)-Carvone 1,8-Cineole (-)-Citronellal Cuminaldehyde P-Cymene (-)-Pinene -Terpinene Phenylpropenes transCinnamaldehyde Eugenol Sesquiterpenes Farnesol (Z,E)-Nerolidol

1000 mg/L Mean gain GIc (%) weight ± SE (mg/larva)b 44.0 ± 1.51a 00.00

2nd larval instar 2000 mg/L Mean gain GI (%) weight ± SE (mg/larva) 44.0 ± 1.51a 00.00

4000 mg/L Mean gain GI (%) weight ± SE (mg/larva) 44.0 ± 1.51a 00.00

1000 mg/L Mean gain GI (%) weight ± SE (mg /larva) 483.3 ±7.70a 00.0

4th larval instar 2000 mg/L Mean gain GI (%) weight ± SE (mg /larva) 483.3 ± 7.70a 00.0

4000 mg/L Mean gain GI (%) weight ± SE (mg/larva) 483.3 ± 7.70a 00.0

4.4 ± 0.15cd 3.3 ± 0.45d 5.9 ± 0.29cd 4.0 ± 0.90cd 5.8 ± 0.24cd 4.7 ± 0.33cd 4.3 ± 0.09cd

90.0 92.6 86.7 90.9 86.9 89.2 90.2

4.4 ± 0.09bc 2.8 ± 0.48c 5.1 ± 0.07bc 5.8 ± 0.44b 5.8 ± 0.29b 6.5 ± 0.09b 4.7 ± 0.12bc

89.9 93.6 88.5 86.9 86.9 85.3 89.4

4.2 ± 0.09bcd 1.4 ± 0.32fg 3.5 ± 0.15cde 2.8 ± 0.23def 5.4 ± 0.39bc 5.8 ± 0.23b 3.5 ± 0.18cde

90.5 96.9 92.0 93.7 87.7 86.9 92.1

60.0 ± 2.98cd 41.8 ± 1.17ef 36.3 ± 2.14f 139.2 ± 4.14b 65.8 ± 0.99c 32.7 ± 1.46f 50.6 ± 5.17de

87.6 91.3 92.5 71.2 86.4 93.2 89.5

60.0 ± 0.71ef 41.8 ± 1.96f 36.3 ± 0.12de 139.2 ± 4.71b 65.8 ± 0.64d 32.7 ± 3.73f 50.6 ± 2.43d

87.6 91.3 92.5 71.2 86.4 93.2 89.5

39.9 ± 2.10de 23.4 ± 1.19f 33.7 ± 2.02def 64.8 ± 5.91b 56.0 ± 1.51bc 28.1 ±2.09ef 36.9 ± 2.02de

91.7 95.2 93.0 86.6 88.4 94.2 92.4

8.8 ± 0.10b 6.3 ± 0.96c

80.0 85.7

5.9 ± 0.63b 2.8 ± 0.52c

86.6 93.7

5.3 ± 0.29bc 2.1 ± 0.52ef

88.0 95.3

73.3 ± 4.44c 69.7 ± 2.71c

84.8 85.6

73.3 ± 2.28c 69.7 ± 5.63c

84.8 85.6

45.9 ± 2.34cd 27.3 ± 2.01f

94.3

3.5 ± 0.26cd 4.5 ± 0.53cd

92.0 89.7

4.2 ± 0.20bc 4.2 ± 0.12bc

90.4 90.5

4.9 ± 0.20bc 6.1 ± 0.24b

88.9 86.2

63.7 ± 0.76cd 58.7 ± 0.47cd

86.8 87.9

63.7 ± 1.77de 58.7 ± 2.21d

86.8 87.9

45.5 ± 2.10cd 44.8 ± 1.72cd

90.6 90.7

a Data

are expressed as mean values ± SE from experiments with four replicates of 10 larvae each. values within a column sharing the same letter are not significantly different at the 0.05 probability level. c Growth inhibition. b Mean

26

Table 6. Values of IC50 of monoterpenes and sesquiterpenes on the Spodoptera littoralis larval acetylcholinesterase (AChE) and adenosine triphosphatases (ATPases) Enzyme

Compound

IC50a (mM) (Confidence limits)

Slope ± SE b

Intercept ± SE c

(X2) d

(−)-Carvone Cuminaldehyde (Z,E)-Nerolidol Methomyl Paraoxon

6.34 (5.38 -7.56) 1.04 (0.89 -1.22) 6.28 (4.68 -8.70) 2.09 µM (1.24 -3.12) 0.75 µM (0.66-0.86)

1.69 ± 1.69 1.52 ± 0.09 2.12 ± 0.18 1.84 ± 0.14 2.39 ± 0.17

-1.36 ± 0.13 -0.03 ± 0.05 -1.70 ± 0.15 -0.59 ± 0.10 0.30 ±0.07

0.47 3.32 11.11 6.39 2.51

(−)-Carvone Cuminaldehyde (Z,E)-Nerolidol

0.22 (0.18-0.27) 0.05 (0.045.-0.07) 0.02 (0.024- 0.061)

1.46 ± 0.14 1.58 ± 0.12 1.38 ± 0.11

-3.43 ± 0.33 -2.74 ± 0.20 -1.8 ± 0.15

2.62 0.72 0.62

AChE

ATPases

a

The concentration causing 50 % enzyme inhibition. Slope of the concentration-inhibition regression line. c Intercept of the regression line ± SE. d Chi square value. b

27

O O H H (-)-Citronellal

trans-Cinnamaldehyde

OH

OCH3 Eugenol

p-Cymene

-Pinene O

H

O (-) Carvone

-Terpinene

Cuminaldehyde

O

OH  Cineole

Farnesol

HO

(Z,E)-Nerolidol Figure 1. The chemical structures of monoterpenes, phenylpropenes and sesquiterpenes.

28

Graphical abstract

O

(-) Carvone O

H

Insecticidal activities Growth inhibtion Biochemical effects AChE, ATPases Cuminaldehyde

Spodoptera littoralis

HO

Nerolidol

Botanical compounds may serve as bioinsecticides for insect management ►Monoterpenes, phenylpropenes and sesquiterpenes showed remarkable insecticidal activity against Spodoptera littoralis ►The tested compounds caused higher fumigant toxicity against 2nd larval instar than 4th larval instar ►The chemical structure and the bioassay methods affect the insecticidal activity of tested compounds ►The selected compounds significantly inhibited acetylcholinesterase and adenosine triphosphatases. ►

29

Conflict of interest

The authors Nagwa M. A. Al-Nagar, Hamdy K. Abou-Taleb,

Mohamed S. Shawir and Samir A. M. Abdelgaleil report no conflicts of interest to be declared.

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