The Efficacy of Egyptian Isolate of Beauveria bassiana Against Red Palm Weevil (Rhynchophorus ferrugineus) Olivier (Coleoptera: Curculionidae): Mortality Rates, Biochemical Responses, and Enzymatic Activity

doi:10.21203/rs.3.rs-5255595/v1

Red palm weevil (RPW) larvae, Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae), were evaluated for susceptibility to an Egyptian isolate of the entomopathogenic fungus Beauveria bassiana. The results showed that R. ferrugineus larvae were more susceptible to B. bassiana at the 5^th and 10^th instars, respectively. A concentration of 10⁸ spores/ml of B. bassiana led to a higher mortality rate, with LT₅₀ and LT₉₀ values of 5.947 and 14.59 days, respectively. However, resistance ratios for concentrations of 10⁷ and 10⁸ spores/ ml were 1.80 and 1.32 times higher in the 10^th instar compared to the 5^th instar larvae, indicating lower susceptibility. Biochemically, after treatment with B. bassiana, significant changes in total protein and carbohydrate activity were observed in both larval stages. After 96 hours, GST activity increased significantly compared to the control. The effects of B. bassiana on alpha esterase and Glutathione-S-transferase (GST) enzymes varied by concentration and duration. Alpha esterase activity slightly decreased at 10⁸ spores/ ml after 48 hours but was not statistically significant after 96 hours. Conversely, at 10⁷ spores/ ml, alpha esterase decreased after 48 hours but increased after 96 hours. GST activity increased at 10⁸ spores/ ml after 48 and 96 hours and rose significantly at 10⁷ spores/ ml after 48 hours, followed by a decline after 96 hours. These findings suggest that B. bassiana could be used as an effective control agent against R. ferrugineus.

Red palm weevil

Rhynchophorus ferrugineus

Beauvaria Bassiana

biochemical studies

enzyme activity.

Rhynchophorus ferrugineus (Red Palm Weevil, RPW) (Olivier, 1790) is a significant pest of palm trees. It inflicts on palm trees, leading to substantial financial losses for farmers, agricultural sectors, and national economies dependent on date palm cultivation. This pest is native to South and Southeast Asia, including countries e.g., India, Indonesia, and Pakistan, RPW was also reported in China in 1990 (Abraham et al., 1998; Burkill & Bulletin, 1917; El-Mergawy & Ajlan, 2011). It has since been observed in 28 countries across Asia, 6 countries in Africa, 1 in North America, 2 in Central America and the Caribbean, 14 in Europe, and 5 in Oceania (Rugman-Jones et al., 2013; Aziz et al., 2022). RPW poses a significant invasive threat due to its cryptic behavior (Giblin-Davis et al., 2013; Mahmoud et al., 2015). Date palm (Phoenix dactylifera) is a critical crop in Egypt, both culturally and economically. Egypt is one of the largest producers of dates globally. However, the infestation of Rhynchophorus ferrugineus poses a severe threat to this production. Even if a tree survives the initial infestation, its ability to bear fruit and produce high-quality dates is significantly reduced. This directly impacts the income of farmers.

The larval stage of the RPW is the most critical to control, as the larvae often cause severe damage to palm trees. In severe infestations, RPW larvae hollow out tree trunks, causing the canopy to collapse (Faleiro, 2006; Abdullah, 2009). These larvae feed on palm trunks, which threaten both wild and cultivated palm trees across various regions. The symptoms of RPW infection in date palm trees vary depending on the stage of infection. These symptoms include the discharge of a brown liquid with a fermented odor, caused by a mixture of palm tissue and larval secretions. Damaged trees release volatile compounds, known as kairomones, which attract male weevils (Dalbon, 2021). The larval feeding destroys internal tissues, ultimately leading to the death of the date palm (Abraham et al., 1998).

RPW has become a highly invasive pest, posing a major threat to palm trees globally, and is difficult to control using conventional methods. Entomopathogenic fungi (EPF) are among the most widely used microbial control agents, and their effectiveness in controlling RPW has been demonstrated in various studies (Devi et al., 2008; Dembilio et al., 2010; Abdel-Samad et al., 2011; Sabbour & Abdel-Rahim, 2014; Sutanto et al., 2023). The fungus Beauveria bassiana, a commonly used microbial pathogen, has shown effectiveness against RPW larvae. Previous studies confirmed its virulence, indicating its potential as a biocontrol agent. B. bassiana conidia can penetrate the insect cuticle and spread through contact with infected hosts (Lacey et al., 1999; Dembilio et al., 2010; Lo Verde et al., 2015). Consequently, microbial control is a viable alternative (Strategy 2022; Yang et al., 2023; Sutanto, 2023). Abdel-Baky (2021) reported a mortality rate of up to 93.3% in RPW larvae treated with B. bassiana after 21 days.

Understanding the immune systems of insects could further enhance the effectiveness of biopesticides and expand knowledge about host-pathogen relationships (Hernandez et al., 1999; De Silva et al., 2000). For example, adults of the green bug Nezara viridula treated with B. bassiana and Metarhizium anisopliae showed a significant reduction in total carbohydrate levels (Nada, 2015). Similar findings were observed in the arm worm Spodoptera litura, which exhibited altered protein levels after treatment with B. bassiana (Vidhya et al., 2016).

Koodalingam et al. (2011) and Polson et al. (2011) also recorded changes in the activity of protective enzymes such as glutathione S-transferase (GST), α-/β-esterases (ESTs), acetylcholinesterase (AChE), acid and alkaline phosphatases (ACP and ALP), and mixed-function oxidases (MFOs), which help organisms metabolize or eliminate endogenous and exogenous compounds.

The losses from R. ferrugineus are not just from the destruction of individual trees but also from the costs associated with pest management, loss of agricultural land productivity, and market value reduction of affected dates. In Egypt, these losses are estimated to be in millions of dollars annually (El-Sabea et al., 2009; FAO, 2017). Given that date palms have a long lifespan, the replacement of infested trees is a slow and expensive process, making the economic impact more severe. Accordingly, in this study, we aim to isolate B. bassiana fungi and evaluate their virulence against the 5^th and 10^th instar larvae of R. ferrugineus. Additionally, we studied physiological modifications in enzymes, including total protein, carbohydrates, nonspecific esterase, phenoloxidase, peroxidase, and GST activity.

1. Red Palm Weevil Source:

Larvae of the red palm weevil (Rhynchophorus ferrugineus) were collected from date palm trees in a cultivated field located in the Quasasine and Salhia district, Ismailia and Sharkia Governorate, Egypt.

2. Insect Source:

Infested palm trees (Phoenix canariensis, 3–5 m in height) were found on private farms in the Ismailia and Sharkia Governorates, Egypt, in October 2023. Heavily infested trees were transported from the field to the laboratory for dissection. Various larval stages, pupae, and adult red palm weevils were carefully extracted from the tree parts for comparative susceptibility studies with selected entomopathogenic fungi (EPFs). The palm trees were removed at their bases, and the trunks were wrapped in heavy plastic sheets sealed with tape to prevent accidental escape of weevil adults during transport. Trunk slices were sequentially cut from the top toward the base. Using head capsule width measurements (Jacas et al., 2011), live larvae were classified as young (second–third instar), medium (fifth–sixth instar), and full-grown larvae. All samples were washed three times with distilled water to remove plant fibers before being tested with EPFs in plastic boxes.

3. Entomopathogenic Fungi Cultures:

Following the method described by Nada (2006), B. bassiana was isolated from soil in Giza Governorate, Egypt. The fungus was cultured on autoclaved Sabouraud dextrose yeast agar (SDAY) containing 1% peptone, 4% dextrose, 1.5% agar, and 0.2% yeast extract dissolved in 1L distilled water at 25±1ºC. After 14 days, spores were harvested by rinsing with an aqueous solution of 0.01% Tween 80 and filtered through cheesecloth to reduce mycelium clumping. Spore concentrations were prepared as 10⁷ and 10⁸ spores/ml using a Haemocytometer (Neubauer improved HBG, Germany 0.10 mm X 0.0025mm²).

4. Effect of Two B. bassiana Concentrations on 5^th and 10^th Instar Larvae of R. ferrugineus:

Newly molted 5^th and 10^th instar larvae were immersed in each conidial suspension (10⁷ and 10⁸ spores/ml) for 10 seconds, after which excess suspension was removed with filter paper. An aqueous solution of 0.01% Tween 80 was used as a control. Treated larvae were placed individually in transparent plastic boxes with ventilation and provided with a saturated cotton wool piece by distilled water and pieces of sugarcane for food. Six replicates, each containing five larvae, were prepared for each concentration and the control. The larvae were incubated at 25±1ºC, and mortality was assessed at 5, 10, 15, and 20 days post-treatment. Dead larvae were transferred to plastic petri dishes with saturated filter papers to allow fungal sporulation. The LT₅₀ and LT₉₀ values were determined.

5. Biochemical Response Comparison of 5^th and 10^th Instar Larvae to B. bassiana:

Surviving larvae from the treatments were used for biochemical analysis. The 5^th and 10^th larval instars were treated with the same two B. bassiana concentrations (10⁷ and 10⁸ spores/ml). Surviving larvae were used for biochemical analysis within 48 and 96 hours of treatment. Larvae were homogenized for analysis in a chilled Teflon tissue homogenizer (ST–2 Mechanic, Preczyina, Poland). Supernatants were stored at -20ºC until used for biochemical assays. Absorbance of colored substances or metabolic compounds was measured using a double-beam ultraviolet/visible spectrophotometer (Spectronic 1201, Milton Roy Co., USA) as described by Amin (1998).

6. Preparation of Insects for Analysis:

Insects were homogenized in distilled water (50 mg/ml). Homogenates were centrifuged at 8,000 R.P.M. for 15 minutes at 2ºC in a refrigerated centrifuge. The supernatants, referred to as enzyme extracts, were stored at 5ºC for up to one week without significant loss of enzyme activity.

6.1. Determination of Total Proteins:

The protein reagent was prepared by dissolving 100 mg of Coomassie Brilliant Blue G-250 in 50 ml of 95% ethanol. Then, 100 ml of 85% phosphoric acid was added, and the solution was diluted to 1 liter. A sample solution (50µl) was used to prepare a standard curve with bovine serum albumin concentrations from 10 to 100µg. The protein reagent (5 ml) was added to each test tube, and absorbance was measured at 595 nm after 2 minutes (Bradford, 1976).

6.2. Determination of Total Carbohydrates:

Total carbohydrates were estimated using the phenol-sulfuric acid reaction (Dubois et al., 1956). Samples were homogenized in 0.3N HClO4, centrifuged, and combined into an acid extract. Absorbance of the characteristic yellow-orange color was measured at 490 nm.

6.3. Determination of Nonspecific Esterase:

Alpha (α) and beta (β) esterase were measured according to Van Asperen (1962) using α- or β-naphthyl acetate as substrates.

6.4. Determination of Phenoloxidase Activity:

Phenoloxidase activity was determined using a reaction mixture containing phosphate buffer, enzyme solution, and catechol. Optical density was measured at 405 nm (Shaaya, 1971).

6.5. Determination of Peroxidase Activity:

Peroxidase activity was measured by adding hydrogen peroxide to a sample cuvette containing pyrogallol and enzyme extract (Hammer Schmidt et al., 1982).

6.6. Determination of Glutathione S-transferase (GST):

Glutathione S-transferase activity was measured using the method of Habig et al. (1974) by detecting the conjugation of reduced glutathione with 1-chloro-2,4-dinitrobenzene.

7. Statistical Analysis:

Mortality percentages were computed according to Finney (1971) to determine LT₅₀and LT₉₀values using "Ldp Line" software (Bakr, 2000). Biochemical analysis data were expressed as means ± standard error and analyzed via one-way ANOVA using SAS software (SAS Institute 1988), with Tukey's HSD test applied at P = 0.05.

1. Comparison between B. bassiana LT Values against Both 5th and 10th Instar Larvae of R. ferrugineus:

Data in Table 1 and Fig. 1 demonstrate that the 5th instar larvae of R. ferrugineus were more susceptible to B. bassiana at a concentration of 10⁸ spores /ml. Their mortality rate was faster, with LT₅₀ and LT₉₀ values of 5.947 and 14.59 days, respectively. At a lower concentration (10⁷ spores/ml), the treatment was 80.86% slower for the 5th instar larvae compared to the 10⁸ spores/ml concentration. Meanwhile, the 10th instar larvae were less susceptible. The resistance ratio (RR) indicated that at 10⁷ spores/ ml, the 5th instar larvae had a 1.80 times higher resistance than those treated with 10⁸ spores/ ml.

Table 1

LT values (days) of entomopathogenic fungi *B. bassiana* against 5th and 10th instar larvae of *R. ferrugineus* at two concentrations (10⁷ and 10⁸ spores/ml).
Age of larval instar	Concentration (spores/ml)	LT values (days)
Age of larval instar	Concentration (spores/ml)	LT₅₀	Lower limit	Upper limit	index	RR	slope	LT90
5th	10⁸	5.95	4.17	7.34	100	1	3.29	14.59
5th	10⁷	7.36	4.67	9.44	80.86	1.24	2.28	26.91
10th	10⁸	7.84	6.08	9.39	75.86	1.32	3.34	18.98
10th	10⁷	10.75	8.19	13.65	55.50	1.80	2.41	36.43

Index compared with 5th instar larvae treated with 10⁸
Resistance Ratio (RR) compared with 5th instar larvae treated with 10⁸

2. Comparison of Biochemical Response of 5th and 10th Instar Larvae to Two B. bassiana Concentrations (10⁷ and 10⁸ Spores/ml):

Results in Table 2 show that total protein levels were significantly decreased in the 10th instar larvae treated with B. bassiana at 10⁷ spores/ml (40.6 and 43.7 mg/g.b.wt, respectively) compared to the control treatment after 96 hours. No significant effect was observed at 10⁸ spores/ml. Table 2 also shows a significant reduction in total carbohydrate activity after 48 hours at 10⁸ spores/ ml compared to the control (45.47 and 68.27 mg/g.b.wt, respectively). After 96 hours, carbohydrate activity dropped sharply (61.8 and 79.23 mg/g.b.wt, respectively). Similar trends were observed for the 10⁷ spores/ml concentration, with reduced carbohydrate activity after 48 and 96 hours compared to the control.

Data in Table 3 showed that after 48 hours, total protein activity in the 5th instar larvae significantly decreased compared to the control (41.97 and 45.47 mg/g.b.wt, respectively). However, in the 10th instar larvae, protein activity did not significantly decrease. After 96 hours, there was a significant reduction in total protein in the 5th instar larvae compared to the control (41.1 and 46.33 mg/g.b.wt, respectively), but no significant change in the 10th instar larvae. Regarding carbohydrate activity, there was a significant increase in the 5th instar larvae after 48 hours compared to the control (74.67 and 70 mg/g.b.wt, respectively), followed by a sharp decrease after 96 hours (38.23 and 97 mg/g.b.wt, respectively). In contrast, total carbohydrate activity significantly decreased in the 10th instar larvae at both 48 and 96 hours.

Table 2

Effect of two concentrations of *B. bassiana* (10⁷ and 10⁸ spores/ ml) on total protein and carbohydrates in the 10th larval instar of *R. ferrugineus*.
Total protein (mg/g.b.wt)
Hours after treatment	10⁸		10⁷		F	P	H.S.D
Hours after treatment	Control ± SE	B. bassiana ± SE	B. bassiana ± SE	Control ± SE	F	P	H.S.D
48	37.07^b ± 0.64	35.83^b ± 0.48	41^a ± 0.77	42.13^a ± 0.52	24.68	0.0002	2.7641
96	39.33^b ± 0.37	40 .17^b ± 0.44	43.7^a ± 0.70	40.6^b ± 0.85	9.39	0.0053	2.8181
Total carbohydrates (mg/g.b.wt)
Hours after treatment	10⁸		10⁷		F	P	H.S.D
Hours after treatment	Control ± SE	B. bassiana ± SE	Control ± SE	B. bassiana ± SE	F	P	H.S.D
48	68.27^a ± 1.36	45.47^c ± 0.82	63.37^a ± 1.48	56.17^b ± 1.44	57.89	˂.0001	5.8952
96	79.23^a ± 1.39	61.8^c ± 1.06	72.33^b ± 1.64	54.43^d ± 1.85	52.88	˂.0001	6.8508

^a Mean, within a row, bearing different subscripts are significantly different.

Table 3

The effect of *B. bassiana* 10⁸ spores/ml treatment on *R. ferrugineus* 5th and 10th larvae instars on Total protein and carbohydrates
Total protein (mg/g.b.wt)
Hours after treatment	10th		5th		F	P	H.S.D
Hours after treatment	Control ± SE	B. bassiana ± SE	Control ± SE	B. bassiana ± SE	F	P	H.S.D
48	37.07^c ± 0.64	35.83^c ± 0.48	45.47^a ± 0.27	41.97 ^b ± 0.47	85.78	˂0.0001	2.1811
96	39.33^b ± 0.37	40.17^b ± 0.44	46.33^a ± 1.01	41.1^b ± 0.50	24.61	0.0002	2.8762
Total carbohydrates (mg/g.b.wt)
Hours after treatment	10th		5th		F	P	H.S.D
Hours after treatment	Control ± SE	B. bassiana ± SE	Control ± SE	B. bassiana ± SE	F	P	H.S.D
48	68.27^b ± 1.35	45.47^c ± 0.82	70^b ± 0.58	74.67 ^a ± 1.53	188.2	˂0.0001	4.3043
96	79.23^b ± 1.39	61.8 ^c ± 1.06	97^a ± 2.52	38.23^d ± 0.72	254.19	˂0.0001	7.1244

^a Mean, within a row, bearing different subscripts are significantly different.

3. Enzymatic Activity Response:

In Table 4, treatment with B. bassiana (10⁸ spores/ml) resulted in a slight but significant decrease in alpha-esterase activity after 48 hours compared to the control (773.67 and 852.67 Ug α-naphthol/min/g.b.wt, respectively), with no significant effect after 96 hours. However, at 10^7 spores/ml, alpha-esterase activity decreased significantly after 48 hours (902.33 and 1342.67 Ug α-naphthol/min/g.b.wt, respectively) and increased significantly after 96 hours compared to the control.

Beta-esterase activity (Table 4) slightly increased when R. ferrugineus was treated with B. bassiana (10⁸ spores/ml) after 48 hours compared to the control (3332.33 and 3006.67 O.D./min/g.b.wt, respectively), but decreased after 96 hours. At 10⁷ spores/ml, beta-esterase activity significantly decreased after 48 hours (2681.7 and 3553.7 O.D./min/g.b.wt, respectively) and increased after 96 hours compared to the control.

For phenoloxidase activity (Table 4), a significant increase was observed after 48 hours at 10⁸ spores/ml compared to the control (10.23 and 9.07 O.D./min/g.b.wt, respectively), but no significant effect was noted after 96 hours. At 10⁷ spores/ml, phenoloxidase activity slightly increased after 48 hours but decreased after 96 hours.

Peroxidase activity significantly decreased at 10⁸ spores/ ml after 48 hours (5.43 and 9.77 O.D./min/g.b.wt, respectively) and then increased after 96 hours. At 10⁷ spores/ ml, there was a slight increase after 48 hours, followed by a significant increase after 96 hours.

Finally, Glutathione S-transferase (GST) activity (Table 4) increased slightly after 48 hours at 10⁸ spores/ ml, but increased significantly after 96 hours. At 10⁷ spores/ ml, GST activity significantly increased after 48 hours, followed by a slight decrease after 96 hours.

The activity of alpha esterase significantly decreased in the 5th larval instar after both 48 and 96 hours compared to the control (1213.33, 1606.67, 1016.67, and 1574.33 Ug α-naphthol/min/g.b.wt, respectively). In contrast, the 10th larval instar showed a non-significant decrease in alpha esterase activity after 48 hours, followed by a non-significant increase after 96 hours compared to the control (Table 5).

Data in Table 5 also show a significant reduction in beta esterase activity in the 10th larval instar after 48 hours compared to the control. Meanwhile, in the 5th larval instar, beta esterase activity showed a non-significant decrease compared to the control (3635.33 and 3763.67 Ug β-naphthol/min/g.b.wt, respectively). After 96 hours, beta esterase activity decreased non-significantly in the 10th instar, while there was a significant decrease in the 5th instar compared to the control (2710 and 3789 Ug β-naphthol/min/g.b.wt, respectively).

Table 5 further reveals a significant decrease in phenoloxidase activity in the 5th larval instar after 48 hours compared to the control (14.8 and 21.33 O.D./min/g.b.wt, respectively), with this significant decrease continuing after 96 hours (15.733 and 21.33 O.D./min/g.b.wt, respectively). In the 10th instar, phenoloxidase activity showed a non-significant increase.

The data in Table 5 also indicate a sharp, significant decrease in peroxidase activity after 48 hours in the 10th larval instar compared to the control (5.43 and 9.77 O.D./min/g.b.wt., respectively), as well as in the 5th larval instar (7.6 and 22.667 O.D./min/g.b.wt., respectively). After 96 hours, peroxidase activity continued to show a significant decrease in the 5th larval instar compared to the control (9.9667 and 22.033 O.D./min/g.b.wt, respectively), while a slight but significant increase was observed in the 10th instar compared to the control.

Lastly, Table 5 shows that Glutathione-S-Transferase (GST) activity significantly increased in the 5th larval instar compared to the control after 48 hours (75.667 and 52 mmol sub. conjugated/min/g.b.wt, respectively), and exhibited a sharp, significant increase after 96 hours (104.667 and 44 mmol sub. conjugated/min/g.b.wt, respectively). In the 10th larval instar, GST activity also showed a significant increase after 96 hours compared to the control.

Several previous studies have shown that R. ferrugineus is highly susceptible to the entomopathogenic fungus B. bassiana, with results indicating a relationship between different larval development stages and susceptibility to B. bassiana.

Table 4

Effect of two concentrations of *B. bassiana* (10⁷ and 10⁸ spores/ml) on enzyme activity in 10th larval instar of *R. ferrugineus*.
Alpha Esterase (Ug α - naphthol/min/g.b.wt)
Hours after treatment	10th		5th		F	P	H.S.D
Hours after treatment	Control ± SE	B. bassiana ± SE	Control ± SE	B. bassiana ± SE	F	P	H.S.D
48	852.67^cb ± 9.33	773.67^c ± 5.37	1342.67^a ± 33.17	902.33 ^b± 30.33	122.17	˂0.0001	104.66
96	1214^b ± 15.88	1254.67^b ± 24.94	1354.3^b ± 32.27	1813.3^a ± 85.11	33.24	˂0.0001	216.72
Beta Esterase (Ug β - naphthol/min/g.b.wt)
Hours after treatment	10th		5th		F	P	H.S.D
Hours after treatment	Control ± SE	B. bassiana ± SE	Control ± SE	B. bassiana ± SE	F	P	H.S.D
48	3006.67bc ± 63.59	3332.33ab ± 74.45	3553.7a± 106.49	2681.7c ± 61.00	23.58	0.0003	355.51
96	3806.33b ± 33.28	3800b ± 57.74	3688.3b± 45.12	4196a ± 113.16	10.25	0.0041	314.44
phenoloxidaes (O.D./min/g.b.wt)
Hours after treatment	10th		5th		F	P	H.S.D
Hours after treatment	Control ± SE	B. bassiana ± SE	Control ± SE	B. bassiana ± SE	F	P	H.S.D
48	9.07b ± 0.15	10.23^a ± 0.20	9.93^ab ± 0.23	9.27^b ± 0.18	8.17	0.0081	0.8705
96	14.47^a ± 0.09	15.67^a ± 0.29	12.70^b ± 0.38	12.43^b ± 0.29	28.89	0.0001	1.2876
peroxidases (O.D./min/g.b.wt)
Hours after treatment	10th		5th		F	P	H.S.D
Hours after treatment	Control ± SE	B. bassiana ± SE	Control ± SE	B. bassiana ± SE	F	P	H.S.D
48	9.77a ± 0.39	5.43b ± 0.29	4.1667c ± 0.09	4.33bc ± 0.10	108.44	˂0.0001	1.1403
96	6.27b ± 0.12	8.27a ± 0.19	5.013c ± 0.11	7.503a ± 0.26	62.91	˂0.0001	0.8153
GST (mmol sub. conjugated/min/g.b.wt)
Hours after treatment	10th		5th		F	P	H.S.D
Hours after treatment	Control ± SE	B. bassiana ± SE	Control ± SE	B. bassiana ± SE	F	P	H.S.D
48	111.33b ± 4.37	117.33b ± 1.86	86c ± 3.21	142a ± 2.08	56.82	˂0.0001	13.815
96	86.33b ± 2.33	105.67a ± 4.70	70.67c ± 2.33	65c ± 1.73	36.90	˂0.0001	13.586

^a Mean, within a row, bearing different subscripts are significantly different.

Table 5

The effect of *B. bassiana* 10⁸ spores/ml treatment on *R. ferrugineus* 5th and 10th larvae instars on different enzymes
Alpha esterase (Ug α- naphthol/min/g.b.wt)
Hours after treatment	10th		5th		F	P	H.S.D
Hours after treatment	Control ± SE	B. bassiana ± SE	Control ± SE	B. bassiana ± SE	F	P	H.S.D
48	825.67^c ± 9.33	773.67^c ± 5.36	1606.67^a ± 17.64	1213.33^b ± 49.10	205.15	˂0.0001	120.63
96	1214^b ± 15.88	1254.67^b ± 24.94	1574.33^a ± 43.15	1016.67^c ± 27.29	61.32	˂0.0001	133.59
Beta esterase (Ug β- naphthol/min/g.b.wt)
Hours after treatment	10th		5th		F	P	H.S.D
Hours after treatment	Control ± SE	B. bassiana ± SE	Control ± SE	B. bassiana ± SE	F	P	H.S.D
48	3006.67c ± 63.59	3332.33b ± 74.45	3763.67a ± 18.35	3635.33a ± 59.27	33.97	˂0.0001	262.42
96	3806.33a ± 33.28	3800a ± 57.74	3789a ± 84.29	2710b ± 47.26	86	˂0.0001	265.8
phenoloxidases (O.D./min/g.b.wt)
Hours after treatment	10th		5th		F	P	H.S.D
Hours after treatment	Control ± SE	B. bassiana ± SE	Control ± SE	B. bassiana ± SE	F	P	H.S.D
48	9.07c ± 0.15	10. 23c ± 0.20	21.33a ± 0.57	14.8b ± 0.25	275.15	˂0.0001	1.519
96	14.47c ± 0.09	15.67c ± 0.29	21.33b ± 0.18	15.733a ± 0.39	134.79	˂0.0001	1.2006
peroxidases (O. D. /min/g.b.wt)
Hours after treatment	10th		5th		F	P	H.S.D
Hours after treatment	Control ± SE	B. bassiana ± SE	Control ± SE	B. bassiana ± SE	F	P	H.S.D
48	9.77b ± 0.39	5.43c ± 0.29	22.667a ± 1.22	7.6 cb ± 0.27	134.06	˂0.0001	3.0267
96	6.27c ± 0.12	8.27bc ± 0.19	22.033a ± 0.69	9.9667b ± 0.58	232.12	˂0.0001	2.1094
GST (mmol sub. conjugated/min/g.b.wt)
Hours after treatment	10th		5th		F	P	H.S.D
Hours after treatment	Control ± SE	B. bassiana ± SE	Control ± SE	B. bassiana ± SE	F	P	H.S.D
48	111.33a ± 4.37	117.33a ± 1.86	52c ± 1.53	75.667b ± 3.38	104.52	˂0.0001	13.649
96	86.33b ± 2.33	105.67a ± 4.70	44c ± 2.08	104.667a ± 3.18	79.26	˂0.0001	14.675

^a Mean, within a row, bearing different subscripts are significantly different.

Several studies have examined the impact of different fungal isolates on the virulence against the destructive red palm weevil (RPW), Rhynchophorus ferrugineus. In this study, we found that the virulence of Beauveria bassiana was concentration-dependent, with the highest concentration (10⁸ spores/ml) showing the greatest pathogenic effect on younger larvae of RPW. This aligns with findings from Tahir et al. (2018), who reported that B. bassiana isolate W6-18 had a higher virulence against the 2nd instar larvae of Helicoverpa armigera compared to 4th instar larvae, likely due to melanisation in the cuticles of older larvae. Melanisation, as noted by Inglis et al. (2001), is a critical defense mechanism that prevents pathogen penetration and produces melanin, which is toxic to microorganisms.

Moreover, Lee and Wilson (2006) demonstrated that higher melanin levels in older larvae of Spodoptera littoralis contributed to their resistance to fungal infection, similar to our observations where younger larvae of R. ferrugineus were more susceptible to fungal treatments. Consistent with Boaventura et al. (2021), who found that the younger instar nymphs of Bemisia tabaci were more vulnerable to fungal infections compared to older nymphs, our findings reinforce the idea that susceptibility decreases with larval age.

Our study found that a higher concentration of spores (10⁸ spores/ ml) was more pathogenic than lower concentrations (10⁷ spores/ml), confirming the results of Torta et al. (2009) and El-Sufty et al. (2009). These researchers similarly reported that indigenous strains of B. bassiana caused significantly higher mortality in RPW younger larvae and adults, with younger larvae being more vulnerable due to the lack of antimicrobial cuticular components (Mazza et al. 2011). The higher pathogenicity at greater spore concentrations is consistent with findings from Hamzah et al. (2021), who showed that the efficacy of B. bassiana and Metarhizium anisopliae against Bacterocera cucurbitae increased with higher spore concentrations and extended time intervals.

Trinh et al. (2020) also supported this trend, showing that increasing concentrations of entomopathogenic fungi (B. bassiana and Verticillium lecanii) enhanced their efficacy against the bean aphid Megoura japonica. Mohamed et al. (2022) further found that younger larvae (L3 stage) of RPW were most susceptible to B. bassiana isolates, with 100% mortality at the highest concentration, corroborating our results, which demonstrated greater vulnerability in younger larvae.

The reduction in total protein activity observed in this study is consistent with Ragheb et al. (2018), who reported a similar decrease in total protein in 4th instar larvae of RPW treated with B. bassiana. This reduction in protein activity could impair several physiological functions, as proteins are vital for enzymatic, hormonal, and immune responses in insects (Fagan et al., 2002). Comparable reductions in total protein activity have been reported in adult Nezara viridula treated with M. anisopliae and B. bassiana (Nada, 2015) and in Spodoptera littura larvae treated with entomopathogenic fungi (Vidhya et al., 2016).

Additionally, our findings related to carbohydrate activity showed a decrease after fungal infection, in agreement with Elbanna et al. (2012), who reported a decrease in carbohydrate levels in Schistocerca gregaria following treatment with M. anisopliae. The role of carbohydrates in insect metabolism and the impact of their reduction on insect vitality is well documented (Wigglesworth, 1972). The reduction in carbohydrate metabolism, due to a decrease in carbohydrate-hydrolyzing enzymes such as amylase, trehalase, and invertase (Khaleil et al., 2016), could significantly affect the energy supply needed for insect survival. Our findings are consistent with those of Nada (2021), who observed a reduction in carbohydrate activity in Graptostethus servus after B. bassiana infection.

In terms of fat storage and its role in fungal resistance, Aldossary (1999) noted that older larvae of RPW were less active and had greater fat accumulation, which may have shielded them from fungal penetration. James et al. (2003) supported this hypothesis, indicating that cuticular lipids in older larvae provide a toxic or inhibitory barrier to fungal conidia, thus increasing their resistance. This concept is reinforced by Butt and Goettel (2000), who noted a positive correlation between higher spore concentrations and rapid mortality, confirming our observation of greater mortality at higher concentrations.

Overall, our results highlight the importance of fungal concentration and larval stage in determining the efficacy of B. bassiana against RPW. The susceptibility of younger larvae, particularly in the early instars, and the rapid mortality seen at high fungal concentrations suggest that targeting these life stages may be an effective strategy for controlling RPW populations. These findings align with El-Husseini (2019), who demonstrated that higher concentrations of B. bassiana led to faster mortality in younger larvae of R. ferrugineus. These findings contribute valuable insights for biological control strategies against the red palm weevil.

RPW Red palm weevil

LT50 Lethal time to kill 50% of population

LT90 Lethal time to kill 90% of population

GST Glutathione-S-transferase

EPF Entomopathogenic fungi

GST Glutathione S-transferase

AChE Acetylcholinesterase

ACP Acid phosphatases

ALP And alkaline phosphatases

MFOs Mixed-function oxidases

SDAY Sabouraud dextrose yeast agar

RR Resistance ratio

Funding

This study was partially funded by the project; Smart Agriculture Clinic-Egyptian Platform for the Farmer.

Availability of data and materials

The data sets used and/or analyzed during the current study are available from the corresponding authors upon reasonable request.

Ethics approval and consent to participate

Not applicable.

Consent for publication

All the authors give their consent to publish the submitted manuscript as “Original paper” in EJBPC.

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No competing interests reported.

The Efficacy of Egyptian Isolate of Beauveria bassiana Against Red Palm Weevil (Rhynchophorus ferrugineus) Olivier (Coleoptera: Curculionidae): Mortality Rates, Biochemical Responses, and Enzymatic Activity

Status:

Version 1

Abstract

Figures

Background

Methods

1. Red Palm Weevil Source:

2. Insect Source:

3. Entomopathogenic Fungi Cultures:

4. Effect of Two B. bassiana Concentrations on 5^th and 10^th Instar Larvae of R. ferrugineus:

5. Biochemical Response Comparison of 5^th and 10^th Instar Larvae to B. bassiana:

6. Preparation of Insects for Analysis:

6.1. Determination of Total Proteins:

6.2. Determination of Total Carbohydrates:

6.3. Determination of Nonspecific Esterase:

6.4. Determination of Phenoloxidase Activity:

6.5. Determination of Peroxidase Activity:

6.6. Determination of Glutathione S-transferase (GST):

7. Statistical Analysis:

Results

3. Enzymatic Activity Response:

Discussion

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1

The Efficacy of Egyptian Isolate of Beauveria bassiana Against Red Palm Weevil (Rhynchophorus ferrugineus) Olivier (Coleoptera: Curculionidae): Mortality Rates, Biochemical Responses, and Enzymatic Activity

Status:

Version 1

Abstract

Figures

Background

Methods

1. Red Palm Weevil Source:

2. Insect Source:

3. Entomopathogenic Fungi Cultures:

4. Effect of Two B. bassiana Concentrations on 5th and 10th Instar Larvae of R. ferrugineus:

5. Biochemical Response Comparison of 5th and 10th Instar Larvae to B. bassiana:

6. Preparation of Insects for Analysis:

6.1. Determination of Total Proteins:

6.2. Determination of Total Carbohydrates:

6.3. Determination of Nonspecific Esterase:

6.4. Determination of Phenoloxidase Activity:

6.5. Determination of Peroxidase Activity:

6.6. Determination of Glutathione S-transferase (GST):

7. Statistical Analysis:

Results

3. Enzymatic Activity Response:

Discussion

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1

4. Effect of Two B. bassiana Concentrations on 5^th and 10^th Instar Larvae of R. ferrugineus:

5. Biochemical Response Comparison of 5^th and 10^th Instar Larvae to B. bassiana: