108 ISJ 18: 108-118, 2021 ISSN 1824-307X RESEARCH REPORT Activity of detoxification enzymes in Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) after exposure to Beauveria bassiana (Balsamo) R Ahmed, S Freed*, A Naeem, M Akmal Department of Entomology, Faculty of Agricultural Sciences and Technology, Bahauddin Zakariya University, Multan, Punjab, Pakistan This is an open access article published under the CC BY license Accepted August 27, 2021 Abstract Rhynchophorus ferrugineus is a devastating pest of palms worldwide. An integrated management strategy largely depends on chemical insecticides but due to concerns about human health risks and environmental pollution, it’s essential to emphasize on the integrated pest management (IPM). In the current research the activities of detoxification enzymes esterases (EST), alkaline phosphatases (ALP), acid phosphatases (ACP), glutathione S-transferases (GST), and acetylcholinesterase (AChE) in R. ferrugineus collected from Punjab, Baluchistan, Sindh and Khyber Pakhtunkhwa (KPK) provinces of Pakistan were estimated after infection of Beauveria bassiana on 3rd-, 5th- and 7th-day post- treatment. The insects were exposed by immersion method with different concentrations of B. bassiana. The significant increase in activities of ALP (6.09), ACP (2.51), AChE (21.28) and EST (8.61) μmol/min/mg protein was observed in KPK population, while a significant increase in the activity of GST (5.23 μmol/min/mg protein) was recorded in Baluchistan population on 7th- day. The detection of elevated activities of detoxification enzymes showed the possibility of the resistance development against B. bassiana in R. ferrugineus. Key Words: date palm; Rhynchophorus ferrugineus; entomopathogenic fungi; biocontrol; biochemical; detoxification enzymes; resistance mechanism Introduction Date palm (Phoenix dactylifera) is probably the oldest tree cultivated by humans and its production in Pakistan ranks at sixth position (Tavakolian et al., 2013; FAO, 2014). Among notable insects damaging date palm, Red Palm Weevil, Rhynchophorus ferrugineus (Coleoptera: Curculionidae) appears to be one of the cryptic insect (Molet et al., 2011; Arab and El-Deeb, 2012). R. ferrugineus infestation was recorded in 50 % of date producing countries (Suma et al., 2014; Wakil et al., 2015). The native range of R. ferrugineus is Melanesia and South Asian countries and dispersal occurs due to transportation of ornamental palms across all continents (El-Mergawy and Al-Ajlan, 2011). R. ferrugineus prefers to attack young palm which are less than the age of 20 years because stem of young palm is juicy, soft, and easily penetrated by the insects. A single female of red palm weevil can give rise to more than approximately _________________________________________ Corresponding author: Shoaib Freed Department of Entomology Faculty of Agricultural Sciences and Technology Bahauddin Zakariya University Multan, Punjab, Pakistan E-mail: sfareed@bzu.edu.pk half billion of grubs in three generations. Moreover, R. ferrugineus is reflected as very disparaging insect of coconut palms (Ferry and Gomez, 2002). Despite huge efforts have been done to protect palm trees via synthetic chemicals, quarantine and other traditional methods (Abd-Elgawad, 1996), R. ferrugineus has proved to be stronger than these control measures and it has been entitled as the AIDS (acquired immune deficiency syndrome) of palm tree (Hanounik, 1998). The growing demand of farmers to reduce chemical insecticides in agriculture, along with the environmental pollution and increased resistance to insecticides has provided huge impetus for the development of alternative control. An entomopathogenic fungus is alternative to the use of chemical insecticides (Sandhu et al., 2012). Entomopathogenic fungi can penetrate the host cuticle and can be transmitted by contact with fungal spores or infected insects (Klein and Lacey, 1999), these are the main insect pathogens infecting beetles, because bacterial and viral diseases are rare among beetles (Hajek and St. Leger, 1994). The entomopathogenic fungi are usually host specific and are known to cause many physiological and biochemical changes in the host that alter the rate of growth, development and food utilization of 109 Table 1 Median lethal time of B. bassiana virulence against R. ferrugineus at the highest tested concentration Provinces LT50 (Days) (95 % FL) Slope χ2 Df P N Punjab 2.849 2.497-3.203 2.20 ± 0.22 6.472 6 0.372 80 Sindh 3.166 2.769-3.586 2.02 ± 0.22 1.314 6 0.970 80 Baluchistan 3.599 3.162-4.099 1.98 ± 0.22 3.972 6 0.680 80 KPK 3.027 2.753-3.300 3.17 ± 0.25 10.381 6 0.109 80 FL=Fiducial limits P-values are based on Chi-square goodness of fit test. N=Number of larvae used in the treatment including control. the host (Butt et al., 2016). B. bassiana is potential fungi against R. ferrugineus (Gindin et al., 2006; Güerri‐Agulló et al., 2010; Ricaño et al., 2013). Insects routinely deal with many toxic substances that may be chemicals or microbial agents. Insects use enzymes including acetylcholinesterase (AChE), esterase (EST), alkaline phosphatases (ALP), acid phosphatases (ACP) and glutathione S-transferases (GST) as their defense mechanism to xenobiotic agents (Zibaee et al., 2009a). Xenobiotic agents are compounds which might penetrate insect body and then enzymes enable insects to escape from these agents. The toxic chemicals are degraded by these enzymes without showing their action (Bogwitz et al., 2005). The entomopathogenic fungi infected insects elevate the expression of GST, EST and AChE. The activation of detoxifying enzyme after fungal infection initiates its rapid degradation, catalyzation, hydroxylation and finally excretion (Wang et al., 2004). The activities of EST and GST increased post treatment with entomopathogens in Dendrolimus tabulaeformis Tsai and Liu (Fan et al., 2013) and elevated EST and GST in Eurygaster integriceps Puton were also detected post-treatment with B. bassiana (Zibaee et al., 2009a). The AChE activity also increased in Nilaparvata lugens (Stål) after treatment with fungal metabolites and botanical insecticides (Nathan et al., 2008). For the management of R. ferrugineus, previous studies just focused on the use of insecticides. However, ecofriendly B. bassiana can challenge the voracious damage against R. ferrugineus (Qayyum et al., 2020), but the role of detoxifying enzymes after its infection remains under explored. Thus, the present study was conducted to assess metabolic resistance development after treating R. ferrugineus with B. bassiana, as a baseline to suggest better management tactics. Materials and methods Insect collection and rearing The adults and larvae of R. ferrugineus were collected from all four provinces of Pakistan i.e., Baluchistan, Punjab, Khyber Pakhtunkhwa (KPK) and Sindh. The insects were later on shifted to sterile cages (60×60×30cm) covered with muslin cloth. Saccharum officinarum was used as diet for adult R. ferrugineus that was refreshed after two days. The larvae were reared on artificial diet made by following the method described by Ahmed and Freed (2021a) which was refreshed after three days. The rearing conditions were maintained at 27 ± 2 °C temperature, 70 ± 5 % relative humidity and 12/12 hours L/D photoperiod. Beauveria bassiana The isolate of B. bassiana tested was Bb-01 and had been maintained in laboratory culture prior to the beginning of the study. Fungal bioassay 3rd instar larvae of R. ferrugineus were subjected to bioassays. For this individual larva was dipped for 10-15s in concentrations of B. bassiana i.e., 3 × 108, 2 × 108, 1 × 108, 1 × 107 and 1 × 106 spores/mL. All concentrations were prepared in 0.1 % Tween 80 solution following the methodology of Alkhaibari et al. (2017). Eighty larvae were treated for each concentration and each concentration was replicated four time. While a total of 480 larvae were treated with different concentrations including a control which was treated with Tween 80 solution only. The larvae after treatment were shifted in Petri plates (2.5 cm diameter) with an artificial diet. The data on enzymatic activity was recorded on 3rd-, 5th- and 7th-days post treatment. The pathogenicity of B. bassiana against KPK, Punjab, Sindh and Baluchistan were statistically non-similar (95 % FLs did not overlap) to each other. Nevertheless, lowest LC50 (1.3×107 spores/ml) was noted in the KPK samples, while samples from Punjab, Sindh and Baluchistan had LC50 values of 1.5 × 107, 5.3 × 107 and 1.02 × 108 spores/ml, respectively (Ahmed and Freed, 2021b). Sample preparation for determining the enzyme activities The samples (n= 4) were taken from the afore- mentioned assays to further assess the enzymatic levels in B. bassiana-treated R. ferrugineus on 3rd-, 5th- and 7th-days as described by Serebrov et al. (2006). Third instar larvae were crushed in 80 µL of 110 Table 2 Mean (± SE) enzyme activities in the R. ferrugineus after infection with B. bassiana across different concentration (Spores/mL), three post infection times and four different locations in Pakistan Beauveria bassiana Treatment GST AChE ACP ALP EST 1×106 1.83 ± 0.08d 3.53 ± 0.39e 0.49 ± 0.02e 2.19 ± 0.09e 2.72 ± 0.21e 1×107 2.15 ± 0.11d 4.76 ± 0.57d 0.94 ± 0.03d 2.65 ± 0.12d 3.27 ± 0.23d 1×10⁸ 2.81 ± 0.14c 5.81 ± 0.68c 1.27 ± 0.04c 3.64 ± 0.11c 4.13 ± 0.27c 2×10⁸ 3.67 ± 0.15b 7.81 ± 1.05b 1.51 ± 0.03b 4.15 ± 0.14b 4.64 ± 0.32b 3×10⁸ 4.17 ± 0.17a 9.51 ± 1.34a 1.92 ± 0.04a 4.76 ± 0.14a 5.37 ± 0.33a Control 1.36 ± 0.04e 1.45 ± 0.04f 0.34 ± 0.02f 1.53 ± 0.05f 1.41 ± 0.03f Location Baluchistan 2.90 ± 0.23a 5.35 ± 0.76b 1.08 ± 0.07b 2.99 ± 0.15bc 3.41 ± 0.27bc KPK 2.64 ± 0.21a 5.83 ± 0.73a 1.25 ± 0.09a 3.67 ± 0.21a 4.16 ± 0.29a Punjab 2.21 ± 0.19b 5.22 ± 0.77b 0.95 ± 0.07c 2.74 ± 0.16c 3.23 ± 0.25c Sindh 2.90 ± 0.22a 5.51 ± 0.73ab 1.03 ± 0.07bc 3.21 ± 0.17b 3.55 ± 0.26b Day 3rd day 2.26 ± 0.10c 2.34 ± 0.10c 0.98 ± 0.07c 2.83 ± 0.14b 2.48 ± 0.13c 5th day 2.62 ± 0.14b 3.05 ± 0.14b 1.07 ± 0.06b 3.21 ± 0.15a 2.97 ± 0.15b 7th day 3.11 ± 0.16a 11.05 ± 0.75a 1.19 ± 0.07a 3.43 ± 0.16a 5.31 ± 0.26a Means with similar alphabets within columns, for each tested variable, are not significantly different (Tukey’s HSD test, p > 0.05) 0.15 M NaCl with a mortar and pestle. The final volumes were adjusted to 900 µL per replication for centrifugation. The samples were spun at 10,000 rpm for 10 min, and supernatants were used to determine enzyme activities. Protein determination Protein contents in B. bassiana-treated larval samples of R. ferrugineus were measured by following the Bradford (1976) procedure. Enzyme Assays The activity of AChE was measured as explained by Ellman et al. (1961) using acetylcholine iodide (0.075 M) as a substrate. The samples were incubated in 0.1 mM of EDTA, 100 mM phosphate buffer (pH 7.2), 10 mM of 5,5′- dithiobis (2-nitrobenzoic acid), and 100 mM of acetyl-choline at 30 °C for 30 min. The variation in absorbance was recorded at λ of 412 nm for 4 min at 30 s interval. ALP and ACP activities were determined by following the method of Serebrov et al. (2006) with slight modification. The samples were mixed with 2.3×10-4 M p-nitrophenylphosphate in 0.05 Tris-HCl, pH, 8.8 for ALP, 0.05 M citrate phosphate buffer, pH, 5.0 for ACP and incubated for 2 h at 30 °C. 500 μL (0.05 M NaOH) was added for color development. The change in absorbance was noted at 410 nm for 4 min and 30 s intervals. GST activity was measured by using chloro-2, 4- dinitrobenzene1 mM with 5 mM reduced glutathione and 0.1 M Tris buffer pH 8.0 (Caballero et al., 2008). The activity of the enzyme was evaluated by monitoring continuous changes in absorbance at 340 nm for 4 min at 25 °C. The extinction coefficient of CDNB (0.0096) was used to determine the total GST’s activity (Rizvi et al., 2018). EST activity was recorded by using 1 mM P-Nitrophenyl acetate and 50 mM phosphate buffer as substrate (Damayanthi and Karunaratne (2005). In each replicate, 100 μL of 0.6 M aNa (or bNa) and 100 μL of phosphate buffer (pH 6.5) were added to 10 μL of R. ferrugineus homogenate. After 30 min incubation, 100 μL solution of Fast Garnett BC was mixed to stop the reaction. The changes were determined at λ of 405 nm as an end point calculated from standard curves of a- and b- Naphtol. Following Rizvi et al. (2018), extinction coefficient of Pnpa (176.47) was used to measure EST activities. Statistical analysis The mortality data of B. bassiana treated R. ferrugineus were examined by POLO Plus software which yielded LT50 values, 95 % confidence limits 111 Table 3 ANOVA results for release activities of detoxification enzyme in the R. ferrugineus after infection with B. bassiana across different concentration (Spores/mL), three post infection times and four different locations in Pakistan Enzyme activity against Beauveria bassiana(μmol/min/mg) Sources df AChE GST ACP ALP EST F P F P F P F P F P Treatment (T) 5 614.2 <0.001 119.31 <0.001 394.87 <0.001 162.85 <0.001 206.92 <0.001 Location (L) 3 7.58 <0.001 15.85 <0.001 26.24 <0.001 24.69 <0.001 25.14 <0.001 Day (D) 2 3390.04 <0.001 36.1 <0.001 23.38 <0.001 19.56 <0.001 466.65 <0.001 T × L 15 0.68 0.7975NS 1.73 0.051 1.25 0.2419 NS 1.92 0.0255 2.11 0.0123 T × D 10 286.22 <0.001 2.23 0.019 0.67 0.7503 NS 0.75 0.6778 NS 20.76 <0.001 L × D 6 4.65 <0.001 1.54 0.1693 NS 1.59 0.1534 NS 1.11 0.3579 NS 1.05 0.3941 NS T × L × D 30 0.73 0.8376 NS 0.46 0.9929 NS 0.2 1.0000 NS 0.3 0.9999 NS 0.33 0.9996 NS Error (df) 144 NS Labelled values are showing non-significant results (p > 0.05) (FL), chi-square values and slope ± SE. Statistical analyses were undertaken with the linear model using a factorial analysis of variance (ANOVA) considering location, concentration effects and post- infection time and their interaction as factor against the dependent responses (i.e., enzyme activity). Further, concentration effects were compared across districts for each post-infection time. The significant (p < 0.05) means for above analyses were compared using Tukey’s Honestly Significant Difference (HSD) multiple comparisons Test. Graphs were prepared by using Graph pad Prism, version 6.02. Results Median lethal time of B. bassiana virulence against R. ferrugineus The infectivity of B. bassiana on R. ferrugineus and its LT50 values were calculated. The lowest LT50 value (2.849 days) was noted in Punjab population, while populations of KPK, Sindh and Baluchistan had values of 3.027, 3.166 and 3.599 days, correspondingly at highest concentration (Table 1). Enzymatic response in R. ferrugineus post infection with B. bassiana The results indicated the significant effects for concentration, location, and post-infection time towards AChE, GST and EST activities in B. bassiana treated R. ferrugineus (Table 2, 3). The activities of enzymes in B. bassiana treated R. ferrugineus increased in a highly concentration- dependent as well as time-dependent manner, i.e., enzyme activities increased after each concentration and post-infection time increase. AChE, GST, EST, ACP and ALP activities were highest for KPK and lowest for Punjab populations. An effect for treatment × day was typically significant towards AChE, GST and EST activities. However, the location × day interaction was typically significant towards AChE activity. AChE, GST, ACP, ALP and EST post infection responses to B. bassiana In B. bassiana treated R. ferrugineus, the post- infection activities of AChE, GST, ACP, ALP and EST increased with increasing post-infection time. The releases were highest for seventh-days post- infection time and typically for the highest exposure concentration (i.e., 3 × 108 spores/mL) (Figure 1-5). AChE The KPK population of R. ferrugineus treated with B. bassiana showed the maximum AChE activities on the seventh-day i.e., 21.28 ± 0.78 μmol/min/mg protein (F = 186.78, df =23, p < 0.001) followed by Sindh, Baluchistan and Punjab populations with maximum AChE activities i.e., 20.95 ± 0.45, 20.61 ± 0.23 and 19.95 ± 0.34 μmol/min/mg protein, respectively, at the highest exposure concentration (Figure 1). ACP The KPK population of R. ferrugineus infected by B. bassiana showed the maximum activity of ACP on the 7th-day i.e., 2.51 ± 0.39 (F = 30.19, df =23, p < 0.001) at the highest concentration in 3×108 spores/mL followed 1.98 ± 0.03, 1.85 ± 0.08 and 1.86 ± 0.06 μmol/min/mg protein in Baluchistan, Punjab and Sindh, respectively (Figure 2). ALP The B. bassiana treatment on R. ferrugineus showed maximum activity of ALP on 7th-day in KPK population i.e., 6.09 ± 0.32 μmol/min/mg protein 112 B a l o c h i s t a n K P K P u n j a b S i n d h 0 1 2 3 4 5 3 r d D a y 1 × 1 0 6 1 × 1 0 7 1 × 1 0 8 2 × 1 0 8 c o n tr o l 3 × 1 0 8 a a b a -c a -e b - e d e a -c a -d a -e b - e d e d e a b a -d a -e c - e d e e a -e b - e a bc - e d e e B a l o c h i s t a n K P K P u n j a b S i n d h 0 2 4 6 8 5 t h D a y a a b a -e a -g b -h g h a - f a -h a -h c -h d -h g h a -e b -h c -he -h f -h h a b a -c a -d a -h c -h f -h B a l o c h i s t a n K P K P u n j a b S i n d h 0 5 1 0 1 5 2 0 2 5 a b c d d - f g h a b c d c - e f g h a b c c d f g h a b c d d - g e - g h 7 t h D a y P r o v i n c e s A C h E a c t iv it y ( µ m o l/ m in m g p r o t e in ) Fig. 1 Mean ( ± SE) activities of AChE in B. bassiana treated R. ferrugineus across three post-infections times for populations from different provinces of Pakistan. SE denotes standard error. Figure panels are showing post- ANOVA statistics for concentration effects according to 3rd, 5th and 7th day of treatment by location interaction. Bars within each panel labelled with similar letters are not significant from one another (F = 12.78, df =23, p < 0.001) at the highest exposure concentration followed by Sindh, Punjab and Baluchistan populations with maximum AChE activities i.e., 5.26 ± 0.32, 5.01 ± 0.51 and 4.51 ± 0.16, respectively (Figure 3). EST B. bassiana-treated R. ferrugineus showed a significant increase in EST activity. The maximum activity of EST was recorded in KPK population at the highest exposure concentration i.e., 8.61 ± 0.48 μmol/min/mg protein (F = 40.13, df =23, p < 0.001) on 7th-day followed by 7.94 ± 0.52, 7.28 ± 0.54 and 7.27 ± 0.19 μmol/min/mg protein in Baluchistan, Sindh and Punjab, respectively (Figure 4). GST These results showed maximum GST activity in Baluchistan population 5.23 ± 0.38 μmol/min/mg protein (F = 11.77, df =5, p = p < 0.001) on 7th-day at the highest exposure concentration followed by Sindh, KPK and Punjab populations with maximum 113 B a l o c h i s t a n K P K P u n j a b S i n d h 0 . 0 0 . 5 1 . 0 1 . 5 2 . 0 2 . 5 a b a -e d - g e - i h -j ij a a -d e -h e - g h -j h -j a -c c - gd - g g - j ij j a -d c - g d - g f - g h -j j 3 r d d a y B a l o c h i s t a n K P K P u n j a b S i n d h 0 . 0 0 . 5 1 . 0 1 . 5 2 . 0 2 . 5 5 t h D a y a b a -d d - f e -h g - j ij a a -d b - e e - g g - j h -j a -c c - f e - g f - i h -j j a -d b - ec - f e -h h -j j B a l o c h i s t a n K P K P u n j a b S i n d h 0 1 2 3 4 a b b - e c - g f - g h i i a b c b - d d - g g - i h i b - d b - f e -h f - i h i i b - d b - e b - f e -h h i i 7 t h d a y A C P a c t iv it y ( µ m o l/ m in m g p r o t ie n ) P r o v i n c e s Fig. 2 Mean ( ± SE) activities of ACP in B. bassiana treated R. ferrugineus across three post-infections times for populations from different provinces of Pakistan. SE denotes standard error. Figure panels are showing post- ANOVA statistics for concentration effects according to 3rd, 5th and 7th day of treatment by location interaction. Bars within each panel labelled with similar letters are not significant from one another AChE activities i.e., 5.17 ± 0.26, 4.87 ± 0.56 and 4.71 ± 0.51 μmol/min/mg protein, respectively (Figure 5). Discussion The enzymatic system is activated prior to infection by entomopathogens and maintains the regular physiological activities of an insect (Jun et al., 2003). In the current study, treatment of larvae of R. ferrugineus with B. bassiana resulted in a significant increase in activities of the enzymes ALP, ACP, AChE, and EST in the KPK population only. In the Baluchistan population, only the activity of the GST enzyme was increased. The increased activities of detoxifying enzymes in insects against fungal infection may be due to activation of the immune response (Moorhouse et al., 1993). The results of our research are consistent with those of Bilal et al. (2018) showing amplified GST and EST 114 B a l o c h i s t a n K P K P u n j a b S i n d h 0 2 4 6 3 r d D a y 1 × 1 0 6 1 × 1 0 7 1 × 1 0 8 2 × 1 0 8 c o n tr o l 3 × 1 0 8 a a b b - f f - g d - g g a -da -e b - g e - g f g g a -d a -e b - f e - ge - g g a -c a -d b - f c - g d - g g B a l o c h i s t a n K P K P u n j a b S i n d h 0 2 4 6 8 5 t h D a y a -d b - db - e e -he -h h a a b a -c b - e d -h g h b - d b - f c - g f -h g h h a -c b - d b - d d -h e -h g h B a l o c h i s t a n K P K P u n j a b S i n d h 0 2 4 6 8 7 t h D a y a -e a -ga - f d - i e - i i a a -c a - f b - i e - i i a -d b - g c - i f - i h i h i a b a -d b -h d - i g - i h i A L P a c t iv it y ( µ m o l/ m in m g p r o t e in ) P r o v i n c e s Fig. 3 Mean ( ± SE) activities of ALP in B. bassiana treated R. ferrugineus across three post-infections times for populations from different provinces of Pakistan. SE denotes standard error. Figure panels are showing post- ANOVA statistics for concentration effects according to 3rd, 5th and 7th day of treatment by location interaction. Bars within each panel labelled with similar letters are not significant from one another levels in Helicoverpa armigera Hübner after B. bassiana infections. Similar results were reported by Serebrov et al. (2006) in Galleria mellonella L. in which the activity of GST and EST increased post fungal infection. Similarly, Naeem et al. (2020) reported increased activity of EST and GST in Diaphorina citri (Kuwayama) post fungal infection. The results of our research also relate to Farooq et al. (2018) who showed maximum GST activity in Musca domestica L. against the combined treatment of B. bassiana and imidacloprid. Enzymes enable insects to escape from infection of microbial agents. The toxic chemicals are degraded by the detoxification enzyme prior to show their effectiveness (Bogwitz et al., 2005). Our results showed that the application of different concentrations of B. bassiana to R. ferrugineus caused a significant increase in EST and GST 115 B a l o c h i s t a n K P K P u n j a b S i n d h 0 2 4 6 8 3 r d D a y 1 × 1 0 6 1 × 1 0 7 1 × 1 0 8 2 × 1 0 8 c o n tr o l 3 × 1 0 8 b c b - e c - e c - e d e e b c b - e b - e c - e c - e e b - d b - e b - e c - e d e e a a b b c b - e c - e d e B a l o c h i s t a n K P K P u n j a b S i n d h 0 2 4 6 8 5 t h D a y a -d b - g b - g d - g d - g g a -c a -d b - f c - g d - g e - g a -d a -e b - g c - g d - g g a a b a -d b - g c - g f g B a l o c h i s t a n K P K P u n j a b S i n d h 0 2 4 6 8 1 0 7 t h D a y a -d b - f c - g e - g g h a b a -d b - f e - g g h a a -c b - e e - gf g h a -d a -d b - f d - g g h P r o v i n c e s E S T a c t iv it y ( µ m o l/ m in m g p r o t e in ) Fig. 4 Mean ( ± SE) activities of EST in B. bassiana treated R. ferrugineus across three post-infections times for populations from different provinces of Pakistan. SE denotes standard error. Figure panels are showing post- ANOVA statistics for concentration effects according to 3rd, 5th and 7th day of treatment by location interaction. Bars within each panel labelled with similar letters are not significant from one another activities. Similar results were reported in E. integriceps which showed increased activities of EST and GST due to post-treatment with B. bassiana (Zibaee et al., 2009a). Similarly, enhancement of GST and EST activities in locust was observed after fungal infections by Dubovskiy et al. (2012). In the current study, AChE activity increased after infection by B. bassiana. Our results are quite similar to the findings of Vidhya et al. (2016) who described elevated activity of AChE in Spodoptera litura (Fabricius) after the treatment of B. bassiana. The results of our study are also consistent with Bilal et al. (2018) who reported increased AChE activity in H. armigera post fungal infections. Contrary to this Cao et al. (2016) reported inhibiting activities of AChE in Locusta migratoria L. after fungal infection. Insects use detoxification enzymes to show resistance against xenobiotics (Zibaee et al., 2009b). Detoxification enzymes e.g., ALP and ACP 116 B a l o c h i s t a n K P K P u n j a b S i n d h 0 2 4 6 3 r d D a y 1 × 1 0 6 1 × 1 0 7 1 × 1 0 8 2 × 1 0 8 c o n tr o l 3 × 1 0 8 a -d a -e b - f d - f e f f a -e d - f b - f f a a b b - f c - f d - f a -d c - f a -c b - f b - f c - f d - f f f B a l o c h i s t a n K P K P u n j a b S i n d h 0 2 4 6 a -da -e a - f e fe f f a - f a - f b - f e f f f a a b a - f b - f e f f a a -c a -e b - f d - f f 5 t h D a y B a l o c h i s t a n K P K P u n j a b S i n d h 0 2 4 6 a b a b a -d b - e c - e d e a b a -e c - e c - e c - e d e a a b a a b a -c b - e c - e c - e c - e e a -e d - e 7 t h D a y P r o v i n c e s G S T a c t iv it y ( µ m o l/ m in m g p r o t ie n ) Fig. 5 Mean ( ± SE) activities of GST in B. bassiana treated R. ferrugineus across three post-infections times for populations from different provinces of Pakistan. SE denotes standard error. Figure panels are showing post- ANOVA statistics for concentration effects according to 3rd, 5th and 7th day of treatment by location interaction. Bars within each panel labelled with similar letters are not significant from one another hydrolyze phosphomonoesters under alkaline and acidic conditions. In the current study, application of different concentrations of B. bassiana on R. ferrugineus showed increase in ALP and ACP activities. Similar enhanced expression of ALP and ACP as a defense mechanism was also reported by Bilal et al. (2017) in H. armigera after treatment with B. bassiana. Our results are also quite similar to the results of Vidhya et al. (2016) who showed an increased activity of ACP and ALP in B. bassiana- treated larvae of S. litura. Moreover, similar results were reported in Schistocerca gregaria post fungal infections (Xia et al., 2000). In conclusion, current study has described that R. ferrugineus infection with B. bassiana sharply increased detoxification enzyme activities mediating 117 detoxification and degradation of B. bassiana. This consequently increased the adaptation ability of insect body, particularly by decreasing their sensitivity to entomopathogenic fungi. This research provided novel options to develop very effective bio- control agents based on entomopathogenic fungi and their effect on R. ferrugineus due to the activities of enzymes. References Abd-Elgawad M. The Indian red palm weevil: modernization of the methods for the pest management. Agric. and Develop. in the Arab Homeland. 15: 36-45, 1996. Abe F, Hata K, Sone K. Life history of the red palm weevil, Rhynchophorus ferrugineus (Coleoptera: Dryophtoridae), in Southern Japan. Fla. Entomol. 92: 421-425, 2009. Ahmed R, Freed S. Biochemical resistance mechanisms against chlorpyrifos, imidacloprid and lambda-cyhalothrin in Rhynchophorus ferrugineus (Olivier)(Coleoptera: Curculionidae). Crop Prot. 143, 105568, 2021a. Ahmed R, Freed S. Virulence of Beauveria bassiana Balsamo to red palm weevil, Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae). Egypt. J. Biol. Pest Control. 3: 1-4, 2021b. Alkhaibari A, Carolino A, Bull J, Samuels R, Butt T. Differential pathogenicity of Metarhizium blastospores and conidia against larvae of three mosquito species. J. Med. Entomol. 54: 696- 704, 2017. Arab YA, El-Deeb HM, The use of endophyte Beauveria bassiana for bioprotection of date palm seedlings against red palm weevil and Rhizoctonia root-rot disease. Sci. J. King Faisal Univ. (Basic Appl. Sci.). 13: 1433, 2012. Bilal M, Freed S, Ashraf MZ, Muhammad S. Enhanced activities of acetylcholinesterase, acid and alkaline phosphatases in Helicoverpa armigera after exposure to entomopathogenic fungi. Invertebr. Surviv. J. 14: 464-476, 2017. Bilal M, Freed S, Ashraf MZ, Zaka SM, Khan MB. Activity of acetylcholinesterase and acid and alkaline phosphatases in different insecticide- treated Helicoverpa armigera (Hübner). Environ. Sci. Pollut. Res. 25: 22903-22910, 2018. Bogwitz MR, Chung H, Magoc L, Rigby S, Wong W, O'Keefe M, et al. Cyp12a4 confers lufenuron resistance in a natural population of Drosophila melanogaster. Proc. Natl. Acad. Sci. 102: 12807-12812, 2005. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254, 1976. Butt T, Coates C, Dubovskiy I, Ratcliffe N. Entomopathogenic fungi: new insights into host–pathogen interactions. Adv. Genet. 94: 307-364, 2016. Caballero RJ, Hoshi T, Kashyap AK. Zombie lending and depressed restructuring in Japan. Am. Econ. Rev. 98: 1943-1977, 2008. Cao G, Jia M, Zhao X, Wang L, Tu X, Wang G, et al. Different effects of Metarhizium anisopliae strains IMI330189 and IBC200614 on enzymes activities and hemocytes of Locusta migratoria L. PloS One 1: e0155257, 2016. Damayanthi B, Karunaratne S. Biochemical characterization of insecticide resistance in insect pests of vegetables and predatory ladybird beetles. J. Natn. Sci. Foundation of Sri Lanka, 33: 115-122, 2005. Dubovskiy I, Slyamova N, Kryukov VY, Yaroslavtseva O, Levchenko M, Belgibaeva A, et al. The activity of nonspecific esterases and glutathione-S-transferase in Locusta migratoria larvae infected with the fungus Metarhizium anisopliae (Ascomycota, Hypocreales). Entomol. Rev. 92: 27-31, 2012. El-Mergawy R, Al-Ajlan A. Red palm weevil, Rhynchophorus ferrugineus (Olivier): economic importance, biology, biogeography and integrated pest management. J. Agric. Sci. Technol. 1: 1-23, 2011. Ellman GL, Courtney KD, Andres JrV, Featherstone RM. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7: 88-95, 1961. Faleiro J. A review of the issues and management of the red palm weevil Rhynchophorus ferrugineus (Coleoptera: Rhynchophoridae) in coconut and date palm during the last one hundred years. Int. J. Trop. Insect Sci. 26: 135- 154, 2006. Faleiro J, Abdallah AB, El-Bellaj M, Al-Ajlan A, Oihabi A. Threat of the red palm weevil, Rhynchophorus ferrugineus (Olivier) to date palm plantations in North Africa. Arab. J. Plant Prot. 30: 274-280, 2012. Fan J, Xie Y, Xue J, Liu R. The effect of Beauveria brongniartii and its secondary metabolites on the detoxification enzymes of the pine caterpillar, Dendrolimus tabulaeformis. J. Insect Sci. 13: 44-57, 2013. FAO, Food and Agriculture Organization of the United Nations. Food and agricultural commodities production for Pakistan for 2012. www. faostat.fao.org/DesktopDefault.aspx?PageID=3 39&lang=en&country=16 5, 2014 Farooq M, Steenberg T, Højland DH, Freed S, Kristensen M. Impact of sequential exposure of Beauveria bassiana and imidacloprid against susceptible and resistant strains of Musca domestica. BioControl 63: 707-718, 2018. Ferry M, Gomez S. The red palm weevil in the Mediterranean area. Palms, 46: 172-178, 2002. Gindin G, Levski S, Glazer I, Soroker V. Evaluation of the entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana against the red palm weevil Rhynchophorus ferrugineus. Phytoparasitica, 34: 370-379, 2006. Güerri‐Agulló B, Gómez‐Vidal S, Asensio L, Barranco P, Lopez‐Llorca LV. Infection of the red palm weevil (Rhynchophorus ferrugineus) by the entomopathogenic fungus Beauveria bassiana: a SEM study. Microsc. Res. Tech. 73: 714-725, 2010. Hajek A, St Leger R. Interactions between fungal pathogens and insect hosts. Annu. Rev. Entomol. 39: 293-322, 1994. 118 Hanounik S. Steinernematids and Heterorhabditids as biological control agents for red palm weevil (Rhynchophorus ferrugineus Oliv.). J. Agri. Mar. Sci. 3: 95-102, 1998. Jun Z, Dunlun S, Jianxin C. Physiological and biochemical changes of the silkworm, Bombyx mori infected by Cordyceps militaris. Kun Chong xue bao. Acta Entomol. Sin. 46: 674- 678, 2003. Kehat M. Threat to date palms in Israel, Jordan and the Palestinian Authority, by the red palm weevil, Rhynchophorus ferrugineus. Phytoparasitica, 27: 241-242, 1999. Klein MG, Lacey LA. An attractant trap for autodissemination of entomopathogenic fungi into populations of the Japanese beetle Popillia japonica (Coleoptera: Scarabaeidae). Biocontrol Sci. Technol. 9: 151-158, 1999. Milosavljević I, El-Shafie HA, Faleiro JR, Hoddle CD, Lewis M, Hoddle MS. Palmageddon: the wasting of ornamental palms by invasive palm weevils, Rhynchophorus spp. J. Pest Sci. 92: 143-156, 2019. Molet T, Roda A, Jackson L. CPHST Pest Datasheet for Rhynchophorus ferrugineus. USDA-APHIS-PPQ-CPHST. Revised Mar 2014, 2011. Moorhouse E, Gillespie A, Charnley A. Laboratory selection of Metarhizium spp. isolates for control of vine weevil larvae (Otiorhynchus sulcatus). J. Invertebr. Pathol. 62: 15-21, 1993. Naeem A, Freed S, Akmal M. Biochemical analysis and pathogenicity of entomopathogenic fungi to Diaphorina citri Kuwayama (Hemiptera: Liviidae). Entomol. Res. 50: 245-254, 2020. Nathan SS, Choi MY, Seo HY, Paik CH, Kalaivani K, Kim JD. Effect of azadirachtin on acetylcholinesterase (AChE) activity and histology of the brown planthopper Nilaparvata lugens (Stål). Ecotoxicol. Environ. Saf. 70: 244-250, 2008. Ricaño J, Güerri-Agulló B, Serna-Sarriás MJ, Rubio- Llorca G, Asensio L, Barranco P, et al. Evaluation of the pathogenicity of multiple isolates of Beauveria bassiana (Hypocreales.: Clavicipitaceae) on Rhynchophorus ferrugineus (Coleoptera: Dryophthoridae) for the assessment of a solid formulation under simulated field conditions. Fla. Entomol. 96: 1311-1324, 2013. Rizvi SAH, Ling S, Tian F, Xie F, Zeng X. Toxicity and enzyme inhibition activities of the essential oil and dominant constituents derived from Artemisia absinthium L. against adult Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Psyllidae). Ind. Crops Prod. 121: 468-475, 2018. Sandhu SS, Sharma AK, Beniwal V, Goel G, Batra P, Kumar A, et al. Myco-biocontrol of insect pests: factors involved, mechanism, and regulation. J. Pathog. pp 1-10, 2012. Serebrov V, Gerber O, Malyarchuk A, Martemyanov V, Alekseev A, Glupov V. Effect of entomopathogenic fungi on detoxification enzyme activity in greater wax moth Galleria mellonella L.(Lepidoptera, Pyralidae) and role of detoxification enzymes in development of insect resistance to entomopathogenic fungi. Biol. Bull. 33: 581–586, 2006. Suma P, Pergola L, Alessandra L, Santi S, Victoria. The use of sniffing dogs for the detection of Rhynchophorus ferrugineus. Phytoparasitica, 42: 269-274, 2014. Tavakolian MS, Silaghi FA, Fabbri A, Molari G, Giunchi A, Guarnieri A, Differentiation of post harvest date fruit varieties non-destructively using FT-NIR spectroscopy. Int. J. Food Sci. Technol. 48: 1282-1288, 2013. Vidhya D, Rajiv P, Padmanabhan N. Impact of entamopathogenic fungal infection on the detoxifying enzyme in cotton leaf worm Spodoptera litura (Fabricius). Int. J. Pharm. BioSci. 7: 943-948, 2016. Wakil W, Faleiro JR, Miller TA. Sustainable pest management in date palm: Current status and emerging challenges. Springer International Publishing AG, Switzerland, 2015. Wang JJ, Cheng WX, Ding W, Zhao ZM. The effect of the insecticide dichlorvos on esterase activity extracted from the psocids, Liposcelis bostrychophila and L. entomophila. J. Insect Sci. 4: 1-5, 2004. Wattanapongsiri A. A revision to the genera Rhynchophorus and Dynamis (Coleoptera: Curculionidae) 1965. Xia Y, Dean P, Judge A, Gillespie J, Clarkson J, Charnley A. Acid phosphatases in the haemolymph of the desert locust, Schistocerca gregaria, infected with the entomopathogenic fungus Metarhizium anisopliae. J. Insect Physiol. 46: 1249-1257, 2000. Zibaee A, Bandani AR, Tork M. Effect of the entomopathogenic fungus, Beauveria bassiana, and its secondary metabolite on detoxifying enzyme activities and acetylcholinesterase (AChE) of the sunn pest, Eurygaster integriceps (Heteroptera: Scutellaridae). Biocontrol Sci. Technol. 19: 485-498, 2009a. Zibaee A, Jalali Sendi J, Ghadamyari M, Alinia F, Etebari K. Diazinon resistance in different selected strains of Chilo suppressalis (Lepidoptera: Crambidae) in northern Iran. J. Econ. Entomol. 102: 1189-1196, 2009b.