Larvicidal Action of Cannabidiol Oil and Neem Oil against Three Stored Product Insect Pests: Effect on Survival Time and in Progeny

Simple Summary Each year, agricultural produce suffers significant loss and quality deterioration upon infestation by stored product insects. Synthetic insecticides represent a ready-to-go, effective pest control solution, albeit with several environmental and health risks. In this light and within the framework of integrated pest management research, the present study focuses on the potential insecticidal effect of two essential oils, neem oil and CBD, against 4th instar larvae of the harmful Tribolium confusum, Oryzaephilus surinamensis and Plodia interpunctella, on wheat, rice and corn seeds. CBD, especially, has been under-researched in this regard. Treatment efficacy was expressed as larval mortality in relation to dosage, time exposure intervals and product. In comparison with the control, the results showed clear dose-dependent pesticidal activity for both oils, expressed as significant larval mortality at high dose application, as high as 100% at the highest dose (90 mg/mL). Moreover, the overall survival time of the tested larvae was also considerably shorter than that of control larvae, while the treatments also produced considerably fewer offspring in the tested insects. Our results reinforce the prospect of incorporating botanical insecticides in Integrated Pest Management programs. Abstract Stored product pests can be detrimental to agricultural produce. As much as chemical pesticides are effective control agents, they involve several environmental and health risks. Within the framework of studies on alternative pest management methods, interest has focused on a plethora of plants whose extracts have demonstrated promising action as insecticides. Azadirachta indica and the derived neem oil have been extensively tested against many harmful insect species. In contrast, Cannabis sativa L. and its main compound, CBD, a highly concentrated cannabinoid, have not been investigated much. The present study examined the potential insecticidal activity of CBD and neem oils against 4th instar larvae of Tribolium confusum, Oryzaephilus surinamensis and Plodia interpunctella on wheat, rice and corn seeds. Treatment efficacy was expressed in terms of larval mortality. Mortality was observed in relation to dosage, time exposure intervals and product types. The results showed clear pesticidal activity for both oils, which at high doses induced significant mortality. The treatments produced significantly fewer offspring in the insect species tested than the control. The efficacy of treatment in progeny suppression was, as expected, dose dependent.

Our idea is to obtain bioactive essential oils from the inflorescences of industrial hemp that usually remain underutilized, in order to manufacture natural insecticides for application in organic agriculture and IPM programs. Indeed, research on this issue is still poor. The objective of the present study was to evaluate the larvicidal effect of CBD oil and neem oil in vitro, against the three major stored product pests, T. confusum, O. surinamensis and P. interpunctella in three different seeds. The potential insecticidal properties of these oils might lead to an increase in pest control efficiency and improve pest management strategies.

Insect Rearing
Three major stored product pests were tested in the present study: O. surinamensis, T. confusum and P. interpunctella. O. surinamensis was reared on whole oats, T. confusum on a mixture of whole wheat flour and dried yeast (1:10), and P. interpunctella was provided with a mixture of honey, glycerin, dried yeast, and sterile fully-grinded flour and wheat bran. The insect colony used was established in 2018 from a stock colony provided by the University of Thessaly, Department of Agrotechnology, Laboratory of Entomology (Larisa, Greece), and it has since then been continuously grown in the Laboratory of Molecular Biology and Immunology, Department of Pharmacy, University of Patras. Insects were placed with 200-250 g of their diet in glass jars (0.45 L capacity) which were then covered with a sterilized muslin cloth. Two weeks later, we carefully sieved out the adult individuals. Insects were contained in a growth chamber (PHC Europe Sanyo Panasonic Biomedical MLR-352-PE), in controlled environmental conditions, at 27.5 • C and 75% R.H.

Bioassay
The experiments were carried out at the Laboratory of Molecular Biology and Immunology, Department of Pharmacy, University of Patras. The CBD oil obtained from the company Elixinol, Enecta CBD oil (Athens, Greece), contains 3% CBD. The neem oil acquired from Laboratoire Alth, Organic neem Oil (Monfort, France) contains 3% azadirachtin. MeOH (1%) was used for the dilution of both oils; the dilution process was carried out inside a laminar flow chamber (Equip Vertical Air Laminar Flow Cabinet Clean Bench, Mechanical Application LTD, Athens, Greece). We tested the effectiveness of the oils against larvae of T. confusum, O. surinamensis and P. interpunctella, at three different doses, 90 mg/mL (hi con), 45 mg/mL (med con), 15 mg/mL (low con). The experimental product was directly sprayed with 2 mL using a Potter spray tower (Burkard Manufacturing Co. Ltd., Rickmansworth, Hertfordshire, U.K.) at 1 kgf cm −2 . After applying the CBD and Neem oils, the product lots were placed back in the petri dish, and they were shaken manually for 30 s to achieve equal distribution of the oil; the product was then air dried for 30 min. The tests were carried out on wheat cv. Mexa which contained 5% cracked kernels, corn cv. Golden Bantam which contained 5% cracked kernels, and rice cv. Japonica which also contained 5% cracked kernels. These products were adjusted at 12% moisture content (m.c.), via storage in ambient conditions for 28 d.
Laboratory-reared 4th instar larvae from each experimental insect species were used for this study. Each batch of ten 4th instar larvae was collected from the rearing jars and then placed in 9-cm diameter petri dishes with 10 g of the desirable product, after the larvae had been starved for 1 h. Ten larvae were used per replication (n = 10), and ten replications were carried out per dose (n = 100); the experiment was replicated ten times (n = 1000). The petri dishes had a plastic lid with a hole in the center covered with fine mesh, while the internal "upper lid" of the petri dishes was covered with Fluon (Northern Products, Woonsocket, RI, USA), to prevent insect individuals from escaping. Then, all petri dishes were placed in incubators set at 27.5 • C and 75% R.H. The control larvae were treated with MeOH and ddH 2 O. Mortality of the exposed larvae was examined after 12, 24, 48, 72, 144, 216 and 288 h.
After 288 h, the petri dishes containing T. confusum, O. surinamensis and P. interpunctella larvae, were shielded with parafilm and placed back in the incubators, in the same conditions, for an additional period of 25 d. After the termination of this interval, the petri dishes were opened, and progeny was recorded. Progeny was recorded separately per life stage of larvae or pupae.

Statistical Analysis
Preceding analysis, we arcsin transformed all values. We analyzed data by five-way ANOVA with the general linear model of the SPSS (SPSS Inc., Armonk, NY, USA, version 25) (IBM, 2019), and we compared means of significant F values with the Bonferroni test. We also applied the Kaplan-Meier method to determine the mean survival time of T. confusum, O. surinamensis and P. interpunctella larvae post treatment application.
The percentage of pupal and adult inhibitions for P. interpunctella, T. confusum and O. surinamensis is expressed as equal to: whereby t denotes the number of pupae or adults in the treatment, and c refers to pupae or adults in the control. We processed data with one-way ANOVA, using the general SPSS linear model (SPSS Inc., Armonk, NY, USA, version 25) (IBM, 2019), and means of significant F values were compared with the Bonferroni test.

Larvicidal Effect of CBD Oil and Neem Oil on Stored Pest in Vitro Assay
Significant differences were detected among treatments in many cases. Oil Dose, Insect Species, Seeds Product and Time were proven to have a significant effect on larval mortality. Factors interactions showed a significant effect; this is indicative of the fact that experimental factors affected the insects' survival time in diverse ways (Table 1). Very low control mortality was recorded among all experimental larvae on all products. Mean control mortality was recorded between 3.33% and 6.67% in all cases. When T. confusum larvae were exposed to the CBD oil, they suffered mortality between 17% and 100% on wheat, 17% and 93% on corn, and 26% and 83% on rice. Similarly, the neem oil caused larval mortality between 13% and 73% on wheat, 23% and 63% on corn, and between 20% and 60% on rice ( Figure 1). In all cases, mortality was always significantly increased as the dose increased. As far as O. surinamensis larvae are concerned, their mortality ranged between 17% and 100% on wheat, 36% and 96% on corn, and 67% and 100% on rice, when they were sprayed with CBD oil. The respective neem oil treatments caused larval mortality between 20% and 77% on wheat, 30% and 77% on corn, and 30% and 83% on rice ( Figure 2). As far as O. surinamensis larvae are concerned, their mortality ranged between 17% and 100% on wheat, 36% and 96% on corn, and 67% and 100% on rice, when they were sprayed with CBD oil. The respective neem oil treatments caused larval mortality between 20% and 77% on wheat, 30% and 77% on corn, and 30% and 83% on rice (Figure 2). Finally, the mortality of P. interpunctella larvae treated with CBD oil was between 16% and 76% on wheat, 13% and 60% on corn, and 33% and 63% on rice. Accordingly, the neem oil caused larval mortality between 36% and 90% on wheat, 26% and 77% on corn, and 36% and 77% on rice ( Figure  3). Finally, the mortality of P. interpunctella larvae treated with CBD oil was between 16% and 76% on wheat, 13% and 60% on corn, and 33% and 63% on rice. Accordingly, the neem oil caused larval mortality between 36% and 90% on wheat, 26% and 77% on corn, and 36% and 77% on rice (Figure 3).

Overall Survival Time of Larvae Exposed to Doses of CBD Oil and Neem Oil
The survival time of T. confusum larvae exposed at overall low, mid and high doses was recorded at 222.667, 159.971 and 63.067 h. Similarly, the survival time of O. surinamensis larvae exposed at overall low, mid and high doses was recorded at 245.667, 146.933 and 58.000 h, respectively (Table

Overall Survival Time of Larvae Exposed to Doses of CBD Oil and Neem Oil
The survival time of T. confusum larvae exposed at overall low, mid and high doses was recorded at 222.667, 159.971 and 63.067 h. Similarly, the survival time of O. surinamensis larvae exposed at overall low, mid and high doses was recorded at 245.667, 146.933 and 58.000 h, respectively (Table 2). Finally, the survival time of P. interpunctella larvae exposed at overall low, mid and high doses was recorded at 244.267, 196.0000 and 87.333 h, respectively. Significant differences were detected between insect and dose ( Table 2). The overall survival time of larvae exposed to the CBD oil at the low, mid and high doses was recorded at 219.156, 180.556 and 75.956 h. Similarly, the overall survival time of larvae exposed to the neem oil at the low, mid and high doses was recorded at 239.244, 204.044 and 119.644 h. Significant differences were detected between both treatments with the control and between the high dose of CBD and the high dose of the neem oil (Table 3).

Effect of CBD Oil and Neem Oil Doses to Pupation and Adult Emergence
Results related to inhibition of metamorphosis showed that a noticeable decrease in percentage achievement of pupal and adult stage was detected when 4th stage larvae of P. interpunctella, T. confusum and O. surinamensis were subjected to high doses of CBD oil and neem oil. At the higher dose of CBD oil, the lowest pupation and adult emergence were 8.43% and 6.13% for P. interpunctella (Table 4); 5.75% and 3.13% for T. confusum (Table 5); and 3.71% and 1.89% for O. surinamensis (Table 6). In parallel, at the higher dose of neem oil the lowest pupation and adult emergence were 10.10% and 9.08% for P. interpunctella (Table 4); 7.10% and 6.04% for T. confusum (Table 5); and 4.10% and 1.13% for O. surinamensis (Table 6). Table 4. Mean effect of CBD oil and neem oil on pupation and adult emergence (% ± sd) of 4th larval instars of P. interpunctella. Means of the same column followed by the same letter are not significantly different (Long Rank test, p = 0.05).    (Table 6).

Discussion
Essential oils have been successfully used to manage a rather wide number of serious insect pests [39,40]. The present study has highlighted substantial insecticidal activity of CBD oil and neem oil against T. confusum, O. surinamensis and P. interpunctella larvae and pupae, as high as 100% at the highest dose (90 mg/mL), whereby the overall survival time of the tested larvae was also considerably shorter than that of control larvae.
Similarly, neem oil caused high mortality to all three insects as well as low survival time when administered at the highest dose. This adds to the existing bibliography on the role of neem oil in providing sustained protection of stored grains [45] and efficient pest control in storage facilities, alone or in combination with other protective measures [46][47][48][49][50][51][52][53][54][55]. Only a few studies have investigated the effect of neem oil on the survival of our experimental insect species. Indicatively, it has been found that neem extracts have caused noteworthy mortality to T. confusum [56,57] and O. surinamensis adults [58], whereas no data have been published on P. interpunctella.

Conclusions
In general terms, both oils displayed noteworthy action against the tested larvae of P. interpunctella, T. confusum and O. surinamensis. As such, our results allow us to state that these stored product pests and the larvae of O. surinamensis, exhibit a clear susceptibility to the tested oils, which warrants further research. As the target of limiting the use of synthetic pesticides becomes more urgent, the prospect of incorporating botanical insecticides in Integrated Pest Management programs comes to the forefront.