Anti-Termitic Activity of Three Plant Extracts, Chlorpyrifos, and a Bioagent Compound (Protecto) against Termite Microcerotermes eugnathus Silvestri (Blattodea: Termitidae) in Egypt

Simple Summary The termite Microcerotermes eugnathus Silvestri (Blattodea, Termitidae) showed structural damage in Bir al-Shaghala cemeteries located in the oasis of Dakhla, Egypt. The mud tubes of this termite spread inside and over the mural painted floors of the tombs. Extracts from Lavandula latifolia, Origanum vulgare, and Syzygium aromaticum were tested for their anti-termitic activity and compared with the bio-insecticide, Bacillus thuringiensis var. kurstaki (Protecto 9.4% WP) and Dursban (Chlorpyrifos 48%). The bioassay experimental showed that the extracts have low activity against M. eugnathus compared to Protecto and Dursban, but the extract from O. vulgare showed promising natural termiticides. Abstract A trend towards environmentally friendly chemicals for use in termite management has been occurring globally. This study examined three naturally occurring plant extracts from Lavandula latifolia (Spike lavender), Origanum vulgare (Marjorum), and Syzygium aromaticum (Clove) against the termite Microcerotermes eugnathus. Plant extract results were compared to two commercially used termite pesticides, the bio-insecticide, Bacillus thuringiensis var. kurstaki (Protecto 9.4% WP) and Dursban (Chlorpyrifos 48%). Gas chromatography–mass spectrometry (GC-MS) analysis was used to identify the main compounds in the three plant extracts. The main compounds in Lavandula Latifolia were linalool (21.49%), lavandulol (12.77%), β-terpinyl acetate (10.49%), and camphor (9.30%). Origanum vulgare extract contained thymol (14.64%), m-cymene (10.63%), linalool (6.75%), and terpinen-4-ol (6.92%) as main compounds. Syzygium aromaticum contained eugenol (99.16%) as the most abundant identified compound. The extract of O. vulgare caused the highest termite death rate, with an LC50 of 770.67 mg/L. Exposure to lavender extract showed a high death rate with an LC50 of 1086.39 mg/L. Clove extract did not show significant insecticidal activity with an LC50 > 2000 mg/L. Significant termiticide effects were found, with LC50 values of 84.09 and 269.98 mg/L for soldiers and workers under the application of Dursban and Protecto, respectively. The LC50 values reported for nymphs were <120, <164.5, and 627.87 mg/L after exposure to Dursban, Protecto, and O. vulgare extract, respectively. The results of the study show that some of the extracts have low toxicity compared to the bioagent and Dursban, and may show promise as natural termiticides, particularly as extracts from O. vulgare.

This research aimed to (1) determine the chemical composition of three plant extracts (lavender, marjoram and clove) with potential anti-termitic activities; (2) evaluate the anti-termitic activity of the three plant extracts against Microcerotermes eugnathus compared with two commercial pesticides used in termite control; the bioagent "Bacillus thuringiensis var. kurstaki" (Protecto 9.4% WP) and the chemical compound Dursban (chlorpyrifos 48%). In addition, the chemical composition of the three extracts were analyzed with using GC-MS apparatus.

Termite Infestation in the Cemeteries of Bir al-Shaghala at the Oases of Dakhla
Termite infestation in the cemeteries of Bir al-Shaghala located at the oases of Dakhla, Egypt was noted by the presence of Microcerotermes eugnathus foraging tubes. Termite tunnels could be observed on the floors and tomb substructure, causing cracks and separation of mortar. This termite species produces winged alates during the months of April and January, the remnants of which (wings) could often be observed around the tombs [59]. Figure 1A,B shows the cemetery termite damage.

Plant Extracts
Three plant extracts from Spike Lavender (Lavandula latifolia Medik., Lamiaceae) dry flower, Marjoram (Origanum vulgare L., Lamiaceae) dry leaves and Clove (Syzygium aromaticum (L.) Merrill and Perry, Mytaceae) dry buds were selected for their potential effect on termites. The extraction process was carried out at the National Research Center in Cairo, Egypt. Plant material was shade air-dried in the laboratory at room temperature for one week, then ground into a fine powder in an electrical blender. About 100 g of the fine powdered material was put into a 2-L flask containing 1000 mL of distilled water (DW) and extracted by the hydrodistillation method using a Clevenger-type apparatus for 3 h [60].

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis of Extracts
The chemical composition of extracts from L. latifolia, O. vulgare, and S. aromaticum were determined using a Trace GC-TSQ Evo 9000 mass spectrometer (Thermo Scientific, Austin, TX, USA) with a TG-5MS direct capillary column (30 m × 0.25 mm × 0.25 µm film thickness). Extracts were diluted in n-hexane solvent in the ratio of 3:1 (3 n-heaxane: 1 extract sample) before being injected to the GC-MS. The column oven temperature was initially held at 50 • C and then increased by 5 • C/min to 250 • C and held for 2 min, then increased to a final temperature of 310 • C by 25 • C/min and held for 2 min. The injector and MS transfer line temperatures were kept at 270 and 260 • C, respectively; helium was used as a carrier gas at a constant flow rate of 1 mL/min. The solvent delay was 3 min, and diluted samples of 2 µL were injected automatically using an Autosampler AS1310 coupled with GC in the splitless mode. EI mass spectra were collected at 70 eV ionization voltages over the m/z range of 50-650 in full scan mode. The ion source temperature was set at 250 • C. The components were identified by comparison of their retention times and mass spectra with those in the WILEY 09 and NIST 11 mass spectral databases [61]. The match factor (MF) between the mass spectrum obtained for each compound and the library mass spectra for each compound was measured and reported; it was accepted if its value was more than or equal to 650 [36,[38][39][40]62,63].

Collecting and Rearing the Termites
Microcerotermes eugnathus castes were collected from soil around cemeteries and kept in a container with a portion of termite nesting material. Briefly, from the experimental location, termites were placed inside a special box (4-L in capacity) for preservation and the box was covered with wool textile to maintain the temperature, because the temperatures in the interior oases are as high as 50 • C. During the transfer to the laboratory in Cairo, the death of the termites occurs due to the different environmental conditions, so it was necessary to warm the termites. Termites' death was also observed during transportation to the laboratory or even in the laboratory during the preparation for the laboratory experiment. Therefore, 10% glucose sugar solution [64,65] was placed where sterile cotton was absorbed into the sugar solution and suspended in a flask above the cardboard feeding medium as a source of nourishment for termites, to give moisture and nutrition. The termites were incubated at 48 ± 3 • C in order to provide a medium close to that of the oasis.

Testing Anti-Termitic Activity (No-Choice Bioassay Method)
A no-choice bioassay method [66,67] was employed to measure the anti-termitic activity of the three plant extracts, Protecto, and Dursban. 1125 pieces of cardboard similar to the laboratory rearing material were prepared and divided into three groups and each group contained 375 pieces and divided to five subgroups (75 pieces). In the first group, each subgroup (75 pieces Untreated control replicates (five replicates) contained 10 workers, five soldiers and five nymphs, who were supplied with pieces of untreated cardboard paper only. Each concentration tested was replicated five times. All the treatments were kept in darkness at 48 ± 3 • C. After seven days, the test was halted, the number of surviving termites was counted, and percent mortality was calculated. The lethal concentration (LC 50 ) expressed as mg/L was calculated from log-concentration mortality regression lines [38].

Discussion
Many plant compounds can incur high mortality in termites. This is related to their mechanism of action on the insect nervous system [69]. Plant compounds may also have neurotoxic (hyperactivity, seizures, and tremors) modes of action [70][71][72]. The data presented in this work clearly show big differences in chemical components among plant samples depending on their origin, even in the same species.
Oxygenated monoterpenes such as thymol, eucalyptol, linalool and carvacrol found in the extracts from the present study are reported to be more toxic against termite workers [92]. For example, thymol and carvacrol from Origanum were shown to have insecticidal activity against larvae of Culex pipiens L. (Diptera: Culicidae) [93]. The extract of Lippia sidoides contained 44.55% thymol and showed high insecticidal activity against the termite Nasutitermes corniger (Blattodea: Termitidae) with 48 h exposure at a dose of 0.27 µg/mg [85].
Our present results show a higher resistance of M. eugnathus against termiticides than those of other termites. In addition, our results suggest that O. vulgare extract caused moderate activity against the termite M. eugnathus compared to commercial termiticides tested. Further research may show that O. vulgare extract or its derivatives could potentially be used to control and manage some termite infestations and lessen or limit the amount of more toxic pesticides currently in use.

Conclusions
Chemical pesticides had the greatest impact on the mortality of the termite M. eugnathus. However, due to the highly toxic nature of Dursban and the fact that it has been withdrawn for use in some countries, the search for less toxic and natural termiticides from plants is of interest. Extract from the plant Origanum vulgare showed some promise as a plant-based toxicant for this termite.