Bioactivity and biochemical efficacy of chitinase and Justicia brandegeana extract against Red Palm Weevil Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae)

Abstract The red palm weevil Rhynchophorus ferrugineus is a large polyphagous insect, and this study was carried out to isolate chitinase from Beauveria bassiana as well as phytochemical screening of Justicia brandegeana to elucidate its effect as biocontrol agents against the red palm weevil and its possible effect on enzymatic bioactivity. It is the first time that the lipoid constituents of J. brandegeana were examined by both gas–liquid chromatography (GLC) and gas chromatography–mass spectrometry (GC/Mass). The results showed that the highest rates of mortality in treated prepupae were 35.0% and 30.0% with the higher concentration of chitinase (25 ppm) and petroleum ether extract of J. brandegeana (1,200 ppm), respectively. Moreover, changes in enzyme activity of ALP, PO, GPT, and GOT in the prepupal stage after treatment with LC50 chitinase and J. brandegeana extract were 36.63 & 14.32, −21.99 & 41.20, −11.02 & 47.05, and −36.00 & 21.43% compared with untreated control, respectively. This study demonstrated effectiveness of chitinase, and the petroleum ether extract of J. brandegeana has potent effect against Rh. ferrugineus due to its disturbance effect on the enzymatic system, protein as well as DNA damage.

Chitinases are the enzymes that breaking down glycosidic bonds in chitin; hydrolytic enzyme (Jollès & Muzzarelli, 1999). Thus, chitinobiosidases cleave diacetylchitobiose units from nonreducing end of the chitin chain and disaccharides released. While, endochitinases cleave glycosidic linkages along the chitin chain producing low molecular mass of oligomers; chitotrioses and diacetylchitobioses (Guthrie, Khalif, & Castle, 2005). Whereas, chitinases of Eukaryotes, fungal chitinase, plant chitinase, and endo-beta-N-acetylglucosaminidases sharing weak amino acid sequence at the certain regions of each enzyme. These regions may assume to be important for catalytic activities of the enzymes (Watanabe et al., 1993). Therefore, previous research by Mubarik, Mahagiani, Anindyaputri, Santoso, and Rusmana (2010) showed that the chitinase of some fungus strain showed their ability to degrade exoskeleton chitin of the whitefly.
Also, it can be used for controlling leaf miner such as aphis gossypii.
Moreover, study of Moussa, Shehawy, Baiomy, Taha, and Ahmed (2014) was isolated chitinase protein of 55kD from B. bassiana and concluded for partially extraction and purification of chitinase enzyme which had a significant potency in two aphid species compared with chemical insecticide Pirimicarb.
Recently, plant extracts used widely in pest control as a safe tool to the environment. Lignans from Justicia. flava were reported to cause ataxia, decrease in motor activity, and decreases muscular tone (Navarro, Alonso, & Navarro, 2011). Furthermore, the elenoside which isolated from leaves of Justicia hyssopifolia showed decrease in the muscular tone at doses of 25, 50, and 100 mg/kg in mice.
On the other hand, many enzymes involved in detoxification; alkaline phosphatase (ALP) is the common hydrolytic enzyme, which play an important role in hydrolyzation of phosphomonoesters under alkaline conditions. ALP is used as enzyme marker for brush border membrane (Wolfersberger, 1984) and is indicator for tissue cytolysis during the insect development (Dadd, 1970). Peganum harmala, Senna alexandrina extract showed an increase in Alkaline phosphatase against Aphis craccivora compared with the control (Shehawy, Khalil, Maklad, & Qari, 2019). Glutamine pyruvic transaminase (GPT) and glutamic oxaloacetic transaminase (GOT) are known as alanine transaminase (ALT) aspartic transferase (AST), respectively. The transaminases are the key enzymes in the process of gluconeogenesis, nonessential amino acids formation, nitrogen compound metabolism, and mainly associated with protein metabolism (Mordue & Goldworthy, 1973). Phenol oxidase (PO) convert phenols to quinones that subsequently polymerize to form melanin in melanogenesis (Söderhäll & Cerenius, 1998). Strengthening of the immune system of insects occurred by increased PO activity to challenge xenobiotics and healing, and phenol oxidase is an important tool against numerous pathogens (Cerenius & Söderhäll, 2004).
During the infection of desert locust by Metarhizium anisopliae, the activity of the enzyme phenoloxidase decreased (Gillespie, Burnett, & Charnley, 2000). Clarifying the role of PO in the immune response of insects to fungi is important in the efficient use of entomopathogenic fungi as biocontrol agents (González-Santoyo & Córdoba-Aguilar, 2012). Accordingly, the current study aimed to isolate chitinase from B. bassiana as well as Phytochemical Screening of J. brandegeana to elucidate its effect as biocontrol agents against Rh. ferrugineus (RPW) and its possible effect on enzymatic bioactivity of ALP, PO, GPT, and GOT as well as on DNA and protein content.

| Fungal growth and chitinase extraction
For chitinase preparation, Beauveria bassiana strain brought from central laboratory, Aljumum University College, Umm Al-Qura University, Saudi Arabia. B. bassiana inoculated into Soluble-Yeast Extract (SYG, pH 6.0) medium and then held on shaking incubator at 150 rpm and 25 ± 1°C for three days. Finally, the chitinase precipitate was collected according to salting out method described by Kim and Je (2010); broth culture centrifuged at 16,000 g at 4°C for 10 min.
After that, the supernatant incorporated with ammonium sulfate crystals 70% (w/v) with stirring until saturation then held overnight at 4°C. and then centrifuged at 16,000 g at 4°C for 10 min. Finally, pellet (chitinase) was dissolved in buffer (0.1 M citrate-phosphate (pH 6)) then filtrated into 0.2 μm sterile filter to ensure its purity.

| Chitinase determination
In order to determine the concentration and molecular weight of chitinase enzyme, the method described by Kim and Je (2010) was performed. The concentration of the protein was determined as described by (Bradford, 1976) using BioRad assay, using Bovine Serum Albumin (BSA) as a standard. The absorbance was registered on UNICO UV-2000 spectrophotometer at 595 nm. After that, the concentration was calculated, then stock solution was stored at −20°C for bioassay experiments.

| Preparation of the petroleum ether extract (lipoidal matter)
The air-dried powder (500 g) of leaves of the plant under investigation was extracted with petroleum ether (60-80°C) in continuous extraction apparatus. The extract was evaporated to dry under vacuum and weighed (7.25 g).

| Fractionation of lipoid matter extract
0.2 g of the extract was taken, and 40 ml KOH 20% was added; the sample was saponified over night at room temperature and then extracted with ether (petroleum ether 40-60°C) in to two layers as follows: 1. Saponifiable matter (ether extract): add few ml of MeOH, then add few ml H 2 SO 4 to get red color, then add few ml of petroleum ether, then washed the petroleum ether extract (H 2 O-NaCl 10%), then dried over anhydrous sodium sulfate, and then make methylation process by CH 2 N 2 .

| Gas Chromatography-Mass Spectrometry (GC/Mass) of fatty acids
The qualitative identification of the different contents of Justcia brandegeana leaves was calculated by comparison of their relative times and mass spectra with those of authentic reference compound (fatty acid methyl ester, purity 98% by GC), and carried out by central Service Laboratory, Faculty of Agriculture, Cairo University, Giza.

| Insect rearing
Adult stages of Rh. ferrugineus (Red palm weevil) were collected from infected date palm tree (Al-Qaseem area, Saudi Arabia). Adults set in groups for mating in the laboratory in small plastic cages (30 × 50 × 50 cm) approximately, on its preferable diet which called sugarcane in order to complete life cycle and giving eggs and larvae under laboratory relative humidity (80 ± 10% R.H.) and temperature of (28 ± 2°C) on sugarcane according to Hussain, Rizwan-ul-Haq, Al-Ayedh, Ahmed, and Al-Jabr (2015).

| Larvae exposure
Each ten prepupal larvae will be placed in plastic poxes lined with filter paper, topically treated individually with different serial doses of the plant extract (J. brandegeana) as well as Chitinase and transferred to boxes (one larva per plastic box 35 × 20×30 mm) compared with untreated control. The boxes incubation at 27°C in darkness for 1-2 weeks. The bioassay will be repeated ten times, and the results will be calculated.

| Enzyme sample preparations for the determination of the ALP, PO, GPT and GOT activities
The hemolymph was collected from prepupal instar after 48-hr treatment. 0.1 ml of hemolymph was drawn into Eppendorf containing phenol oxidase inhibitor (few milligrams) to prevent tanning, then diluted with saline solution 0.7%. In order to rupture the hemocytes, the diluted hemolymph was frozen for 20 s. After that, the hemolymph specimen was centrifuged at 3200 g for 5 min at 4°C. Then, the supernatant was used for assay directly.

| Fat body
Fat bodies were collected from prepupal instar after 48-hr treatment and then homogenized in a saline solution (the fat body of one insect/1 ml saline solution 0.7%) using an electric homogenizer for 2 min. Then, the homogenates were centrifuged at 4,000 rpm for 15 min at 4°C. The supernatant was frozen at −20°C until use.
For biochemical investigation, ALP activity was evaluated according to Klein, Read, and Babson (1960) and the enzyme activity was recorded using spectrophotometer at 550 nm. Phenol oxidase (PO) activity was determined using the method of Oppenoort and Welling (1976). 20 μL of samples was added to microplate wells containing 180 μL 10 mmol/L catechol. At 27°C, the reaction was measured every 1 min for an hour at 420 nm. The activity of the enzyme was measured as the absorbance change rate per min. GOT and GPT activity was measured according to Harold (1975), and the enzyme activity was recorded at 546 nm using spectrophotometer. The protein content estimation was conducted according to Qari and Shehawy (2020). Bovine serum albumin (BSA) was used as a standard. DNA was extracted as Baeshin, Qari, and Sabir

| Data analysis
For bioassays, LdP-Line ® software was used to determine the median lethal concentration (LC 50 ) by probity analysis. SPSS software was used in analysis of variance (ANOVA) of ALP, PO, GPT, and GOT bioactivity in prepupal instar. Comparison among means carried out using the least significant difference test at p < .05.

| Preliminary phytochemical screening
The phytochemical screening results were illustrated in Table 1. As shown in Table 1, it can be concluded that the leaves of J. brandegeana contain carbohydrates, glycosides, coumarins, saponins, flavonoids, phenolics, volatile oils, and triterpenes, whereas anthraquinones was absent.

| Investigation of lipoidal matter
There is no information about investigation of lipoidal matter of Justicia brandegeana before, so it was deemed of interest to investigate the lipoidal content of this plant. As presented in Table 2, the total lipoidal matter separated was representing 1.45% among which 62% unsaponifiable matter and 27.5% representing the total fatty acids.

| GLC of unsaponifiable matter
Data illustrated in Table 3 and Figure 1 show the relative retention time (RRT) of separated compounds as well as their relative percentages. Also, results of GLC of unsaponifiable matter in Table 3 Confirmed that the percentage of total hydrocarbon compound representing 63.311%; (Heneicosane (C 21 ) is the major component (43.824%) in hydrocarbon). Whereas, the percentage of total sterol is 36.688%; (Stigmasterol is the major component (19.156%) in sterol).
While, unidentified compounds representing 1.53% of the oil.

| Insect bioassay
As shown in

| Enzyme activity
In general, it was found that the activities of ALP, PO, GPT, and GOT are greater in hemolymph than in fat body in untreated control larvae of Rh. ferrugineus.

| Alkaline phosphatase
As shown in Table 6

| Phenol oxidase (PO) activity
Moreover, as shown in

| Glutamic pyruvic transaminase (GPT)
Results illustrated in Table 7 showed that the GPT activity after treat-

| Glutamic oxaloacetic transaminase activity (GOT)
As shown in

| DNA and total protein
Results illustrated in

| Phytochemical screening for Justicia brandegeana
As mentioned before the plants are the most common sources of potential compounds which may have toxic or therapeutic effect. Many phytochemicals have been shown to be active against may resistant pathogenic bacteria (Gopalakrishnan, George, & Benny, 2010 (Kavitha et al., 2014). Moreover, Bachheti, Pandey, Archana, and Vikas (2011)

| Biological activity of chitinase and J. brandegeana against red palm weevil larvae
Chitinases considered as the key chitin degradation enzymes, which regulate the growth and development of the insect. These are the potential target compound for insect pest management.
In the current study, it was found that chitinase has toxic effect against red palm weevil larvae by degrading the cuticle of it, and this is the first study exhibiting the results of Chitinase and J. brandegeana extract against red palm weevil larvae. However, the current study shows the great potential effect of chitinase and J. brandegeana extract against red palm weevil insect. On the other hand, the results of this study are in the same line or agree with those of previous studies recorded against various insect species, chitinases are generally found in many organisms which was needs to reshape their own chitin during molting process (Jump up Sámi et al., 2001), or dissolve and digest the chitin of insects.

| Enzymatic activity in Red Palm Weevil Larvae after treatment with of chitinase and J. brandegeana
Alkaline Phosphatase enzyme increased by chitinase in both tissues and plant extract with no exception according to the concentration which were used. Also, PO increase is indicator for tissues cytolysis during the insect development (Dadd, 1970). In our study, Alkaline ing (Chang, Rahmawaty, & Chang, 2013). Phenol oxidase is an important tool against numerous pathogens (Cerenius & Söderhäll, 2004).).
During the infection of desert locust by M. anisopliae, the activity of the enzyme phenol oxidase decreased (Gillespie et al., 2000).
As before, Glutamic pyruvic transaminase (GPT) and Glutamic oxaloacetic transaminase activity (GOT) decreased after treatment with chitinase may be due to that the transaminases are the key enzymes in the process of gluconeogenesis, nonessential amino acids formation, nitrogen compound metabolism and mainly associated with protein metabolism problem as reported by Mordue and Goldworthy (1973). While, the increase in GPT after treatment with Note: Conc. concentration; mean ± SD followed with the same letter is not significantly different (p > .05), mean ± SD followed with the different letter is not significantly different (p < .05).
J. brandegeana extract is in agree with that of Ghoneim, Hamadah, and El-Hela (2016a)  with that of Mótyán, Tóth, and Tőzsér (2013) who reported that, the decrease in total protein was occurred due to the increase of Proteolytic enzymes which required for insecticides detoxification.
Also, changes in the protein content reflect changes in synthesis and degradation balance in functional and structural protein during metamorphosis and pesticide detoxification (Ghoneim et al., 2014).
Meanwhile, DNA content decreased significantly after treatment with LC 50 of the different natural compounds against R. dominica, due to DNA damage by such extracts compared with the control (Qari, Abdel-Fattah, & Shehawy, 2017). Note: Conc. concentration; mean ± SD followed with the same letter is not significantly different (p > .05), mean ± SD followed with the different letter is not significantly different (p < .05), activity ration = protein in treated stage/ protein in untreated stage.

| CON CLUS ION
This work was supported financially by the Deanship of Scientific Research at Umm Al-Qura University (Grant cod: 18-SCI-1-01-0005).

CO N FLI C T O F I NTE R E S T
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Sameer H. Qari https://orcid.org/0000-0001-9424-5217