The Insecticidal and Antifeedant Activity of Calotropis procera Latex and Foliar Extracts Against the Red Palm Weevil, Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae)

The red palm weevil, Rhynchophorus ferrugineus (Oliver) (Coleopter: Curculionidae), is one of the most threatening and most important pests that infect date palms in Egypt. Many strategies have been employed to control this pest, among them, the employment of botanical extracts. The present study aimed to evaluate the insecticidal and antifeedant activity of Calotropis procera latex and foliar extracts against the 3 rd and 5 th instar larvae of R. ferrugineus . Results showed high insecticidal activity against both larval instars, however, the latex extract was more toxic than the leaves extract. Results also showed that both leaves and latex extracts have a high antifeedant index


INTRODUCTION
The red palm weevil, Rhynchophorus ferrugineus (Oliver) (Coleopter: Curculionidae), is one of the most threatening and most important pests that infect date palms (Faleiro, 2006;Rochat et al., 1991). This is due to the insect's capability to develop and feed causing damage inside the palm stem tissues (El-Juhany, 2010;Giblin-Davis et al., 1996;Gonzalez et al., 2019;Milosavljević et al., 2019). Managing this pest has always represented a challenge since the 1990s, as its control is based on insecticide spraying and injection, combined with the removal of infected palms (Gonzalez et al., 2019). Red palm weevil has been evidenced as a major and serious pest in many locations in the Mediterranean; Egypt, Saudi Arabia, and the United Arab of Emirates (Azmi et al., 2017;Ishak et al., 2020;Orfali et al., 2020). All red palm weevil' stages are found in the same host palm. The female lays about 200-300 eggs in an isolation cavity. Formed eggs crosshatch in 2-5 days, and larvae holes in the inner host palm nourishing at soft succulent tissues, abandoning total fibrous materials (Muhammad et al., 2019). Infested date palms exhibit several symptoms depending on the stage of the attack, such as producing of brown fluid with a fermented odor that is mixed with palm tissue exerted by feeding larvae, tunneling of palm tissue, presence of adults and pupae at the base of fronds, dried of infested offshoots, pupae around the palm base, dried outer leaves, and topping off the trunk in cases of severe and extensive tissue The red palm weevil, Rhynchophorus ferrugineus (Oliver) (Coleopter: Curculionidae), is one of the most threatening and most important pests that infect date palms in Egypt. Many strategies have been employed to control this pest, among them, the employment of botanical extracts. The present study aimed to evaluate the insecticidal and antifeedant activity of Calotropis procera latex and foliar extracts against the 3 rd and 5 th instar larvae of R. ferrugineus. Results showed high insecticidal activity against both larval instars, however, the latex extract was more toxic than the leaves extract. Results also showed that both leaves and latex extracts have a high antifeedant index which was confirmed by the decreased mean larval weight. Finally, we can conclude that C. procera either used as leaves extract or latex extract represents an excellent alternative for conventional management procedures. More studies are considered on the effect of this plant on the different biological and biochemical parameters.
damage (Al Dawsari, 2020). Several strategies have been employed to control and manage the red palm weevil and its damage, of these strategies is the use of insecticides. The extensive application of conventional insecticides led to the development of insect resistance to these chemical compounds besides the harm hazards of these compounds to the environment and ecosystem leading to the need to develop new control approaches safe for the environment and effective against this pest (Ferry et al., 2004). Screening of plant extracts for deleterious effects on insects is one of the approaches in the search for novel biological insecticides (Carlini & Grossi-De-Sá, 2002). Insecticidal activity of many plants against several insects has been demonstrated (Carlini & Grossi-De-Sá, 2002;Khatter & Abuldahab, 2012). Calotropis procera is a soft-wooded, perennial shrub of the family Apocynaceae and subfamily Asclepiadaceae (the milkweed family). The plant is erect, tall, large, branched and perennial with milky latex throughout. It is an evergreen xerophytic plant, generally found in arid and semi-arid habitats (Al-Rowaily et al., 2020;Kaur et al., 2021). It is known by various common names such as apple of Sodom, calotrope, giant milkweed, Indian milkweed, wild cotton, rubber tree, ushar, etc., in different parts of the world. Its subspecies, C. procera subsp. procera and C. procera subsp. hamiltonii, vary from each other in fruit morphology (Dhileepan, 2014). It is a multipurpose plant, which provides a wide range of provisioning ecosystem services. It has been widely used in traditional medicinal systems in North Africa, Middle East Asia, South Asia, and South-East Asia (Al Sulaibi et al., 2020). It has also been utilized for fiber, fuel, fodder, and timber purposes since antiquity (Batool et al., 2020). C. procera has many alkaloids, flavonoids, terpenes, terpenoids, enzymes, and other inorganic elements (Quazi et al., 2013). These compounds also exhibit insecticidal properties apart from other bioactivities. Numerous reports are claiming the insecticidal properties of plant extract and essential oils of C. procera (de Morais et al., 2007;Esmaeily et al., 2012;Nighat et al., 2013). The latex of C. procera has shown larvicidal efficacy against all three important vector species, viz. Aedes aegypti, Anopheles stephensi, and Culex quinqefasciatus, are vectors of dengue, malaria, and lymphatic filariasis, respectively (Conti et al., 2010;Shahi et al., 2010). Latex of this plant has complex chemical constituents such as N-acetyl-β-d-glucosamines (Ramos et al., 2006). Although the insecticidal activities of C. procera have been extensively studied on various pests, mosquitoes, larvae, stored grain pests, etc., several authors have studied various parts such as latex, flowers, and stem, roots, and barks for insecticidal properties (Kumar et al., 2022). Given the vast usage and importance of the plant as insecticidal activities, it is highly required to bring the majority of the available data to one platform (Kumar et al., 2022). In this context, the present study aimed to evaluate the insecticidal and antifeedant effect of Calotropis procera latex and leaves extract against the young larval instars of red palm weevil, Rhynchophorus ferrugineus, under laboratory conditions as a new strategy for managing and controlling this pest.

Tested Insects:
Adults of red palm weevil, R. ferrugineus, were collected from infected date palms in Al Qasasin Center, Sharkia Governorate. They were then transferred to the laboratory in plastic containers (10×15×30 cm) that were perforated from the top and provided with sugarcane cuttings (6 cm long) (supplied from the local market) for feeding. Insects were reared at 27 ± 1 °C and 75±5% RH, and L:D cycle of 10:14 h, at the Wood and tree scavenger research department, Plant Protection Research Institute, Agricultural Research Center, Dokki, Giza, Egypt. Adults were fed until egg oviposition. Larvae were reared until the 3 rd and 5 th instar larvae for further investigation.

Plant Source:
Plant parts of C. procera were collected from areas surrounding Cairo Airport region and from Ain Sokhna Road, Red Sea Governorate. The collected plant parts were then identified at the department of botany, Faculty of Science, Cairo University. Latex was collected freshly by cutting the tip-stem junction and allowing the latex to flow into dark glass bottles containing ethanol 95%. Leaves, branches, and flowers were also collected and transferred to the laboratory for further analysis.

Extraction of Collected Plant Parts:
The crude extract either from leaves or latex was prepared according to the procedure described by Seiber et al. (1982) with few modifications. The leaves were washed with distilled water and left in the shadows to dry separately at room temperature for one week. The dried leaves were ground finely to powder using an electrical mill. The fine leaves powder (400 gm) was extracted by the addition of 2000 ml of 50% ethanol for 48 h. at 40-50° C and then filtered using a Buchner funnel. The obtained filtrate was then treated with lead acetate ((CH3COO)2Pb) to help precipitate all undesirable materials except for the cardenolide glycosides. The resulting solution was filtered, and the filtrate was treated with hydrogen sulphide gas (H2S) to precipitate the excess of (CH3COO)2Pb as lead sulphide (PbS), then filtered and the obtained filtrate was concentrated to about one-third of its volume at 40-50° C using a rotary evaporator.
The obtained filtrate was then soaked in 1L of ethanol 95% for five days, followed by sonication for 30 min. and centrifugation for 10 min. at 5000 r.p.m. the supernatant was then collected and evaporated to dryness using a rotary evaporator with high pressure at a temperature ranging from 45-50° C. The obtained crude ethanol extract was kept in the refrigerator until use (Al-Rajhy et al., 2003;Al-Sarar et al., 2012;Seiber et al., 1982). 150 ml of collected latex was coagulated with 150 ml of 95% ethanol and then filtered on a Buchner funnel. The coagulate was then shaken with 150 ml of 50% ethanol and filtrated. The obtained mixture of the two filtrates was treated with an excess concentrated solution of (CH3COO)2Pb and filtered on a Buchner funnel. The formed filtrate was then treated with H2S gas to precipitate the excess of (CH3COO)2Pb as lead sulphide (PbS), then filtered and the obtained filtrate was concentrated to about one-third of its volume at 40-50° C using a rotary evaporator. The stock solution of either leaf extract or latex extract was prepared by dissolving 5 gm of the extract in 10 ml of distilled water and in the addition of 5 ml of Tween 80. The formed suspension was then completed to 100 ml with distilled water. The stock solution was used for further dilution.

Larvicidal Activity of Leaves and Latex Extracts:
Newly molted 3 rd and 5 th instar larvae were selected for investigating the larvicidal activity of latex and leaves extracts. Sugarcane stems were cut into cuttings of 6 cm long and 2.5 cm in diameter, then a longitudinal chamber was made (3 cm long × 1.5 cm diameter). Four suspensions were prepared from the stock solution (described in the previous section), 1, 2, 4, and 6%, in 100 ml of distilled water. A solution of tween 80 in distilled water was prepared as the control. Five replicates were set for each concentration, each replicate had five larvae. The sugarcane cuttings were then soaked in the prepared suspensions for 5 min. and then left on clean filter papers and left to air dry. The treated sugarcane cuttings were offered to larvae for 48 h. then replaced with clean cuttings. The larval mortality was recorded on daily basis until the death of all larvae. The mortality percentage was corrected according to Abbott's formula (Abbott, 1925).

The Antifeedant Activity of Leaves and Latex Extracts:
To study the antifeedant effect of latex and leaves extracts against the 3 rd and 5 th instar larvae, the sugarcane cuttings were weighed before being offered to larvae and after larvae been fed on them both in treated and untreated. Sugarcane cuttings have emerged for 5 min. in 6% suspension prepared from the stock solution. The larvae were fed on the treated cuttings for 48h. then the cuttings were replaced by fresh clean ones. In addition, the mean larval weight before treatment and after treatment was also recorded daily. The percent of the antifeedant index was calculated using the following formula (Ben Jannet et al., 2000): (1) where, C and T represent the amount of food eaten by the larvae on control and treated cuttings, respectively. Statistical Analysis: The LC50 values for latex and leaves extracts were determined according to Finney (1971) using "LdPLine ® " software. [http://embakr.tripod.com/ldpline/ldpline.htm]. The obtained data were presented as mean ± S.E. One-way analysis of variance (ANOVA) and significant differences between treatments were determined by Tukey's multiple range tests (Snedecor & Cochran, 1980) (P ≤ 0.05) using SPSS statistics 23.0 release 23 software.

Larvicidal Activity of Leaves and Latex Extracts of C. Procera Against Red Palm Weevil Larvae:
The toxicity of leaves and latex extracts of C. procera were represented in the LC50 values and shown in the Figures (1-4). Results showed that the LC50 values for leaves extract against 3 rd and 5 th instar larvae were 1.406, and 3.696 gm/ml, respectively. Furthermore, the 2.132, and LC50 values for latex extract against 3 rd instar larvae were 2.132 gm/ml and for 5 th instar larvae were 2.648 gm/ml. The obtained results confirmed the toxic effect of C. procera against the larval stage of red palm weevil either treated with leaves or latex extract. Results also revealed that the larval mortality increased as the concentrations increased. C. procera is known to contain some chemical constituents which proved to exhibit insecticidal activity such as Calotropin and Calotoxin (Dubey et al., 2007;Kumar et al., 2022;Shahi et al., 2010) and they are the main cause of larval mortality. The larvicidal activity of both latex and leaves extracts was previously recorded against mosquito larvae (Nighat et al., 2013;Ramos et al., 2006;Shahi et al., 2010;Singh et al., 2005) and red palm weevil (Orfali et al., 2020). Results in Table (1) showed the antifeedant index of leaves and latex extracts of C. procera against the 3 rd and 5 th instar larvae of R. ferrugineus after feeding larvae on treated sugarcane cuttings for 48h. Results showed both leaves and latex extracts had a high antifeedant index for the 3 rd instar larvae; 80.3 and 98.3 %, respectively. This may be due to the high toxicity of both extracts against the 3 rd instar larvae. Furthermore, the antifeedant index for leaves and latex extracts for the 5 th instar larvae was less than of the 3 rd instar but still high (78.3 and 80.6 %, respectively). This could be related to the that the 3 rd instar larvae are more susceptible than the 5 th ones. Both the extracts showed antifeedant activity leading to weight loss and reduced size of the treated larvae as compared to control ones ( Table 2). The antifeedant property of C. procera may be due to the different compounds present in the extract possessing different bioactivities (Nighat et al., 2013). The deleterious effect of plant extracts or pure natural/ synthetic compounds on insects can be manifested in several manners including toxicity, mortality, antifeedant, growth inhibitor, suppression of reproductive behavior, and reduction of fecundity and fertility. The antifeedant effect of a plant is related to the phytochemicals produced by this plant. These phytochemicals help in the protection of plants from insect-pests attacks (Ahmad, 2007;Prabhu et al., 2018). The production of phytochemicals is stimulated as a response to insect attacks (Ahmad, 2007;Prabhu et al., 2018) leading to disturbed feeding habits and fertility issues (Ahmad, 2007).  Table 2: Mean larval weight of the 3 rd and 5 th instar larvae of Rhynchophorus ferrugineus during the experiment period after treatment with leaves and latex extracts at 6% concertation (Mean ± S. E.). Treatment 3 rd instar larvae (gm) 5 th instar larvae (gm) Leaves extract 0.75±0.11 a 1.60±0.01 a Latex extract 0.66±0.10 a 1.45±0.02 a Control 2.23±0.03 b 5.48±0.01 b Means followed by the same letters are insignificantly different at P < 0.05

Conclusion:
In the present study, we evaluated the insecticidal activity of Calotropis procera leaves and latex extracts against the young larval instars of Rhynchophorus ferrugineus under laboratory conditions. The present study proved the high larvicidal property of the extracts. In the same context, results also confirmed the antifeedant characteristic of C. procera against larvae and this led to weight loss for treated larvae. C. procera either used as leaves extract or latex extract represents an excellent alternative for conventional management procedures. More studies are considered on the effect of this plant on the different biological and biochemical parameters.