Abstract

The Crude Extract and powder of Lepidium sativum Linn. seeds were evaluated for a toxicity effect on 5^th instar larvae of the Trogoderma granarium beetle by vapor and diet incorporation methods, respectively, as well as the repellent indicators of the powder at concentrations of 2, 0.9, 0.5, and 0.1% (w/w); the toxicity assay of essential oil was evaluated at 20, 15, 10, and 5 µl/L air. The results show that a variety of 5^th larvae responses proved the effectiveness, where the essential oil vapors showed a high fumigate toxicity at 28.64 µl/L air, where the LC50 was identified after 72 hours of exposure. Additionally, the powder showed a toxicity effect at the concentrations of 2, 0.9 and 0.5% (w/w), which caused 100 and 96% mortality, respectively, and the LC50 was identified at 0.26% after 10 days of treatment. The exhibited repellent percentages of the powder were weak and at the same time had high acceptance and larval attractivity to the wheat grains treated. The attractive percentage was determined at -53.3% and -66.6% at the lowest and highest applied concentrations of 0.1% and 2% after 72 hours of exposure. The presence of the inverse relationship between the attractive and toxicity properties of the same material (L. sativum seeds) is a promising sign to the safe modeling of toxic baits, which attract and then kill larvae.

1. Introduction

The Kingdom of Saudi Arabia is characterized by a breadth and diversity of the terrain, the weather and its agricultural features. This has resulted in diversity in the vegetation cover and thus diversity of the food culture between its regions and food culture among its nomadic, rural and urban populations ¹. It is obvious that this diversity is followed by species richness in the plantations used in drugs, folk medicine and even in cooking. This case particularly appears in most Asian countries. Some of these plants have accidentally caught the attention of farmers and the general public to control bacteria ², viruses and insects ³. These simple observations are considered as initial and preliminary indications that have led a large number of researchers, since the beginning of the twentieth century, to the feasibility of applying them in a deliberate manner against insects ^{4, 5, 6, 7}. Lepidium sativum: Cruciferae is a popular medicine plant that is widespread in different regions ^{1, 8}. Lepidium sativum is considered to be a multi-use plant ^{9, 10}, particularly the seeds, which have been given to children to strengthen their bones and heal bone fractures ^{10, 11}.

It is also characterized as one of the medicines given for bronchitis ^{12, 13}. Moreover, it is traditionally used as repellent against some of the insects carrying viral and bacterial diseases, such as mosquitoes ¹⁴. Similarly, ^{15, 16, 17} reported the richness and diversity of Lepidium sativum seeds with biochemically active compounds that are well suited as antioxidants and antibacterial. All these properties encouraged the carrying out of this study to evaluate the repellency and toxicity activity of this plant on 5^th instar larvae of Trogoderma granarium: Dermestidae, which is classified as a highly stored grain pest and severely tolerant to insecticidals and fumigants ^{18, 19, 20}, and the adult and immature stages are destructive to more than 30% of the stored product ^{21, 22, 23}.

T. granarium larvae continue feeding the stored wheat grains until the endosperm of the seed, thus decreasing the quantity and quality ^{20, 24, 25}, and the 5^th instar larvae is the most harmful instars larvae and eater of the stored product ^{23, 26}. It is known for its gluttonous eating habits and its high resistance to pesticides ²⁷ in addition to the possession of thick hairs covering its entire body, which provide it with a kind of physical protection from impurities and pesticides mixed in with wheat grains ²⁸, as well as its ability to withstand high temperature and humidity conditions ²⁹.

2. Materials and Methods

In accordance with ³⁰, to prepare homogeneous populations of the insect, the different Trogoderma granarium Everts stages of adults, larvae and eggs were obtained from samples of many storage products, for example, wheat grains, barley, broccoli, rice, pasta, nuts and dried fruit, which were infected by this pest. Then, these stages were bred on common wheat grains, Triticum aestivum, sterilized by freezing the wheat grains in a deep freezer at - 40°C for approximately three hours. Finally, the insect cultures were kept in an incubator at a temperature of 25 ± 2°C and relative humidity 70-60%, with consideration of renewing the culture after each generation to avoid the formation a sensitive strain, and then the scientific experiments were conducted.

Lepidium sativum Linn. belongs to the family Cruciferae, with a common name of “thefa” and “hab al rashad”, and the seeds used in this study were purchased from the local market. The plant used was classified by a specialized plant classification and then cleaned of impurities and air-dried in the shade. Finally, seeds were ground in a high-voltage electric blinder, and the powder was well sifted to obtain a fine powder.

Accordingly ³¹, after drying the seeds, they were put into a device to extract the crude oil by distillation: 100 grams of dry seeds were put in water (1:10 w/v) for three hours, and then the oil mixed with a hexane solvent was separated by a separator funnel. The oil and hexane mixture was dried by anhydrous sodium sulfate. The solvent was then disposed of using a rotary evaporator, and the oil was preserved in dark glass bottles and stored in the refrigerator at a temperature of 4 °C until used.

To determine the attractive and repellent activity of the seed powder on 5^th instar larvae, we followed ³², an assay by using a wide glass dish 15 cm and 2.5 cm in diameter and height, respectively, and placed the powder in a small dish, which represents a treatment dish (TD), and a constant mix of wheat grains with determined concentrations of 0.1, 0.5, 0.9, and 2% of the fine seed powder in a weight/weight ratio in the center. Every concentration was repeated six times. Additionally, a control without any treatment and ten newly molted 5^th instar larvae were evenly distributed over three replicates. Then, the wide dishes were closed by muslin cloths and rubber bands in the same culture condition. According to the ³³ method, the attractive and repellent percentages were determined by counting the larvae that stayed (C) or got out of the treatment dish (TD), respectively, for the two percentages at the exposure times of 24, 48 and 72 hours. The repellence percentage was calculated using the following equation:

PR = percentage of repellence

C = percentage of 5^th instar larvae that stayed in the treatment dish (TD)

PR becomes positive if C is more than 50%, and the treatment by plant powder has a repellent efficiency. Conversely, if C is less than 50%, PR becomes negative, and the plant powder has an attractive effect.

The 0.1, 0.5, 0.9, and 2% weight/weight ratios of the fine L. sativum seed powder were well mixed with 10 g of the sterile wheat grains and placed in sterile Petri dishes with a diameter of 10 cm, and then 10 newly molted fifth instar larvae were introduced for every concentration, each repeated for six replicates. For the control treatment, the 5th instar larvae were fed non-treated wheat grains with the same conditions of the treatments mentioned above. In both the treatment and control experiments, the counts of the mortality were recorded after 4, 7, and 10 days with any changes in the behavior, morphology and activity of the 5^th instar larvae. According to ³⁴ the formula, the correct mortality percentages were detected using the mortality in the controls at the 10th day of treatment.

With some modifications, the activity of the fumigants for the crude oil of L. sativum seed powder was conducted in accordance with ³⁵ on 10 newly molted 5th instar larvae. One microliter of the crude oil of the L. sativum seed powder was placed on a 20 g cotton ball with a diameter of 2 cm inside a 500 ml glass jar for each concentration of 0.1, 0.5, 0.9, and 2 (µ1/L air) of the crude oil. The control cotton ball was treated with hexane only. After the cotton balls were left to dry in a fume hood, the larvae were introduced into the assay jars, and food (a few wheat grains) was placed in the treatment and control containers with six replicates for each concentration and control. After 24, 48 and 72 hours of exposure time, the mortality number was recorded in a completely randomized design by average and accumulative percentages, respectively.

The mortality data were corrected with the control mortality following the formula given in Abbot. All data were statistically analyzed using a one-way analysis of variance (ANOVA, SPSS statistical analysis software, version 23). The means were compared using the least significant difference (L.S.D) at the 0.10 level.

3. Results

As shown in Table 1, the results indicated the slight effectiveness of the repellent activity of the L. sativum seed powder toward 5^th instar larvae. For the 5^th instar larvae recorded as escaped from the food media treated by 3.67% and 3.33% at the highest concentrations of 2 and 5 (w/w) after 72 hours of exposure, the repellency percentage was zero in contrast to the attraction effect percentages (-53.3, -46.6%, -53.3% and -66.6%, respectively) for the concentrations of 0.1%, 0.5%, 0.9% and 2% after 72 hours of exposure (Table 1). The results of the statistical analysis showed no significant difference in the effect of the powders used in the repellency percentage. The results showed a progressive relationship: the higher powder concentrations of 2% and 5% achieved greater attractive effect percentages compared with the untreated larvae in the control, where no abnormal results or behavioral changes were recorded. The statistical analysis showed that there were no significant effect differences on the Repellency percentage at a significant level (P<0.10), with significant effect of the attractive percentage

The toxic effect of L. sativum seed powder was tested on the mortality percentage of 5^th instar larvae. The results (Table 2) showed extermination effectiveness ranges from medium to high for all applied concentrations. The 2% and 0.1% concentrations recorded 43.3% and 10% mortality after the fourth day of the treatment, respectively, and after 10 days of treatment, it was completely a 100% and 68%, respectively. Additionally, the concentrations of 0.9 and 0.5%, with the progress of the treatment time, provided a steady mortality percentage from 33.3% until completely 96.4% at the fourth and tenth days, respectively.

In general, the results presented in Table 2 show an ascending relationship between the concentrations on the one hand and the time of exposure on the other hand with the mortality percentage of the 5^th instar larvae. The statistical analysis showed that there were significant effect differences on the mortality percentage at a significant level (P<0.10). Additionally, the LC50 of the seed powder was detected at the 10th day of exposure, and it was 0.26% closer to the concentrations of 0.1% and 0.5%, which both provided 50% the mortality percentage of the 5^th instar larvae.

Table 3 shows the toxicity results of the crude oil fumigants on the 5^th instar larvae. The significant toxicity of the 20 µl concentration was recorded after 48 hours of exposure with 40% mortality; notably, the concentrations of 5 µl and 10 µl provided 33.3% and 27% mortality, respectively, after 72 hours of exposure. While after 72 hours, the concentrations of 15 µl and 20 µl were superior in killing, and the mortality percentage was increased until 100%; moreover, the most prominent changes observed in the larvae treated with oil vapors were that they were weak and slow in movement. The statistical analysis showed that there were significant effect differences on the mortality percentage at a significant level (P<0.10).

4. Discussion

The findings of the present study showed the distinguished effects of the L. sativum seed powder on the 5^th instar larvae. The more attractive activities were the larvicidal effect of the mixed powder and crude oil, and acting as a fumigant when wheat grains were provided to the treated larvae, respectively.

The double efficiency (repellency and toxicity) is described as valuable and precious because the highly f attractive effect of a toxic substance to the targeted pest is a successful strategy characterized by toxic bait. Certainly, this variation of the effects is because the L. sativum seed powder possesses the chemical composition to act as an attractor to insects because its odor was accepted by the larvae.

Based on the opinion of ³⁶, insects are always looking for a plant and its real and suitable host for food to lay eggs on, for meeting and mating, and for the small larvae that hatch. Therefore, insects accept some plants and reject others. This acceptance and rejection is significant behavior due to the differences in the chemical compounds contained in plants.

Reference ³⁷ mentioned that some plants parts are acceptable to some insects, even if they are not their food, but only after they reach the larval body. Reference ³⁷ also mentioned that the accepted chemical compounds at the beginning of the treatment are released, the toxic compounds that kill the treated larvae either gradually kill if the concentrations reached are low or directly kill if these concentrations are high, such as the complete mortality (100%) that was provided by the concentrations of 2% and 20 µl of the powder, and the essential oil fumigants contrasted to the 100% attractive percentage of the seed powder.

Generally, most of the increase in the toxicity or repellency activity for any treatment is expected with an increased concentration and/or exposure time ³⁸. According to ^{39, 40, 41}, the essential oils of many plants have insecticidal and repellency properties due to the mixture of compounds insecticidal in nature.

The chemical analysis of the essential oil of L. sativum seeds showed that the seeds contained a high proportion of compounds that have biological activity and medicinal sources ^{10, 42}.

In addition, the Cruciferae family to which L. sativum belongs is one of the most distinguished plant families that contain glycoside and sulfur compounds ⁴³, which are considered to be defensive compounds used by the plants against herbivores.

Reference ⁴⁴ found that the L. sativum seeds contain the amino acid glutamic acid (19.3%), leucine (8.21%) and protein (12%) that has an effect on the circulatory and digestive systems of larvae treated with powder and plant oil and confuse the function of those systems.

Reference ^{14, 45} revealed that the major components with high biological activity in the essential oil from L. sativum seeds were B-caryophyllene, eugenol, eucalyptol, sulfur, α- terpiny acetate and X-terpiny acetate.

These compounds provided the larvacidal effect against mosquito larvae, especially the eugenol ⁴⁶. Additionally, for eucalyptol ⁴⁷, the bio-efficacy of eucalyptol(1,8-cineole) at LC50 was reported as 101 and 642 µg/µl against blow fly, Chrysomya megacephala (F.), and house fly, Musca domestica L., larvae, respectively.

Moreover, ⁴⁸ confirmed the toxic and physiological effects of eucalyptol (1,8-cineole)(eucalyptol) on the larvae of two stored pests: the rice weevil, Sitophilus oryzae, and the red flour beetle, Tribolium castaneum.

Reference ⁴⁹ reported that the 1, 8-cineole (eucalyptol) has fumigant toxicity toward Callosobruchus maculatus during the first hours of treatment.

According to ^{26, 50}, it is possible due to the arrival of the terpenoids and glycosides, which are the effective and main compounds of the L. sativum seed, after evaporation, that it has great ability to penetrate the outer wall of the larvae body because the compounds were lipophilic and had insecticidal activity ^{49, 51, 52}.

Reference ⁵³ found out that the L. sativum seed aqueous extract killed 71% of the small stages of Bemisia tabaci nymphs. Similarly, ⁵⁴ emphasized that the high pesticide effect or the activity of the crude oil fumigants of Artemisia plants on Callosobruchus maculates , Sitophilus oryzae and Tribolium castanium beetles was LC50.

Reference ⁵⁵ indicated that the traditional use of L. sativum seed powder of applying and placing it on the skin to repel mosquitoes, domestic flies, ticks, and pest mites was effective due to its high repellency and exterminating activity.

The bio-activity and “phytotoxicity” of the L. sativum seed crude oil was evaluated ⁶ and was characterized by 42% biologically active compounds, as well as an effective exterminating activity, which resulted in high mortalities for Rhyzopertha dominica by testing the toxicity on contact.

The results of this study, in addition to previous studies, indicate that the powder and oil of L. sativum seeds are characterized by the high insecticidal activity on the 5^th instar larvae of the Trogoderma granarium beetle.

5. Conclusions

The results of this study agreed with a large number of previous studies that emphasized the importance of integrating plants, their parts and extracts, whether using oil, water or alcohol extracts, into integrated pest management. Clearly, the seeds of the L. sativum plant are indicated for their desirable signs of fighting pest larvae that represent a real economic problem in most of Asia and the Middle East. It is possible to use L. sativum powder and oil as poisonous bait in grain stores, and its use will be safer than manufactured bait. These seeds can be recommended as a good alternative to synthetic pesticides against grain pests.

One of the most important factors responsible for the insecticidal effect is the presence of chemical compounds in any form.

Acknowledgements

I would like to thank M. Al-rwais from the Prince Sattam Bin Abdul-Aziz University, the College of Science and Humanities for technical assistance, and the Department of Biology for their invaluable help in completing this study.

Statement of Competing Interests

The authors have no competing interests.

List of Abbreviations

PR = percentage of repellence

C = percentage of 5^th instar larvae that stayed in the treatment dish

TD = treatment dish

References