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Article

Immune Responses of Rhynchophorus ferrugineus to a New Strain of Beauveria bassiana

by
Mohsen Mohamed Elsharkawy
1,2,*,
Fatimah O. Alotibi
3,
Abdulaziz A. Al-Askar
3,
Muhammad Kamran
4,
Said I. Behiry
5,
Salam S. Alasharari
6,
Fatma H. Galal
6,7,
Muhammad Adnan
8 and
Ahmed Abdelkhalek
9
1
Palms and Dates Center, Ministry of Environment, Water and Agriculture, Al-Ahsa 31982, Saudi Arabia
2
Department of Agricultural Botany, Faculty of Agriculture, Kafrelsheikh University, Kafr El-Sheikh 33516, Egypt
3
Department of Botany and Microbiology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia
4
School of Agriculture, Food and Wine, The University of Adelaide, Adelaide, SA 5005, Australia
5
Agricultural Botany Department, Faculty of Agriculture (Saba Basha), Alexandria University, Alexandria 21531, Egypt
6
Biology Department, College of Science, Jouf University, Sakaka P.O. Box 72341, Saudi Arabia
7
Entomology Department, Faculty of Science, Cairo University, Cairo 12613, Egypt
8
Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing, MI 48824, USA
9
Plant Protection and Biomolecular Diagnosis Department, ALCRI, City of Scientific Research and Technological Applications, New Borg El Arab City 21934, Egypt
*
Author to whom correspondence should be addressed.
Sustainability 2022, 14(20), 13002; https://doi.org/10.3390/su142013002
Submission received: 23 August 2022 / Revised: 8 October 2022 / Accepted: 9 October 2022 / Published: 11 October 2022
(This article belongs to the Special Issue Mitigate Hunger through Enhancing Plant Resistance to Biotic and Abiotic Stresses)
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Abstract

:
Evaluating a novel fungal strain’s pathogenicity to important pests and their involved immune responses may give crucial data on a broad scale for future use in pest management strategies. Date palms are mostly destroyed by invading populations of red palm weevils; thus, developing natural biopesticides for them requires a comprehensive screening program of plant secondary metabolites. In this research, we examined the pathogenicity of a new strain of Beauveria bassiana on an important agricultural pest, Rhynchophorus ferrugineus, by measuring the relative activity of defensive enzymes and detoxifying enzymes in certain larval instars. Our findings reveal that the B. bassiana strain may infect the instars of R. ferrugineus, and its pathogenicity to the larvae steadily increases as the spore concentration increases. Seven days after inoculation, the LC50 (the median lethal concentration) of B. bassiana was 490.42 × 105 and 2974.47 × 108 spores/mL for the second and fourth instar R. ferrugineus, respectively, and the LC50 of B. bassiana for each R. ferrugineus instar decreased with infection time, indicating a significant dose effect. Infected R. ferrugineus larvae of the second instars showed considerable changes in the activity of both protecting and detoxifying enzymes (peroxidase, catalase, superoxide dismutase, Cytochrome P450, glutathione S transferase (GST), and esterase) as infection time progressed. In addition, R. ferrugineus larvae that were infected with B. bassiana had enzyme activity that persisted from 24 to 48 h, which was much longer than in the control group. Lethality of B. bassiana resulted in elevated expressions of GST, Esterase, and Cytochrome P450 responsive genes. In conclusion, the results of this research indicate that B. bassiana may be utilized as a bio-insecticide to suppress young larvae of R. ferrugineus in an integrated pest management program.

1. Introduction

Entomopathogenic fungi may naturally regulate pest populations, minimize the need for pesticides, and preserve ecological balance. Numerous entomopathogenic fungi, especially those from the genera Beauveria, Lecanicillium, Isaria, and Metarhizium, have been widely explored for their ability to manage a variety of pests [1,2,3,4]. To assess entomopathogenic fungi for pest management on a wide basis, it is necessary to have a better knowledge of their pathogenicity. Therefore, prior to the creation of an effective biological control program, it is important to evaluate the virulence of entomopathogenic fungi. Beauveria bassiana is an entomopathogenic fungus that has been marketed and utilized extensively in greenhouse and field conditions [5,6,7]. Numerous studies have shown that B. bassiana is fatal to a wide range of insect pests, including Ostrinia furnacalis [8], Cylas formicarius [9], Bemisia tabaci [10], Lycorma delicatula [5], Frankliniella occidentalis [6], and Leptinotarsa decemlineata [4]. There are significant differences in the virulence of various B. bassiana strains against pests on different hosts [11,12,13]. Clarifying a novel B. bassiana strain’s virulence against pests and the involved immune mechanisms, therefore, gives crucial information on a broad scale for future use in pest management.
Insects are often exposed to xenobiotics in their environment, making an effective mechanism for their bodies to detoxify and remove these compounds necessary for appropriate adaptation to these environmental threats. For instance, B. bassiana infection alters the physiological and metabolic processes that normally occur in insects [14]; it damages the free amino acids present in the hemolymph and interferes with a number of crucial metabolic enzymes, including glutathione S-transferases (GST) [15], carboxylesterase (CarE) [16], and Cytochrome P450 (CYP450) [17], which are crucial for the detoxification of consumed xenobiotics [18,19].
On the defense response of the red palm weevil (Rhynchophorus ferrugineus) against entomopathogenic fungi, there is little information available. The defensive enzyme system found in insects, which is mostly comprised of the enzymes catalase (CAT), peroxidase (POX), and superoxide dismutase (SOD), might decrease oxidative damage by reducing H2O2 [20]. Toxic reactive oxygen species (ROS) are produced and stored by an organism’s innate immune cells when insects are under stress, such as entomopathogenic fungi or chemical pesticides [21,22,23]. Protective enzymes may scavenge a variety of ROS in order to preserve normal cellular function and avoid oxidative damage [24,25]. Consequently, it is important for pest management studies to know about the resistance mechanism and how insect control changes during entomopathogenic infection.
B. bassiana, like other entomopathogenic fungi of the order Hypocreales, penetrates primarily via the insect cuticle, which serves as an initial point of contact and a protective barrier between the fungus and host [26]. Upon the adhesion to and recognition of the insect surface, B. bassiana deploys a combination of biochemical and mechanical tools to make its way through the insect integument into the hemocoel [27]. When the fungus enters the nutrient-rich environment, the mycelium changes into yeast-like cell phenotype; these cells are sometimes referred to as hyphal bodies or blastospores when they are experimentally grown in a culture medium. Despite the fact that the immune system (cellular and humoral) has been activated in an attempt to defeat the fungus, the host has very little possibility of surviving after fungal infection at this stage [28]. Consequently, the success of fungal infection relies on several factors, including the development of harmful secondary metabolites that may either assist the fungal invasion or function as immunosuppressive chemicals, fighting against the host’s natural defensive mechanisms [29,30,31]. Nonribosomal peptides and polyketides are examples of secondary metabolites that may have a wide range of chemical properties. In Metarhizium, the main products are destruxins, which are cyclic hexadepsipeptides, while in Beauveria, the main products are beauvericin, bassianolide, and various beauverolides, which are all cyclic peptides, as well as oosporein, bassiatin, and tenellin [26,29,31].
The objectives of this study are to examine the pathogenicity of a novel B. bassiana strain on R. ferrugineus larvae of various instars and to identify the protective enzymes and mechanisms in the immunological resistance of R. ferrugineus larvae. Additionally, quantitative real-time polymerase chain reaction (qRT-PCR) analysis of the genes encoding detoxification enzymes such as Cytochrome P450, glutathione S transferase (GST), and esterase was used to study the effects of the red palm weevil’s detoxification mechanism against B. bassiana and serve as a starting point for the creation of more secure natural biopesticides to combat red palm weevil infestations.

2. Materials and Methods

2.1. Entomopathogenic Fungus

The red palm weevil (RPW), R. ferrugineus, larvae and adults, were collected from infected date palms. R. ferrugineus cultures were developed in a growth chamber using sterile plastic cages (20 × 50 × 50 cm) wrapped with muslin material. The raising of RPW was done in controlled conditions (28 ± 1 °C and 65–75% RH, with photoperiod control (13:11 h L:D) to establish the insect colony. The sugarcane stems served as a food source of the RPW.
The entomopathogenic fungus B. bassiana strain utilized in this investigation was obtained from naturally infected dead Galleria mellonella L. larvae in Al Ahsa, Saudi Arabia. Larvae were collected and treated with 1% Na-hypochlorite to prevent any exterior saprophytic fungi that would grow on the dead larvae. To detect the presence of muscardine, dead larvae were maintained in Petri dishes coated with a single layer of moist filter paper. The fungus spores were grown on potato dextrose agar (PDA) and incubated from 6 days at 24 °C. The multispore cultures were separated into single spore cultures for further purification. Prior to the experiment, the strain was grown for 15 days at 25 ± 1 °C, with 80 ± 120 5% RH, and a photoperiod of 0:24 h (L:D) on PDA medium (200 g potato, 20 g glucose, 15 g agar, 1000 mL water, and natural pH). The fungal conidia were collected from the surface of the culture medium after being cultivated for 15 days. Mycelia of B. bassiana were further harvested from the mycelia subculture developed by a microbiological loop and immediately suspended in a 0.01% tween 80 solution and vortexed for 30s. After being incubated for 5 days in complete darkness, a 20 µL aliquot of the mycelia suspension was then transferred to PDA medium. The presence of pure B. bassiana mycelia on the surface of the medium was validated by comparing it to the mycelia development of the B. bassiana control strain. Once the B. bassiana isolate had grown on PDA, the mycelia were scraped off the surface using a sterile scalpel and placed in a clean Petri dish. Mycelium was then put into 100 mL of potato dextrose broth medium after being sliced into tiny pieces using a sterile blade. The pure mycelia of the fungal isolate were centrifuged after 1 week and frozen for further use. The fungus cultivated in PDA was also stained with lactophenol cotton blue to confirm its species [32,33].

2.2. Identification of the Isolated Fungi

A solution containing 150 μL of sodium dodecyl sulfate (10%, w/v), 500 mL of Tris buffer (100 mm Tris HCL, pH 8.0, 40 mM ethylene diamine tetra acetic acid [EDTA]), and 300 μL of benzoyl chloride was used to suspend 0.005 g of dried, pure mycelia in a 1.5 mL centrifuge tube and incubated at 56 °C for 30 min. Proteins were then eliminated by further processing with RNase (1 mg/mL) and phenol, chloroform, and isoamyl alcohol (25:24:1). The tube was then vortexed at 10,000 g for 1 min to separate the supernatant. Subsequently, by adding 2.5 volumes of 100% ice-cold ethanol to the supernatant, DNA was precipitated. The DNA pellet was then carefully cleaned twice with 70% ethanol before being re-suspended in distilled water [34]. Polymerase chain reaction (PCR) and sequencing were done as described by Mondal and Baksi [32]. After denatured with HiDi-Formamide at 95 °C for 3 min, the samples were examined on a 3730 DNA Analyzer after being filtered to eliminate excess salt (Applied Biosystems, Carlsbad, CA, USA). The acquired sequences were aligned and consensus sequences were determined and deposited in Genbank. Utilizing MEGA-11 version 11.0.2, molecular phylogenetic reconstruction was carried out [35]. A phylogenetic tree was constructed using neighbor-joining technique and data derived from whole nucleotide sequences [35].

2.3. Virulence of B. bassiana on Rhynchophorus ferrugineus Larvae

To assess the pathogenicity of B. bassiana on two R. ferrugineus larval instars, we used five conidial concentrations (treatments), with Tween-80 (0.05%) as a control. The conidial concentration was measured using a hemocytometer after making the conidial suspensions and adjusted to 1 × 105, 1 × 106, 1 × 107, 1 × 108, and 2 × 108 spores/mL using Tween-80 (0.05%) solution. Each treatment consisted of 30 individuals and five repetitions. The R. ferrugineus larvae were put on a Petri dish and individually submerged in the solution for 10 s for each treatment. Only the Tween 80 aqueous solution was utilized in the control group. The larvae were monitored for 7 days at 28 ± 1 °C, with 65–75% RH, and a photoperiod of 13:11 h (L:D). It was considered dead if a larva did not react to a brush’s contact. Larvae deaths were counted, and correct mortality was determined as:
Corrected   Mortality % = ( 1 − n u m b e r   o f   i n s e c t s   i n   t r e a t m e n t   g r o u p   m o r t a l i t y n u m b e r   i n   i n s e c t s   i n   c o n t r o l   g r o u p   m o r t a l i t y )   ×   100
The median lethal concentration (LC50) of R. ferrugineus larvae was determined. On days 3, 5, and 7 after treatment, mortalities were reported. The dead larvae were collected and placed in a culture plate sterilized with 75% EtOH.

2.4. Measurement of Enzyme Activities in R. ferrugineus Larvae

The R. ferrugineus larvae of various instars were treated using the immersion technique with Tween-80 (0.05%) and 2 × 108 spores/mL of B. bassiana as the treatment group and control group, respectively. The treated R. ferrugineus larvae were reared the same as above. Random samples of the treated larvae were taken throughout the culture process at 24 and 48 h after treatment with B. bassiana and Tween-80. There were 50 samples (2nd instars) and 10 samples (4th instars) for each test group at each time point, respectively. The insect samples were homogenized with 0.1 mol/L PBS (samples weight:PBS = 1 g:9 mL). The tissue homogenate was then centrifuged for 15 min at 12,000 rpm and 4 °C. The supernatant was transferred to fresh tubes as the source of the enzyme and kept at −20 °C. The enzymatic activities related to the observed larvae were assessed in three repetitions of each test.
An optical wavelength of 340 nm was used to measure the glutathione-S-transferase enzyme activity. Glutathione peroxidase (GPx), Catalase (CAT), Superoxide Dismutase (SOD) activity assays of each enzyme were carried out by following protocol of Ahmed and El-Sobki [36]. The quantity of 1 mol H2O2 degraded per milligram of tissue protein per second as 1 unit is used to measure CAT activity. The standard for measuring POX activity was 1 U per mg of tissue protein to catalyze 1 g of substrate per minute at 37 °C. For the purpose of measuring SOD activity, 1 U of SOD was chosen to represent a 50% SOD inhibition rate per mg of tissue protein in 1 mL of reaction solution.

2.5. Quantitative Real-Time PCR

The larvae were dissected in saline, and their midguts were ground separately in liquid nitrogen for total RNA extraction using RNA purification kit (Thermo Scientific, Fermentas, # K0731, Waltham, MA, USA). Each sample’s total cDNA was utilized as a template for a single RT-qPCR experiment (using Thermo Scientific, Fermentas, # EP0451, Waltham, MA, USA). The following were the RT-qPCR conditions: Hot-start DNA polymerase was activated at 95 °C for 2 min, then 40 cycles of 95 °C for 15 sec and 60 °C for 1 min and the dissociation at 95 °C was the last stage. Using SYBR Green (Thermo Scientific, USA, # K0221), three primers for the detoxification genes (GST, Cytochrome P450, and Esterase) were utilized (Table 1). The five replicates’ means were calculated. The internal control in RT-qPCR was β-actin. Using the 2−∆∆Ct approach, we normalized our data to the amplification of β-actin [37].

2.6. Statistical Analysis

SPSS 17.0 for Windows (SPSS Inc., Chicago, IL, USA) was used for all statistical analyses. A one-way analysis of variance was used to analyze the data. Probit analysis was used to determine the regression equation, LT50, LT90, and their 95% confidence intervals. To compare various means, Tukey’s honestly significant difference (HSD) test (p ≤ 0.05) was utilized.

3. Results

3.1. Identification of Fungal Isolate

The obtained isolate, according to nucleotide database blasting utilizing a nucleotide query approach, is Beauveria bassiana. With the use of amplicon generated by PCR, the isolate was identified. The sequences for B. bassiana have been submitted to NCBI, with accession number OP108819. Nucleotide alignment revealed that the sequences were strongly connected with B. bassiana strain SD-1 from China (100% similarity) when assessed according to BLASTn (Figure 1).

3.2. Virulence of B. bassiana against R. ferrugineus

To determine the virulence of B. bassiana on R. ferrugineus larvae, LC50 values and virulence indices were calculated for weevil populations. After 7 days of treatment, the LC50 of spore concentrations increased with instar stage of R. ferrugineus (Table 2). The second instar had the lowest LC50 value (490.42 × 105 spores/mL), followed by the fourth instar with LC50 value of 2974.47, respectively.

3.3. Effect of B. bassiana on the Mortality of R. ferrugineus Larvae

Table 3 demonstrates the predicted dosage impact of B. bassiana -infected R. ferrugineus larvae. Mortality of R. ferrugineus larvae on each instar increased gradually with the increase of the spore concentration of B. bassiana. For example, the mortality rate of the second instar larvae increased from 54.3% at day 3 to 88.9% at day 7 after being treated at 2 × 108 spores/mL (Table 3). A similar mortality pattern was also detected for the fourth instar (Table 3). After 7 days of treatment, the corrected mortality of the second instar larvae treated with 1.0 × 107 and 1.0 × 108 spores/mL suspensions were 71.3% and 80.5%, respectively, which are significantly higher than those treated with the control (17.6%) (Table 3). Obviously, the mortality gradually reduced with the increase of larval instar (Table 3). The deadly concentration declined with increasing time, whereas the dosage effect increased. The time impact grew as the fatal concentration increased. The lethal concentration for the second and fourth instar larvae was rather high.

3.4. Effect of B. bassiana on Enzyme Activities in R. ferrugineus Larvae

The second and fourth instars of R. ferrugineus infected by B. bassiana had considerably higher CAT activity after 24 and 48 h of the infection. CAT activity was significantly increased at 24 and 48 h in the second instar compared to the control (Figure 2), and at 48 h in the fourth instar larvae (Figure 3). On the other hand, CAT activity in the fourth instar larvae was not significantly different at 24 h after infection with B. bassiana compared with the control treatment.
The SOD activity in the second instars was significantly changed at 24 and 48 h of the infection time, while in the fourth instar, it did not significantly change over the infection time (Figure 2 and Figure 3).
POX activity in R. ferrugineus second and fourth-instar larvae infected with B. bassiana changed significantly after the infection. No significant difference was observed between infected second instar larvae and the control at 48 h after infection with B. bassiana (Figure 2). POX activity was significantly increased at 24 and 48 h in the fourth instar larvae compared to the control (Figure 3), and at 24 h in the second instar (Figure 2).

3.5. The Expression of Detoxification Genes of Red Palm Weevils

B. bassiana induced different levels of GST expressions, resulting in significant differences in their quantitative expressions. The GST expressions in the second instar larvae significantly changed at 12 and 24 h after the infection time, while in the fourth instar it did not significant vary at 12 h after the infection time (Figure 4 and Figure 5).
B. bassiana failed to induce esterase expression of the fourth instar red palm weevil larvae and remained nonsignificant at the lowest level (Figure 5). The esterase expression in the second instar was significantly higher at 24 h after infection time, while it did not significantly vary at 12 h after infection time (Figure 4).
B. bassiana induced different expression levels of the Cytochrome P450 gene (Figure 4 and Figure 5). The quantitative expression of Cytochrome P450 revealed significant differences in the second and fourth instar larvae at 12 and 24 h of the infection.

4. Discussion

The red palm weevil, R. ferrugineus (Olivier), is the most destructive invasive pest and the principal cause of the devastation of date palms in several countries. Red palm weevil grubs have cryptic, trunk-dwelling lives, creating considerable management issues. Soil treatments, trunk injections, tree fumigations, wound dressings, and crown drenching of infested palm trees are the principal application methods of pesticides to prevent the spread of red palm weevils [38]. However, pesticide residues in dates are mostly due to the careless use of synthetic pesticides [39], environmental pollution [40], resistance development [41], and detrimental effects on wildlife [42]. Hence, pesticide usage is discouraged, and the idea of developing natural, environmentally friendly insecticides is gaining traction.
The genus Beauveria has a total of 23 species, the majority of which are morphologically indistinguishable [43,44]. The use of a DNA-based method to the identification of fungal species has revolutionized the field of mycology. Beauveria currently includes species that demonstrate typical conidial morphology and/or are phylogenetically connected to teleomorphs categorized as Cordyceps [45]. The morphological similarity between B. rudraprayagi sp. nov. and B. bassiana is intriguing and suggests that the DNA-based technique is effective in sorting out the Beauveria species complex. The strain is reported using a single isolate, as was indicated in the prior reports of B. vermicola and B. kipukae [45,46].
A potential biological insect pest control agent is Beauveria bassiana. Our findings in this study show that B. bassiana, a strain isolated from Galleria mellonella larvae, had strong pathogenicity to the young R. ferrugineus larvae, and the protective enzymes and detoxifying enzymes in R. ferrugineus larvae play important roles in suppressing the infection of B. bassiana, especially in the older larvae. Our findings confirm that the pathogenicity of B. bassiana on the non-target host R. ferrugineus, as well as the immune resistance’s protecting enzymes and detoxifying genes, are stage-dependent. In entomopathogenic fungi, virulence is an important indicator of pathogenicity. The capacity of entomopathogenic fungi to cause insect death, despite host resistance, is strongly correlated with the level of virulence. The present study’s findings demonstrate that B. bassiana, a strain isolated from G. mellonella larvae, displayed considerable virulence against R. ferrugineus second- and fourth-instar larvae despite a steady decline in pathogenicity with increasing larval instars. Similar findings have also been shown when B. bassiana infection of other pest insects or pathogenic fungus was obtained from other host species [38,47,48]. For instance, applying B. bassiana to young Indarbela dea larvae has a greater impact [49], indicating that younger larvae are more susceptible to B. bassiana than older ones. The initial defense against pathogenic fungal infection is provided by the insect epidermis. Due to the increasing melanin content in the epidermis and midgut of older larvae, which prevents fungal budding, the pathogenicity of entomogenous fungi to pests often declines as larval instars grow [50]. The composition of the larval body wall is also directly connected to the pathogenicity of entomogenous fungus to pests [51] because the body wall of immature larvae is comparatively thin, but the waxy coating of the body wall progressively increases with the growth of the instar stage, preventing the invasion of B. bassiana [52]. Insects may avoid various toxins by controlling alterations in the body’s detoxifying and protecting enzymes [53,54]. This research found that B. bassiana was less harmful to older R. ferrugineus larvae, which suggests that the enzyme and defense systems in the host may be included in the pathogenicity of B. bassiana.
After the pathogen successfully enters the host, the host’s defense mechanism is activated, changing the defensive enzymes, particularly the SOD, POX, and CAT. It is generally known that when insects experience stress brought on by harmful stimuli, a significant amount of active oxygen gathers in their bodies, which causes an antioxidant enzyme response [19,55]. Previous research has demonstrated an inverse relationship between the SOD and CAT activities and the larval instars [55]. Our findings in this work demonstrate that distinct larval instars’ protective enzyme activity altered in diverse ways after B. bassiana infection. These data suggest that B. bassiana sensitivity is higher in young larvae. The protective enzyme activity greatly increased in the second and fourth larval instars before declining. In Mythimna separata, Bemisia tabaci, and Xylotrechus rusticus, similar findings have been found [19,56,57]. The capacity of infected larvae to scavenge free radicals in the body is diminished due to pathogenic fungus infection in the inhabitation of antioxidant enzyme activity in the late instar stages of R. ferrugineus. Previous research demonstrates that when insects are exposed to B. bassiana, the body’s oxygen balance is quickly disrupted [21,58]. Exogenous chemicals may activate SOD to convert O2 into H2O2; however, the CAT can breakdown H2O2 into H2O and O2 if the concentration of H2O2 is too high, while POX decomposes it when the concentration of H2O2 is low [59,60]. We hypothesize that B. bassiana has weaker stimulation in older hosts and lower H2O2 concentration after infection, leading to a minimal change in the activity of defensive enzymes.
Substance toxicity speeds up the host’s metabolism, which in turn speeds up the regulation of detoxification-related genes. Genes involved in host defense are well-known to play an important role in host detoxification systems; examples include cytochrome P 450, glutathione S-transferase, and esterase. Previous research has shown that feeding resistant populations of red palm weevils a synthetic diet containing cypermethrin increases GST expression [41]. Cytochrome P450, glutathione S-transferase, and esterase gene expression levels were measured in the present investigation. In response to the B. bassiana infection, we found that all genes involved in detoxification were highly expressed. GST was the most active detoxifying gene against B. bassiana among all the assessed genes, particularly in second instar larvae. Recent research on the regulation of the host detoxification system in response to an essential plant secondary metabolite was confirmed by the increased GST response of red palm weevils given a diet integrated with fewer toxic substances [47].

5. Conclusions

Our results show that all larval instars of R. ferrugineus may become infected by B. bassiana strain Elsharkawy, which causes greater death rates, particularly for the second instar larvae. Protective enzymes and the expression of detoxification genes in R. ferrugineus larvae were activated. Our findings show that B. bassiana is significantly pathogenic to R. ferrugineus; however, this pathogenicity is stage-dependent. In conclusion, our findings imply that B. bassiana strain Elsharkawy may be employed in an integrated pest management program as a bio-insecticide to control young R. ferrugineus larvae. Future studies should concentrate on a number of issues, including formulation, safety, and application, in order to maximize the effectiveness of B. bassiana for controlling red palm weevil infestations.

Author Contributions

Conceptualization, M.M.E.; methodology, M.M.E.; software, M.M.E.; validation, M.M.E., A.A., S.I.B., M.K., A.A.A.-A., S.S.A. and F.H.G.; formal analysis, M.M.E.; investigation, M.M.E.; resources, M.M.E., A.A., S.I.B., M.K., A.A.A.-A. and M.A.; data curation, M.M.E.; writing—original draft preparation, M.M.E.; writing—review and editing, M.M.E., M.A. and F.O.A.; visualization, M.M.E., F.O.A., A.A., S.I.B., M.K., M.A., A.A.A.-A., S.S.A. and F.H.G.; supervision, M.M.E.; project administration, M.M.E.; funding acquisition, A.A.A.-A. and F.O.A. All authors have read and agreed to the published version of the manuscript.

Funding

This research was financially supported by the Researchers Supporting Project number (RSP-2021/114), King Saud University, Riyadh, Saudi Arabia.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

The authors would like to extend their appreciation to the Researchers Supporting Project number (RSP-2021/114), King Saud University, Riyadh, Saudi Arabia.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Phylogenetic dendrogram showing the position of Beauveria bassiana among phylogenetic neighbors.
Figure 1. Phylogenetic dendrogram showing the position of Beauveria bassiana among phylogenetic neighbors.
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Figure 2. Effect of B. bassiana on enzyme activities (peroxidase, catalase, superoxide dismutase) of R. ferrugineus second instar larvae at 24 and 48 h after infection. The means sharing same letters in the same time point are not significantly different (p ≤ 0.05).
Figure 2. Effect of B. bassiana on enzyme activities (peroxidase, catalase, superoxide dismutase) of R. ferrugineus second instar larvae at 24 and 48 h after infection. The means sharing same letters in the same time point are not significantly different (p ≤ 0.05).
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Figure 3. Effect of B. bassiana on enzyme activities (peroxidase, catalase, superoxide dismutase) of R. ferrugineus fourth instar larvae at 24 and 48 h after infection. The means sharing same letters in the same time point are not significantly different (p ≤ 0.05).
Figure 3. Effect of B. bassiana on enzyme activities (peroxidase, catalase, superoxide dismutase) of R. ferrugineus fourth instar larvae at 24 and 48 h after infection. The means sharing same letters in the same time point are not significantly different (p ≤ 0.05).
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Figure 4. Effect of B. bassiana on the expression of detoxification genes in Rhynchophorus ferrugineus second instar larvae at 12 and 24 h after infection. The means sharing same letters in the same time point are not significantly different (p ≤ 0.05).
Figure 4. Effect of B. bassiana on the expression of detoxification genes in Rhynchophorus ferrugineus second instar larvae at 12 and 24 h after infection. The means sharing same letters in the same time point are not significantly different (p ≤ 0.05).
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Figure 5. Effect of B. bassiana on the expression of detoxification genes in Rhynchophorus ferrugineus fourth instar larvae at 12 and 24 h after infection. The means sharing same letters in the same time point are not significantly different (p ≤ 0.05).
Figure 5. Effect of B. bassiana on the expression of detoxification genes in Rhynchophorus ferrugineus fourth instar larvae at 12 and 24 h after infection. The means sharing same letters in the same time point are not significantly different (p ≤ 0.05).
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Table
Table 1. Primers of detoxification genes used for RT-qPCR.
Table 1. Primers of detoxification genes used for RT-qPCR.
PrimerForwardReverseAccession Number
GSTATAGCCAACCACCACTGTCGCGTTCCTCTTGCCGCTAGTTKR902496
EsteraseACCTACAAGAATCCGACGCCACTCCGAAACTTTGGGCCATKT748822
Cytochrome P450TGGAGAAACACCCGCAAGAACGGCGATTTTGCCTACCAAGKT748789
β-ActinAAAGGTTCCGTTGCCCTGAATGGCGTACAAGTCCTTCCTGKM438516
Table
Table 2. Virulence of B. bassiana to the second and fourth instar larvae of R. ferrugineus.
Table 2. Virulence of B. bassiana to the second and fourth instar larvae of R. ferrugineus.
InstarRegression Equationχ2PLC50 (×105
Spores/mL)		95% Confidence
Limit (Spores/mL)
Secondy = 0.473x − 3.124 *1.140.52490.422.1 × 106~1.1 × 108
Fourthy = 0.413x − 3.6520.590.732974.476.3 × 107~6.9 × 109
* The mortality and concentration log are represented by the variables y and x, respectively.
Table
Table 3. Mortality percentages of Rhynchophorus ferrugineus at 3, 5, and 7 days after treatment with different concentrations of Beauveria bassiana spores.
Table 3. Mortality percentages of Rhynchophorus ferrugineus at 3, 5, and 7 days after treatment with different concentrations of Beauveria bassiana spores.
Concentrations
(Spores/mL)		Second InstarFourth Instar
3 d5 d7 d3 d5 d7 d
1 × 105 spores/mL 30.6 d39.6 d54.6 e27.4 d31.4 e45.2 e
1 × 106 spores/mL 41.2 c46.7 c63.4 d34.6 c38.9 d49.8 d
1 × 107 spores/mL 47.5 b49.6 c71.3 c39.5 b43.7 c55.7 c
1 × 108 spores/mL 49.8 b58.6 b80.5 b40.2 b49.6 b60.2 b
2 × 108 spores/mL 54.3 a70.3 a88.9 a44.6 a59.5 a71.2 a
Control11.6 e15.6 e17.6 f10.1 e12.5 f14.7 f
The means sharing same letters in the same column are not significantly different (p ≤ 0.05).
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Elsharkawy, M.M.; Alotibi, F.O.; Al-Askar, A.A.; Kamran, M.; Behiry, S.I.; Alasharari, S.S.; Galal, F.H.; Adnan, M.; Abdelkhalek, A. Immune Responses of Rhynchophorus ferrugineus to a New Strain of Beauveria bassiana. Sustainability 2022, 14, 13002. https://doi.org/10.3390/su142013002

AMA Style

Elsharkawy MM, Alotibi FO, Al-Askar AA, Kamran M, Behiry SI, Alasharari SS, Galal FH, Adnan M, Abdelkhalek A. Immune Responses of Rhynchophorus ferrugineus to a New Strain of Beauveria bassiana. Sustainability. 2022; 14(20):13002. https://doi.org/10.3390/su142013002

Chicago/Turabian Style

Elsharkawy, Mohsen Mohamed, Fatimah O. Alotibi, Abdulaziz A. Al-Askar, Muhammad Kamran, Said I. Behiry, Salam S. Alasharari, Fatma H. Galal, Muhammad Adnan, and Ahmed Abdelkhalek. 2022. "Immune Responses of Rhynchophorus ferrugineus to a New Strain of Beauveria bassiana" Sustainability 14, no. 20: 13002. https://doi.org/10.3390/su142013002

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