Screening for insecticidal efficacy of two Algerian essential oils with special concern to their impact on biological parameters of Ephestia kuehniella (Zeller) (Lepidoptera: Pyralidae)

Chemical composition of Algerian Mentha rotundifolia and Myrtus communis essential oils, their insecticidal activities and their impact on some biological parameters of the Mediterranean flour moth Ephestia kuehniella were assessed. Results showed that M. rotundifolia essential oil contained piperitenone oxide (46.06%), d-limonene (9.10%), cis-piperitone oxide (6.81%), and endo-borneol (4.64%) as major compounds, while M. communis oil was rich in α-pinene (29.08%), 1,8-cineole (36.82%), α-terpineol (6.42%), geranyl acetate (4.38%), and β-linalool (4.04%). The fumigant potential and contact toxicity tests against E. kuehniella demonstrated the effectiveness of M. rotundifolia essential oil (LC50 = 0.54 μL/L air, LC50 = 0.004 μL/cm2) compared to M. communis oil (LC50 = 2.91 μL/L air, LC50 = 0.025 μL/cm2). Moreover, results revealed that all biological parameters were significantly affected (fecundity: 6 eggs/female, oviposition deterrence: 96.62%, log fertility: 0, hatching rate: 0%, copulation rate: 0% for M. rotundifolia oil against fecundity: 93 eggs/female, percentage of oviposition deterrence: 47.85%, log fertility: 6.7, hatching rate: 57%, copulation rate: 53.33% for M. communis oil). This work supports the use of botanical insecticide as active pest control agents under storage conditions.


Introduction
Agricultural products are often subject to biotic and abiotic damage during production and conservation. Contamination of food commodities by insect pests is an important quality control issue of concern for the food industries (Rajendran and Sriranjini 2008). About 35% on the field and 14% in storage (around 50% in total) crops are lost annually because of insect pests, which adversely affected the world food production during crop growth, harvest and storage (Jitendra et al. 2009). Among damaging insects, the Mediterranean flour moth Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) is one of the major pests in industrial flour mills (Jacob and Cox 1977), in Algeria (Hami et al. 2005) and Tunisia (Jarraya 2003). It is a worldwide pest that causes major economic losses in the Mediterranean basin and near East regions (Al-Izzi et al. 1985). Its main habitats are flour and grout mills, corn milling plants, bakeries and any other place used for processing grains or preparing flour products (Ben Achour et al. 2008). Larvae of E. kuehniella reduce both quantitative and qualitative quality of the products by feeding, by their presence and by the production of feces and webbing (Johnson et al. 1997). Last years, the overuse of synthetic insecticides and fumigants such as phosphine for grain storage has resulted in a number of problems, including the development of insecticide resistance among insect pests of stored grains (Sousa et al. 2009). Currently, stored-product pest control strategies tend to emphasize the non-chemical aspects of pest control with the judicious use of pesticides (Titouhi et al. 2017). In this respect, plant extracts are safe, ecofriendly and more compatible with environmental components compared to synthetic pesticides and so ranked under Green pesticides category (Rahman et al. 2016).
Besides, Kim et al. (2003) showed that plant extracts are often active against a limited number of target insects, are biodegradable, and are potentially suitable for use in integrated pest management. These authors reported also that plant extracts could lead to the improvement in new classes of nontoxic insect control. Plant essential oils have been recently qualified as ecological alternatives to chemical pesticides due to their multifunctional efficacy including anti-insect pest activity (Isman and Grieneisen 2014;Mediouni Ben Jemâa 2014). Furthermore, essential oils possess important contact and fumigant toxicity (Liu and Ho 1999;Abdelgaleil et al. 2009;Kasrati et al. 2015;Quan et al. 2018), antifeedant activity (Huang et al. 1997;Ebadollahi 2013), repellent activity (Hori 2003;Prajapati et al. 2005;Nerio et al. 2009;Chang et al. 2017) as well as development and growth inhibitory activity (Waliwitiya et al. 2009;Sangha et al. 2017) against various insect species and are regarded as environmentally compatible pesticides (Cetin et al. 2004).
Their lipophilic nature facilitates their interference with basic metabolic, biochemical, physiological and behavioral functions of insects (Nishimura 2001). Commonly, essential oils with anti-insect pest ability are known to possess insecticidal and repellent effects by neurotoxic mode of action (Kostyukovsky et al. 2002). Mentha rotundifolia (L.) huds (Lamiales: Lamiaceae) and Myrtus communis (L.) (Myrtales: Myrtaceae) are commonly known under the vernacular names of round leaf and common myrtle. These aromatic plant species, grow wild in northern Algeria, are widely used by the population in traditional phytotherapy for their therapeutic virtues. The essential oils extracted from the leaves of Myrtus communis have shown insecticidal activity against E. kuehniella, Plodia interpunctella, Acanthoscelides obtectus (Ayvaz et al. 2010), Tribulium castaneum (Senfi et al. 2013), Callosobruchus maculatus (Khani and Farzaneh 2012), and Trogoderma granarium (Tayoub et al. 2012). Few studies have reported the insecticidal potential of M. rotundifolia essential oil on stored-product pests (El Arch et al. 2003;Brahmi et al. 2016). Nevertheless, no study has been reported before on the insecticidal activity of M. rotundifolia against E. kuehniella adults.
This work aimed to assess the fumigant and contact toxicity of M. rotundifolia and M. communis essential oils from Algeria as well as their effects on longevity, fecundity, fertility, copulation rate and hatching rate against new emerged adults of a Tunisian strain of the Mediterranean flour moth E. kuehniella.

Insect rearing
A rearing colony of E. kuehniella was maintained on wheat flour and semolina under laboratory-controlled condition (temperature of 25 °C ± 1 °C, a relative humidity of 65 ± 5% and darkness). Insects were maintained in 2-L plastic storage boxes. Unsexed adults aged between 24 and 48 h were used for the bioassays.

Extraction of the essential oils
100 g of dry leaves of each plant was hydrodistilled during 90 min using a Clevenger apparatus. The crude-extracted essential oils were stored in opaque flasks in a refrigerator at 4 °C. Essential oils' yields obtained from each plant species were evaluated according to AFNOR (1986) formula: oil% (w/w) = weight of essential oil (g)/weight of plant material (g) × 100.

Gas chromatography-mass spectrometry (GC/MS) analysis
The analysis was carried out using an Agilent 7890A gas chromatograph combined to an Agilent 5972C mass spectrometer with electron impact ionization (70 eV). The mass spectrometer was equipped with a capillary column HP-5 MS (19091S-433), length 30 m, diameter 250 μm, and 2.5 μm film thicknesses (5% phenyl methyl silicone, 95% dimethylpolysiloxane; Hewlett-Packard, CA, USA). The column temperature was automated to rise from 50 to 250 °C at a rate of 7 °C/min. The transfer line temperature was 250 °C. The flow rate of helium (carrier gas) was 1 mL/min. A sample of 2 μL was manually injected with a constant pressure of 7.65 psi using split mode (split ratio 50:1). Scan time and mass range were 1 s and of 45-400 m/z, respectively. The essential oils components were identified by comparing their retention indices (RIs) relative to n-alkanes with those of authentic compounds published in the literature or available in our laboratory. Furthermore, the identification was confirmed by matching their mass spectra with those recorded in Wiley Registry 9th Edition/NIST 2011 edition mass spectral library. The composition of the essential oils was stated as a relative percentage of total peak area.

Fumigant toxicity
The insecticidal activity of M. rotundifolia and M. communis essential oils was evaluated by the fumigant test according to Titouhi et al. (2017). Ten adults of E. kuehniella were placed in Plexiglas bottles of 38 mL volume. The bottom surface of the screw caps was lined with Whatman n° 1 paper disks (2 cm diameter with a 3 cm length fixing tab). Using a micropipette, a series of essential oils doses (without the use of any solvent) were deposited on the filter paper disks. Doses used in the essay were 0.05, 0.125, and 0.5 μL giving equivalent concentrations of 1.31, 3.28, and 13.16 μL/L air. The caps were quickly screwed tightly onto the bottles. Three repetitions were carried out for each concentration of each essential oil including a negative control without essential oil and maintained under the same conditions described for breeding. Mortality was assessed by direct observation of insects every hour until total death. Insects are considered dead when no leg or antenna movements have been observed. Mortality was adjusted via the Abbott correction formula (Abbott 1925). Probit analysis (IBM SPSS V22.0) was used to estimate lethal concentrations LC 50 and LC 95 values and lethal time LT 50 .

Contact toxicity
The contact bioassay of M. communis and M. rotundifolia essential oils against E. kuehniella adults was done according to Mami Maazoun et al. (2017). A Whatman filter paper disks, having 9 cm of diameter, were treated with three doses 0.05, 0.125, and 0.5 μL dissolved in acetone to give concentrations equivalent to 9 × 10 −4 , 24 × 10 −4 , and 99 × 10 −4 μL/ cm 2 . Acetone was used as a negative control. The solvent was allowed to evaporate completely at room temperature for 5 min. Each filter paper disk was then placed in a glass Petri dish, and 10 adults of E. kuehniella were placed into the center of each Petri dish. Each hour, the number of dead insects was recorded. Tests were done in triplicate. The percentage of mortality was calculated using Abbott's correction formula (1925). Insects are considered dead when no leg or antenna movements have been observed. The LC 50 , LC 95 , and LT 50 values were calculated by using Probit analysis (IBM SPSS V20.0).

Impact on biological parameters
The insecticidal effects of M. communis and M. rotundifolia essential oils through fumigation were studied by assessing their impact on longevity, fecundity, fertility, hatching rate, percentage of oviposition deterrence, and copulation rate of E. kuehniella. Five pairs of unsexed adults (0-24 h old) were exposed at the lethal concentration LC 15 . The longevity of males and females, the total number of eggs laid per female (fecundity), the percentage of oviposition deterrence according to Pascual Villalobos and Robledo (1998), fertility, and the percentage of egg hatching were determined. Non-exposed adults kept under the same conditions served as a control. Experiments were replicated three times. For the copulation rate study, prior to exposure, virgin females were obtained by rearing last instar larvae of E. kuehniella separately in plastic boxes. Female's bursa copulatrix were dissected out under a binocular microscope in a saline solution (10% NaCl) and observed for the presence of spermatophores as a criterion of successful mating.

Data analysis
For each biological parameter (longevity, percentage of oviposition deterrence, fecundity, fertility, hatching rate, and copulation rate of E. kuehniella), data were subjected to two-way ANOVA, with essential oils and exposure time as main fixed factors plus with essential oils * exposure time interaction term. Differences in values of each essential oil and exposure time were tested by one-way ANOVA followed by Duncan test to detect significant differences in means at 0.05 percent level. In some cases, certain data have been transformed into a common logarithm or square root in order to obtain a normal distribution of the variables. All data values represented the mean of three replications and were expressed as the mean ± standard deviation. All statistical analyses were achieved using SPSS statistical software version 22.0.

Essential oil fumigant toxicity
Fumigant toxicity results are presented in Fig. 1 as a percentage of mortality of E. Kuehniella adult's strains from Tunisia exposed to M. rotundifolia and M. communis essential oils collected from Algeria. Data revealed that fumigant toxicity depended significantly on plant species (F 1,36 = 33.02, p < 0.01), concentrations (F 2,36 = 20.01, p < 0.01), and exposure time (F 1,36 = 4.82, p < 0.01). The mortality values increased depending on the increasing essential oil concentrations. Furthermore, M. rotundifolia essential oil was more toxic than M. communis. In fact, for the lowest concentration (1.31 μL/L air), percentage mortality of E. kuehniella after 48 h attained, respectively, 100% and 41.66% with M. rotundifolia and M. communis essential oils. For the highest concentration (13.16 μL/L air), M. rotundifolia and M. communis essential oils caused 100% mortality after, respectively, 18 and 24 h of exposure. Moreover, essential oil of M. rotundifolia caused significantly the highest adulticidal effects at all tested concentrations. Moreover, results indicated that M. rotundifolia essential oil showed 100% effectiveness at 3.28 μL/L air after only 18 h of treatment.
Probit analyses have also confirmed that E. kuehniella was more susceptible to M. rotundifolia oil (Table 2). Additionally, the LC 50 and LC 95 values proved that M. rotundifolia essential oil was more toxic than M. communis. The LC 50 values reached 0.54 μL/L air for M. rotundifolia and 2.91 μL/L air for M. communis. The LC 95 was evaluated to be, respectively, 2.11 μL/L air and 5.29 μL/L air for M. rotundifolia and M. communis essential oils. Furthermore, the LT 50 values ranged between 1.03 and 20.76 h for M. rotundifolia and from 20.19 h to 105.09 h for M. communis (Table 3).

Essential oil contact toxicity
Fumigant toxicity data are illustrated in Fig. 2 as percentage of mortality of E. kuehniella adults. Statistical analysis revealed significant differences in percentage of mortality as a function of essential oils treatments (F 1,12 = 10.08, p < 0.01) and concentrations (F 2,12 = 7.58, p < 0.01) after 72 h of exposure. But no significant differences were detected in percentage of mortality after 120 h of exposure time [essential oils treatments (F 1,12 = 4.00, p > 0.05) and concentrations (F 2,12 = 1.75, p > 0.05)]. Results indicated the sensitivity of E. kuehniella adults to both essential oils

Biological parameters
Longevity Essential oils significantly reduced adults' longevity at LC 15 concentration (Fig. 3). Statistical analysis showed highly significant differences in longevity as a function of essential oils treatments for both sex [males (F 2,24

Fecundity, oviposition deterrence, and hatching rate
According to the data presented in Fig. 4, the fecundity of females was highly dependent on essential oil and exposure time. In fact, the number of eggs laid increased over time, whether in the control or in M. communis essential oil treatment. Based on statistical analysis, there is significant differences in fecundity between control and treated females (F 2,6 = 412.81, p < 0.01). The mean number of eggs bequeathed by the untreated females was 178 eggs compared to 93 eggs for the essential oil of M. communis and 6 eggs for M. rotundifolia. As it can be perceived in Fig. 4

Fertility and copulation rate of E. kuehniella females
Results of fertility and copulation rate of E. kuehniella females are illustrated in Table 6. Data showed that fertility and copulation rate significantly depended on essential oil. Statistical analyses indicated a significant reduction in fertility between control and treated insects (F 2,6 = 14.90, p < 0.01). Significant differences were detected in the copulation rate among control and treatments (F 2,6 = 169, p < 0.01). In control, 100% of females had been fertilized by the males and had filled spermathecae. Almost 50% of the females who were treated with M. communis oil have been fertilized. Thus, the mating rate had been halved by the action of this oil. Mating rate in females treated with essential oil of M. rotundifolia was null (0%).

Discussion
The present study showed that the essential oils of M. rotundifolia and M. communis collected from the northeast of Algeria are a promising source of phytochemicals. Mentha rotundifolia yielded 1.29% which is in accordance with the results reported for the same species in Tunisia 1.26%, (Riahi et al. 2013). Contrary, M. rotundifolia from other locations of Algeria have higher essential oil yields 1.6-1.8% (Brada et al. 2007) and 1.65% (Benabdallah et al. 2018) but lower than those reported in Morocco 1.54% (Derwich et al. 2009) and 4.33% (Derwich et al. 2010). In our case, the yield of M. communis essential oil was 0.64%. This result was in agreement with the studies of Barhouchi et al. (2016) in Algeria (0.6%) and Aidi Wannes et al. (2007) in Tunisia (0.61%) but seemed to be higher than those found in Greece 0.28-0.3% (Gardeli et al. 2008) and Morocco 0.3-0.4% (Satrani et al. 2006).
As we can observe, there are several chemotypes of Myrtus communis and M. rotundifolia essential oils over the world; this difference in the chemical composition is closely linked to several factors like adaptive metabolism of plant (Nikšić et al. 2014), geographical position, temperature, day length, nutrients, maturation stage, polyploidy (Scora 1973), air movement, rainfall (Boukhebti et al. 2011), harvest period, distillation method, and time of extraction (Rajendran and Sriranjini 2008). 100.00 ± 0.00 c 6.69 ± 0.14 c Plant extracts have been traditionally used around the world to protect stored products, but the recent increased interest in essential oil was observed with the emergence of scientific evidences on their fumigant and contact insecticidal toxicity to a large range of pests (Isman 2000). In fact, essential oils represent excellent alternatives to chemicals because of their low mammalian toxicity, fast degradation, and local obtainability (Rajendran and Sriranjini 2008). Essential oils could be regarded among the new generation of chemicals that interrupt insects' life cycle and thus seen as more target-oriented softer technique of control with less impact on the environment. The essential oils used in the current work showed a potent toxicity to E. kuehniella adults, especially M. rotundifolia. Our results demonstrated a high fumigant and contact toxicity and exceptional disruptive effects on the biological parameters of E. kuehniella. Brahmi et al. (2016) andEl Arch et al. (2003) have reported the insecticidal activity of M. rotundifolia against pest beetles. Furthermore, context of KarabÖrkulü et al. (2011) evaluated the fumigant action of eight essential oils from Turkish aromatic plants including M. communis and demonstrated that this oil showed a moderate toxicity with LC 50 value of 15.15 μL/L air. In another study, Ayvaz et al. (2010) testified the insecticidal activity (fumigant test) of M. communis (LC 50 = 12.75 μL/L air). This is in contradiction with our results, since we demonstrate that myrtle oil was more efficient with a low lethal concentration (LC 50 = 2.21 μL/L air). Mediouni Ben Jemâa et al. (2012) studied the impact of seasonal variations on the chemical composition and the fumigant bioassay of five Eucalyptus species essential oils against E. kuehniella and confirmed that all species were characterized by the same major compounds: 1, 8-cineole and α-pinene, and Eucalyptus camaldulensis was the toxic one with LC 50 = 26.73 μL/L air and LT 50 = 30.46 h. Also, 100% mortality of E. kuehniella larvae was reached by Satureja hortensis essential oil at 228.5 μL/L air after 12 h of exposure (Maedeh et al. 2011). Similarly, in the study of Emamjomeh et al. (2014), Zataria multiflora exhibited high fumigant toxicity against E. kuehniella adults and larvae at 0.98 μL/L air and 20.67 μL/L air LC 50 , respectively. Also, fumigant toxicity screening on different development stages of E. kuehniella showed the effectiveness of Mentha spicata essential oil with LC 50 value of 0.5 μL/L air for adults and eggs mortality reaching 50-60% (Eliopoulos et al. 2015). In addition, Kheirkhah et al. (2015) noted that E. kuehniella adults were more sensitive to the fumigant effect of Ziziphora clinopodioides oil than larvae (LC 50 = 1.39 μL, 42.17 μL/L air). Mediouni Ben Jemâa et al. (2013) conducted fumigant assays using Laurus nobilis essential oil from Algeria and Tunisia against E. kuehniella adults and recorded the superiority of the Algerian oil (LC 50 = 20.77, 33.75 μL/L air). Likewise, Ecran et al. (2013) observed that Prangos ferulacea oil was more toxic to E. kuehniella adults (LC 50 = 1) than eggs (LC 50 = 320. 37 μL/L air). Pandir and Baş. (2016) recorded that paprica, basil, papermint, and rosemary essential oils were toxic to eggs, larvae, and adults E. kuehniella. Ben Chaaban et al. (2019) proved the fumigant efficacy of Mentha pulegium against E. kuehniella adults (LC 50 = 0.3 μL/L air).
Inhibition of oviposition using essential oils represents a pertinent criterion to control pest infestation and manage stored products (Singh and Pandey 2018). In this respect, Ulukanli et al. (2014) pointed out that essential oils extracted from cortex of Pinus pinea and Pinus brutia presented ovicidal activity on E. kuehniella eggs (LC 50 = 343. 57, 299.9 μL/L air). According to Tunç et al. (2000), cumin and anise induced 100% mortality of E. kuehniella eggs. Besides, Bachrouch et al. (2010) cited that Pistacia lentiscus was toxic against E. kuehniella (LC 50 = 1.84 μL/L air) and reduced adults' longevity, fecundity, hatching rate, and copulation rate. Generally, as recorded by Delimi et al. (2013) and Taibi et al. (2018), topical bioassay of Artemisia herbaalba and Origanum vulgaris oils on E. kuehniella chrysalis causes disorders of nymphal duration, disrupts adult reproduction, reduces the longevity of females, and therefore decreases their fecundity.
As reported in this work, strong contact and fumigant toxicities were exhibited by both Algerian essential oils. The round-leaved mint M. rotundifolia showed most promising results in terms of adult mortality and impact on biological parameters of the target pest E. kuehniella. Indeed, a complete inhibition of egg hatching and copulation rate was accomplished. Consequently, our results clearly support the application of this oil as a potential biocontrol agent that may be an appropriate alternative for achieving a safer and more sustainable management of stored commodities in Algeria. Therefore, further research including the efficacy of this oil on other stored-product pests (coleopteran species) and its impact on food quality are warranted.

Compliance with ethical standards
Conflict of interest The author declares that they have no conflict of interest.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.